Parvovirus antibodies for veterinary use

Abstract

To provide parvovirus antibodies including caninized, felinized, and chimeric antibodies that bind to canine and / or feline parvovirus and have improved expression characteristics.SOLUTION: In various embodiments, parvovirus antibodies have ADCC, ADCP, and / or CDC effector functions. In various embodiments, such monoclonal parvovirus antibodies can be used in methods to prevent and / or treat parvovirus infection in subjects such as dogs and cats. For example, the provided parvovirus antibodies can be used to provide passive immunity against infection with a canine or feline parvovirus.SELECTED DRAWING: Figure 3A

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims the benefit of U.S. Provisional Application No. 62 / 880,650, filed Jul. 30, 2019; U.S. Provisional Application No. 62 / 968,970, filed Jan. 31, 2020; and U.S. Provisional Application No. 63 / 030,123, filed May 26, 2020, each of which is hereby incorporated by reference in its entirety for all purposes.

[0002] [Field of the Invention] This invention relates to, for example, isolated canine parvovirus antibodies having improved recombinant productivity and binding neutralizing activity against canine and / or feline parvovirus, and to methods of using said antibodies, for example, to provide passive immunity against infection by canine or feline parvovirus and / or to treat parvovirus infections in companion animals such as dogs and cats.

Background Art

[0003] Canine parvovirus (CPV) is the most important enteric virus infecting dogs worldwide. 1 . CPV virions are non - enveloped DNA viruses. There are multiple variants of the original CPV, including CPV - 2a, CPV - 2b, and CPV - 2c. The variants of CPV - 2 differ from each other by only a few amino acids. 2 . Since it is highly persistent in the environment, CPV can remain infectious for months. CPV spreads through direct and indirect contact. 3 . When in contact with the oral mucosa, CPV starts replicating in local lymphatics and spreads throughout the body. With an incubation period of 4 to 14 days, CPV targets rapidly dividing cells in the body, including bone marrow, lymphocytes, and intestinal crypt epithelial cells.

[0004] Clinical disease manifests as fever and depression, followed by vomiting, diarrhea (profuse and bloody), lymphopenia, dehydration, and quite frequently secondary sepsis and death. The mortality rate can exceed 70% in puppies.3 Infections are most prevalent in weaned puppies because maternal antibodies begin to wane. Vaccines are readily available and effectively protect against all variants of CPV-2. Vaccination at the appropriate time is protective, but parvovirus infection remains a problem. Major populations of susceptible dogs include puppies in the susceptible period before vaccination, when maternally-derived antibodies have waned to non-protective levels (see Figure 1). Puppies under 24 weeks of age are at highest risk for CPV disease. In addition, puppies in which passive transfer of maternally-derived antibodies has failed are another vulnerable population. Outbreaks continue to occur in naive kennel and shelter populations.

[0005] Treatment of CPV infection up to this point has been mostly supportive. Treatment typically includes intravenous fluids, anti-emetics, and broad-spectrum antibiotics to protect against sepsis. Various other treatments, including antiviral drugs and hyperimmune plasma, have been tried and failed. 4 Hospitalization is typically recommended, but it is costly for many owners and can lead to the decision to euthanize. Strict isolation protocols are required for hospitalized CPV-infected dogs. This has been found to be a logistics challenge for veterinary facilities and staff. 5 Thus, there is an unmet medical need for preventive and therapeutic measures.

[0006] Currently, there are no USDA- or FDA-approved treatments, particularly for CPV. The parvovirus monoclonal antibodies described herein will be administered as a therapeutic intervention to dogs affected with active CPV infection to reduce the severity of CPV or eliminate the morbidity and mortality associated with CPV. In addition, the parvovirus monoclonal antibodies described herein will be utilized as a prophylactic treatment for dogs exposed to CPV-infected dogs to prevent the onset of CPV infection. SUMMARY OF THE INVENTION

