Canine antibody against human NGF

Abstract

The present invention provides caninized anti-human NGF antibodies that have high binding affinity for canine NGF. The present invention also relates to the use of these antibodies in the treatment of pain in dogs and other companion animals.

Description

[Technical field]

[0001] REFERENCE TO ELECTRONICALLY SUBMITTED SEQUENCE LISTING This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML file was created on December 6, 2022 and is named 25370.xml. This Sequence Listing, submitted via EFS-Web, is a part of the present specification and is incorporated herein by reference in its entirety.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 USC § 119(e) to U.S. Provisional Patent Application No. 63 / 327,076, filed April 4, 2022, and U.S. Patent Application No. 63 / 290,264, filed December 16, 2021, the subject matter of which is incorporated herein by reference in its entirety.

[0003] The present invention relates to an antibody against a protein involved in pain.More specifically, the present invention further relates to a caninized antibody against human NGF that has high binding affinity to canine NGF.The present invention also relates to the use of the antibody of the present invention in the treatment of pain in dogs, including dogs with osteoarthritis. [Background technology]

[0004] Nerve growth factor (NGF) is a well-characterized secreted protein that plays an important role in the development of the nervous system. In addition, NGF has also been shown to have biological effects on tissues, including non-neuronal cells and cells of the immune system. NGF was initially isolated in mouse submandibular glands as a complex composed of three non-covalently linked subunits. While the alpha and gamma subunits of NGF belong to the kallikrein family of serine proteases, the beta subunit of the NGF complex exhibits the biological activity attributed to NGF. NGF (also called beta-NGF) is produced as a prepropeptide with an 18 amino acid residue signal peptide [Wiesmann and de Vos, CLMS:58,748-759,(2001)]. Recombinant human beta-NGF is a homodimer of two 120 amino acid polypeptides. The C-terminal 120 amino acids of human NGF have approximately 98% homology to the predicted C-terminus of NGF from other species, including dogs and cats.

[0005] Many studies have shown that NGF plays an important role in pain transmission. For example, in humans, NGF levels are elevated in synovial fluid from patients with some arthritic conditions [Aloe, et al., Arch. Rheum., 35: 351-355 (1992)]. Furthermore, elevated levels of canine NGF expression have been found in the synovial fluid of dogs with osteoarthritis [Isola, et al., Vet Comp. Orthop. Traumatol., 4: 279 (2011)]. It has also been demonstrated that agents that inhibit the function of NGF, such as neutralizing antibodies, prevent hyperalgesia and allodynia in animal models of neuropathic pain [see, for example, Ramer et al., Eur. J. Neurosci. 11: 837-846 (1999) and Ro et al., Pain, 79: 265-274 (1999)]. The recognition that NGF is involved in pain transmission in certain inflammatory and non-inflammatory conditions, such as osteoarthritis and cancer, has led to interest in developing antibodies that can neutralize the biological activity of NGF [examples of anti-NGF antibodies known in the art include WO 01 / 78698, WO 01 / 64247, WO 02 / 096458, U.S. Pat. No. 7,601,818 B2, and Gearing et al., BMC Veterinary Research, 9:226, (2013)].

[0006] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO 01 / 78698 [Patent Document 2] WO 01 / 64247 [Patent Document 3] WO 02 / 096458 [Patent Document 4] US Patent No. 7,601,818 B2 [Non-patent literature]

[0008] [Non-Patent Document 1] Wiesmann and de Vos,CLMS:58,748-759,(2001) [Non-Patent Document 2] Aloe,et al.,Arch.Rheum.,35:351-355(1992) [Non-Patent Document 3] Isola,et al.,Vet Comp.Orthop.Traumatol.,4:279(2011) [Non-Patent Document 4] Ramer et al.,Eur.J.Neurosci.11:837-846(1999) [Non-Patent Document 5] Ro et al.,Pain,79:265-274(1999) [Non-Patent Document 6] Gearing et al.,BMC Veterinary Research,9:226,(2013) Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention relates to a caninized anti-human nerve growth factor (NGF) antibody, which not only has specific binding affinity to canine NGF, but also has the ability to block the binding of canine NGF to canine NGF receptor.The present invention includes the use of such an antibody in the treatment of hyperalgesia and allodynia in animals.The antibody can also be used to treat pain in dogs with osteoarthritis.

[0010] Thus, the present invention provides a novel caninized antibody or antigen-binding fragment thereof capable of binding and neutralizing canine NGF, wherein the caninized antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain. The heavy chain of the caninized antibody comprises a variable region (VH) and three constant regions including a canine fragment crystallizable region (cFc or cFc region). The light chain also comprises a variable region (VL), but has only one constant region. Each of the variable regions of the heavy and light chains comprises three hypervariable regions, i.e., complementarity determining regions (CDRs). Thus, the light chain comprises three light chain complementarity determining regions (CDRs), each comprising an amino acid sequence: CDR light 1 (CDRL1), CDR light 2 (CDRL2), and CDR light 3 (CDRL3), and the heavy chain comprises three heavy chain CDRs, each comprising an amino acid sequence: CDR heavy 1 (CDRH1), CDR heavy 2 (CDRH2), and CDR heavy 3 (CDRH3). CDRH1 comprises the amino acid sequence of SEQ ID NO:1, CDRH2 comprises the amino acid sequence of SEQ ID NO:2, and CDRH3 comprises the amino acid sequence of SEQ ID NO:3, whereas CDRL1 comprises the amino acid sequence of SEQ ID NO:4, CDRL2 comprises the amino acid sequence of SEQ ID NO:5, and CDRL3 comprises the amino acid sequence of SEQ ID NO:6.

[0011] The caninized antibody also comprises a hinge region. The hinge region is preferably a canine hinge region. In certain embodiments, the hinge region comprises an amino acid sequence that comprises at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 45. In other embodiments, the hinge region comprises an amino acid sequence that comprises at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 46. In yet other embodiments, the hinge region comprises an amino acid sequence that comprises at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 47. In yet other embodiments, the hinge region comprises an amino acid sequence that comprises at least 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 48. The invention further provides antigen-binding fragments of all of these antibodies.

[0012] The caninized antibodies of the present invention comprise a canine fragment crystallizable region (cFc region). In one embodiment, the canine cFc region comprises an amino acid sequence that comprises at least 90%, 95%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 49. In another embodiment, the canine cFc region comprises an amino acid sequence that comprises at least 90%, 95%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 50. In yet another embodiment, the canine cFc region comprises an amino acid sequence that comprises at least 90%, 95%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 52. In yet another embodiment, the canine cFc region comprises an amino acid sequence that comprises at least 90%, 95%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 53. In yet another embodiment, the canine cFc region is an IgG-Bm comprising an amino acid sequence comprising at least 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:20 or SEQ ID NO:51, wherein both the aspartic acid residue (D) at position 31 of SEQ ID NO:50 and the asparagine residue (N) at position 63 of SEQ ID NO:50 are substituted with an alanine residue (A). The invention further provides all antigen-binding fragments of these antibodies.

[0013] In certain embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27. In other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 28. In related embodiments, the caninized antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 29. In other related embodiments, the caninized antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30. The invention further provides antigen-binding fragments of all of these antibodies.

[0014] In certain embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27, and the caninized antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 29. In other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27, and the caninized antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30. In yet other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 28, and the caninized antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 29. In yet other embodiments, the caninized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 28, and the caninized antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30. The invention further provides antigen-binding fragments of all of these antibodies.

[0015] In some embodiments, the caninized antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 38. In other embodiments, the caninized antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 39. In yet other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 36. In yet other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 37. The invention further provides antigen-binding fragments of all of these antibodies.

[0016] In certain embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and comprises a light chain comprising the amino acid sequence of SEQ ID NO: 38. In other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and comprises a light chain comprising the amino acid sequence of SEQ ID NO: 39. In yet other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 37 and comprises a light chain comprising the amino acid sequence of SEQ ID NO: 38. In yet other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 37 and comprises a light chain comprising the amino acid sequence of SEQ ID NO: 39. The invention further provides antigen-binding fragments of all of these antibodies.

[0017] The present invention also provides nucleic acids, including isolated nucleic acids, encoding any of the caninized antibodies and antigen-binding fragments thereof of the present invention. Thus, the present invention provides nucleic acids (including isolated nucleic acids) encoding any one of the light chain variable regions of the caninized antibodies of the present invention. The present invention also provides nucleic acids encoding any one of the light chains of the caninized antibodies of the present invention. Similarly, the present invention further provides nucleic acids encoding any one of the heavy chain variable regions of the caninized antibodies of the present invention. In addition, the present invention further provides nucleic acids encoding any one of the heavy chains of the caninized antibodies of the present invention. The present invention further provides nucleic acids encoding any one of the antigen-binding fragments of the antibodies of the present invention. In certain embodiments, the nucleic acid encodes a light chain comprising the amino acid sequence of SEQ ID NO: 38. In other embodiments, the nucleic acid encodes a light chain comprising the amino acid sequence of SEQ ID NO: 39. In related embodiments, the nucleic acid encodes a heavy chain comprising the amino acid sequence of SEQ ID NO: 36. In other embodiments, the nucleic acid encodes a heavy chain comprising the amino acid sequence of SEQ ID NO: 37. The present invention further provides nucleic acid pairs, one of the nucleic acid pairs comprising a nucleotide sequence encoding a heavy chain of a particular caninized antibody of the present invention, and the other of the nucleic acid pairs comprising a nucleotide sequence encoding a light chain of said particular caninized antibody.

[0018] Thus, the present invention provides nucleic acids encoding the heavy chain variable region of a caninized antibody or antigen-binding fragment thereof; the heavy chain of a caninized antibody or antigen-binding fragment thereof, the light chain variable region of a caninized antibody or antigen-binding fragment thereof, and / or the light chain of a caninized antibody or antigen-binding fragment thereof. The present invention further provides a nucleic acid pair, one of which comprises a nucleotide sequence encoding a light chain of a specific caninized antibody of any one of the antibodies of the present invention, and the other of which comprises a nucleotide sequence encoding a heavy chain of that (said) specific caninized antibody. The present invention also provides expression vectors comprising such a nucleic acid pair, or individual nucleic acids of the present invention. In addition, the present invention provides an expression vector pair, one of which comprises a nucleic acid comprising a nucleotide sequence encoding a light chain of a specific caninized antibody of any one of the caninized antibodies of the present invention, and the other of which comprises a nucleic acid comprising a nucleotide sequence encoding a heavy chain of that (said) specific caninized antibody. Thus, the present invention provides nucleic acids encoding the heavy chain variable region of a caninized antibody or antigen-binding fragment thereof of the present invention. The present invention further provides a nucleic acid encoding a heavy chain of a caninized antibody or antigen-binding fragment thereof of the present invention. The present invention also provides a nucleic acid encoding a light chain variable region of a caninized antibody or antigen-binding fragment thereof of the present invention. The present invention also provides a nucleic acid encoding a light chain of a caninized antibody or antigen-binding fragment thereof of the present invention. In certain embodiments, a nucleic acid encoding a heavy chain variable region encodes the heavy chain of a caninized antibody, and a corresponding nucleic acid encoding a light chain variable region encodes the light chain of the caninized antibody.

[0019] The present invention further provides a pair of nucleic acids encoding a set of three heavy chain CDRs and a corresponding set of three light chain CDRs. In certain embodiments, the nucleic acid encoding the set of three heavy chain CDRs encodes the heavy chain variable region of a caninized antibody, and the corresponding nucleic acid encoding the set of three light chain CDRs encodes the light chain variable region of the (said) caninized antibody. The present invention also provides a kit comprising the two nucleic acid pairs. In certain embodiments, the nucleic acid encoding the set of three heavy chain CDRs encodes the heavy chain of a caninized antibody, and the corresponding nucleic acid encoding the set of three light chain CDRs encodes the light chain of the caninized antibody.

[0020] In certain embodiments, the nucleic acid encodes a caninized antibody heavy chain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 3. In a related embodiment, the nucleic acid encodes a caninized antibody light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 6. The invention further provides a pair of nucleic acids encoding a caninized antibody heavy chain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 3, and a pair of nucleic acids encoding a caninized antibody light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 6. The invention also provides a kit comprising the two nucleic acid pairs.

[0021] In certain embodiments, a nucleic acid of the invention encodes a heavy chain variable region of a caninized antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 27. In a related embodiment, a nucleic acid of the invention encodes a light chain variable region of a caninized antibody or antigen-binding fragment thereof, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 29. The invention further provides a nucleic acid pair, one of the nucleic acid pair comprising a nucleotide sequence encoding a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27, and the other of the nucleic acid pair comprising a nucleotide sequence encoding a light chain variable region comprising the amino acid sequence of SEQ ID NO: 29. The invention also provides a kit comprising the two nucleic acid pairs. In certain embodiments, the nucleic acid encoding the heavy chain variable region encodes the heavy chain of a caninized antibody, and the corresponding nucleic acid encoding the light chain variable region encodes the light chain of the caninized antibody.

[0022] In other embodiments, the nucleic acid of the invention encodes a heavy chain variable region of a caninized antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 27. In a related embodiment, the nucleic acid of the invention encodes a light chain variable region of a caninized antibody or antigen-binding fragment thereof, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 30. The invention further provides a nucleic acid pair, one of the nucleic acid pair comprising a nucleotide sequence encoding a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27, and the other of the nucleic acid pair comprising a nucleotide sequence encoding a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30. The invention also provides a kit comprising the two nucleic acid pairs. In certain embodiments, the nucleic acid encoding the heavy chain variable region encodes the heavy chain of a caninized antibody, and the corresponding nucleic acid encoding the light chain variable region encodes the light chain of the caninized antibody.

[0023] In other embodiments, the nucleic acid of the invention encodes a heavy chain variable region of a caninized antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 28. In a related embodiment, the nucleic acid of the invention encodes a light chain variable region of a caninized antibody or antigen-binding fragment thereof, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 29. The invention further provides a nucleic acid pair, one of the nucleic acid pair comprising a nucleotide sequence encoding a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 28, and the other of the nucleic acid pair comprising a nucleotide sequence encoding a light chain variable region comprising the amino acid sequence of SEQ ID NO: 29. The invention also provides a kit comprising the two nucleic acid pairs. In certain embodiments, the nucleic acid encoding the heavy chain variable region encodes the heavy chain of a caninized antibody, and the corresponding nucleic acid encoding the light chain variable region encodes the light chain of the caninized antibody.