[0007] Embodiment 1. An isolated antibody that binds to canine parvovirus and / or feline parvovirus, comprising: (a) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, and (iv) an HC-FR1 sequence of SEQ ID NO: 7 or SEQ ID NO: 8; or (b) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 42, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43, (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 44, and (iv) an HC-FR1 sequence of SEQ ID NO: 45 or SEQ ID NO: 46. Embodiment 2. An isolated antibody that binds to canine parvovirus and / or feline parvovirus, comprising: (a) (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and (iv) an LC-FR1 sequence of SEQ ID NO: 16; or (b) (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54, and (iv) an LC-FR1 sequence of SEQ ID NO: 55 or SEQ ID NO: 56. Embodiment 3. The isolated antibody of Embodiment 1, wherein the antibody of (a) comprises the HC-FR4 sequence of SEQ ID NO: 12. Embodiment 4. The isolated antibody of Embodiment 1, wherein the antibody of (b) comprises the HC-FR2 sequence of SEQ ID NO: 48. Embodiment 5. The isolated antibody of Embodiment 1 or Embodiment 4, wherein the antibody of (b) comprises the HC-FR3 sequence of SEQ ID NO: 50. Embodiment 6. The isolated antibody of Embodiment 2, wherein the antibody of (b) comprises the HC-FR3 sequence of SEQ ID NO: 59. Embodiment 7. The isolated antibody of Embodiment 2 or Embodiment 6, wherein the antibody of (b) comprises the HC-F4 sequence of SEQ ID NO: 61. Embodiment 8. An isolated antibody that binds to canine parvovirus and / or feline parvovirus, the antibody being a caninized or felinized antibody comprising: a) a heavy chain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; or b) a heavy chain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 42; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 44. Embodiment 9. An isolated antibody that binds to canine parvovirus and / or feline parvovirus, the antibody being a caninized or felinized antibody comprising: a) a light chain comprising (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; or b) a light chain comprising (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54. Embodiment 10. An isolated antibody that binds to canine parvovirus and / or feline parvovirus, the antibody being a caninized or felinized antibody comprising: a) a heavy chain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; and b) a light chain comprising (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15. Embodiment 11. An isolated antibody that binds to canine parvovirus and / or feline parvovirus, the antibody being a canineized or felineized antibody comprising: a) a heavy chain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 42; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 44; and b) a light chain comprising (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54. Embodiment 12. The antibody according to any one of the preceding embodiments, wherein the antibody is a chimeric antibody. Embodiment 13. The antibody according to any one of the preceding embodiments, wherein the antibody comprises a canine or feline constant heavy chain region or a canine or feline constant light chain region. Embodiment 14. The antibody is (a) a canine heavy chain constant region selected from the IgG-A, IgG-B, IgG-C, and IgG-D constant regions; or (b) a feline heavy chain constant region selected from the IgG1, IgG2a, and IgG2b constant regions The antibody according to any one of the preceding embodiments. Embodiment 15. The antibody according to any one of the preceding embodiments, wherein the antibody comprises a wild-type or variant IgG Fc having complement binding activity. Embodiment 16. The antibody according to any one of the preceding embodiments, wherein the antibody comprises a wild-type or variant IgG Fc having antibody-dependent cell-mediated cytotoxicity (ADCC) activity. Embodiment 17. The antibody according to any one of the preceding embodiments, wherein the antibody comprises a wild-type or variant IgG Fc having antibody-dependent cell phagocytosis (ADCP) activity. Embodiment 18. An antibody wherein: a) there is aspartic acid or glutamic acid at the position corresponding to position 10 of SEQ ID NO: 91; b) there is aspartic acid or glutamic acid at position 10 of SEQ ID NO: 91; c) there is aspartic acid or glutamic acid at the position corresponding to position 103 of SEQ ID NO: 91; d) there is aspartic acid or glutamic acid at position 103 of SEQ ID NO: 91; e) there is aspartic acid or glutamic acid at the position corresponding to position 10 and / or 103 of SEQ ID NO: 91; f) there is aspartic acid or glutamic acid at position 10 and / or 103 of SEQ ID NO: 91; or g) the antibody of any one of the previous embodiments, comprising the amino acid sequence of SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, or SEQ ID NO: 99. Embodiment 19. An antibody of any one of the previous embodiments, wherein the antibody comprises a canine κ light chain constant region or a feline κ light chain constant region. Embodiment 20. An antibody of any one of the previous embodiments, wherein the antibody comprises a feline κ light chain constant region having no one or more N-glycosylation sites. Embodiment 21. An antibody of any one of the previous embodiments, wherein the antibody binds to an epitope comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3. Embodiment 22. The antibody binds to canine parvovirus and / or feline parvovirus with a dissociation constant (Kd) of less than 5×10 -6 M, less than 1×10 -6 M, less than 5×10 -7 M, less than 1×10 -7 M, less than 5×10 -8 M, less than 1×10 -8 M, less than 5×10 -9 M, less than 1×10 -9 M, less than 5×10 -10 M, less than 1×10 -10 M, less than 5×10 -11 M, less than 1×10 -11 M, less than 5×10 -12 M, less than 1×10 -12 M, less than 5×10 -13 M, or less than 1×10 -13 M, as measured by biolayer interferometry, and is an antibody of any one of the previous embodiments that binds to canine parvovirus and / or feline parvovirus. Embodiment 23. An antibody according to any one of the previous embodiments, wherein the antibody is determined by immunoblot analysis and / or biolayer interferometry to bind to canine parvovirus or feline parvovirus. Embodiment 24. An antibody according to any one of the previous embodiments, wherein the antibody at a concentration of 200 μg / mL has a hemagglutination inhibition value of at least 8000, at least 16000, or at least 32000. Embodiment 25. An antibody according to any one of the previous embodiments, wherein the antibody is a monoclonal antibody. Embodiment 26. An antibody according to any one of the previous embodiments, comprising one or more of: (a) the (HC-FR1) sequence of SEQ ID NO: 7, 8, 45, or 46; (b) the HC-FR2 sequence of SEQ ID NO: 9, 47, or 48; (c) the HC-FR3 sequence of SEQ ID NO: 10, 49, or 50; (d) the HC-FR4 sequence of SEQ ID NO: 11, 12, or 51; (e) the variable region light chain framework 1 (LC-FR1) sequence of SEQ ID NO: 16, 55, or 56; (f) the LC-FR2 sequence of SEQ ID NO: 17 or 57; (g) the LC-FR3 sequence of SEQ ID NO: 18, 58, or 59; or (h) the LC-FR4 sequence of SEQ ID NO: 19, 60, or 61. Embodiment 27. An antibody according to any one of the previous embodiments, wherein the antibody comprises: (a) the variable heavy chain sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 40, SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO: 88; and / or (b) the variable light chain sequence of SEQ ID NO: 22, SEQ ID NO: 87, SEQ ID NO: 41, SEQ ID NO: 64, SEQ ID NO: 65, or SEQ ID NO: 89. Embodiment 28. An antibody according to any one of the previous embodiments, wherein the antibody comprises (a) a variable heavy chain sequence of SEQ ID NO: 20 and a variable light chain sequence of SEQ ID NO: 22; (b) a variable heavy chain sequence of SEQ ID NO: 21 and a variable light chain sequence of SEQ ID NO: 22; (c) a variable heavy chain sequence of SEQ ID NO: 85 and a variable light chain sequence of SEQ ID NO: 87; (d) a variable heavy chain sequence of SEQ ID NO: 86 and a variable light chain sequence of SEQ ID NO: 87; (e) a variable heavy chain sequence of SEQ ID NO: 40 and a variable light chain sequence of SEQ ID NO: 41; (f) a variable heavy chain sequence of SEQ ID NO: 62 and a variable light chain sequence of SEQ ID NO: 64 or SEQ ID NO: 65; (g) a variable heavy chain sequence of SEQ ID NO: 63 and a variable light chain sequence of SEQ ID NO: 64 or SEQ ID NO: 65; or (h) a variable heavy chain sequence of SEQ ID NO: 88 and a variable light chain sequence of SEQ ID NO: 89. Embodiment 29. An antibody according to any one of the previous embodiments, wherein the antibody comprises (a) (i) a heavy chain sequence of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 79; and / or (ii) a light chain sequence of SEQ ID NO: 25, SEQ ID NO: 39, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 80; or (b) (i) a heavy chain sequence of SEQ ID NO: 31, SEQ ID NO: 40, or SEQ ID NO: 74; and / or (ii) a light chain sequence of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 41, SEQ ID NO: 75, or SEQ ID NO: 76. Embodiment 30. An antibody according to any one of the previous embodiments, wherein the antibody comprises (a) a heavy chain sequence of SEQ ID NO: 23 or SEQ ID NO: 24 and a light chain sequence of SEQ ID NO: 25; (b) a heavy chain sequence of SEQ ID NO: 31 and a light chain sequence of SEQ ID NO: 32 or SEQ ID NO: 33; (c) a heavy chain sequence of SEQ ID NO: 37 or SEQ ID NO: 38 and a light chain sequence of SEQ ID NO: 39; (d) a heavy chain sequence of SEQ ID NO: 66 or SEQ ID NO: 67 and a light chain sequence of SEQ ID NO: 68 or SEQ ID NO: 69; (e) a heavy chain sequence of SEQ ID NO: 66 and a light chain sequence of SEQ ID NO: 68; (f) a heavy chain sequence of SEQ ID NO: 67 and a light chain sequence of SEQ ID NO: 69; (g) a heavy chain sequence of SEQ ID NO: 74 and a light chain sequence of SEQ ID NO: 75 or SEQ ID NO: 76; or (h) a heavy chain sequence of SEQ ID NO: 79 and a light chain sequence of SEQ ID NO: 80. Embodiment 31. An antibody according to any one of the previous embodiments, wherein the antibody comprises a heavy chain sequence of SEQ ID NO: 24 and a light chain sequence of SEQ ID NO: 25. Embodiment 32. An isolated nucleic acid encoding any one of the antibodies of the previous embodiments. Embodiment 33. A host cell containing the nucleic acid of Embodiment 31. Embodiment 34. A method for producing an antibody, comprising culturing the host cell of Embodiment 32 and isolating the antibody. Embodiment 35. A pharmaceutical composition comprising any one or more of the antibodies of Embodiments 1 to 31 and a pharmaceutically acceptable carrier. Embodiment 36. The pharmaceutical composition of Embodiment 35, wherein the pharmaceutically acceptable carrier is phosphate buffered saline. Embodiment 37. A method for providing passive immunity to a subject against infection with canine or feline parvovirus, comprising administering to the subject a therapeutically effective amount of a monoclonal antibody that binds to canine or feline parvovirus. Embodiment 38. The method of Embodiment 37, wherein the monoclonal antibody is administered prior to exposure to canine or feline parvovirus. Embodiment 39. The method of Embodiment 37 or Embodiment 38, wherein the monoclonal antibody is administered after exposure to canine or feline parvovirus. Embodiment 40. The method of any one of Embodiments 37 to 39, wherein the monoclonal antibody is administered after infection with canine or feline parvovirus. Embodiment 41. The method of any one of Embodiments 37 to 40, wherein the monoclonal antibody is administered after the subject exhibits at least one symptom selected from fever, vomiting, diarrhea, lymphopenia, and sepsis. Embodiment 42. The method of any one of Embodiments 37 to 41, wherein the monoclonal antibody is administered after canine or feline parvovirus is detected in the feces, as determined, for example, by a positive cage-side SNAP test. Embodiment 43. The method of any one of Embodiments 37 to 42, wherein the subject has previously been administered a parvovirus vaccine. Embodiment 44. The method of any one of Embodiments 37 to 42, wherein the subject has not previously been administered a parvovirus vaccine. Embodiment 45. Any one of the methods of Embodiments 37 to 44, wherein the subject is not protected at birth due to the absence of maternally-derived antibodies against canine or feline parvovirus or the failure of passive transfer of antibodies against canine or feline parvovirus. Embodiment 46. Any one of the methods of Embodiments 37 to 45, wherein the subject is hand-reared, the subject's mother does not produce milk, or the subject is unable to produce antibodies against parvovirus. Embodiment 47. Any one of the methods of Embodiments 37 to 46, wherein the subject lives in an environment contaminated with canine or feline parvovirus. Embodiment 48. A method of treating canine or feline parvovirus infection in a subject, comprising administering to the subject a therapeutically effective amount of a monoclonal antibody that binds to canine or feline parvovirus. Embodiment 49. The method of Embodiment 48, wherein the monoclonal antibody is administered after the subject exhibits at least one symptom selected from fever, vomiting, diarrhea, lymphopenia, and sepsis. Embodiment 50. The method of Embodiment 48 or Embodiment 49, wherein the monoclonal antibody is administered after canine or feline parvovirus is detected in the feces, as determined by, for example, a positive cage-side SNAP test. Embodiment 51. Any one of the methods of Embodiments 48 to 50, wherein the subject has previously been administered a parvovirus vaccine. Embodiment 52. Any one of the methods of Embodiments 48 to 50, wherein the subject has not previously been administered a parvovirus vaccine. Embodiment 53. Any one of the methods of Embodiments 48 to 52, wherein the subject lives in an environment contaminated with canine or feline parvovirus. Embodiment 54. Any one of the methods of Embodiments 48 to 53, wherein the subject is a dog or a cat. Embodiment 55. Any one of the methods of Embodiments 48 to 53, wherein the subject is a human. Embodiment 56. The method according to any one of embodiments 37 to 55, comprising administering to a subject a therapeutically effective amount of a monoclonal antibody that binds to an epitope comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3. Embodiment 57. The method according to any one of embodiments 37 to 56, comprising administering to a subject a therapeutically effective amount of a monoclonal antibody comprising: (a) a heavy chain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 42; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 44; and (b) a light chain comprising (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54. Embodiment 58. The method according to any one of embodiments 37 to 57, comprising administering to a subject a therapeutically effective amount of an antibody according to any one of embodiments 1 to 31 or a pharmaceutical composition according to embodiment 35 or embodiment 36. Embodiment 59. The method according to any one of embodiments 37 to 58, wherein the antibody or pharmaceutical composition is administered parenterally. Embodiment 60. The method according to any one of embodiments 37 to 59, wherein the antibody or pharmaceutical composition is administered by intramuscular, intraperitoneal, intrathecal, subcutaneous, intraarterial, intrasynovial, intrathecal, intravenous, or inhalation routes. Embodiment 61. The method according to any one of embodiments 37 to 60, wherein the antibody or pharmaceutical composition is administered intravenously. Embodiment 62. The method according to any one of embodiments 37 to 60, wherein the antibody or pharmaceutical composition is administered subcutaneously. Embodiment 63. The method of any one of Embodiments 37 to 62, wherein the subject is less than 1 week old, less than 2 weeks old, less than 3 weeks old, less than 4 weeks old, less than 5 weeks old, less than 6 weeks old, less than 6 weeks old, less than 7 weeks old, less than 8 weeks old, less than 9 weeks old, less than 10 weeks old, less than 11 weeks old, less than 12 weeks old, less than 6 months old, from 0 to 12 weeks old, from 0 to 10 weeks old, from 0 to 8 weeks old, from 0 to 6 weeks old, from 0 to 4 weeks old, from 0 to 2 weeks old, from 4 to 12 weeks old, from 6 to 12 weeks old, from 10 to 12 weeks old, from 4 weeks to 6 months old, from 2 months to 6 months old, from 4 months to 6 months old, from 6 months to 1 year old, older than 13 weeks old, or older than 1 year old. Embodiment 64. The method of any one of Embodiments 37 to 63, wherein the subject is 13 weeks old or older. Embodiment 65. The method of any one of Embodiments 37 to 64, wherein the antibody is administered in an amount in the range of 0.01 mg / kg body weight to 100 mg / kg body weight per dose. Embodiment 66. The method of any one of Embodiments 37 to 65, wherein the antibody is administered in an amount of 5 mg / kg body weight per dose. Embodiment 67. The method of any one of Embodiments 37 to 66, wherein the antibody or pharmaceutical composition is administered as a single dose. Embodiment 68. The method of any one of Embodiments 37 to 67, wherein the antibody or pharmaceutical composition is repeatedly administered, such as once a week for at least 2 weeks or 3 weeks in a row. Embodiment 69. The method of any one of Embodiments 37 to 68, wherein the method comprises administering to the subject two or more different antibodies of any one of Embodiments 1 to 31 in a therapeutically effective amount, and the two or more different antibodies are administered simultaneously or sequentially, and in some cases, the administrations of the two or more different antibodies are separated by one or more days. Embodiment 70. The method of any one of Embodiments 37 to 69, wherein, as determined by a hemagglutination inhibition assay before administration of the antibody or pharmaceutical composition, the subject has a hemagglutination inhibition titer of less than 20. Embodiment 71. The method of any one of Embodiments 37 to 70, wherein, as determined by a hemagglutination inhibition assay before administration of the antibody or pharmaceutical composition, the subject is parvovirus titer negative. Embodiment 72. Any one of the methods according to Embodiments 37 to 71, wherein after administration of the antibody or pharmaceutical composition, the subject survives infection by canine or feline parvovirus. Embodiment 73. A method for reducing parvovirus infection of cells, comprising exposing the cells to any one of the antibodies according to Embodiments 1 to 31 or the pharmaceutical composition according to Embodiment 35 or Embodiment 36 under conditions that permit binding of the antibody to parvovirus. Embodiment 74. The method according to Embodiment 73, wherein the cells are exposed to the antibody or pharmaceutical composition in vitro. Embodiment 75. The method according to Embodiment 73 or Embodiment 74, wherein the cells are mammalian cells, human cells, canine cells, or feline cells. Embodiment 76. A method for detecting parvovirus infection in a sample from a subject, comprising contacting the sample with any one of the antibodies according to Embodiments 1 to 31 or the pharmaceutical composition according to Embodiment 35 or Embodiment 36 under conditions that permit binding of the antibody to parvovirus, and detecting whether a complex is formed between the antibody and parvovirus in the sample. Embodiment 77. The method according to Embodiment 76, wherein the sample is a biological sample obtained from a dog, a cat, or a human. Embodiment 78. A variant IgG Fc polypeptide comprising: a) aspartic acid or glutamic acid at a position corresponding to position 10 of SEQ ID NO: 91; b) aspartic acid or glutamic acid at position 10 of SEQ ID NO: 91; c) aspartic acid or glutamic acid at a position corresponding to position 103 of SEQ ID NO: 91; d) aspartic acid or glutamic acid at position 103 of SEQ ID NO: 91; e) aspartic acid or glutamic acid at a position corresponding to position 10 and / or 103 of SEQ ID NO: 91; or f) aspartic acid or glutamic acid at position 10 and / or 103 of SEQ ID NO: 91. Embodiment 79. A polypeptide comprising the variant IgG Fc polypeptide according to Embodiment 78. Embodiment 80. A polypeptide comprising the amino acid sequence of SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, or SEQ ID NO: 99. Embodiment 81. An isolated nucleic acid encoding the polypeptide of any one of Embodiments 78 to 80. Embodiment 82. A host cell comprising the nucleic acid of Embodiment 81.

Brief Description of the Drawings

[0008]

Figure 1

[0009]

Figure 2

[0010]

Figure 3A

[0011]

Figure 3B

[0012]

Figure 3C

[0013]

Figure 4A

[0014]

Figure 4B

[0015]

Figure 5

[0016] Table 1 provides a list of the predetermined arrays referred to herein.

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Mode for Carrying Out the Invention

[0018] Antibodies that bind to canine parvovirus and / or feline parvovirus are provided. Antibody heavy and light chains capable of forming antibodies that bind to canine parvovirus and / or feline parvovirus are also provided. In addition, antibodies, heavy chains, and light chains comprising one or more specific complementarity determining regions (CDRs) are provided. Polynucleotides encoding antibodies against canine parvovirus and / or feline parvovirus are provided. Methods for producing or purifying antibodies against canine parvovirus and / or feline parvovirus are also provided. Methods for providing passive immunity against infection with canine or feline parvovirus and / or methods for treating parvovirus infection using antibodies against canine parvovirus and / or feline parvovirus are provided.

[0019] For the convenience of the reader, the following definitions are provided for the terms used herein.

[0020] As used herein, numerical terms such as Kd are calculated based on scientific measurements and are thus subject to the effects of appropriate measurement errors. In some cases, numerical terms may include values rounded to the nearest significant digit.

[0021] As used herein, "a" or "an" means "at least one" or "one or more" unless otherwise specified. As used herein, the term "or" means "and / or" unless otherwise expressly stated. In the context of claims that depend on multiple items, the use of "or" in citing other claims refers to the citation of only the alternative claim.

[0022] [Exemplary Parvovirus Antibodies] Novel antibodies against parvovirus are provided, such as antibodies that bind to canine parvovirus and / or feline parvovirus. The parvovirus antibodies provided herein include, but are not limited to, monoclonal antibodies, chimeric antibodies, canineized antibodies, and felineized antibodies.

[0023] Also provided herein is the amino acid sequence of a monoclonal parvovirus antibody. For example, the variable heavy chain CDRs (SEQ ID NOs: 4-6), variable light chain CDRs (SEQ ID NOs: 13-15), variable region heavy chain framework sequences (SEQ ID NOs: 7 and 8-11), and variable region light chain framework sequences (SEQ ID NOs: 16-19) for Mab A are provided. Also provided are the variable heavy chain CDRs (SEQ ID NOs: 42-44), variable light chain CDRs (SEQ ID NOs: 52-54), variable region heavy chain framework sequences (SEQ ID NOs: 45, 47, 49, and 51), and variable region light chain framework sequences (SEQ ID NOs: 55, 57, 58, and 60) for Mab B. The amino acid sequences of the variable heavy and light chains of Mab A are provided (SEQ ID NOs: 20 and 22, respectively). The amino acid sequences of the variable heavy and light chains of Mab B are provided (SEQ ID NOs: 62 and 64, respectively).

[0024] In addition, the CDRs, framework sequences, and amino acid sequences of the variable heavy and light chains of variant Mab A and Mab B antibodies are provided. The variable heavy chain CDRs (SEQ ID NOs: 4-6), variable heavy chain framework regions (SEQ ID NOs: 8-10, and 12), variable heavy chain (SEQ ID NO: 21), variable light chain CDRs (SEQ ID NOs: 13-15), variable light chain framework regions (SEQ ID NOs: 16-19), and variable light chain (SEQ ID NO: 22) of Mab A variant 2 (v2) are provided. The variable heavy chain CDRs (SEQ ID NOs: 42-44), variable heavy chain framework regions (SEQ ID NOs: 46, 48, 50, and 51), variable heavy chain (SEQ ID NO: 63), variable light chain CDRs (SEQ ID NOs: 52-54), variable light chain framework regions (SEQ ID NOs: 56, 57, 59, and 61), and variable light chain (SEQ ID NO: 65) of Mab B variant 2 (v2) are provided.

[0025] Also provided herein are chimeric, chimeric cat, canineized, and felineized antibodies derived from Mab A, Mab B, Mab A v2, and Mab B v2. In some embodiments, the amino acid sequences of canineized and felineized Mab A, Mab B, Mab A v2, and Mab B v2, such as SEQ ID NOs: 34-41, 88, 77-80, 88, and 89, are provided. In some embodiments, the amino acid sequences of chimeric antibodies derived from Mab A, Mab B, Mab A v2, and Mab B v2, such as SEQ ID NOs: 23-25, 28-33, and 66-76, are provided.

[0026] The term "antibody" as used herein is used in the broadest sense and includes, without limitation, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific (e.g., bispecific T cell engagers) and trispecific antibodies), and antibody fragments (such as Fab, F(ab’)2, ScFv, minibodies, diabodies, triabodies, and tetra-bodies) as long as they exhibit the desired antigen-binding activity. Dogs, cats, and horses have different classes of antibodies that are shared by many mammals.

[0027] The term "antibody" includes, but is not limited to, fragments capable of binding to an antigen, such as Fv, single-chain Fv (scFv), Fab, Fab’, di-scFv, sdAb (single-domain antibody), and (Fab’)2 (including chemically conjugated F(ab’)2). Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and the remaining "Fc" fragment, whose name reflects its ability to readily crystallize. Pepsin treatment produces an F(ab’)2 fragment that has two antigen-binding sites and can still crosslink antigens. The term "antibody" also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies from various species such as mouse, human, cynomolgus monkey, dog, cat, horse, etc. Furthermore, for all antibody constructs provided herein, variants having sequences from other organisms are also contemplated. Thus, when a mouse form of an antibody is disclosed, one of ordinary skill in the art will know how to convert an antibody based on a mouse sequence to a sequence such as a cat, dog, horse, etc. Antibody fragments also include single-chain scFv, tandem di-scFv, diabody, tandem tri-sdcFv, minibody, etc. in any orientation. Antibody fragments also include nanobodies (antibodies having a single monomeric domain such as sdAb, variable domain pair of a heavy chain without a light chain). Antibody fragments can be said to be of a particular species in some embodiments (e.g., mouse scFv or dog scFv). This indicates the sequence of at least a portion of the non-CDR regions, rather than the construct source. In some embodiments, the antibody includes a label or is conjugated to a second moiety.