[0024] In yet other embodiments, the nucleic acid of the invention encodes a heavy chain variable region of a caninized antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 28. In a related embodiment, the nucleic acid of the invention encodes a light chain variable region of a caninized antibody or antigen-binding fragment thereof, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 30. The invention further provides a nucleic acid pair, one of the nucleic acid pair comprising a nucleotide sequence encoding a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 28, and the other of the nucleic acid pair comprising a nucleotide sequence encoding a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30. The invention also provides a kit comprising the two nucleic acid pairs. In certain embodiments, the nucleic acid encoding the heavy chain variable region encodes the heavy chain of a caninized antibody, and the corresponding nucleic acid encoding the light chain variable region encodes the light chain of the caninized antibody.

[0025] In yet other specific embodiments, a nucleic acid of the invention encodes a heavy chain of a caninized antibody or antigen-binding fragment thereof, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 36. In a related embodiment, a nucleic acid of the invention encodes a light chain of a caninized antibody or antigen-binding fragment thereof, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 38. The invention further provides a nucleic acid pair, one of the nucleic acid pair comprises a nucleotide sequence encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 36, and the other of the nucleic acid pair comprises a nucleotide sequence encoding a light chain comprising the amino acid sequence of SEQ ID NO: 38. The invention also provides a kit comprising the two nucleic acid pairs.

[0026] In yet other embodiments, a nucleic acid of the invention encodes a heavy chain of a caninized antibody or antigen-binding fragment thereof, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 36. In a related embodiment, a nucleic acid of the invention encodes a light chain of a caninized antibody or antigen-binding fragment thereof, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 39. The invention further provides a nucleic acid pair, one of the nucleic acid pair comprises a nucleotide sequence encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 36, and the other of the nucleic acid pair comprises a nucleotide sequence encoding a light chain comprising the amino acid sequence of SEQ ID NO: 39. The invention also provides a kit comprising the two nucleic acid pairs.

[0027] In certain embodiments, a nucleic acid of the invention encodes a heavy chain of a caninized antibody or antigen-binding fragment thereof, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 37. In a related embodiment, a nucleic acid of the invention encodes a light chain of a caninized antibody or antigen-binding fragment thereof, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 38. The invention further provides a nucleic acid pair, one of the nucleic acid pair comprises a nucleotide sequence encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 37, and the other of the nucleic acid pair comprises a nucleotide sequence encoding a light chain comprising the amino acid sequence of SEQ ID NO: 38. The invention also provides a kit comprising the two nucleic acid pairs.

[0028] In certain embodiments, a nucleic acid of the invention encodes a heavy chain of a caninized antibody or antigen-binding fragment thereof, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 37. In a related embodiment, a nucleic acid of the invention encodes a light chain of a caninized antibody or antigen-binding fragment thereof, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 39. The invention further provides a nucleic acid pair, one of the nucleic acid pair comprises a nucleotide sequence encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 37, and the other of the nucleic acid pair comprises a nucleotide sequence encoding a light chain comprising the amino acid sequence of SEQ ID NO: 39. The invention also provides a kit comprising the two nucleic acid pairs.

[0029] The present invention further provides an expression vector that contains and expresses one or more of the nucleic acids of the present invention. In certain embodiments, the expression vector contains and expresses a nucleic acid encoding a heavy chain of a caninized antibody of the present invention and a nucleic acid encoding a light chain of the caninized antibody. The present invention also provides a host cell that contains one or more expression vectors of the present invention.

[0030] The present invention also provides a pharmaceutical composition comprising a caninized antibody and / or antigen-binding fragment of the antibody and a pharma- ceutically acceptable carrier or diluent. Additionally, a pharmaceutical composition is provided comprising a nucleic acid encoding a heavy chain of a caninized antibody of the present invention, a nucleic acid encoding a light chain of the caninized antibody, and a pharma- ceutically acceptable carrier or diluent. In other embodiments, the pharmaceutical composition comprises a nucleic acid encoding both a heavy chain of a caninized antibody of the present invention and a light chain of the caninized antibody. In yet other embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier or diluent and an expression vector capable of expressing a caninized antibody and / or antigen-binding fragment of the antibody of the present invention in vivo by comprising one or more nucleic acids encoding a heavy chain of a caninized antibody of the present invention and a light chain of the caninized antibody.

[0031] The present invention further provides a method of treating a condition associated with pain in an animal subject. The method of treatment may comprise administering a therapeutically effective amount of a pharmaceutical composition of the present invention to an animal subject in need thereof. In certain embodiments, the method is used to treat osteoarthritis. In other embodiments, the method is used to treat hyperalgesia. In yet other embodiments, the method is used to treat allodynia. In yet other embodiments, the method is used to treat pain. In yet other embodiments, the method is used to treat any combination of osteoarthritis, hyperalgesia, allodynia, and / or pain. The animal subject is preferably a dog.

[0032] The present invention also provides a method for making a caninized antibody or an antigen-binding fragment thereof that binds to canine NGF. In certain embodiments, the method comprises culturing one or more host cells comprising one or more expression vectors of the present invention encoding and expressing the caninized antibody light chain of the present invention and / or the caninized antibody heavy chain under conditions in which the one or more nucleic acids are expressed, thereby producing a polypeptide comprising the caninized antibody light chain of the present invention and the caninized antibody heavy chain. The polypeptide is then recovered from the one or more host cells and / or culture medium. In certain embodiments, a polypeptide comprising the caninized antibody light chain of the present invention and a polypeptide comprising the caninized antibody heavy chain of the present invention are combined with each other under conditions favoring the formation of a caninized antibody.

[0033] The present invention further provides a pair of host cells, one of which contains an expression vector comprising one of a pair of nucleic acids comprising a nucleotide sequence encoding a heavy chain of a particular caninized antibody of the present invention, and the other of the pair of host cells comprises an expression vector comprising the other of the pair of nucleic acids comprising a nucleotide sequence encoding a light chain of said particular caninized antibody. The present invention further provides a method of making a caninized antibody of the present invention that binds to canine NGF, comprising culturing each of the pair of host cells, individually or in combination, in a culture medium under conditions in which the nucleic acid is expressed, thereby producing a polypeptide comprising the caninized antibody light chain, the caninized antibody heavy chain, or both, and then recovering the caninized antibody light chain, the caninized antibody heavy chain, or both, from the pair of host cells or the culture medium.

[0034] These and other aspects of the present invention will be better understood by reference to the following brief description and detailed description of the drawings. [Brief description of the drawings]

[0035] [Figure 1] 1 shows binding of human-canine chimeric furanumab (Ful Chimeric) and caninized furanumab (cFul) antibody variants to canine NGF.

[0036] Ful Chimeric (●), cFulVH1L1 (■), cFulVH1L2 (▲), cFulVH2L1 (▼), cFulVH2L2 (◆), and mab control (○). [Diagram 2] Binding of human-canine chimeric fasinumab (Fas Chimeric) and individual caninized fasinumab (cFas) antibody variants to canine NGF. Fas Chimeric (●), cFasVH2L2 (■), cFasVH2L3 (▲), and mab control (○). [Diagram 3] Binding of canine NGF to the canine TrkA receptor. Binding of canine NGF to the canine NGF receptor (TrkA) was determined by ELISA. Canine NGF (●) [Figure 4] Inhibition of canine NGF binding to the canine TrkA receptor by human-canine chimeric furanumab or individual caninized antibodies. Ful Chimeric (●), cFulVH1L1 (■), cFulVH1L2 (▲), cFulVH2L1 (▼), cFulVH2L2 (◆), and mab control (○). [Diagram 5] Stimulation of TF-1 cell proliferation by canine NGF. [Canine NGF (●)] [Figure 6] Inhibition of TF-1 cell proliferation by human-canine chimeric furanumab (Ful Chimeric) or individual caninized anti-NGF antibodies. Ful Chimeric (●), cFulVH1L1 (■), cFulVH1L2 (▲), cFulVH2L1 (▼), cFulVH2L2 (◆), and mab control (○). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] In response to the demand for better therapy for pain in dogs, the present invention provides a formulation and methodology that can achieve significant effect in reducing pain associated with NGF and / or pain caused by NGF.Therefore, it is surprisingly found that a caninized antibody that comprises a set of CDRs derived from one antibody that was originally raised against human NGF can strongly bind to canine NGF and block the binding of canine NGF to canine TrkA receptor, while a caninized antibody that comprises a set of CDRs derived from another antibody that was originally raised against human NGF cannot measurably bind to canine NGF.This is true even though both corresponding human-canine chimeric constructs can strongly bind to canine NGF.

[0038] Abbreviation The following abbreviations are used throughout the detailed description and examples of the present invention:

[0039] ADCC antibody-dependent cytotoxicity CDC Complement-dependent cytotoxicity CDR Complementarity determining region in an immunoglobulin variable region, as defined using the Kabat numbering system EC50 Concentration that results in 50% efficacy or binding ELISA Enzyme-Linked Immunosorbent Assay FR Antibody framework region: immunoglobulin variable region excluding the CDR regions. IC50 Concentration that produces 50% inhibition IgG Immunoglobulin G Immunoglobulin alignment and numbering system originated by Elvin A. Kabat [Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)] mAb Monoclonal antibody (also Mab or MAb) V region Segments of IgG chains whose sequences are variable among different antibodies VH immunoglobulin heavy chain variable region VL immunoglobulin light chain variable region

[0040] definition So that the present invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0041] As used in this specification, including the appended claims, the singular forms of words such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0042] The "activity" of a molecule can describe or refer to the binding of the molecule to a ligand or receptor, catalytic activity; the ability to stimulate gene expression or cell signaling, differentiation, or maturation; antigenic activity, modulation of the activity of other molecules, and the like. The "activity" of a molecule can also refer to activity that modulates or maintains cell-cell interactions, such as adhesion, or activity that maintains the structure of a cell, such as the cell membrane or cytoskeleton. "Activity" can also mean specific activity, such as [catalytic activity] / [mg protein], or [immune activity] / [mg protein], concentration in a biological compartment, and the like. "Activity" can refer to modulation of a component of the innate or adaptive immune system.

[0043] "Administration" and "treatment," as applied to an animal, e.g., a canine subject, a cell, a tissue, an organ, or a bodily fluid, refer to the contact of an exogenous medicinal, therapeutic, diagnostic, or composition with the animal, e.g., a canine subject, a cell, a tissue, an organ, or a bodily fluid. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid in contact with the cell.

[0044] "Administration" and "treatment" also refer to in vitro and ex vivo treatments, e.g., of a cell, with a reagent, diagnostic, binding compound, or another cell. The term "subject" includes any organism, preferably a non-human animal, more preferably a mammal (e.g., a dog or cat), most preferably a dog.

[0045] "Treat" or "treating" means administering a composition containing a therapeutic agent, such as any of the antibodies of the present invention, internally or externally to a canine subject or patient having or suspected of having one or more symptoms for which the agent has therapeutic activity. Typically, the agent is administered in an amount effective to alleviate and / or ameliorate one or more disease / condition symptoms in the treated subject or population by inducing regression of such symptoms to any clinically measurable extent or by inhibiting the progression of such symptom(s). The amount of therapeutic agent effective to alleviate any particular disease / condition symptom (also referred to as a "therapeutically effective amount") may vary depending on factors such as the disease / condition stage, age and weight of the patient (e.g., dog), and the ability of the pharmaceutical composition to elicit a desired response in the subject. Whether a disease / condition symptom has been alleviated or improved can be assessed by any clinical measurement normally used by a veterinarian or other skilled health care provider to assess the severity or progression of the condition. An embodiment of the invention (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the target disease / condition symptom(s) in all subjects, but it should alleviate the target disease / condition symptom(s) in a statistically significant number of subjects as determined by any statistical test known in the art, e.g., Student's t-test, chi-squared test, Mann-Whitney U test, Kruskal-Wallis test (H test), Jonkheel-Tapstra test, and Wilcoxon test.

[0046] "Treatment," when applied to a veterinary subject (e.g., a dog) or a research subject, refers to therapeutic treatment as well as research and diagnostic uses. When applied to a veterinary subject (e.g., a dog), or a research subject, or a cell, tissue, or organ, "treatment" encompasses contacting an antibody of the invention with, for example, a dog or other animal subject (e.g., a cat), a cell, tissue, physiological compartment, or physiological fluid.

[0047] As used herein, the term "dog" includes all domestic dogs, Canis lupus familiaris or Canis familiaris, unless otherwise specified.

[0048] As used herein, the term "cat" refers to any member of the Felidae family. Members of this family include wild, zoo, and domestic members, including domestic cats, purebred and / or mixed breed companion cats, show cats, laboratory cats, cloned cats, and wild or natural cats.

[0049] As used herein, the term "canine frame" refers to the amino acid sequences of the heavy and light chains of a canine antibody other than the hypervariable region residues defined herein as CDR residues. For caninized antibodies, in most embodiments, the amino acid sequences of the native canine CDRs are replaced in both chains with the corresponding foreign CDRs (e.g., of mouse or human origin). The heavy and / or light chains of the canine antibody may contain some foreign non-CDR residues to preserve the conformation of the foreign CDRs in the caninized antibody and / or to modify Fc region function, for example, as exemplified below and / or as disclosed in U.S. Pat. No. 10,106,607 B2, which is incorporated herein by reference in its entirety.

[0050] The "fragment crystallizable region", abbreviated as "Fc" or used interchangeably with "Fc region", corresponds to the CH3-CH2 portion of an antibody that interacts with cell surface receptors called Fc receptors. The canine fragment crystallizable region (cFc region) of each of the four canine IgGs was first described by Tang et al. [Vet. Immunol. Immunopathol. 80:259-270 (2001); see also Bergeron et al., Vet. Immunol. Immunopathol. 157:31-41 (2014) and U.S. Patent No. 10,106,607 B2].