[0028] The terms "label" and "detectable label" mean a moiety attached to an antibody or its analyte to enable detection of a reaction (e.g., binding) between members of a specific binding pair. The labeled member of the specific binding pair is referred to as "detectably labeled". Thus, the term "labeled binding protein" refers to a protein into which a label has been introduced that results in the identification of the binding protein. In some embodiments, the label is a detectable marker that can generate a signal detectable by visual or instrumental means, e.g., incorporation of a radiolabeled amino acid or attachment of a biotinyl moiety to a polypeptide that can be detected by the attachment of a labeled avidin (e.g., streptavidin containing a fluorescent marker or enzyme activity detectable optically or colorimetrically). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153 Sm); chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites of secondary antibodies, metal binding domains, epitope tags); and magnetic agents such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include a moiety that produces light, e.g., an acridinium compound, and a moiety that produces fluorescence, e.g., fluorescein. In this regard, the moiety itself is not detectably labeled but may become detectable upon reaction with yet another moiety.

[0029] The term "monoclonal antibody" refers to a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are produced against a single antigenic site. Further, in contrast to polyclonal antibody preparations, which typically include different antibodies against different determinants (epitopes), each monoclonal antibody is produced against a single determinant on the antigen. Thus, a sample of monoclonal antibodies can bind to the same epitope on the antigen. The modifier "monoclonal" indicates the property of the antibody being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies can be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries made using, for example, the techniques described in McCafferty et al., 1990, Nature 348:552-554.

[0030] In some embodiments, the monoclonal antibody is Mab A, Mab A v2, Mab B, or Mab B v2.

[0031] "Amino acid sequence" means the sequence of amino acid residues in a peptide or protein. The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may include natural or unnatural amino acid residues and include, without limitation, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are included in the definition. The term also includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Further, for the purposes of the present disclosure, "polypeptide" refers to a protein that includes modifications such as deletions, additions, and substitutions (generally conservative in nature) with respect to the native sequence, as long as the protein maintains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as by mutations in the host producing the protein or errors during PCR amplification.

[0032] As used herein, "parvovirus" refers to any naturally occurring parvovirus or parvovirus variant, including canine parvovirus (CPV) such as CPV-2a, CPV-2b, and CPV-2c, and feline parvovirus (panleukopenia virus).

[0033] As used herein, the term "epitope" means a site on a target molecule (e.g., an antigen, e.g., a protein, nucleic acid, sugar or lipid) to which an antigen-binding molecule (e.g., an antibody, antibody fragment, or scaffold protein containing an antibody-binding region) binds. Epitopes often include chemically active surface groups of molecules such as amino acids, polypeptides, or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be formed from both contiguous or juxtaposed non-contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of the target molecule. Epitopes formed from contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes can include, but are not limited to, at least 3 residues, at least 5 residues, or 8-10 residues (e.g., amino acids or nucleotides). In some examples, the epitope length is less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues or less than 12 residues. Two antibodies can bind to the same epitope within an antigen if they exhibit competitive binding to the antigen. In some embodiments, an epitope can be identified at a predetermined minimum distance from CDR residues on an antigen-binding molecule. In some embodiments, an epitope can be identified by the above distance and is further limited to those residues involved in a bond (e.g., a hydrogen bond) between an antibody residue and an antigen residue. Epitopes can also be identified by various scans, e.g., alanine or arginine scans can indicate one or more residues with which an antigen-binding molecule can interact. Unless explicitly indicated otherwise, a set of residues as an epitope does not exclude other residues from being part of the epitope for a particular antibody. Rather, the presence of such a set represents the minimum series (or set of species) of the epitope. Thus, in some embodiments, a set of residues identified as an epitope represents the minimum epitope associated with the antigen rather than an exclusive list of the residues of the epitope on the antigen.

[0034] In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3.

[0035] The term "CDR" means a complementarity determining region defined by at least one method of identification by those skilled in the art. In some embodiments, the CDR can be defined according to any of the Chothia numbering scheme, Kabat numbering scheme, combination of Kabat and Chothia, AbM definition, contact definition, or combination of Kabat, Chothia, AbM, or contact definition. The various CDRs within an antibody can be represented by their appropriate numbers and chain types including, but not limited to, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The term "CDR" is used herein to also encompass "hypervariable region" or HVR, which includes hypervariable loops.

[0036] In some embodiments, the parvovirus antibody comprises a heavy chain comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; or (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the parvovirus antibody comprises a light chain comprising (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14; or (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15.

[0037] In some embodiments, the parvovirus antibody comprises a heavy chain comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 42; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43; or (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 44. In some embodiments, the parvovirus antibody comprises a light chain comprising (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53; or (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54.

[0038] In some embodiments, the parvovirus antibody comprises a heavy chain comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof in which 1, 2, or 3 amino acids of CDR-H1 are substituted with different amino acids; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof in which 1, 2, or 3 amino acids of CDR-H2 are substituted with different amino acids; and / or (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof in which 1, 2, or 3 amino acids of CDR-H3 are substituted with different amino acids. In some embodiments, the parvovirus antibody comprises a light chain comprising (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof in which 1, 2, or 3 amino acids of CDR-L1 are substituted with different amino acids; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof in which 1, 2, or 3 amino acids of CDR-L2 are substituted with different amino acids; and / or (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof in which 1, 2, or 3 amino acids of CDR-L3 are substituted with different amino acids.

[0039] In some embodiments, the parvovirus antibody comprises a heavy chain comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof in which 1, 2, or 3 amino acids of CDR-H1 are substituted with different amino acids; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof in which 1, 2, or 3 amino acids of CDR-H2 are substituted with different amino acids; and / or (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof in which 1, 2, or 3 amino acids of CDR-H3 are substituted with different amino acids. In some embodiments, the parvovirus antibody comprises a light chain comprising (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, or a variant thereof in which 1, 2, or 3 amino acids of CDR-L1 are substituted with different amino acids; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, or a variant thereof in which 1, 2, or 3 amino acids of CDR-L2 are substituted with different amino acids; and / or (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54, or a variant thereof in which 1, 2, or 3 amino acids of CDR-L3 are substituted with different amino acids.

[0040] As used herein, the term "variable region" means a region that includes at least three CDRs. In some embodiments, the variable region includes three CDRs and at least one framework region ("FR"). The terms "heavy chain variable region" or "variable heavy chain" are used interchangeably and mean a region that includes at least three heavy chain CDRs. The terms "light chain variable region" or "variable light chain" are used interchangeably and mean a region that includes at least three light chain CDRs. In some embodiments, the variable heavy chain or variable light chain includes at least one framework region. In some embodiments, the antibody includes at least one heavy chain framework region selected from HC-FR1, HC-FR2, HC-FR3, and HC-FR4. In some embodiments, the antibody includes at least one light chain framework region selected from LC-FR1, LC-FR2, LC-FR3, and LC-FR4. The framework region may be juxtaposed between light chain CDRs or between heavy chain CDRs. For example, the antibody may include a variable heavy chain having the following structure: (HC-FR1)-(CDR-H1)-(HC-FR2)-(CDR-H2)-(HC-FR3)-(CDR-H3)-(HC-FR4). The antibody may include a variable heavy chain having the following structure: (CDR-H1)-(HC-FR2)-(CDR-H2)-(HC-FR3)-(CDR-H3). The antibody may include a variable light chain having the following structure: (LC-FR1)-(CDR-L1)-(LC-FR2)-(CDR-L2)-(LC-FR3)-(CDR-L3)-(LC-FR4). The antibody may also include a variable light chain having the following structure: (CDR-L1)-(LC-FR2)-(CDR-L2)-(LC-FR3)-(CDR-L3).

[0041] In some embodiments, the parvovirus antibody comprises one or more of: (a) the variable region heavy chain framework 1 (HC-FR1) sequence of SEQ ID NO: 7 or SEQ ID NO: 8; (b) the HC-FR2 sequence of SEQ ID NO: 9; (c) the HC-FR3 sequence of SEQ ID NO: 10; (d) the HC-FR4 sequence of SEQ ID NO: 11 or SEQ ID NO: 12; (e) the variable region light chain framework 1 (LC-FR1) sequence of SEQ ID NO: 16; (f) the LC-FR2 sequence of SEQ ID NO: 17; (g) the LC-FR3 sequence of SEQ ID NO: 18; or (h) the LC-FR4 sequence of SEQ ID NO: 19.

[0042] In some embodiments, the parvovirus antibody comprises one or more of: (a) the variable region heavy chain framework 1 (HC-FR1) sequence of SEQ ID NO: 45 or SEQ ID NO: 46; (b) the HC-FR2 sequence of SEQ ID NO: 47 or SEQ ID NO: 48; (c) the HC-FR3 sequence of SEQ ID NO: 49 or SEQ ID NO: 50; (d) the HC-FR4 sequence of SEQ ID NO: 51; (e) the variable region light chain framework 1 (LC-FR1) sequence of SEQ ID NO: 55 or SEQ ID NO: 56; (f) the LC-FR2 sequence of SEQ ID NO: 57; (g) the LC-FR3 sequence of SEQ ID NO: 58 or SEQ ID NO: 59; or (h) the LC-FR4 sequence of SEQ ID NO: 60.

[0043] In some embodiments, the parvovirus antibody comprises the variable light chain sequence of SEQ ID NO: 22, SEQ ID NO: 87, SEQ ID NO: 41, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 89, SEQ ID NO: 82, or SEQ ID NO: 84. In some embodiments, the parvovirus antibody comprises the variable heavy chain sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 40, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 88, SEQ ID NO: 81, or SEQ ID NO: 83.

[0044] In some embodiments, the parvovirus antibody comprises the variable heavy chain sequence of SEQ ID NO: 20 and the variable light chain sequence of SEQ ID NO: 22. In some embodiments, the parvovirus antibody comprises the variable heavy chain sequence of SEQ ID NO: 21 and the variable light chain sequence of SEQ ID NO: 22. In some embodiments, the parvovirus antibody comprises the variable heavy chain sequence of SEQ ID NO: 85 and the variable light chain sequence of SEQ ID NO: 87. In some embodiments, the parvovirus antibody comprises the variable heavy chain sequence of SEQ ID NO: 86 and the variable light chain sequence of SEQ ID NO: 87. In some embodiments, the parvovirus antibody comprises the variable heavy chain sequence of SEQ ID NO: 40 and the variable light chain sequence of SEQ ID NO: 41. In some embodiments, the parvovirus antibody comprises the variable heavy chain sequence of SEQ ID NO: 62 and the variable light chain sequence of SEQ ID NO: 64 or SEQ ID NO: 65. In some embodiments, the parvovirus antibody comprises the variable heavy chain sequence of SEQ ID NO: 63 and the variable light chain sequence of SEQ ID NO: 64 or SEQ ID NO: 65. In some embodiments, the parvovirus antibody comprises the variable heavy chain sequence of SEQ ID NO: 88 and the variable light chain sequence of SEQ ID NO: 89.

[0045] As used herein, the term "constant region" means a region that includes at least three constant domains. The terms "heavy chain constant region" or "constant heavy chain" are used interchangeably and mean a region that includes at least three heavy chain constant domains, CH1, CH2, and CH3. Non-limiting exemplary heavy chain constant regions include γ, δ, α, ε, and μ. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody that includes a γ constant region is an IgG antibody, an antibody that includes a δ constant region is an IgD antibody, an antibody that includes an α constant region is an IgA antibody, an antibody that includes a μ constant region is an IgM antibody, and an antibody that includes an ε constant region is an IgE antibody. A given isotype can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (including the γ1 constant region), IgG2 (including the γ2 constant region), IgG3 (including the γ3 constant region), and IgG4 (including the γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (including the α1 constant region) and IgA2 (including the α2 constant region) antibodies; IgM antibodies include, but are not limited to, IgM1 and IgM2. The terms "light chain constant region" or "constant light chain" are used interchangeably and mean a region that includes the light chain constant domain CL. Non-limiting exemplary light chain constant regions include λ and κ. Deletions and changes that do not alter the function within a domain are included within the scope of the term "constant region" unless otherwise specified. Dogs and cats have antibody classes such as IgG, IgA, IgD, IgE, and IgM. Among the dog IgG antibody classes are IgG-A, IgG-B, IgG-C, and IgG-D. Among the cat IgG antibody classes are IgG1, IgG2a, and IgG2b.

[0046] The term "chimeric antibody" or "chimeric" means an antibody in which a portion of the heavy or light chain is derived from a particular source or species, while at least a portion of the remaining heavy or light chain is derived from a different source or species. In some embodiments, a chimeric antibody means an antibody comprising at least one variable region from a first species (e.g., mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (e.g., human, dog, cat, horse, etc.). In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one dog constant region. In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one cat constant region. In some embodiments, all of the variable regions of the chimeric antibody are from the first species and all of the constant regions of the chimeric antibody are from the second species. In some embodiments, a chimeric antibody comprises a constant heavy chain region or a constant light chain region from a companion animal. In some embodiments, a chimeric antibody comprises mouse variable heavy and light chains, and constant heavy and light chains of a companion animal. For example, a chimeric antibody can comprise mouse variable heavy and light chains, and dog constant heavy and light chains; a chimeric antibody can comprise mouse variable heavy and light chains, and cat constant heavy and light chains; or a chimeric antibody can comprise mouse variable heavy and light chains, and horse constant heavy and light chains.

[0047] In some embodiments, the parvovirus antibody comprises a chimeric antibody comprising: (a) (i) the heavy chain amino acid sequence of SEQ ID NO: 23; (ii) the light chain amino acid sequence of SEQ ID NO: 25; or (iii) a heavy chain amino acid sequence as in (i) and a light chain sequence as in (ii); (b) (i) the heavy chain amino acid sequence of SEQ ID NO: 24; (ii) the light chain amino acid sequence of SEQ ID NO: 25; or (iii) a heavy chain amino acid sequence as in (i) and a light chain sequence as in (ii); (c) (i) the heavy chain amino acid sequence of SEQ ID NO: 31; (ii) the light chain amino acid sequence of SEQ ID NO: 32; or (iii) a heavy chain amino acid sequence as in (i) and a light chain sequence as in (ii); (d) (i) the heavy chain amino acid sequence of SEQ ID NO: 31; (ii) the light chain amino acid sequence of SEQ ID NO: 33; or (iii) a heavy chain amino acid sequence as in (i) and a light chain sequence as in (ii); (e) (i) the heavy chain amino acid sequence of SEQ ID NO: 66; (ii) the light chain amino acid sequence of SEQ ID NO: 68; or (iii) a heavy chain amino acid sequence as in (i) and a light chain sequence as in (ii); (f) (i) the heavy chain amino acid sequence of SEQ ID NO: 67; (ii) the light chain amino acid sequence of SEQ ID NO: 69; or (iii) a heavy chain amino acid sequence as in (i) and a light chain sequence as in (ii); (g) (i) the heavy chain amino acid sequence of SEQ ID NO: 74; (ii) the light chain amino acid sequence of SEQ ID NO: 75; or (iii) a heavy chain amino acid sequence as in (i) and a light chain sequence as in (ii); or (h) (i) the heavy chain amino acid sequence of SEQ ID NO: 74; (ii) the light chain amino acid sequence of SEQ ID NO: 75; or (iii) a heavy chain amino acid sequence as in (i) and a light chain sequence as in (ii).

[0048] "Canine chimeric", "chimeric canine", or "canine chimeric antibody" means a chimeric antibody having at least a portion of a heavy chain or a portion of a light chain derived from a dog. "Feline chimeric", "chimeric feline", or "feline chimeric antibody" means a chimeric antibody having at least a portion of a heavy chain or a portion of a light chain derived from a cat. In some embodiments, the canine chimeric antibody comprises mouse or rat variable heavy and light chains and canine constant heavy and light chains. In some embodiments, the feline chimeric antibody comprises mouse or rat variable heavy and light chains and feline constant heavy and light chains.

[0049] In some embodiments, the parvovirus antibody comprises a canine heavy chain constant region selected from IgG-A, IgG-B, IgG-C, and IgG-D constant regions.

[0050] In some embodiments, the parvovirus antibody comprises a feline heavy chain constant region selected from IgG1, IgG2a, and IgG2b constant regions.

[0051] "Caninized antibody" means an antibody in which at least one amino acid in a portion of the non-canine variable region has been replaced with the corresponding amino acid from a canine variable region. In some embodiments, the caninized antibody comprises at least one canine constant region (e.g., γ constant region, α constant region, δ constant region, ε constant region, μ constant region, etc.) or a fragment thereof. In some embodiments, the caninized antibody is an antibody fragment, such as Fab, scFv, (Fab')2, etc. The term "caninized" also refers to a form of a non-canine (e.g., mouse) antibody that comprises a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of an antibody) that contains a minimal sequence of a non-canine immunoglobulin. A caninized antibody can comprise a canine immunoglobulin (recipient antibody) in which residues from the CDRs of a recipient have been replaced by residues from the CDRs of a non-canine species (donor antibody), such as a mouse, rat, or rabbit, that have the desired specificity, affinity, and potency. In some cases, residues of the Fv framework region (FR) of the canine immunoglobulin are replaced by the corresponding non-canine residues. Further, the caninized antibody can contain residues that are not found in the transferred CDRs or framework sequences of the recipient antibody but are included to further improve and optimize antibody performance.

[0052] In some embodiments, at least one amino acid residue in a portion of a rat or mouse variable heavy chain or a rat or mouse variable light chain has been replaced with the corresponding amino acid from a canine variable region. In some embodiments, the modified chain is fused to a canine constant heavy chain or a canine constant light chain.

[0053] "Catized antibody" means an antibody in which at least one amino acid in a part of the non-cat variable region is replaced with the corresponding amino acid from the cat variable region. In some embodiments, the catized antibody comprises at least one cat constant region (e.g., γ constant region, α constant region, δ constant region, ε constant region, μ constant region, etc.) or a fragment thereof. In some embodiments, the catized antibody is an antibody fragment such as Fab, scFv, (Fab')2, etc. The term "catized" also refers to the form of a non-cat (e.g., mouse) antibody that comprises a chimeric immunoglobulin, an immunoglobulin chain, or a fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of an antibody) that contains the minimal sequence of a non-cat immunoglobulin. A catized antibody can comprise a cat immunoglobulin (recipient antibody) in which residues from the CDRs of a recipient have been replaced by residues from the CDRs of a non-cat species (donor antibody) such as a mouse, rat, or rabbit that have the desired specificity, affinity, and potency. In some cases, the Fv framework region (FR) residues of the cat immunoglobulin are replaced by the corresponding non-cat residues. Furthermore, the catized antibody can contain residues that are not found in the CDRs or framework sequences that have also been transferred into the recipient antibody, but are included to further improve and optimize antibody performance.