[0051] As used herein, canine Fc (cFc) "IgG-Bm" is a canine IgG-B Fc that contains two amino acid residue substitutions, D31A and N63A, as in the amino acid sequence of SEQ ID NO:20 of IgG-B (see below), and preferably does not contain the c-terminal lysine ("K"), i.e., SEQ ID NO:51). The aspartic acid residue (D) at position 31 of SEQ ID NO:50 and the asparagine residue (N) at position 63 of SEQ ID NO:50 are both replaced with an alanine residue (A) in IgG-Bm. These two amino acid residue substitutions serve to significantly reduce the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of native canine IgG-B [see U.S. Patent No. 10,106,607 B2, the entire contents of which are incorporated herein by reference]. Further amino acid substitutions to IgG-Bm are also envisioned, which correspond to those that may be made in IgG-B. The amino acid sequence of IgG-B, SEQ ID NO:50: [ka]

[0052] The amino acid sequence of IgG-Bm, SEQ ID NO:51, is provided below. TIFF2025500806000003.tif46151

[0053] Amino acid sequence of IgG-Bm with C-terminal lysine (K), SEQ ID NO:20: TIFF2025500806000004.tif46151

[0054] As used herein, "substitution of an amino acid residue" in the amino acid sequence of an antibody by another amino acid residue is equivalent to, for example, "exchange of an amino acid residue" by another amino acid residue, and indicates that a particular amino acid residue at a particular position in the amino acid sequence is replaced (or substituted) by a different amino acid residue. Such substitutions can be specifically designed, i.e., an alanine can be purposely replaced with a serine at a particular position in the amino acid sequence, for example by recombinant DNA techniques. Alternatively, a particular amino acid residue or string of amino acid residues of an antibody can be replaced by one or more amino acid residues through a natural selection process, for example, based on the ability of an antibody produced by a cell to bind to a given region on its antigen, e.g., one that contains an epitope or a portion thereof, and / or because the antibody contains a particular CDR that retains the same canonical structure as the CDR that is replaced. Such substitutions / exchanges can result in "variant" CDRs and / or variant antibodies.

[0055] As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. Antibodies can be monomeric, dimeric, or larger multimeric. It is therefore used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), caninized antibodies, fully caninized antibodies, chimeric antibodies, and camelized single domain antibodies. A "parent antibody" is an antibody obtained by exposing the immune system to an antigen prior to modification of the antibody for the intended use, e.g., caninization of the antibody for use as a canine therapeutic antibody.

[0056] As used herein, an antibody of the invention that "blocks" or "blocking", or, for example, "blocks the binding" of a canine ligand to its binding partner (e.g., its receptor), is an antibody that blocks (partially or completely) the binding of a canine ligand to its receptor, and vice versa, as determined by standard binding assays (e.g., BIACore®, ELISA or flow cytometry).

[0057] Typically, the antibody or antigen-binding fragment of the present invention retains at least 10% of its canine antigen-binding activity (when compared to the parent antibody) when the activity is expressed on a molar basis. Preferably, the antibody or antigen-binding fragment of the present invention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the canine antigen-binding affinity of the parent antibody. It is also contemplated that the antibody or antigen-binding fragment of the present invention may contain conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "function-conservative variants" of the antibody) that do not substantially change its biological activity.

[0058] "Isolated antibody" refers to a purified state, meaning in such a context that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium. In general, the term "isolated" is not intended to refer to the total absence of such materials, or the absence of water, buffers, or salts, unless present in amounts that would substantially interfere with the experimental or therapeutic use of the binding compounds described herein.

[0059] As used herein, an antibody is said to specifically bind to a polypeptide that contains a given antigen sequence (in this case, a portion of the amino acid sequence of canine NGF) if it binds to a polypeptide that contains that portion of the amino acid sequence of canine NGF, but does not bind to other canine proteins that lack that portion of the sequence of canine NGF. For example, an antibody that specifically binds to a polypeptide that contains canine NGF may bind to a FLAG®-tagged form of canine NGF, but will not bind to other FLAG®-tagged canine proteins.

[0060] As used herein, unless otherwise indicated, "antibody fragment" or "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen (e.g., canine NGF) bound by the full-length antibody, e.g., a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include Fab, Fab', F(ab') 2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.

[0061] An antibody, or a binding compound derived from the antigen-binding site of an antibody, binds "specifically" to the canine antigen or variant or mutein thereof if it has an affinity for the canine antigen or variant or mutein thereof that is at least 10 times, more preferably at least 20 times, and even more preferably at least 100 times greater than its affinity for any other canine antigen tested. An antibody that binds "specifically" to canine NGF may also bind to NGF from another species (e.g., feline NGF and / or human NGF).

[0062] As used herein, a "chimeric antibody" is an antibody that has a variable domain from a first antibody and a constant domain from a second antibody, the first and second antibodies being from different species. [U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)]. Typically, the variable domain is obtained from an antibody "parent antibody" from a laboratory animal, such as a rodent (or a rodent that contains a human immune system), and the constant domain sequence is obtained from an animal subject antibody, such as a dog, so that the resulting chimeric antibody will be less likely to induce an adverse immune response in each canine subject than the parent (e.g., rodent) antibody.

[0063] As used herein, the term "caninized antibody" refers to a form of an antibody that contains sequences from both canine and non-canine (e.g., mouse or human) antibodies. In general, a caninized antibody contains substantially all of at least one or more, typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-canine immunoglobulin (e.g., including six CDRs as exemplified below) and all or substantially all of the framework (FR) regions (and typically all or substantially all of the remaining frame) being from a canine immunoglobulin sequence. A caninized antibody can contain, for example, both three heavy chain CDRs and three light chain CDRSs from a human anti-human NGF antibody, together with a canine frame or modified canine frame. The modified canine frame contains one or more amino acid changes exemplified herein that further optimize the effectiveness of the caninized antibody, for example, increasing its binding to a canine antigen and / or its ability to block the binding of the canine antigen to its natural binding partner.

[0064] The variable regions of each light / heavy chain pair form the antibody binding site. Thus, in general, a complete antibody has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites are generally the same. Typically, both heavy and light chain variable domains contain three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions, allowing binding to a specific epitope. Generally, from the N-terminus to the C-terminus, both light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain generally follows the definitions in Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat, Adv. Prot. Chem. 32:1-75 (1978); Kabat, et al., J. Biol. Chem. 252:6609-6616 (1977); Chothia, et al., J. Mol. Biol. 196:901-917 (1987) or Chothia, et al., Nature 342:878-883 (1989)].

[0065] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable region includes amino acid residues from "complementarity determining region" or "CDR" (i.e., LCDR1 or CDRL1, LCDR2 or CRDL2, and LCDR3 or CDRL3 in the light chain variable domain, and HCDR1 or CDRH1, HCDR2 or CDRH2, and HCDR3 or CDRH3 in the heavy chain variable domain). [See Kabat et al. Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), which defines the CDR region of an antibody by sequence; also see Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), which defines the CDR region of an antibody by structure]. As used herein, the terms "framework" or "FR" residues refer to variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0066] There are four known IgG heavy chain subtypes of canine IgG, called IgG-A or IgGA, IgG-B or IgGB, IgG-C or IgGC, and IgG-D or IgGD. The two known canine light chain subtypes are called lambda and kappa. Each of the two heavy chains consists of one variable domain (VH) and three constant domains called CH-1, CH-2, and CH-3. The CH-1 domain is connected to the CH-2 domain via an amino acid sequence called the "hinge" or alternatively the "hinge region."

[0067] In certain embodiments of the invention, in addition to binding canine NGF, canine or caninized antibodies to that antigen of the invention optimally have two properties: 1. Absence of effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC); and 2. It is easily purified on a large scale using industry standard techniques, such as techniques based on Protein A chromatography.

[0068] There are no native canine IgG isotypes that meet both criteria. For example, IgG-B can be purified using Protein A, but has high levels of ADCC activity. On the other hand, IgG-A binds weakly to Protein A, but also shows ADCC activity. Furthermore, IgG-D does not show ADCC activity, but neither IgG-C nor IgG-D can be purified on a Protein A column. (IgG-C has significant ADCC activity). One way in which the present invention addresses these issues in certain embodiments is by providing modified canine IgG-B antibodies of the present invention specific for the antigens of the present invention that lack effector functions such as ADCC and can be easily purified using industry standard Protein A chromatography.

[0069] "Homology" as used herein refers to the sequence similarity between two polynucleotide sequences or two polypeptide sequences when they are optimally aligned. If a position in both of the two compared sequences is occupied by the same base or amino acid residue, for example, if the position in each of the two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percentage of homology is the number of homologous positions shared by the two sequences divided by the total number of positions compared x 100. For example, if 6 out of 10 positions in the two sequences are identical or homologous when the sequences are optimally aligned, the two sequences are 60% homologous. Generally, the comparison is performed when the two sequences are aligned to obtain the maximum percentage of homology. Sequence identity refers to the degree to which the amino acids of two polypeptides are the same at equivalent positions when the two sequences are optimally aligned. As used herein, one amino acid sequence is 100% "identical" to a second amino acid sequence if all amino acid residues in both sequences are identical.

[0070] Thus, an amino acid sequence is 50% "identical" to a second amino acid sequence if 50% of the amino acid residues of the two amino acid sequences are identical. Sequence comparison is performed over a contiguous block of amino acid residues contained in a given protein, e.g., a protein or portion of a polypeptide being compared. In certain embodiments, selected deletions or insertions that may otherwise change the correspondence between the two amino acid sequences are taken into account. Sequence similarity includes identical residues and non-identical biochemically related amino acids, e.g., biochemically related amino acids that share similar properties and may be interchangeable.

[0071] "Conservatively modified variants" or "conservative substitutions" refer to the replacement of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.), which allows frequent modifications to be made without changing the biological activity of the protein. Those skilled in the art generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially change biological activity [see, for example, Watson et al. Molecular Biology of the Gene, The Benjamin / Cummings Pub.Co., p.224 (4th ed.; 1987)]. Moreover, substitution of structurally or functionally similar amino acids is unlikely to destroy biological activity. Exemplary conservative substitutions are shown in Table A immediately below.

[0072] [Table 1]

[0073] The present invention also contemplates functionally conservative variants of the antibodies of the present invention.As used herein, "functionally conservative variant" refers to an antibody or fragment in which one or more amino acid residues are changed without changing the desired properties, such as antigen affinity and / or specificity.Such variants include, but are not limited to, the substitution of amino acids with those having similar properties, such as the conservative amino acid substitutions in Table A above.

[0074] An "isolated nucleic acid molecule" means DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or any combination thereof, which is not related to all or a portion of the polynucleotide with which the isolated polynucleotide is found in nature or linked to a polynucleotide with which it is not naturally linked. For purposes of this disclosure, it should be understood that a "nucleic acid molecule comprising" a particular nucleotide sequence does not encompass an entire chromosome. An isolated nucleic acid molecule that "comprises" a specified nucleic acid sequence may, in addition to the specified sequence, include coding sequences for up to 10 or even up to 20 or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequence, and / or may include vector sequences.

[0075] The invention provides isolated caninized antibodies of the invention, methods of using the antibodies in treating conditions, such as treating osteoarthritis in dogs.

[0076] The nucleic acid and amino acid sequences of these four heavy chains were first identified by Tang et al. [Vet. Immunol. Immunopathol. 80:259-270 (2001)]. The amino acid and nucleic acid sequences of these heavy chains are also available from the GenBank database. For example, the amino acid sequence of IgGA heavy chain has accession number AAL35301.1, IgGB has accession number AAL35302.1, IgGC has accession number AAL35303.1, and IgGD has accession number (AAL35304.1). Canine antibodies also contain two types of light chains, kappa and lambda. The DNA and amino acid sequences of these light chains can be obtained from the GenBank database. For example, the kappa light chain amino acid sequence has accession number ABY 57289.1, and the lambda light chain has accession number ABY 55569.1.

[0077] The known amino acid sequences of the four unmodified canine Fc's are as follows:

[0078] cIgG-A [SEQ ID NO: 49] TIFF2025500806000006.tif47151

[0079] cIgG-B [SEQ ID NO: 50] TIFF2025500806000007.tif45150

[0080] cIgG-C [SEQ ID NO:52] TIFF2025500806000008.tif48152

[0081] cIgG-D [SEQ ID NO:53] TIFF2025500806000009.tif48151

[0082] In the present invention, the amino acid sequence of each of the four canine IgG Fc regions is based on the identified boundaries of CH1 and CH2 domains as determined by Tang et al., supra. The caninized mammalian (e.g., mouse or human) anti-human NGF antibody of the present invention that binds to canine NGF includes, but is not limited to, the antibody of the present invention that comprises canine IgG-A, IgG-B, IgG-C and IgG-D heavy chain and / or canine kappa or lambda light chain together with anti-human NGF CDR. Thus, the present invention provides the caninized mouse or human antibody of the present invention (including isolated caninized mouse or human anti-human NGF antibody) that binds to canine NGF and preferably also blocks the binding of said canine NGF to canine TrkA.

[0083] Accordingly, the invention further provides caninized NGF antibodies and methods of using the caninized antibodies of the invention in treating pain, such as osteoarthritis, in dogs.

[0084] The invention further provides full-length caninized heavy chains that can be combined with a corresponding light chain to make a caninized antibody. Thus, the invention further provides caninized mouse or human anti-NGF antibodies of the invention (including isolated caninized human anti-human NGF antibodies), as well as methods of using the antibodies of the invention in treating conditions, such as treating pain in dogs.

[0085] The invention also provides an antibody of the invention comprising a canine fragment crystallizable region (cFc region), the cFc region being engineered to enhance, reduce or eliminate one or more effector functions. In one aspect of the invention, the engineered cFc region reduces or eliminates one or more effector functions. In another aspect of the invention, the engineered cFc region enhances one or more effector functions. In certain embodiments, the engineered cFc region is an engineered canine IgGB Fc region. In another such embodiment, the engineered cFc region is an engineered canine IgGC Fc region. In certain embodiments, the effector function is antibody-dependent cellular cytotoxicity (ADCC) that is enhanced, reduced or eliminated. In another embodiment, the effector function is complement-dependent cytotoxicity (CDC) that is enhanced, reduced or eliminated. In yet another embodiment, the cFc region is engineered to enhance, reduce or eliminate both ADCC and CDC.

[0086] To generate variants of dog IgG that lack effector functions, a number of mutant dog IgGB heavy chains were generated. These variants may contain one or more of the following substitutions in the Fc portion of the heavy chain amino acid sequence, either single or in combination: P4A, D31A, N63A, G64P, T65A, A93G, and P95A. The variant heavy chains (i.e., containing such amino acid substitutions) are cloned into an expression plasmid and transfected into HEK 293 cells together with a plasmid containing a gene encoding a light chain. Complete antibodies can be expressed and purified from HEK 293 cells and then evaluated for binding to FcγRI and C1q to evaluate their potential for mediating immune effector functions. [See U.S. Patent No. 10,106,607 B2, the entire contents of which are incorporated herein by reference].

[0087] The present invention also provides modified canine IgG-D, which comprises, in place of its native IgG-D hinge region, a hinge region from: TIFF2025500806000010.tif31150

[0088] Alternatively, the IgG-D hinge region can be engineered by replacing the serine residue with a proline residue, i.e., PKESTCKCIPPCPVPES SEQ ID NO:48 (replacement of the natural serine residue with the bolded proline residue (P)). Such modifications can result in a canine IgG-D lacking Fab arm exchange. Modified canine IgG-D can be constructed using standard methods of recombinant DNA technology [e.g., Maniatis et al., Molecular Cloning, A Laboratory Manual (1982)]. To construct these variants, a nucleic acid encoding the amino acid sequence of canine IgG-D can be modified to encode the modified IgG-D. The modified nucleic acid sequence is then cloned into an expression plasmid for protein expression.

[0089] The six complementarity determining regions (CDRs) of a caninized mouse or human anti-NGF antibody as described herein can include a kappa (k) or lambda (l) light chain of the canine antibody, which includes mouse light chains LCDR1, LCDR2 and LCDR3, and a heavy chain of the canine antibody, which includes mouse or human heavy chains HCDR1, HCDR2 and HCDR3.

[0090] nucleic acid The invention also includes nucleic acids encoding the antibodies of the invention (see, eg, the Examples below).

[0091] The present invention also includes nucleic acids encoding immunoglobulin polypeptides comprising an amino acid sequence that is at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical, and most preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the amino acid sequence of the caninized antibody provided herein, excluding the unchanged CDRs, when compared by a BLAST algorithm, the parameters of the algorithm being selected to give the greatest match between the respective sequences over the entire length of the respective reference sequences. The present invention further provides nucleic acids encoding immunoglobulin polypeptides comprising an amino acid sequence that is at least about 70% similar, preferably at least about 80% similar, more preferably at least about 90% similar, and most preferably at least about 95% similar (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to any of the reference amino acid sequences, when compared by a BLAST algorithm, the parameters of the algorithm being selected to give the greatest match between the respective sequences over the entire length of the respective reference sequences, further included in the present invention.

[0092] As used herein, the percent identity of nucleotide and amino acid sequences can be determined using the C, MacVector (MacVector, Inc. Cary, NC 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996), and Clustal W algorithms using alignment default parameters and identity default parameters.These commercially available programs can also be used to determine sequence similarity using the same or similar default parameters.Alternatively, Advanced Blast search can be used under default filter conditions, for example, using the GCG (Genetics Computer Group, Program Manual for GCG Package, Version 7, Madison, Wisconsin) pileup program using default parameters.

[0093] The following references relate to the BLAST algorithm, which is often used for sequence analysis: BLAST Algorithm: Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1990); Gish, W., et al., Nature Genet. 3:266-272 (1993); Madden, T. L., et al., Meth. Enzymol. 266:131-141 (1996); Altschul, S. F., et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang, J., et al., Genome Res. 7:649-656 (1997); Wootton, J. C., et al., Comput. Chem. 17:149-163 (1993); Hancock, J. Met al., Comput. Appl. Biosci. 10:67-70(1994);Alignment scoring systems: Dayhoff, MO, et al., "A model of evolutionary change in proteins." (Atlas of Protein Sequence and Structure, vol. 5, suppl. 3) MODayhoff(ed.), pp. 345-352, (1978); Natl. Biomed. Res. Found., Washington, DC; Schwartz, RM, et al., "Matrices for detecting distant relationships." (Atlas of Protein Sequence and Structure, vol. 5, suppl. 3) (1978), MODayhoff(ed.), pp. 353-358(1978), Natl. Biomed. Res. Found., Washington, DC; Altschul, SF, J. Mol. Biol. 219:555-565(1991); States, DJ, et al. al., Methods 3:66-70(1991);Henikoff, S., et al., Proc.Natl.Acad.Sci.USA 89:10915-10919(1992);Altschul, SF, et al., J.Mol.Evol.36:290-300 (1993);Alignment statistics: Karlin, S., et al., Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990); Karlin, S., et al., Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); Dembo, A., et al., Ann. Prob. 22:2022-2039 (1994); and Altschul, S. F. "Evaluating the statistical significance of multiple distinct local alignments." (In Theoretical and Computational Methods in Genome Research) (S. Suhai, ed.), pp. 1-14, Plenum, New York (1997).

[0094] Antibody Protein Engineering By way of example and not limitation, as set forth above, the dog heavy chain constant region can be derived from IgG-A, IgG-B, IgG-C, IgG-D, the corresponding cFc can be a modified cFc, e.g., IgG-Bm for the IgG-B heavy chain constant region used herein [see U.S. Pat. No. 10,106,607 B2, which is incorporated herein by reference in its entirety], and the dog light chain can include a constant region derived from kappa or lambda.

[0095] Antibodies can be engineered to contain modifications to the canine framework and / or canine frame residues within the variable domains of a parent (eg, human) monoclonal antibody, e.g., to improve the properties of the antibody.

[0096] The construction of caninized anti-NGF monoclonal antibodies can be carried out by determining the DNA sequence encoding the heavy and light chains of canine IgG. The DNA and protein sequences of canine heavy and light chains are known in the art and can be obtained by searching the NCBI gene and protein database. As mentioned above, for canine antibodies, there are four known IgG subtypes: IgG-A, IgG-B, IgG-C and IgG-D, and two types of light chains: kappa and lambda.

[0097] Caninized human anti-NGF antibodies can be recombinantly produced by methods known in the art. Mammalian cell lines available as hosts for the expression of the antibodies or fragments disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and several other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other cell lines that can be used are insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells. When recombinant expression vectors encoding the heavy chain or antigen-binding portion or fragment thereof, the light chain and / or antigen-binding fragment thereof are introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow for expression of the antibody in the host cell, or more preferably, secretion of the antibody into the culture medium in which the host cell is growing.

[0098] The antibody can be recovered from the culture medium using standard protein purification methods. Furthermore, the expression of the antibody of the present invention (or other moieties derived therefrom) from the manufacturing cell line can be enhanced using several known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common approach to enhance expression under certain conditions. The GS system is discussed in whole or in part in connection with European Patent Nos. 0216846, 0256055, and 0323997, and European Patent Application No. 89303964.4.

[0099] Thus, in certain embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region, e.g., a canine constant region, e.g., an IgG-A, IgG-B, IgG-C, and IgG-D canine heavy chain region or a variant thereof. In certain embodiments, the antibody or antigen-binding fragment comprises a light chain constant region, e.g., a canine light chain constant region, e.g., a lambda or kappa canine light chain region or a variant thereof. By way of example and not limitation, the canine heavy chain constant region can be derived from IgG-B and the canine light chain constant region can be derived from kappa.

[0100] Caninized mammalian (e.g., mouse or human) anti-human NGF antibodies of the present invention that bind to canine NGF include, but are not limited to, antibodies of the present invention that comprise canine IgG-A, IgG-B, IgG-C and IgG-D heavy chains and / or canine kappa or lambda light chains together with anti-human NGF CDRs.Thus, the present invention provides caninized mouse or human antibodies of the present invention (including isolated caninized mouse or human anti-human NGF antibodies) that bind to canine NGF and preferably also block the binding of said canine NGF to canine TrkA.

[0101] The invention further provides caninized NGF antibodies and methods of using the caninized antibodies of the invention in treating pain, such as osteoarthritis, in dogs.

[0102] The invention further provides full-length caninized heavy chains that can be combined with a corresponding light chain to make a caninized antibody. Thus, the invention further provides caninized mouse or human anti-NGF antibodies of the invention, including isolated caninized human anti-human NGF antibodies.

[0103] Pharmaceutical Compositions and Administration To prepare pharmaceutical or sterile compositions containing the antibodies of the invention, the antibodies can be mixed with a pharma- ceutically acceptable carrier or excipient. [See, e.g., Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984)].

[0104] Formulations of therapeutic and diagnostic agents can be prepared, for example, by mixing with acceptable carriers, excipients, or stabilizers in the form of lyophilized powders, slurries, aqueous solutions, or suspensions [see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: See, for example, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY; Weiner and Kotkoskie (2000) Excipient Forms: Disperse Systems, Marcel Dekker, NY. In one embodiment, the antibodies of the invention are diluted to the appropriate concentration in sodium acetate solution (pH 5-6), with NaCl or sucrose added for tonicity. Additional agents, such as polysorbate 20 or polysorbate 80, can be added to enhance stability.

[0105] The toxicity and therapeutic efficacy of an antibody composition administered alone or in combination with another agent may be determined, for example, by the LD 50 (a dose lethal to 50% of the population) and ED 50 The dose that is therapeutically effective in 50% of a population can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is called the therapeutic index (LD50 / ED 50 In certain embodiments, antibodies that exhibit a high therapeutic index are desirable. The data obtained from these cell culture assays and animal studies can be used to formulate a range of dosages for use in dogs. The dosage of such compounds is preferably within the ED with little or no toxicity. 50 The dosage may vary within this range depending upon the dosage form and route of administration used.

[0106] The mode of administration may vary. Suitable routes of administration include oral, rectal, mucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intracerebroventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, dermal, transdermal, or intraarterial. In certain embodiments, the antibody of the present invention can be administered by an invasive route, such as injection. In further embodiments of the present invention, the antibody of the present invention or a pharmaceutical composition thereof is administered intravenously, subcutaneously, intramuscularly, intraarterially, or by aerosol delivery by inhalation. Administration by non-invasive routes (e.g., oral; e.g., pill, capsule, or tablet) is also within the scope of the present invention.

[0107] Composition can be administered using medical devices known in the art.For example, pharmaceutical composition of the present invention can be administered by injection with hypodermic needle, including for example prefilled syringe or autoinjector.Pharmaceutical composition disclosed herein can also be administered using needleless hypodermic injection device, for example, the device disclosed in U.S. Patent No. 6,620,135;6,096,002;5,399,163;5,383,851;5,312,335;5,064,413;4,941,880;4,790,824 or 4,596,556.

[0108] The pharmaceutical composition disclosed herein can also be administered by injection.The examples of well-known implant and module forms for administering pharmaceutical compositions include: US Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; US Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at a precise infusion rate; US Patent No. 4,447,224, which discloses a variable flow rate implantable infusion device for continuous drug delivery; US Patent No. 4,439,196, which discloses an osmotic drug delivery system with multi-chamber compartments.Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0109] Alternatively, the antibodies of the invention can be administered locally rather than systemically, often in a depot or sustained release formulation.

[0110] The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of the target cells in the biological matrix. Preferably, the administration regimen delivers enough therapeutic antibody to bring about improvement of the target disease / condition, while minimizing undesirable side effects. Thus, the amount of biologic delivered depends, in part, on the particular therapeutic antibody and the severity of the condition being treated. Guidance in the selection of appropriate doses of therapeutic antibodies is available [e.g., Wawrzynczak Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK (1996); Kresina (ed.) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY (1991); Bach (ed.) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY (1993); Baert, et al. New Engl. J. Med. 348:601-608 (2003); Milgrom et al. New Engl. J. Med. 341:1966-1973 (1999); Slamon et al. New Engl. J. Med. 344:783-792 (2001); Beniaminovitz et al. New Engl. J. Med. 344:783-792 (2001); Engl.J.Med.342:613-619(2000);Ghosh et al.New Engl.J.Med.348:24-32(2003);Lipsky et al.New Engl.J.Med.343:1594-1602(2000)].

[0111] The determination of the appropriate dose is made by a veterinarian, for example, using parameters or factors known or suspected in the art to affect treatment. Generally, the dose is started at a dose somewhat less than the optimal dose, and then increased in small increments until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms.

[0112] Antibodies provided herein can be provided by continuous infusion or by doses administered, for example, daily, 1 to 7 times per week, weekly, biweekly, monthly, bimonthly, quarterly, semi-annually, yearly, etc. Doses can be provided, for example, intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. The total weekly dose is generally at least 0.05 μg / kg body weight, more generally at least 0.2 μg / kg, 0.5 μg / kg, 1 μg / kg, 10 μg / kg, 100 μg / kg, 0.25 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 5.0 mg / ml, 10 mg / kg, 25 mg / kg, 50 mg / kg or more [e.g., Yang, et al. New Engl. J. Med. 349:427-434 (2003); Herold, et al. New Engl. J. Med. 346:1692-1698 (2002); Liu, et al. J. Neurol. Neurosurg. Psych. 67:451-456 (1999); Portielji, et al. Cancer See Immunol. Immunother. 52:133-144 (2003)]. Dosages may also be provided to achieve a predetermined target concentration of the antibody of the invention in the serum of the dog, e.g., 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg / ml or more. In other embodiments, the antibody of the invention is administered subcutaneously or intravenously at 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg per subject on a weekly, biweekly, "every four weeks", monthly, bimonthly or quarterly basis.

[0113] As used herein, "inhibit" or "treat" or "treatment" includes postponing the onset of symptoms associated with a disorder and / or reducing the severity of symptoms of such a disorder. The term further includes ameliorating existing uncontrolled or undesirable symptoms, preventing further symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, these terms mean that a beneficial result is provided to a vertebrate subject (e.g., a dog) having a disorder, disease and / or condition or having a potential for developing such a disorder, disease or condition.

[0114] As used herein, the terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to an amount of an antibody of the invention that is effective when administered alone or in combination with an additional therapeutic agent to a cell, tissue or subject, e.g., a dog, to cause a measurable improvement in one or more symptoms of a disease or condition or the progression of such a disease or condition. A therapeutically effective dose further refers to an amount of an antibody sufficient to cause at least a partial improvement of a symptom, e.g., treatment, cure, prevention or amelioration of an associated medical condition, or an increase in the rate of treatment, cure, prevention or amelioration of such a condition. When applied to a combination, a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent will result in an improvement of at least 10%, usually at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50% of a diagnostic measure or parameter. An effective amount can also result in an improvement in a subjective measure when a subjective measure is used to assess the severity of a symptom, e.g., pain.

[0115] [Example] [Example 1] Prior art antibodies to human NGF that are reactive with canine NGF In an effort to develop a treatment for pain (e.g., osteoarthritis) in companion animals such as dogs, cats and horses, an investigation was conducted to see whether two known human or humanized antibodies against human NGF (see, for example, U.S. Pat. No. 7,601,818 B2 (furanumab, abbreviated herein as ful), U.S. Pat. No. 7,988,967 B2 (fasinumab, abbreviated herein as fas)) could also bind to NGF from dogs, cats or horses. Both human / humanized monoclonal antibodies that bind human NGF were found to bind to canine NGF. The six prior art CDR sets for these two previously disclosed antibodies are provided in Tables 1A and 1B below.