[0054] In some embodiments, at least one amino acid residue in a part of the mouse variable heavy chain or mouse variable light chain is replaced with the corresponding amino acid from the cat variable region. In some embodiments, the modified chain is fused to a cat constant heavy chain or a dog constant light chain.

[0055] A "fragment crystallizable polypeptide" or "Fc polypeptide" is a portion of an antibody molecule that interacts with effector molecules and cells. It includes the C-terminal portion of the immunoglobulin heavy chain. As used herein, an Fc polypeptide includes a fragment of an Fc domain that has one or more biological activities of the entire Fc polypeptide. The "effector function" of an Fc polypeptide is an action or activity that is carried out in whole or in part by any antibody in response to stimulation and can include complement binding and / or induction of ADCC (antibody-dependent cell-mediated cytotoxicity) and / or ADCP (antibody-dependent cell phagocytosis).

[0056] In some embodiments, the biological activity of the Fc polypeptide is the ability to bind to FcRn. In some embodiments, the biological activity of the Fc polypeptide is the ability to bind to C1q. In some embodiments, the biological activity of the Fc polypeptide is the ability to bind to CD16. In some embodiments, the biological activity of the Fc polypeptide is the ability to bind to protein A.

[0057] The term "IgX Fc" means that the Fc region is derived from a specific antibody isotype (e.g., IgG, IgA, IgD, IgE, IgM, etc.), where "X" means the antibody isotype. Thus, "IgG Fc" means the Fc region of the γ chain, "IgA Fc" means the Fc region of the α chain, "IgD Fc" means the Fc region of the δ chain, "IgE Fc" means the Fc region of the ε chain, "IgM Fc" means the Fc region of the μ chain, and so on. In some embodiments, the IgG Fc region includes CH1, the hinge, CH2, CH3, and CL1. "IgX-N-Fc" means that the Fc region is derived from a specific subclass of an antibody isotype (e.g., canine IgG subclasses A, B, C, or D; or feline IgG subclasses 1, 2a, or 2b), where "N" means the subclass. In some embodiments, the IgX Fc or IgX-N-Fc region is derived from a companion animal such as a dog or a cat. In some embodiments, the IgG Fc region is isolated from a canine γ heavy chain such as IgG-A, IgG-B, IgG-C, or IgG-D. In some examples, the IgG Fc region is isolated from a feline γ heavy chain such as IgG1, IgG2a, or IgG2b. Antibodies comprising the Fc region of IgG-A, IgG-B, IgG-C, or IgG-D can provide high expression levels in recombinant production systems.

[0058] The terms "IgX Fc" and "IgX Fc polypeptide" include wild-type IgX Fc polypeptides and variant IgX Fc polypeptides, unless otherwise indicated.

[0059] In some embodiments, the variant IgG Fc polypeptide comprises a variant IgG Fc polypeptide of a companion animal species. In some embodiments, the variant IgG Fc polypeptide comprises a variant canine IgG Fc polypeptide or a feline IgG Fc polypeptide. In some embodiments, the variant IgG Fc polypeptide (e.g., a variant canine IgG-A Fc polypeptide, a variant canine IgG-C Fc polypeptide, or a variant canine IgG-D Fc polypeptide, a variant feline IgG1a Fc polypeptide, a variant feline IgG1b Fc polypeptide, or a variant feline IgG2 Fc polypeptide) has an activity that is substantially lacking in the reference (e.g., wild-type) polypeptide.

[0060] Antibodies can be modified to extend or shorten their half-life. In some embodiments that include higher doses of an antibody, a shorter half-life may be desirable for acute treatment. In some embodiments that include lower doses of an antibody, a longer half-life may be desirable for long-term treatment. For example, as discussed below, mutations in the IgG Fc that affect the FcRn interaction can be introduced.

[0061] In some embodiments, the parvovirus antibody comprises a wild-type or variant IgG Fc having complement binding activity (or complement-dependent cytotoxicity (CDC)). In some embodiments, the parvovirus antibody comprises a wild-type or variant IgG Fc having antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In some embodiments, the parvovirus antibody comprises a wild-type or variant IgG Fc having antibody-dependent cell phagocytosis (ADCP) activity. In some embodiments, the parvovirus antibody comprises a wild-type or variant IgG Fc having complement binding activity and / or ADCC activity and / or ADCP activity. The IgG Fc polypeptide can be modified to have effector function or to have enhanced effector function.

[0062] In some embodiments, the parvovirus antibody comprises a wild-type or variant IgG Fc that binds to canine FcRn at low pH. In some embodiments, the parvovirus comprises a wild-type or variant IgG Fc that binds to C1q. In some embodiments, the parvovirus comprises a wild-type or variant IgG Fc that binds to CD16. In some embodiments, the parvovirus comprises a variant IgG Fc comprising one or more defucosylated glycans.

[0063] In some embodiments, a variant IgG Fc (e.g., a variant canine IgG Fc polypeptide or a variant feline IgG Fc polypeptide) has an altered FcRn binding affinity compared to a reference polypeptide. In some embodiments, the variant IgG Fc has an increased FcRn binding affinity at acidic pH (e.g., a pH in the range of about 5.0 to about 6.5, such as a pH of about 5.0, a pH of about 5.5, a pH of about 6.0, or a pH of about 6.5) compared to the reference polypeptide. Exemplary variant IgG Fc polypeptides with increased FcRn binding affinity are disclosed in International Publication No. WO 2020 / 082048, which is hereby incorporated by reference in its entirety.

[0064] In some embodiments, a variant IgG Fc (e.g., a variant canine IgG Fc polypeptide or a variant feline IgG Fc polypeptide) has an altered C1q binding affinity compared to a reference polypeptide. In some embodiments, the variant IgG Fc has an increased C1q binding affinity compared to the reference polypeptide. Exemplary variant IgG Fc polypeptides with increased C1q binding affinity are disclosed in International Publication No. WO 2020 / 139984 (e.g., Example 2), which is hereby incorporated by reference in its entirety.

[0065] In some embodiments, the variant IgG Fc (e.g., variant canine IgG Fc polypeptide or variant feline IgG Fc polypeptide) has an altered CD16 binding affinity as compared to a reference polypeptide. In some embodiments, the variant IgG Fc has an increased CD16 binding affinity as compared to the reference polypeptide. Exemplary variant IgG Fc polypeptides having increased CD16 binding affinity are disclosed in International Publication No. WO 2020 / 139984 (e.g., Example 2), which is hereby incorporated by reference in its entirety.

[0066] In some embodiments, the variant canine IgG Fc has an enhanced CD16 binding affinity as compared to the reference polypeptide. In some embodiments, the variant IgG Fc comprises: a) aspartic acid or glutamic acid at a position corresponding to position 10 of SEQ ID NO: 91; b) aspartic acid or glutamic acid at position 10 of SEQ ID NO: 91; c) aspartic acid or glutamic acid at a position corresponding to position 103 of SEQ ID NO: 91; d) aspartic acid or glutamic acid at position 103 of SEQ ID NO: 91; e) aspartic acid or glutamic acid at a position corresponding to position 10 and / or 103 of SEQ ID NO: 91; f) aspartic acid or glutamic acid at position 10 and / or 103 of SEQ ID NO: 91. In some embodiments, the variant IgG Fc comprises the amino acid sequence of SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, or SEQ ID NO: 99.

[0067] In some embodiments, the variant IgG Fc (e.g., variant canine IgG Fc polypeptide or variant feline IgG Fc polypeptide) has an altered protein A binding affinity as compared to a reference polypeptide. In some embodiments, the variant IgG Fc has an increased protein A binding affinity as compared to the reference polypeptide. Exemplary variant IgG Fc polypeptides having increased protein A binding affinity are disclosed in International Publication No. WO 2020 / 139984 (e.g., Example 2), which is hereby incorporated by reference in its entirety.

[0068] The term "affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (K D ). Affinity can be measured by common methods known in the art, such as immunoblotting, ELISA KD, KinEx A, biolayer interferometry (BLI), or surface plasmon resonance apparatus.

[0069] The terms "K D ", "K d ", "Kd", or "Kd value" are used interchangeably to refer to the equilibrium dissociation constant of an antibody-antigen interaction. In some embodiments, the K D of an antibody is measured by using a biolayer interferometry assay that uses a biosensor such as the Octet® system (Pall ForteBio LLC, Fremont, CA) according to the supplier's instructions. Briefly, a biotinylated antigen is bound to the sensor tip, the binding of the antibody is monitored for 90 seconds, and the dissociation is monitored for 600 seconds. The buffer for the dilution and binding steps is 20 mM phosphate, 150 mM NaCl, pH 7.2. To correct for drift, a blank curve of buffer alone is subtracted. The data is fit to a 2:1 binding model using ForteBio data analysis software to determine the association rate constant (k on ), the dissociation rate constant (k off ), and K d . The equilibrium dissociation constant (K d ) is calculated as the ratio of k off / k on . The term "kon" refers to the rate constant when an antibody binds to an antigen, and the term "koff" refers to the rate constant when an antibody dissociates from an antibody / antigen complex.

[0070] The term "binds" to an antigen or epitope is a term well understood in the art, and methods for determining such binding are also well known in the art. A molecule is said to exhibit "binding" if it reacts with, associates with, or has an affinity for a particular cell or substance, and the reaction, association, or affinity is detectable by, for example, one or more methods known in the art, such as immunoblotting, ELISA KD, KinEx A, biolayer interferometry (BLI), surface plasmon resonance apparatus, etc.

[0071] "Surface plasmon resonance" is, for example, BIAcore TM system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, N.J.) to detect changes in protein concentration within a biosensor matrix, indicating an optical phenomenon that enables real-time analysis of biomolecular specific interactions. For further explanation, see Jonsson et al (1993) Ann. Biol. Clin. 51: 19-26.