[0116] [Table 2] [Table 3]

[0117] [Example 2] Canine NGF and canine NGF TRKA receptor The amino acid sequence of the canine NGF protein is available at the National Center for Biotechnology Information (NCBI) under the accession number NP_001181879.1 [SEQ ID NO: 13]. To facilitate purification and site-specific biotinylation of the NGF protein having the amino acid sequence of SEQ ID NO: 14, the canine NGF-HIS-Avi protein was generated as a fusion protein of canine NGF by C-terminal addition of six histidine residues and an Avi tag sequence. The predicted amino acid sequence of the high affinity canine nerve growth factor receptor (TrkA) is available at the National Center for Biotechnology Information (NCBI) under the accession number XP_038527745. The amino acid sequence of TrkA is SEQ ID NO: 15. The cNGF-hFc fusion protein has the amino acid sequence of SEQ ID NO: 16. For the canine NGF receptor TrkA ECD-canine Fc fusion protein, the predicted amino acid sequence of TrkA ECD was generated as a fusion protein with the C-terminal addition of cFc from canine IgG-B. The sequence of this fusion protein is shown in SEQ ID NO:17.

[0118] [Table 4] TIFF2025500806000014.tif55152

[0119] [Example 3] Generation of human-canine chimeric NGF antibodies Using the VH and VL sequences previously disclosed [see Table 3 below], chimeric human-dog antibodies were constructed and then tested against canine NGF. Briefly, each of the VH and VL of the selected antibody group was genetically linked (fused) with the canine IgG-B heavy chain constant region (CH1-CH3) and light chain (kappa) constant region, respectively [see Table 4 for more details]. The human / humanized VH and VL regions of the human-dog (HC) chimeras listed in Table 4 were transiently expressed in HEK293 cells and then purified using a protein A column. The binding activity of each chimeric antibody was tested in an ELISA plate coated with canine NGF, as described in Example 4 below.

[0120] [Table 5]

[0121] [Table 6]

[0122] [Example 4] Generation of caninized NGF antibodies Caninized antibodies were constructed using two sets of six CDRs provided in Tables 1A-1B. The binding activity of the chimeric and caninized antibodies to canine NGF was compared by ELISA (see Example 5 below). As shown in Figures 1 and 2, both chimeric antibodies show strong affinity to canine NGF. In sharp contrast, a control caninized monoclonal antibody (having a set of six CDRs obtained from a mouse antibody raised against an unrelated canine antigen) did not bind at all.

[0123] Thus, Figure 1 shows plots of binding of human-canine chimeric furanumab (Ful Chim), and caninized variants containing CDRs from furanumab, and isotype control mAb (mAb ctrl) as determined by ELISA. Chimeric furanumab bound to canine NGF had an EC50 of 22 pM, whereas caninized variants of furanumab bound to canine NGF had EC50s ranging from 32 to 49 pM. These results demonstrate that these caninized antibodies have strong binding affinity for canine NGF, thereby making them suitable for development as drugs for the treatment of pain in dogs.

[0124] Figure 2 shows a plot of binding of human-canine chimeric fasinumab (Fas Chim), and caninized variants containing CDRs from fasinumab, and isotype control mAb (mAb ctrl) determined by ELISA. However, surprisingly, chimeric fasinumab bound to canine NGF with an EC50 of 122 nM, whereas the binding affinity of the corresponding caninized variants containing CDRs from fasinumab to canine NGF was too low to measure. This makes the caninized fasinumab antibody unsuitable for development for the treatment of pain in dogs. This demonstrates that it is unpredictable whether a caninized antibody encoding a set of CDRs from a given antibody to human NGF will also bind to canine NGF, even if the corresponding human-canine chimera binds well.

[0125] [Table 7]

[0126] Caninized antibody heavy and light chains

[0127] cFulVH1-cIgGB (SEQ ID NO: 36) TIFF2025500806000018.tif102150

[0128] cFulVH2-cIgGB (SEQ ID NO: 37) TIFF2025500806000019.tif103151

[0129] cFulVL1-cCk (SEQ ID NO: 38) TIFF2025500806000020.tif55151

[0130] cFulVL2-cCk (SEQ ID NO: 39) TIFF2025500806000021.tif56151

[0131] cFAS-VH1-cIgGB (SEQ ID NO: 40) TIFF2025500806000022.tif102151

[0132] cFAS-VH2-cIgGB (SEQ ID NO: 41) TIFF2025500806000023.tif104151

[0133] cFAS-VL1-cCk (SEQ ID NO: 42) TIFF2025500806000024.tif55150

[0134] cFAS-VL2-cCk (SEQ ID NO: 43) TIFF2025500806000025.tif54150

[0135] cFAS-VL3-cCk (SEQ ID NO: 44) TIFF2025500806000026.tif54150

[0136] [Example 5] Chimeric antibodies and caninized anti-human Binding of NGF antibodies to canine NGF Binding of the chimeric and caninized antibodies to canine NGF was determined by ELISA as follows: 1. Coat 100 ng of canine NGF per well on immunoplates and incubate plates at 4° C. overnight. 2. Wash the plate three times with PBS containing 0.05% Tween 20 (PBST). 3. Block plates with 0.5% BSA in PBS for 45-60 minutes at room temperature. 4. Wash the plate 3 times with PBST. 5. Dilute the antibody 3-fold in each row or column of the dilution plate. 6. Transfer the diluted antibodies to each column or row of the immunoplate and incubate the plate at room temperature for 45-60 minutes. 7. Wash the plate 3 times with PBST. 8. Add horseradish peroxidase-labeled anti-dog IgG Fc diluted 1:2000 to each well of the plate and incubate the plate at room temperature for 45-60 minutes. 9. Wash the plate 3 times with PBST. 10. Add TMB substrate to each well of the plate and incubate the plate at room temperature for 10-15 minutes for color development. 11. Stop the reaction by adding 100 μL of 1.5 M phosphoric acid to each well. 12. Read the plate at 450 nm with a reference wavelength of 540 nm.

[0137] Figure 3 shows the binding of canine NGF to canine TrkA receptor. To develop an assay to measure the ability of caninized anti-canine NGF antibody to block the binding of canine NGF to its TrkA receptor, the binding of canine NGF to canine NGF receptor (TrkA) was determined by ELISA. As shown in Figure 3, canine NGF binds to its canine TrkA receptor in a dose-dependent manner, with an EC50 of 54nM.

[0138] [Example 6] Blocking activity of chimeric and caninized antibodies Chimeric anti-NGF antibodies were tested for blocking binding of canine NGF to the canine NGF receptor (TrkA) as follows: 1. Coat 100 ng of canine TrkA-IgGBFc fusion protein per well on immunoplates and incubate plates at 4° C. overnight. 2. Wash the plate three times with PBS containing 0.05% Tween 20 (PBST). 3. Block plates with 0.5% BSA in PBS for 45-60 minutes at room temperature. 4. Wash the plate 3 times with PBST. 5. Make 3-fold dilutions of antibody in each column or row of the dilution plate, then add 100 ng of biotinylated canine NGF per well and mix with the antibody. 6. Transfer the diluted antibody and canine NGF mixture to each column or row of the immunoplate and incubate the plate at room temperature for 45-60 minutes. 7. Wash the plate 3 times with PBST. 8. Add horseradish peroxidase-conjugated streptavidin diluted 1:2000 to each well of the plate and incubate the plate at room temperature for 45-60 min. 9. Wash the plate 3 times with PBST. 10. Add TMB substrate to each well of the plate and incubate the plate at room temperature for 10-15 minutes for color development. 11. Stop the reaction by adding 100 μL of 1.5 M phosphoric acid to each well. 12. Read the plate at 450 nm with a reference wavelength of 540 nm.

[0139] FIG. 4 shows inhibition of canine NGF binding to the canine TrkA receptor by caninized antibodies and the corresponding human-canine chimeric furanumab. The ability of human-canine chimeric furanumab (Ful Chim), and caninized variants containing CDRs from furanumab, to block canine NGF binding to its TrkA receptor was determined by ELISA. As shown, chimeric furanumab, and caninized variants containing CDRs from furanumab, specifically and dose-dependently inhibited canine NGF binding to its TrkA receptor with IC50s ranging from 10 nM to 87 nM. In contrast, isotype control mAb (mAb ctrl) did not. These results indicate that caninized antibodies are suitable for development for the treatment of pain in dogs.

[0140] [Example 7] Inhibition of canine NGF bioactivity in TF-1 cells TF-1 cell-based assay TF-1 is a human erythroleukemia cell line that expresses human TrkA and proliferates in response to NGF from a variety of species. The effect of canine NGF on the proliferation of TF-1 cells and the ability of chimeric and caninized anti-NGF antibodies to block proliferation of TF-1 cells were assessed as follows:

[0141] material TF1 cell line (CRL-2003) Growth medium: RPMI-1640 (ThermoFisher catalog number 11875-085), 10% FBS and 2ng / mL rhGM-CSF (R&D, 7954-GM) Assay medium: RPMI-1640 containing 10% FBS (ThermoFisher catalog number 11875-085) CELLTITER-GLO® One Solution Assay (Promega Catalog No. G8461)

[0142] TF-1 cell culture: 1. In a T75 flask in a cell culture incubator with 5% CO 2 Incubate the cells at 37°C with >80% relative humidity. Add 4–8 × 10 cells to growth medium. 4 If seeded as cells / mL, passage the cells every 3-4 days. 2. Passage the cells one day before performing the cell proliferation assay.

[0143] Canine beta-NGF assay for TF-1 cell proliferation: 1. Add 50 μL of assay medium to each well of a 96-well plate. 2. Prepare 900 nM recombinant canine beta-NGF (cNGF) in assay medium. Add 25 μL of cNGF to the first well. Dilute 3-fold across the plate in duplicate and discard the final 25 μL volume. 3. Harvest TF-1 cells and wash three times with assay medium. Cells are diluted to 0.5-1 × 10 6 Resuspend in assay medium to a concentration of cells / mL. 4. Add 50 μL of TF-1 cells to each well of the assay plate. 5. Incubate the plate in a cell culture incubator for 48 hours (± 8 hours) at 37°C with 5% CO2 and >80% relative humidity. 6. Add 100 μL / well of CELLITER-GLO ONE SOLUTION ASSAY to the plate. Mix contents on an orbital shaker for 2 minutes and incubate at room temperature for 15 minutes (± 5 minutes). 7. Measure the luminescence intensity using a plate reader.

[0144] Inhibition of cNGF-mediated TF-1 cell proliferation by anti-cNGF antibodies: 1. Add 50 μL of assay medium to each well of a 96-well plate. 2. Prepare 1800 nM antibody in assay medium. Add 25 μL of antibody to the first well. Dilute 3-fold across the plate in duplicate and discard the final 25 μL volume. Include mAb isocontrols, wells containing assay medium and cells only. 3. Prepare 60 nM cNGF in assay medium. Mix an equal volume of cNGF with diluted antibody. 4. Harvest TF-1 cells and wash three times with assay medium. Cells are diluted to 0.5-1 × 10 6 Resuspend in assay medium to a concentration of cells / mL. 5. Add 50 μL of TF-1 cells to each well of a new 96-well plate. Transfer 50 μL of the mixed cNGF / antibody to each well of the cell plate. 6. Incubate the plate in a cell culture incubator for 48 hours (± 8 hours) at 37°C with 5% CO2 and relative humidity >80%. 7. Add 100 μL / well of CELLITER-GLO ONE SOLUTION ASSAY to the plate. Mix contents on an orbital shaker for 2 minutes and incubate at room temperature for 15 minutes (± 5 minutes). 8. Measure the luminescence intensity using a plate reader.

[0145] FIG. 5 shows stimulation of TF-1 cell proliferation by canine NGF. To develop an assay to measure the ability of caninized anti-canine NGF antibodies to block downstream signaling and inhibit cell proliferation induced by canine NGF binding to the TrkA receptor in TF-1 cells, the ability of canine NGF to stimulate TF-1 cell proliferation was determined by bioassay. As shown, canine NGF binds to the endogenous TrkA receptor expressed by TF-1 cells in a dose-dependent manner with an EC50 of 28 nM and stimulates TF-1 cell proliferation. This result indicates that the TF-1 cell-based assay can be used to test the blocking activity of anti-canine NGF antibodies.

[0146] Figure 6 shows inhibition of TF-1 cell proliferation by caninized anti-NGF antibodies. The ability of human-canine chimeric furanumab (Ful Chim), caninized variants containing the CDRS from furanumab identified in Figure 6, and an isotype control mAb (mAb ctrl) to block TF-1 cell proliferation was determined in a bioassay using TF-1 cells. As shown, the chimeric, and the caninized variants containing the CDRs from furanumab specifically and dose-dependently inhibited TF-1 cell proliferation with IC50s ranging from 0.26 to 0.4 nM. These results demonstrate that caninized antibodies are suitable for development for the treatment of pain in dogs.

[0147] (Sequence Listing) ST.25 SEQUENCE LISTING FROM PRIORITY FILING SEQUENCE LISTING <110> Intervet Inc. Intervet International BV Morsey, Mohammed Zhang, Yuanzheng <120> Caninized Antibodies to Human NGF <130> 25370-US-PSP <160> 53 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Homo sapiens <400> 1 See Tyr See Met Asn 1 5 <210> 2 <211> 17 <212> PRT <213> Homo sapiens <400> 2 Tyr Ile Ser Arg Ser Ser His Thr Ile Phe Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 14 <212> PRT <213> Homo sapiens <400> 3 Val Tyr Ser Ser Gly Trp His Val Ser Asp Tyr Phe Asp Tyr 1 5 10 <210> 4 <211> 11 <212> PRT <213> Homo sapiens <400> 4 Arg Ala Ser Gln Gly Ile Ser Ser Ala Leu Ala 1 5 10 <210> 5 <211> 7 <212> PRT <213> Homo sapiens <400> 5 Asp Ala Ser Ser Leu Glu Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Homo sapiens <400> 6 Gln Gln Phe Asn Ser Tyr Pro Leu Thr 1 5 <210> 7 <211> 5 <212> PRT <213> Homo sapiens <400> 7 Glu Leu Ser Ile His 1 5 <210> 8 <211> 17 <212> PRT <213> Homo sapiens <400> 8 Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 9 <211> 9 <212> PRT <213> Homo sapiens <400> 9 Ile Gly Val Val Thr Asn Phe Asp Asn 1 5 <210> 10 <211> 11 <212> PRT <213> Homo sapiens <400> 10 Arg Ala Ser Gln Ala Ile Arg Asn Asp Leu Gly 1 5 10 <210> 11 <211> 7 <212> PRT <213> Homo sapiens <400> 11 Ala Ala Phe Asn Leu Gln Ser 1 5 <210> 12 <211> 9 <212> PRT <213> Homo sapiens <400> 12 Gln Gln Tyr Asn Arg Tyr Pro Trp Thr 1 5 <210> 13 <211> 223 <212> PRT <213> Canis familiaris <400> 13 Glu Pro His Pro Glu Ser His Val Pro Ala Gly His Ala Ile Pro His 1 5 10 15 Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu Arg Arg 20 25 30 Ala Arg Ser Ala Pro Ala Gly Ala Ile Ala Ala Arg Val Thr Gly Gln 35 40 45 Thr Arg Asn Ile Thr Val Asp Pro Lys Leu Phe Lys Lys Arg Arg Leu 50 55 60 Arg Ser Pro Arg Val Leu Phe Ser Thr His Pro Pro Pro Val Ala Ala 65 70 75 80 Asp Ala Gln Asp Leu Asp Leu Glu Ala Gly Ser Thr Ala Ser Val Asn 85 90 95 Arg Thr His Arg Ser Lys Arg Ser Ser Ser His Pro Val Phe His Arg 100 105 110 Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp Lys 115 120 125 Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu 130 135 140 Val Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys 145 150 155 160 Cys Arg Asp Pro Thr Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser 165 170 175 Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala 180 185 190 Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp 195 200 205 Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Gly Arg Arg Ala 210 215 220 <210> 14 <211> 244 <212> PRT <213> Artificial Sequence <220> <223> modified canine <400> 14 Glu Pro His Pro Glu Ser His Val Pro Ala Gly His Ala Ile Pro His 1 5 10 15 Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu Arg Arg 20 25 30 Ala Arg Ser Ala Pro Ala Gly Ala Ile Ala Ala Arg Val Thr Gly Gln 35 40 45 Thr Arg Asn Ile Thr Val Asp Pro Lys Leu Phe Lys Lys Arg Arg Leu 50 55 60 Arg Ser Pro Arg Val Leu Phe Ser Thr His Pro Pro Pro Val Ala Ala 65 70 75 80 Asp Ala Gln Asp Leu Asp Leu Glu Ala Gly Ser Thr Ala Ser Val Asn 85 90 95 Arg Thr His Arg Ser Lys Arg Ser Ser Ser His Pro Val Phe His Arg 100 105 110 Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp Lys 115 120 125 Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu 130 135 140 Val Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys 145 150 155 160 Cys Arg Asp Pro Thr Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser 165 170 175 Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala 180 185 190 Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp 195 200 205 Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Gly Arg Arg Ala His 210 215 220 His His His His His Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile 225 230 235 240 Glu Trp His Glu <210> 15 <211> 796 <212> PRT <213> Canis familiaris <400> 15 Met Leu Arg Gly Gly Arg Leu Gly Gln Arg Gly Gly His Gly Arg Ala 1 5 10 15 Ala Gly Pro Gly Ser Leu Leu Ala Trp Leu Val Leu Ala Ser Ala Gly 20 25 30 Ala Ala Pro Cys Pro Asp Val Cys Cys Pro His Gly Pro Ser Gly Leu 35 40 45 Arg Cys Thr Arg Ala Gly Ala Leu Gln Ser Leu His Arg Leu Pro Gly 50 55 60 Val Glu Asn Leu Thr Glu Leu Tyr Ile Asp Asn Gln Glu His Leu Gln 65 70 75 80 His Leu Asp Ala Val His Leu Lys Gly Leu Gly Met Leu Arg Asp Leu 85 90 95 Thr Ile Val Lys Ser Gly Leu Arg Ser Val Ala Pro Asp Ala Phe His 100 105 110 Phe Thr Pro Arg Leu Arg Arg Leu Asn Leu Ser Phe Asn Ala Leu Glu 115 120 125 Ser Leu Ser Trp Lys Thr Val Gln Gly Leu Pro Leu Gln Glu Leu Val 130 135 140 Leu Ser Gly Asn Pro Leu His Cys Ser Cys Ala Leu His Trp Leu Leu 145 150 155 160 Arg Trp Glu Glu Glu Gly Leu Gly Gly Val Arg Gly Gln Arg Leu Gln 165 170 175 Cys Pro Gly Gln Gly Pro Leu Ala Leu Leu Ser Asn Ala Ser Cys Gly 180 185 190 Val Pro Val Leu Lys Val Gln Met Pro Asn Ala Ser Val Glu Val Gly 195 200 205 Asp Asp Val Leu Leu Gln Cys Gln Val Glu Gly Gln Gly Leu Glu Arg 210 215 220 Ala Gly Trp Ile Leu Pro Glu Val Glu Glu Leu Ala Thr Val Thr Gln 225 230 235 240 Ser Gly Asp Leu Pro Ser Leu Gly Leu Thr Leu Ala Asn Val Thr Ser 245 250 255 Asp Leu Asn Arg Lys Asn Val Thr Cys Trp Ala Glu Asn Asp Val Gly 260 265 270 Arg Ala Glu Val Ser Val Gln Val Asn Val Ser Phe Pro Ala Ser Val 275 280 285 Gln Leu His Glu Ala Val Glu Leu His His Trp Cys Ile Pro Phe Ser 290 295 300 Val Asp Gly Gln Pro Ala Pro Ser Leu Arg Trp Leu Phe Asn Gly Ser 305 310 315 320 Val Leu Asn Glu Thr Ser Phe Ile Phe Thr Glu Phe Leu Glu Pro Val 325 330 335 Ala Asn Glu Thr Val Arg His Gly Cys Leu Arg Leu Asn Gln Pro Thr 340 345 350 His Val Asn Asn Gly Asn Tyr Thr Leu Leu Ala Ala Asn Pro Ser Gly 355 360 365 Arg Ala Ala Ala Phe Val Met Ala Ala Phe Met Asp Asn Pro Phe Glu 370 375 380 Phe Asn Pro Glu Asp Pro Ile Pro Val Ser Phe Ser Pro Val Asp Thr 385 390 395 400 Asn Ser Thr Ser Gly Asp Pro Val Glu Lys Lys Asp Glu Thr Pro Phe 405 410 415 Gly Val Ser Val Ala Val Gly Leu Ala Val Phe Ala Cys Leu Phe Leu 420 425 430 Ser Thr Leu Phe Leu Ala Leu Asn Lys Cys Gly Arg Arg Asn Lys Phe 435 440 445 Gly Gly Asn Arg Ala Val Val Leu Ala Pro Glu Asp Gly Leu Ala Met 450 455 460 Ser Leu His Phe Met Thr Leu Gly Gly Ser Ser Leu Ser Pro Thr Glu 465 470 475 480 Gly Lys Gly Ser Gly Leu Gln Gly His Ile Ile Glu Asn Pro Gln Tyr 485 490 495 Phe Ser Asp Ala Cys Val His His Ile Lys Arg Gln Asp Ile Val Leu 500 505 510 Lys Trp Glu Leu Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu 515 520 525 Cys His Asn Leu Leu Pro Glu Gln Asp Lys Met Leu Val Ala Val Lys 530 535 540 Ala Leu Lys Glu Val Ser Glu Ser Ala Arg Gln Asp Phe Gln Arg Glu 545 550 555 560 Ala Gln Leu Leu Thr Met Leu Gln His Gln His Ile Val Arg Phe Phe 565 570 575 Gly Val Cys Thr Glu Gly Arg Pro Leu Leu Met Val Phe Glu Tyr Met 580 585 590 Arg His Gly Asp Leu Asn Arg Phe Leu Arg Ser His Gly Pro Asp Ala 595 600 605 Lys Leu Leu Ala Gly Gly Glu Asp Val Ala Pro Gly Pro Leu Gly Leu 610 615 620 Gly Gln Leu Leu Ala Val Ala Ser Gln Val Ala Ala Gly Met Val Tyr 625 630 635 640 Leu Ala Gly Leu His Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys 645 650 655 Leu Val Gly Gln Gly Leu Val Val Lys Ile Gly Asp Phe Gly Met Ser 660 665 670 Arg Asp Ile Tyr Ser Thr Asp Tyr Tyr Arg Val Gly Gly Arg Thr Met 675 680 685 Leu Pro Ile Arg Trp Met Pro Pro Glu Ser Ile Leu Tyr Arg Lys Phe 690 695 700 Thr Thr Glu Ser Asp Val Trp Ser Phe Gly Val Val Leu Trp Glu Ile 705 710 715 720 Phe Thr Tyr Gly Lys Gln Pro Trp Tyr Gln Leu Ser Asn Thr Glu Ala 725 730 735 Ile Glu Cys Ile Thr Gln Gly Arg Glu Leu Glu Arg Pro Arg Ala Cys 740 745 750 Pro Pro Glu Val Tyr Ala Ile Met Arg Gly Cys Trp Gln Arg Glu Pro 755 760 765 Gln Gln Arg His Ser Ile Lys Asp Val His Ala Arg Leu Gln Ala Leu 770 775 780 Ala Gln Ala Pro Pro Val Tyr Leu Asp Val Leu Gly 785 790 795 <210> 16 <211> 455 <212> PRT <213> Artificial Sequence <220> <223> canine-human fusion protein <400> 16 Glu Pro His Pro Glu Ser His Val Pro Ala Gly His Ala Ile Pro His 1 5 10 15 Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu Arg Arg 20 25 30 Ala Arg Ser Ala Pro Ala Gly Ala Ile Ala Ala Arg Val Thr Gly Gln 35 40 45 Thr Arg Asn Ile Thr Val Asp Pro Lys Leu Phe Lys Lys Arg Arg Leu 50 55 60 Arg Ser Pro Arg Val Leu Phe Ser Thr His Pro Pro Pro Val Ala Ala 65 70 75 80 Asp Ala Gln Asp Leu Asp Leu Glu Ala Gly Ser Thr Ala Ser Val Asn 85 90 95 Arg Thr His Arg Ser Lys Arg Ser Ser Ser His Pro Val Phe His Arg 100 105 110 Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp Lys 115 120 125 Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu 130 135 140 Val Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys 145 150 155 160 Cys Arg Asp Pro Thr Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser 165 170 175 Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala 180 185 190 Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp 195 200 205 Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Gly Arg Arg Ala Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 17 <211> 629 <212> PRT <213> Canis familiaris <400> 17 Ala Ala Pro Cys Pro Asp Val Cys Cys Pro His Gly Pro Ser Gly Leu 1 5 10 15 Arg Cys Thr Arg Ala Gly Ala Leu Gln Ser Leu His Arg Leu Pro Gly 20 25 30 Val Glu Asn Leu Thr Glu Leu Tyr Ile Asp Asn Gln Glu His Leu Gln 35 40 45 His Leu Asp Ala Val His Leu Lys Gly Leu Gly Met Leu Arg Asp Leu 50 55 60 Thr Ile Val Lys Ser Gly Leu Arg Ser Val Ala Pro Asp Ala Phe His 65 70 75 80 Phe Thr Pro Arg Leu Arg Arg Leu Asn Leu Ser Phe Asn Ala Leu Glu 85 90 95 Ser Leu Ser Trp Lys Thr Val Gln Gly Leu Pro Leu Gln Glu Leu Val 100 105 110 Leu Ser Gly Asn Pro Leu His Cys Ser Cys Ala Leu His Trp Leu Leu 115 120 125 Arg Trp Glu Glu Glu Gly Leu Gly Gly Val Arg Gly Gln Arg Leu Gln 130 135 140 Cys Pro Gly Gln Gly Pro Leu Ala Leu Leu Ser Asn Ala Ser Cys Gly 145 150 155 160 Val Pro Val Leu Lys Val Gln Met Pro Asn Ala Ser Val Glu Val Gly 165 170 175 Asp Asp Val Leu Leu Gln Cys Gln Val Glu Gly Arg Gly Leu Glu Arg 180 185 190 Ala Gly Trp Ile Leu Pro Glu Val Glu Glu Leu Ala Thr Val Thr Gln 195 200 205 Ser Gly Asp Leu Pro Ser Leu Gly Leu Thr Leu Ala Asn Val Thr Ser 210 215 220 Asp Leu Asn Arg Lys Asn Val Thr Cys Trp Ala Glu Asn Asp Val Gly 225 230 235 240 Arg Ala Glu Val Ser Val Gln Val Asn Val Ser Phe Pro Ala Ser Val 245 250 255 Gln Leu His Glu Ala Val Glu Leu His His Trp Cys Ile Pro Phe Ser 260 265 270 Val Asp Gly Gln Pro Ala Pro Ser Leu Arg Trp Leu Phe Asn Gly Ser 275 280 285 Val Leu Asn Glu Thr Ser Phe Ile Phe Thr Glu Phe Leu Glu Pro Val 290 295 300 Ala Asn Glu Thr Val Arg His Gly Cys Leu Arg Leu Asn Gln Pro Thr 305 310 315 320 His Val Asn Asn Gly Asn Tyr Thr Leu Leu Ala Ala Asn Pro Ser Gly 325 330 335 Arg Ala Ala Ala Phe Val Met Ala Ala Phe Met Asp Asn Pro Phe Glu 340 345 350 Phe Asn Pro Glu Asp Pro Ile Pro Val Ser Phe Ser Pro Val Asp Thr 355 360 365 Asn Ser Thr Ser Gly Asp Pro Val Glu Lys Lys Asp Glu Thr Pro Phe 370 375 380 Gly Val Ser Val Ala Val Gly Val Pro Lys Arg Glu Asn Gly Arg Val 385 390 395 400 Pro Arg Pro Pro Asp Cys Pro Lys Cys Pro Ala Pro Glu Met Leu Gly 405 410 415 Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Leu 420 425 430 Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Leu Asp Pro 435 440 445 Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys Gln Met 450 455 460 Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn Gly Thr Tyr 465 470 475 480 Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu Lys Gly 485 490 495 Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser Pro Ile 500 505 510 Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro Ser Val 515 520 525 Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr Val Ser 530 535 540 Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp Val Glu 545 550 555 560 Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg Thr Thr 565 570 575 Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu 580 585 590 Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile Cys Ala 595 600 605 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser 610 615 620 His Ser Pro Gly Lys 625 <210> 18 <211> 123 <212> PRT <213> Homo sapiens <400> 18 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Arg Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Arg Ser Ser His Thr Ile Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Asp Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Val Tyr Ser Ser Gly Trp His Val Ser Asp Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Ile Leu Val Thr Val Ser Ser 115 120 <210> 19 <211> 107 <212> PRT <213> Homo sapiens <400> 19 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 20 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> modified canine <400> 20 Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr 1 5 10 15 Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Leu 20 25 30 Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys 35 40 45 Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Ala Gly 50 55 60 Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu 65 70 75 80 Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser 85 90 95 Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro 100 105 110 Ser Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr 115 120 125 Val Ser Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp 130 135 140 Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg 145 150 155 160 Thr Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser 165 170 175 Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile 180 185 190 Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser 195 200 205 Leu Ser His Ser Pro Gly Lys 210 215 <210> 21 <211> 118 <212> PRT <213> Homo sapiens <400> 21 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Phe Thr Leu Thr Glu Leu 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Thr Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Thr Ile Gly Val Val Thr Asn Phe Asp Asn Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 22 <211> 107 <212> PRT <213> Homo sapiens <400> 22 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Ala Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ala Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Phe Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Leu Ala Ser Tyr Tyr Cys Gln Gln Tyr Asn Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 23 <211> 458 <212> PRT <213> Artificial Sequence <220> <223> Human canine fusion protein <400> 23 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Arg Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Arg Ser Ser His Thr Ile Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Asp Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Val Tyr Ser Ser Gly Trp His Val Ser Asp Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Ile Leu Val Thr Val Ser Ser Ala Ser Thr Thr Ala 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Cys Gly Ser Thr Ser Gly Ser 130 135 140 Thr Val Ala Leu Ala Cys Leu Val Ser Gly Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ser Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ser Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Met Val 180 185 190 Thr Val Pro Ser Ser Arg Trp Pro Ser Glu Thr Phe Thr Cys Asn Val 195 200 205 Ala His Pro Ala Ser Lys Thr Lys Val Asp Lys Pro Val Pro Lys Arg 210 215 220 Glu Asn Gly Arg Val Pro Arg Pro Pro Asp Cys Pro Lys Cys Pro Ala 225 230 235 240 Pro Glu Met Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro 245 250 255 Lys Asp Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val 260 265 270 Val Asp Leu Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val 275 280 285 Asp Gly Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln 290 295 300 Phe Asn Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln 305 310 315 320 Asp Trp Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala 325 330 335 Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala 340 345 350 His Gln Pro Ser Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser 355 360 365 Lys Asn Thr Val Ser Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro 370 375 380 Asp Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser 385 390 395 400 Lys Tyr Arg Thr Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe 405 410 415 Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp 420 425 430 Thr Phe Ile Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr 435 440 445 Gln Glu Ser Leu Ser His Ser Pro Gly Lys 450 455 <210> 24 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Human canine fusion protein <400> 24 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Asn Asp Ala Gln 100 105 110 Pro Ala Val Tyr Leu Phe Gln Pro Ser Pro Asp Gln Leu His Thr Gly 115 120 125 Ser Ala Ser Val Val Cys Leu Leu Asn Ser Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Val Asp Gly Val Ile Gln Asp Thr Gly Ile Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Met Ser Ser Thr Glu Tyr Leu Ser His Glu Leu Tyr Ser 180 185 190 Cys Glu Ile Thr His Lys Ser Leu Pro Ser Thr Leu Ile Lys Ser Phe 195 200 205 Gln Arg Ser Glu Cys Gln Arg Val Asp 210 215 <210> 25 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Human canine fusion protein <400> 25 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Phe Thr Leu Thr Glu Leu 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Thr Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Thr Ile Gly Val Val Thr Asn Phe Asp Asn Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Cys Gly Ser Thr Ser Gly Ser Thr Val Ala Leu Ala 130 135 140 Cys Leu Val Ser Gly Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ser Leu Thr Ser Gly Val His Thr Phe Pro Ser Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Met Val Thr Val Pro Ser Ser 180 185 190 Arg Trp Pro Ser Glu Thr Phe Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Lys Thr Lys Val Asp Lys Pro Val Pro Lys Arg Glu Asn Gly Arg Val 210 215 220 Pro Arg Pro Pro Asp Cys Pro Lys Cys Pro Ala Pro Glu Met Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Leu 245 250 255 Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Leu Asp Pro 260 265 270 Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys Gln Met 275 280 285 Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn Gly Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu Lys Gly 305 310 315 320 Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser Pro Ile 325 330 335 Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro Ser Val 340 345 350 Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr Val Ser 355 360 365 Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp Val Glu 370 375 380 Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg Thr Thr 385 390 395 400 Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu 405 410 415 Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile Cys Ala 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser 435 440 445 His Ser Pro Gly Lys 450 <210> 26 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Human canine fusion protein <400> 26 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Ala Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ala Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Phe Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Leu Ala Ser Tyr Tyr Cys Gln Gln Tyr Asn Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Asn Asp Ala Gln 100 105 110 Pro Ala Val Tyr Leu Phe Gln Pro Ser Pro Asp Gln Leu His Thr Gly 115 120 125 Ser Ala Ser Val Val Cys Leu Leu Asn Ser Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Val Asp Gly Val Ile Gln Asp Thr Gly Ile Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Met Ser Ser Thr Glu Tyr Leu Ser His Glu Leu Tyr Ser 180 185 190 Cys Glu Ile Thr His Lys Ser Leu Pro Ser Thr Leu Ile Lys Ser Phe 195 200 205 Gln Arg Ser Glu Cys Gln Arg Val Asp 210 215 <210> 27 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Caninized human <400> 27 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val 35 40 45 Ser Tyr Ile Ser Arg Ser Ser His Thr Ile Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Tyr Ser Ser Gly Trp His Val Ser Asp Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 28 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Caninized human <400> 28 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Leu Arg Ser Tyr 20 25 30 Ser Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val 35 40 45 Ser Tyr Ile Ser Arg Ser Ser His Thr Ile Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Tyr Ser Ser Gly Trp His Val Ser Asp Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Ile Leu Val Thr Val Ser Ser 115 120 <210> 29 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Caninized human <400> 29 