[0072] "Biolayer interferometry" refers to an optical analysis technique that analyzes the interference pattern of light reflected from an immobilized protein layer on a biosensor tip and an internal reference layer. Changes in the number of molecules bound to the biosensor tip cause a shift in the interference pattern that can be measured in real time. A non-limiting and exemplary device for biolayer interferometry is the Octet® system (Pall ForteBio LLC). See, for example, Abdiche et al, 2008, Anal. Biochem. 377: 209-277.

[0073] In some embodiments, the parvovirus antibody, as measured by biolayer interferometry, is less than 5×10 -6 M, less than 1×10 -6 M, less than 5×10 -7 M, less than 1×10 -7 M, less than 5×10-8 less than M, 1×10 -8 less than M, 5×10 -9 less than M, 1×10 -9 less than M, 5×10 -10 less than M, 1×10 -10 less than M, 5×10 -11 less than M, 1×10 -11 less than M, 5×10 -12 less than M, or 1×10 -12 binds to canine parvovirus or feline parvovirus with a dissociation constant (Kd) of less than M. In some embodiments, the parvovirus antibody, as measured by biolayer interferometry, is between 5×10 -6 M and 1×10 -6 M, between 5×10 -6 M and 5×10 -7 M, between 5×10 -6 M and 1×10 -7 M, between 5×10 -6 M and 5×10 -8 M, between 5×10 -6 M and 1×10 -8 M, between 5×10 -6 M and 5×10 -9 M, between 5×10 -6 M and 1×10 -9 M, between 5×10 -6 M and 5×10 -10 M, between 5×10 -6 M and 1×10 -10 M, between 5×10 -6 M and 5×10 -11 M, between 5×10 -6 M and 1×10 -11 M, between 5×10 -6 M and 5×10 -12 M, between 5×10 -6 M and 1×10 -12 M, between 1×10 -6 M and 5×10 -7 M, between 1×10 -6 M and 1×10 -7 M, between 1×10 -6 M and 5×10 -8 M, between 1×10 -6 M and 1×10 -8 M, between 1×10 -6 M and 5×10 -9Between M, 1×10 -6 Between M and 1×10 -9 Between M, 1×10 -6 Between M and 5×10 -10 Between M, 1×10 -6 Between M and 1×10 -10 Between M, 1×10 -6 Between M and 5×10 -11 Between M, 1×10 -6 Between M and 1×10 -11 Between M, 1×10 -6 Between M and 5×10 -12 Between M, 1×10 -6 Between M and 1×10 -12 Between M, 5×10 -7 Between M and 1×10 -7 Between M, 5×10 -7 Between M and 5×10 -8 Between M, 5×10 -7 Between M and 1×10 -8 Between M, 5×10 -7 Between M and 5×10 -9 Between M, 5×10 -7 Between M and 1×10 -9 Between M, 5×10 -7 Between M and 5×10 -10 Between M, 5×10 -7 Between M and 1×10 -10 Between M, 5×10 -7 Between M and 5×10 -11 Between M, 5×10 -7 Between M and 1×10 -11 Between M, 5×10 -7 Between M and 5×10 -12 Between M, 5×10 -7 Between M and 1×10 -12 Between M, 1×10 -7 Between M and 5×10 -8 Between M, 1×10 -7 Between M and 1×10 -8 Between M, 1×10 -7 Between M and 5×10 -9 Between M, 1×10 -7 Between M and 1×10 -9 Between M, 1×10 -7 Between M and 5×10 -10 Between M, 1×10 -7 Between M and 1×10 -10 Between M, 1×10 -7 Between M and 5×10 -11Between M, 1×10 -7 Between M and 1×10 -11 Between M, 1×10 -7 Between M and 5×10 -12 Between M, 1×10 -7 Between M and 1×10 -12 Between M, 5×10 -8 Between M and 1×10 -8 Between M, 5×10 -8 Between M and 5×10 -9 Between M, 5×10 -8 Between M and 1×10 -9 Between M, 5×10 -8 Between M and 5×10 -10 Between M, 5×10 -8 Between M and 1×10 -10 Between M, 5×10 -8 Between M and 5×10 -11 Between M, 5×10 -8 Between M and 1×10 -11 Between M, 5×10 -8 Between M and 5×10 -12 Between M, 5×10 -8 Between M and 1×10 -12 Between M, 1×10 -8 Between M and 5×10 -9 Between M, 1×10 -8 Between M and 1×10 -9 Between M, 1×10 -8 Between M and 5×10 -10 Between M, 1×10 -8 Between M and 1×10 -10 Between M, 1×10 -8 Between M and 5×10 -11 Between M, 1×10 -8 Between M and 1×10 -11 Between M, 1×10 -8 Between M and 5×10 -12 Between M, 1×10 -8 Between M and 1×10 -12 Between M, 5×10 -9 Between M and 1×10 -9 Between M, 5×10 -9 Between M and 5×10 -10 Between M, 5×10 -9 Between M and 1×10 -10 Between M, 5×10 -9 Between M and 5×10 -11 Between M, 5×10 -9 Between M and 1×10 -11Between M, 5×10 -9 Between M and 5×10 -12 Between M, 5×10 -9 Between M and 1×10 -12 Between M, 1×10 -9 Between M and 5×10 -10 Between M, 1×10 -9 Between M and 1×10 -10 Between M, 1×10 -9 Between M and 5×10 -11 Between M, 1×10 -9 Between M and 1×10 -11 Between M, 1×10 -9 Between M and 5×10 -12 Between M, 1×10 -9 Between M and 1×10 -12 Between M, 5×10 -10 Between M and 1×10 -10 Between M, 5×10 -10 Between M and 5×10 -11 Between M, 1×10 -10 Between M and 5×10 -11 Between M, 1×10 -10 Between M and 1×10 -11 Between M, 1×10 -10 Between M and 5×10 -12 Between M, 1×10 -10 Between M and 1×10 -12 Between M, 5×10 -11 Between M and 1×10 -12 Between M, 5×10 -11 Between M and 5×10 -12 Between M, 5×10 -11 Between M and 1×10 -12 Between M, 1×10 -11 Between M and 5×10 -12 Between M, or 1×10 -11 Between M and 1×10 -12 Binds to canine parvovirus or feline parvovirus at a Kd between M. In some embodiments, the parvovirus antibody binds to canine parvovirus or feline parvovirus as determined by immunoblot analysis.

[0074] "Wild-type" refers to the non-mutated form of a naturally-occurring polypeptide, or a fragment thereof. A wild-type polypeptide may be produced recombinantly.

[0075] "Variant" means a biologically active polypeptide having at least about 50% amino acid sequence identity with a native sequence polypeptide, after aligning the sequences, introducing gaps as necessary to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added to or deleted from the N-terminus or C-terminus of the polypeptide.

[0076] In some embodiments, the variant has at least 1, 2, 3, 4, or 5 amino acids substituted by different amino acids.

[0077] In some embodiments, the variant has at least about 50% sequence identity with a reference nucleic acid molecule or polypeptide, after aligning the sequences, introducing gaps as necessary to achieve the maximum percent sequence identity, and not considering conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added to or deleted from the N-terminus or C-terminus of the polypeptide. In some embodiments, the variant has at least about 50% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity with the sequence of a reference nucleic acid or polypeptide.

[0078] As used herein, the "position corresponding to position n" (where n is any number) refers to the amino acid position of the subject polypeptide that aligns with position n of the reference polypeptide after aligning the amino acid sequences of the subject and reference polypeptides and introducing gaps. Alignment for the purpose of determining whether a position in the subject polypeptide corresponds to position n of the reference polypeptide can be achieved in a variety of ways within the skill of the art using publicly available computer software such as, for example, BLAST, BLAST-2, CLUSTAL OMEGA, ALIGN, or MEGALIGN TM (DNASTAR) software, and can be achieved in a variety of ways within the skill of the art. One of ordinary skill in the art can determine appropriate parameters for alignment, including any parameters necessary to achieve a maximum alignment over the entire length of the two sequences being compared. In some embodiments, the subject polypeptide and the reference polypeptide are of different lengths.

[0079] A "point mutation" is a mutation that involves a single amino acid residue. The mutation can be a loss of an amino acid, a substitution of one amino acid residue with another, or an insertion of an additional amino acid residue.

[0080] An "amino acid substitution" refers to the replacement of one amino acid in a polypeptide with another amino acid. In some embodiments, the amino acid substitution is a conservative substitution. Non-limiting exemplary conservative amino acid substitutions are shown in Table 2. Amino acid substitutions can be introduced into the molecule of interest, and the product can be screened for retention / improvement of the desired activity, such as antigen-binding, reduction of immunogenicity, or improvement of ADCC or CDC or enhancement of pharmacokinetics.

[0081] JPEG2025102776000014.jpg121155

[0082] Amino acids can be grouped according to common side-chain characteristics: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0083] Non-conservative substitutions involve exchanging one member of these classes for another.

[0084] The term "vector" is used to describe a polynucleotide that can be manipulated to include a cloned polynucleotide or a polynucleotide capable of growing in a host cell. A vector may contain one or more of the following elements: an origin of replication, one or more control sequences (e.g., a promoter or enhancer) that regulate the expression of the polypeptide of interest, or one or more selectable marker genes (e.g., an antibiotic resistance gene and a gene usable in colorimetric analysis, such as β-galactosidase). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0085] A "host cell" can be a recipient of a vector or an isolated polynucleotide, or a cell that was a recipient. A host cell can be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting and exemplary mammalian cells include, but are not limited to, NS0 cells, PER.C6® cells (Crucell), 293 cells, and CHO cells, and their derivatives, such as 293-6E, DG44, CHO-S, and CHO-K cells. A host cell includes the progeny of a single host cell, and the progeny are not necessarily identical (in morphology or genomic DNA complementary strand) to the original parent cell due to natural, accidental, or intentional mutations. A host cell includes a cell transfected in vivo with a polynucleotide encoding the amino acid sequence provided herein.