Glu Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ser Leu Ser Gln Glu 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ser Phe Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 30 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Caninized human <400> 30 Glu Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ser Leu Ser Gln Glu 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 31 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Caninized human <400> 31 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Ile Glu Leu 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Ala Gly Leu Asp Trp Met 35 40 45 Gly Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ala Gly Asp Ile Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Gly Val Val Thr Asn Phe Asp Asn Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 32 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Caninized human <400> 32 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu Leu 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Met 35 40 45 Gly Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ala Gly Asp Ile Ala Val Tyr Tyr Cys 85 90 95 Ser Thr Ile Gly Val Val Thr Asn Phe Asp Asn Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 33 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Caninized human <400> 33 Glu Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ser Leu Ser Gln Glu 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ala Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Phe Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ser Phe Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 34 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Caninized human <400> 34 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Thr Ala Ser Ile Ser Cys Arg Ala Ser Gln Ala Ile Arg Asn Asp 20 25 30 Leu Gly Trp Phe Arg Gln Lys Pro Gly Gln Ser Pro Gln Arg Leu Ile 35 40 45 Tyr Ala Ala Phe Asn Leu Gln Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile Ser Arg Val Glu Ala 65 70 75 80 Asp Asp Thr Gly Val Tyr Tyr Cys Gln Gln Tyr Asn Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 35 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> caninized human <400> 35 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Thr Ala Ser Ile Ser Cys Arg Ala Ser Gln Ala Ile Arg Asn Asp 20 25 30 Leu Gly Trp Phe Arg Gln Lys Pro Gly Lys Ser Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Phe Asn Leu Gln Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Glu Ala 65 70 75 80 Asp Asp Thr Gly Val Tyr Tyr Cys Gln Gln Tyr Asn Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 36 <211> 458 <212> PRT <213> Artificial Sequence <220> <223> Caninized human <400> 36 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val 35 40 45 Ser Tyr Ile Ser Arg Ser Ser His Thr Ile Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Tyr Ser Ser Gly Trp His Val Ser Asp Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Thr Ala 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Cys Gly Ser Thr Ser Gly Ser 130 135 140 Thr Val Ala Leu Ala Cys Leu Val Ser Gly Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ser Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ser Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Met Val 180 185 190 Thr Val Pro Ser Ser Arg Trp Pro Ser Glu Thr Phe Thr Cys Asn Val 195 200 205 Ala His Pro Ala Ser Lys Thr Lys Val Asp Lys Pro Val Pro Lys Arg 210 215 220 Glu Asn Gly Arg Val Pro Arg Pro Pro Asp Cys Pro Lys Cys Pro Ala 225 230 235 240 Pro Glu Met Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro 245 250 255 Lys Asp Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val 260 265 270 Val Asp Leu Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val 275 280 285 Asp Gly Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln 290 295 300 Phe Asn Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln 305 310 315 320 Asp Trp Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala 325 330 335 Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala 340 345 350 His Gln Pro Ser Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser 355 360 365 Lys Asn Thr Val Ser Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro 370 375 380 Asp Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser 385 390 395 400 Lys Tyr Arg Thr Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe 405 410 415 Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp 420 425 430 Thr Phe Ile Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr 435 440 445 Gln Glu Ser Leu Ser His Ser Pro Gly Lys 450 455 <210> 37 <211> 458 <212> PRT <213> Artificial Sequence <220> <223> Caninized human <400> 37 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Leu Arg Ser Tyr 20 25 30 Ser Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val 35 40 45 Ser Tyr Ile Ser Arg Ser Ser His Thr Ile Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Tyr Ser Ser Gly Trp His Val Ser Asp Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Ile Leu Val Thr Val Ser Ser Ala Ser Thr Thr Ala 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Cys Gly Ser Thr Ser Gly Ser 130 135 140 Thr Val Ala Leu Ala Cys Leu Val Ser Gly Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ser Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ser Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Met Val 180 185 190 Thr Val Pro Ser Ser Arg Trp Pro Ser Glu Thr Phe Thr Cys Asn Val 195 200 205 Ala His Pro Ala Ser Lys Thr Lys Val Asp Lys Pro Val Pro Lys Arg 210 215 220 Glu Asn Gly Arg Val Pro Arg Pro Pro Asp Cys Pro Lys Cys Pro Ala 225 230 235 240 Pro Glu Met Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro 245 250 255 Lys Asp Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val 260 265 270 Val Asp Leu Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val 275 280 285 Asp Gly Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln 290 295 300 Phe Asn Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln 305 310 315 320 Asp Trp Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala 325 330 335 Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala 340 345 350 His Gln Pro Ser Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser 355 360 365 Lys Asn Thr Val Ser Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro 370 375 380 Asp Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser 385 390 395 400 Lys Tyr Arg Thr Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe 405 410 415 Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp 420 425 430 Thr Phe Ile Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr 435 440 445 Gln Glu Ser Leu Ser His Ser Pro Gly Lys 450 455 <210> 38 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Caninized human <400> 38 Glu Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ser Leu Ser Gln Glu 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ser Phe Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Asn Asp Ala Gln 100 105 110 Pro Ala Val Tyr Leu Phe Gln Pro Ser Pro Asp Gln Leu His Thr Gly 115 120 125 Ser Ala Ser Val Val Cys Leu Leu Asn Ser Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Val Asp Gly Val Ile Gln Asp Thr Gly Ile Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Met Ser Ser Thr Glu Tyr Leu Ser His Glu Leu Tyr Ser 180 185 190 Cys Glu Ile Thr His Lys Ser Leu Pro Ser Thr Leu Ile Lys Ser Phe 195 200 205 Gln Arg Ser Glu Cys Gln Arg Val Asp 210 215 <210> 39 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Caninized human <400> 39 Glu Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ser Leu Ser Gln Glu 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Asn Asp Ala Gln 100 105 110 Pro Ala Val Tyr Leu Phe Gln Pro Ser Pro Asp Gln Leu His Thr Gly 115 120 125 Ser Ala Ser Val Val Cys Leu Leu Asn Ser Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Val Asp Gly Val Ile Gln Asp Thr Gly Ile Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Met Ser Ser Thr Glu Tyr Leu Ser His Glu Leu Tyr Ser 180 185 190 Cys Glu Ile Thr His Lys Ser Leu Pro Ser Thr Leu Ile Lys Ser Phe 195 200 205 Gln Arg Ser Glu Cys Gln Arg Val Asp 210 215 <210> 40 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Caninized human <400> 40 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Ile Glu Leu 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Ala Gly Leu Asp Trp Met 35 40 45 Gly Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ala Gly Asp Ile Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Gly Val Val Thr Asn Phe Asp Asn Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Cys Gly Ser Thr Ser Gly Ser Thr Val Ala Leu Ala 130 135 140 Cys Leu Val Ser Gly Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ser Leu Thr Ser Gly Val His Thr Phe Pro Ser Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Met Val Thr Val Pro Ser Ser 180 185 190 Arg Trp Pro Ser Glu Thr Phe Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Lys Thr Lys Val Asp Lys Pro Val Pro Lys Arg Glu Asn Gly Arg Val 210 215 220 Pro Arg Pro Pro Asp Cys Pro Lys Cys Pro Ala Pro Glu Met Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Leu 245 250 255 Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Leu Asp Pro 260 265 270 Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys Gln Met 275 280 285 Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn Gly Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu Lys Gly 305 310 315 320 Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser Pro Ile 325 330 335 Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro Ser Val 340 345 350 Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr Val Ser 355 360 365 Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp Val Glu 370 375 380 Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg Thr Thr 385 390 395 400 Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu 405 410 415 Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile Cys Ala 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser 435 440 445 His Ser Pro Gly Lys 450 <210> 41 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Caninized human <400> 41 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu Leu 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Met 35 40 45 Gly Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ala Gly Asp Ile Ala Val Tyr Tyr Cys 85 90 95 Ser Thr Ile Gly Val Val Thr Asn Phe Asp Asn Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Cys Gly Ser Thr Ser Gly Ser Thr Val Ala Leu Ala 130 135 140 Cys Leu Val Ser Gly Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ser Leu Thr Ser Gly Val His Thr Phe Pro Ser Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Met Val Thr Val Pro Ser Ser 180 185 190 Arg Trp Pro Ser Glu Thr Phe Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Lys Thr Lys Val Asp Lys Pro Val Pro Lys Arg Glu Asn Gly Arg Val 210 215 220 Pro Arg Pro Pro Asp Cys Pro Lys Cys Pro Ala Pro Glu Met Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Leu 245 250 255 Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Leu Asp Pro 260 265 270 Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys Gln Met 275 280 285 Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn Gly Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu Lys Gly 305 310 315 320 Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser Pro Ile 325 330 335 Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro Ser Val 340 345 350 Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr Val Ser 355 360 365 Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp Val Glu 370 375 380 Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg Thr Thr 385 390 395 400 Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu 405 410 415 Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile Cys Ala 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser 435 440 445 His Ser Pro Gly Lys 450 <210> 42 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Caninized human <400> 42 Glu Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ser Leu Ser Gln Glu 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ala Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Phe Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ser Phe Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Asn Asp Ala Gln 100 105 110 Pro Ala Val Tyr Leu Phe Gln Pro Ser Pro Asp Gln Leu His Thr Gly 115 120 125 Ser Ala Ser Val Val Cys Leu Leu Asn Ser Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Val Asp Gly Val Ile Gln Asp Thr Gly Ile Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Met Ser Ser Thr Glu Tyr Leu Ser His Glu Leu Tyr Ser 180 185 190 Cys Glu Ile Thr His Lys Ser Leu Pro Ser Thr Leu Ile Lys Ser Phe 195 200 205 Gln Arg Ser Glu Cys Gln Arg Val Asp 210 215 <210> 43 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Caninized human <400> 43 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Thr Ala Ser Ile Ser Cys Arg Ala Ser Gln Ala Ile Arg Asn Asp 20 25 30 Leu Gly Trp Phe Arg Gln Lys Pro Gly Gln Ser Pro Gln Arg Leu Ile 35 40 45 Tyr Ala Ala Phe Asn Leu Gln Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile Ser Arg Val Glu Ala 65 70 75 80 Asp Asp Thr Gly Val Tyr Tyr Cys Gln Gln Tyr Asn Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Asn Asp Ala Gln 100 105 110 Pro Ala Val Tyr Leu Phe Gln Pro Ser Pro Asp Gln Leu His Thr Gly 115 120 125 Ser Ala Ser Val Val Cys Leu Leu Asn Ser Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Val Asp Gly Val Ile Gln Asp Thr Gly Ile Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Met Ser Ser Thr Glu Tyr Leu Ser His Glu Leu Tyr Ser 180 185 190 Cys Glu Ile Thr His Lys Ser Leu Pro Ser Thr Leu Ile Lys Ser Phe 195 200 205 Gln Arg Ser Glu Cys Gln Arg Val Asp 210 215 <210> 44 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Caninized human <400> 44 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Thr Ala Ser Ile Ser Cys Arg Ala Ser Gln Ala Ile Arg Asn Asp 20 25 30 Leu Gly Trp Phe Arg Gln Lys Pro Gly Lys Ser Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Phe Asn Leu Gln Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Glu Ala 65 70 75 80 Asp Asp Thr Gly Val Tyr Tyr Cys Gln Gln Tyr Asn Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Asn Asp Ala Gln 100 105 110 Pro Ala Val Tyr Leu Phe Gln Pro Ser Pro Asp Gln Leu His Thr Gly 115 120 125 Ser Ala Ser Val Val Cys Leu Leu Asn Ser Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Val Asp Gly Val Ile Gln Asp Thr Gly Ile Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Met Ser Ser Thr Glu Tyr Leu Ser His Glu Leu Tyr Ser 180 185 190 Cys Glu Ile Thr His Lys Ser Leu Pro Ser Thr Leu Ile Lys Ser Phe 195 200 205 Gln Arg Ser Glu Cys Gln Arg Val Asp 210 215 <210> 45 <211> 17 <212> PRT <213> Canis familiaris <400> 45 Phe Asn Glu Cys Arg Cys Thr Asp Thr Pro Pro Cys Pro Val Pro Glu 1 5 10 15 Pro <210> 46 <211> 22 <212> PRT <213> Canis familiaris <400> 46 Pro Lys Arg Glu Asn Gly Arg Val Pro Arg Pro Pro Asp Cys Pro Lys 1 5 10 15 Cys Pro Ala Pro Glu Met 20 <210> 47 <211> 20 <212> PRT <213> Canis familiaris <400> 47 Ala Lys Glu Cys Glu Cys Lys Cys Asn Cys Asn Asn Cys Pro Cys Pro 1 5 10 15 Gly Cys Gly Leu 20 <210> 48 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> modified canine <400> 48 Pro Lys Glu Ser Thr Cys Lys Cys Ile Pro Pro Cys Pro Val Pro Glu 1 5 10 15 Ser <210> 49 <211> 216 <212> PRT <213> Canis familiaris <400> 49 Leu Gly Gly Pro Ser Val Leu Ile Phe Pro Pro Lys Pro Lys Asp Ile 1 5 10 15 Leu Arg Ile Thr Arg Thr Pro Glu Val Thr Cys Val Val Leu Asp Leu 20 25 30 Gly Arg Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys 35 40 45 Glu Val His Thr Ala Lys Thr Gln Ser Arg Glu Gln Gln Phe Asn Gly 50 55 60 Thr Tyr Arg Val Val Ser Val Leu Pro Ile Glu His Gln Asp Trp Leu 65 70 75 80 Thr Gly Lys Glu Phe Lys Cys Arg Val Asn His Ile Asp Leu Pro Ser 85 90 95 Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Arg Ala His Lys Pro 100 105 110 Ser Val Tyr Val Leu Pro Pro Ser Pro Lys Glu Leu Ser Ser Ser Asp 115 120 125 Thr Val Ser Ile Thr Cys Leu Ile Lys Asp Phe Tyr Pro Pro Asp Ile 130 135 140 Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Arg Lys His 145 150 155 160 Arg Met Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr 165 170 175 Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Gln Gly Asp Pro Phe 180 185 190 Thr Cys Ala Val Met His Glu Thr Leu Gln Asn His Tyr Thr Asp Leu 195 200 205 Ser Leu Ser His Ser Pro Gly Lys 210 215 <210> 50 <211> 215 <212> PRT <213> Canis familiaris <400> 50 Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr 1 5 10 15 Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Leu 20 25 30 Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys 35 40 45 Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn Gly 50 55 60 Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu 65 70 75 80 Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser 85 90 95 Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro 100 105 110 Ser Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr 115 120 125 Val Ser Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp 130 135 140 Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg 145 150 155 160 Thr Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser 165 170 175 Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile 180 185 190 Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser 195 200 205 Leu Ser His Ser Pro Gly Lys 210 215 <210> 51 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> modified canine <400> 51 Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr 1 5 10 15 Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Leu 20 25 30 Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys 35 40 45 Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Ala Gly 50 55 60 Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu 65 70 75 80 Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser 85 90 95 Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro 100 105 110 Ser Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr 115 120 125 Val Ser Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp 130 135 140 Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg 145 150 155 160 Thr Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser 165 170 175 Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile 180 185 190 Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser 195 200 205 Leu Ser His Ser Pro Gly 210 <210> 52 <211> 215 <212> PRT <213> Canis familiaris <400> 52 Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Ile 1 5 10 15 Leu Val Thr Ala Arg Thr Pro Thr Val Thr Cys Val Val Val Asp Leu 20 25 30 Asp Pro Glu Asn Pro Glu Val Gln Ile Ser Trp Phe Val Asp Ser Lys 35 40 45 Gln Val Gln Thr Ala Asn Thr Gln Pro Arg Glu Glu Gln Ser Asn Gly 50 55 60 Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu 65 70 75 80 Ser Gly Lys Gln Phe Lys Cys Lys Val Asn Asn Lys Ala Leu Pro Ser 85 90 95 Pro Ile Glu Glu Ile Ile Ser Lys Thr Pro Gly Gln Ala His Gln Pro 100 105 110 Asn Val Tyr Val Leu Pro Pro Ser Arg Asp Glu Met Ser Lys Asn Thr 115 120 125 Val Thr Leu Thr Cys Leu Val Lys Asp Phe Phe Pro Pro Glu Ile Asp 130 135 140 Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg 145 150 155 160 Met Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser 165 170 175 Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile 180 185 190 Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Ile Ser 195 200 205 Leu Ser His Ser Pro Gly Lys 210 215 <210> 53 <211> 216 <212> PRT <213> Canis familiaris <400> 53 Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Ile 1 5 10 15 Leu Arg Ile Thr Arg Thr Pro Glu Ile Thr Cys Val Val Leu Asp Leu 20 25 30 Gly Arg Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys 35 40 45 Glu Val His Thr Ala Lys Thr Gln Pro Arg Glu Gln Gln Phe Asn Ser 50 55 60 Thr Tyr Arg Val Val Ser Val Leu Pro Ile Glu His Gln Asp Trp Leu 65 70 75 80 Thr Gly Lys Glu Phe Lys Cys Arg Val Asn His Ile Gly Leu Pro Ser 85 90 95 Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro 100 105 110 Ser Val Tyr Val Leu Pro Pro Ser Pro Lys Glu Leu Ser Ser Ser Asp 115 120 125 Thr Val Thr Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Glu Ile 130 135 140 Asp Val Glu Trp Gln Ser Asn Gly Gln Pro Glu Pro Glu Ser Lys Tyr 145 150 155 160 His Thr Thr Ala Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr 165 170 175 Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Gln Gly Asp Thr Phe 180 185 190 Thr Cys Ala Val Met His Glu Ala Leu Gln Asn His Tyr Thr Asp Leu 195 200 205 Ser Leu Ser His Ser Pro Gly Lys 210 215

Claims

1. A caninized antibody or antigen-binding fragment thereof that binds to canine nerve growth factor (NGF), comprising a heavy chain and a light chain, wherein the light chain comprises three light chain complementarity determining regions (CDRs): CDR light 1 (CDRL1), CDR light 2 (CDRL2), and CDR light 3 (CDRL3), and wherein the heavy chain comprises three heavy chain CDRs: CDR heavy 1 (CDRH1), CDR heavy 2 (CDRH2), and CDR heavy 3 (CDRH3); (a) CDRH1 comprises the amino acid sequence of SEQ ID NO: 1; (b) CDRH2 comprises the amino acid sequence of SEQ ID NO:2; (c) CDRH3 comprises the amino acid sequence of SEQ ID NO: 3; (d) CDRL1 comprises the amino acid sequence of SEQ ID NO:4; (e) CDRL2 comprises the amino acid sequence of SEQ ID NO:5, and (f) A caninized antibody or antigen-binding fragment thereof, wherein CDRL3 comprises the amino acid sequence of SEQ ID NO:

6.

2. The caninized antibody or antigen-binding fragment thereof of claim 1, comprising a hinge region comprising an amino acid sequence having at least 90% or 95% identity, or 100% identity, to an amino acid sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO:

48.

3. The caninized antibody or antigen-binding fragment thereof of claim 1, comprising a caninized fragment crystallizable region (cFc region), wherein the cFc region comprises an amino acid sequence having at least 90%, 95%, 98%, 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:20, and SEQ ID NO:

51.

4. The caninized antibody or antigen-binding fragment thereof of claim 3, wherein the cFc region comprises an amino acid sequence having at least 90%, 95%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 20 or the amino acid sequence of SEQ ID NO: 51, wherein both the aspartic acid residue (D) at position 31 of the amino acid sequence of SEQ ID NO: 50 and the asparagine residue (N) at position 63 of the amino acid sequence of SEQ ID NO: 50 are substituted with alanine residues (A).

5. the caninized antibody or antigen-binding fragment thereof is (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 27 and SEQ ID NO: 28; (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 30; (c) both a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:27 and SEQ ID NO:28, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:29 and SEQ ID NO:30; The caninized antibody or antigen-binding fragment thereof of claim 1, comprising:

6. (a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:27, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:29; (b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:27, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:30; (c) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:28 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:29; or (d) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 28, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

30. The caninized antibody or antigen-binding fragment thereof according to claim 5.

7. (a) the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO:

39. (b) the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 36 and SEQ ID NO: 37; or (c) the heavy chain is i. comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 36 and comprising the amino acid sequence of SEQ ID NO: 27; or ii. comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 37 and comprising the amino acid sequence of SEQ ID NO: 28; (d) the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO: 39, and the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 36 and SEQ ID NO:

37. (e) the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO: 39, and the heavy chain comprises i. comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 36 and comprising the amino acid sequence of SEQ ID NO: 27; or ii. comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 37 and comprising the amino acid sequence of SEQ ID NO: 28; The caninized antibody or antigen-binding fragment thereof according to claim 1.

8. the heavy chain comprises the amino acid sequence of SEQ ID NO: 37 and the light chain comprises the amino acid sequence of SEQ ID NO: 39; the heavy chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 37 and comprising the amino acid sequence of SEQ ID NO: 28, and the light chain comprises the amino acid sequence of SEQ ID NO: 39; the heavy chain comprises the amino acid sequence of SEQ ID NO: 37 and the light chain comprises the amino acid sequence of SEQ ID NO: 38; the heavy chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:37 and comprising the amino acid sequence of SEQ ID NO:28, and the light chain comprises the amino acid sequence of SEQ ID NO:38; the heavy chain comprises the amino acid sequence of SEQ ID NO: 36 and the light chain comprises the amino acid sequence of SEQ ID NO: 39; the heavy chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 36 and comprising the amino acid sequence of SEQ ID NO: 27, and the light chain comprises the amino acid sequence of SEQ ID NO: 39; the heavy chain comprises the amino acid sequence of SEQ ID NO: 36 and the light chain comprises the amino acid sequence of SEQ ID NO: 38; or the heavy chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 36 and comprising the amino acid sequence of SEQ ID NO: 27, and the light chain comprises the amino acid sequence of SEQ ID NO: 38; The caninized antibody or antigen-binding fragment thereof according to claim 7.

9. The caninized antibody or antigen-binding fragment thereof of claim 8, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 37 and the light chain comprises the amino acid sequence of SEQ ID NO:

39.

10. A nucleic acid encoding the caninized antibody or antigen-binding fragment thereof of any one of claims 1 to 9: (a) the light chain; (b) a light chain variable region; (c) the heavy chain; (d) a heavy chain variable region; (e) both the light and heavy chains; (f) Both the light chain variable region and the heavy chain variable region.

11. 11. An expression vector comprising one or more of the nucleic acids of claim 10.

12. 12. An expression vector system comprising a pair of expression vectors according to claim 11, wherein: (a) one of a pair of expression vectors encodes the heavy chain of the caninized antibody, and the other of the pair of expression vectors encodes the light chain of the caninized antibody; or (b) one of the pair of expression vectors encodes the heavy chain variable region of the caninized antibody, and the other of the pair of expression vectors encodes the light chain variable region of the caninized antibody; Expression vector system.

13. A host cell comprising one or more of the expression vectors of claim 11.

14. 13. A pair of host cells comprising the expression vector system of claim 12, wherein one of the pair of host cells comprises an expression vector encoding the heavy chain of the caninized antibody, and the other of the pair of host cells comprises an expression vector encoding the light chain of the caninized antibody.

15. 13. A pharmaceutical composition comprising the caninized antibody or antigen-binding fragment thereof of claim 1, or the expression vector system of claim 12, or any combination thereof, and a pharmaceutically acceptable carrier or diluent.

16. 16. Use of a pharmaceutical composition according to claim 15 for the preparation of a medicament to aid in the treatment of pain-related conditions in dogs.

17. 17. The use of claim 16, wherein the condition is osteoarthritis, hyperalgesia, allodynia, pain, or any combination thereof.

18. A method for producing a caninized antibody or antigen-binding fragment thereof that binds to canine NGF, comprising: a. culturing each of the pair of host cells of claim 14, individually or in combination, in a culture medium under conditions in which the expression vector is expressed, thereby producing a polypeptide comprising the light chain of the caninized antibody, the heavy chain of the caninized antibody, or both; and b. recovering the light chain of the caninized antibody, the heavy chain of the caninized antibody, or both, from the host cell or culture medium.

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