[0086] As used herein, the term "isolated" refers to a molecule that is separated from at least some of the components in which it is typically found or produced in nature. For example, a polypeptide is called "isolated" if it is separated from at least some of the components of the cell in which it is produced. When a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered "isolating" the polypeptide. Similarly, a polynucleotide is called "isolated" if it is not part of a larger polynucleotide typically found in nature (e.g., genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide) or, in the case of an RNA polynucleotide, if it is separated from at least some of the components of the cell in which it is produced. Thus, a DNA polynucleotide contained in a vector within a host cell may be called "isolated". In some embodiments, parvovirus antibodies are purified using chromatography such as size exclusion chromatography, ion exchange chromatography, protein A column chromatography, hydrophobic interaction chromatography, and CHT chromatography.

[0087] "Reduce" or "inhibit" means to cause a decrease, reduction, or cessation in activity, function, or amount as compared to a reference. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall decrease of 20% or more. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall decrease of 50% or more. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or more. In some embodiments, the above amounts are inhibited or reduced over a period of time as compared to a control dose (such as a placebo) over the same period of time. As used herein, "reference" refers to any sample, standard, or level used for comparison purposes. A reference can be obtained from a healthy or non-afflicted sample. In some examples, the reference is obtained from a non-afflicted or untreated sample of a companion animal. In some examples, the reference is obtained from one or more healthy animals of a particular species that are not the animals being tested or treated.

[0088] As used herein, the term "substantially reduced" means a sufficiently high reduction between a numerical value and a reference value such that one of ordinary skill in the art would consider the difference between the two values to be statistically significant in the context of the biological property being measured by the value. In some embodiments, the numerically substantially reduced value is reduced by more than any one of approximately 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% as compared to the reference value.

[0089] In some embodiments, the parvovirus antibody can reduce the parvovirus titer in dogs or cats by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% as measured by a hemagglutination inhibition (HI) assay or a virus neutralization (VN) assay and compared to the parvovirus titer in the absence of the antibody.In some embodiments, the reduction in parvovirus titer is between 10% and 15%, between 10% and 20%, between 10% and 25%, between 10% and 30%, between 10% and 35%, between 10% and 40%, between 10% and 45%, between 10% and 50%, between 10% and 60%, between 10% and 70%, between 10% and 80%, between 10% and 90%, between 10% and 100%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 15% and 35%, between 15% and 40%, between 15% and 45%, between 15% and 50%, between 15% and 60%, between 15% and 70%, between 15% and 80%, between 15% and 90%, between 15% and 100%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 20% and 40%, between 20% and 45%, between 20% and 50%, between 20% and 60%, between 20% and 70%, between 20% and 80%, between 20% and 90%, between 20% and 100%, between 25% and 30%, between 25% and 35%, between 25% and 40%, between 25% and 45%, between 25% and 50%, between 25% and 60%, between 25% and 70%, between 25% and 80%, between 25% and 90%, between 25% and 100%, between 30% and 35%, between 30% and 40%, between 30% and 45%, between 30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and 80%, between 30% and 90%, between 30% and 100%, between 35% and 40%, between 35% and 45%, between 35% and 50%, between 35% and 60%, between 35% and 70%, between 35% and 80%, between 35% and 90%, between 35% and 100%, between 40% and 45%, between 40% and 50%, between 40% and 60%, between 40% and 70%, between 40% and 80%, between 40% and 90%, between 40% and 100%, between 45% and 50%, between 45% and 60%, between 45% and 70%, between 45% and 80%, between 45% and 90%, between 45% and 100%, between 50% and 60%, between 50% and 70%, between 50% and 80%, between 50% and 90%, between 50% and 100%, between 60% and 70%, between 60% and 80%, between 60% and 90%, between 60% and 100%, between 70% and 80%, between 70% and 90%, between 70% and 100%, between 80% and 90%, between 80% and 100%, or between 90% and 100%.

[0090] [Exemplary Pharmaceutical Compositions] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation in a form that enables the biological activity of the active ingredient to be effective and that contains no additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0091] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier common in the art for use with a therapeutic agent that together with the "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosages and concentrations employed and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation used. Examples of pharmaceutically acceptable carriers include, but are not limited to, alumina; aluminum stearate; lecithin; serum proteins such as human serum albumin, canine or other animal albumin; buffers such as phosphate, citrate, tromethamine, or HEPES buffer; glycine; sorbic acid; potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water; salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, or magnesium trisilicate; polyvinylpyrrolidone, cellulose-based substances; polyethylene glycol; sucrose; mannitol; or amino acids including arginine.

[0092] The pharmaceutical composition can be stored in a lyophilized form. Thus, in some embodiments, the preparation method includes a lyophilization step. Next, the lyophilized composition can be reformulated, typically as an aqueous composition suitable for parenteral administration, prior to administration to dogs, cats, or horses. In other embodiments, particularly when the antibody is very stable to thermal and oxidative denaturation, the pharmaceutical composition can be stored as a liquid for administration to dogs, cats, or horses either directly or with appropriate dilution, i.e., as an aqueous composition. The lyophilized composition can be reconstituted with water for injection (WFI). An antibacterial agent (e.g., a bacteriostatic reagent such as benzyl alcohol) may be included. Accordingly, the present invention provides a pharmaceutical composition in solid or liquid form.

[0093] The pH of the pharmaceutical composition can range from about pH 5 to about pH 8 when administered. The compositions of the present invention are sterile when used for therapeutic purposes. Sterility can be achieved by any of several means known in the art, including filtration through a sterilizing filter membrane (e.g., a 0.2 micron membrane). Sterility can be maintained regardless of the presence or absence of antibacterial agents.

[0094] In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition has a pH of 5.0 to 6.2, 5.0 to 6.0, or 5.3 to 5.7. In some embodiments, the pharmaceutical carrier is phosphate buffered saline, pH 7.2. In some embodiments, the pharmaceutical carrier is 50 mM sodium citrate pH 7, 150 mM NaCl.

[0095] In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition includes an antibacterial agent.

[0096] [Exemplary Uses of Antibodies and Pharmaceutical Compositions] The antibody or pharmaceutical composition comprising the antibody of the present invention may be useful for providing passive immunity against parvovirus infection and / or for treating parvovirus infections. As used herein, "parvovirus infection" means a condition associated with, caused by, or characterized by parvovirus infection. Such conditions include, but are not limited to, infections confirmed by cage-side ELISA testing, hemagglutination assay (HA), histopathological diagnosis, virus isolation or viral titer, or PCR. Infections with parvovirus often include fever, vomiting, diarrhea, lymphopenia, dehydration, and / or secondary sepsis.

[0097] As used herein, "treatment" is an approach for obtaining a beneficial or desirable clinical outcome. As used herein, "treatment" encompasses any administration or application of a therapeutic agent for a disease in a subject such as a mammal including humans and companion animals (e.g., dogs or cats). For the purposes of this disclosure, beneficial or desirable clinical outcomes include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, prevention of death, reduction in the extent and severity of the disease, prevention or delay of the spread of the disease, elimination or shortening of the period of viral shedding, prevention or delay of recurrence of the disease, prevention or reduction of the cytopathic activity of the virus, delay or slowing of disease progression, improvement of the medical condition, inhibition of the disease or disease progression, resolution of the clinical signs of the disease, inhibition or slowing of the disease or its progression, suppression of its occurrence, and remission (partial or complete). Also included within "treatment" is a reduction in the pathological consequences of a proliferative disease. The methods provided herein contemplate any one or more of these aspects of treatment. Consistent with the foregoing, the term treatment does not require 100 percent removal of all aspects of the disorder.

[0098] In some embodiments, parvovirus antibodies or pharmaceutical compositions containing the same can be utilized according to the methods herein to provide passive immunity against an infecting parvovirus and / or to treat parvovirus infection. In some embodiments, the parvovirus antibodies or pharmaceutical compositions are administered to a subject such as a companion animal (e.g., dog or cat) or a human to provide passive immunity against infection with parvovirus and / or to treat parvovirus infection.

[0099] A “therapeutically effective amount” of a substance / molecule, agonist or antagonist can vary depending on factors such as the type of disease being treated, the disease state, the immune status of the individual subject, the encountered toxic virus load, the severity and extent of viremia, the severity and course of the disease, the type of treatment objective, previous treatment methods, clinical history, response to previous treatment, the state of passive transfer of maternally-derived antibodies, the previous immune status of the individual animal, the discretion of the attending physician, the age, sex, and weight of the subject, and the ability of the substance / molecule, agonist or antagonist to induce a desired response in the subject. A therapeutically effective amount is also an amount where the therapeutically beneficial effects exceed the toxic or detrimental effects of the substance / molecule, agonist or antagonist. A therapeutically effective amount can be delivered in one or multiple administrations. A therapeutically effective amount refers to an amount effective at the required dosage and duration to achieve the desired therapeutic or prophylactic result.

[0100] In some embodiments, the parvovirus antibodies or pharmaceutical compositions containing the parvovirus antibodies are administered parenterally by subcutaneous administration, intravenous infusion, or intramuscular injection. In some embodiments, the parvovirus antibodies or pharmaceutical compositions containing the parvovirus antibodies are administered as a single dose or multiple dose bolus injection. In some embodiments, the parvovirus antibodies or pharmaceutical compositions containing the parvovirus antibodies are administered by intramuscular, intravenous, intraperitoneal, intrathecal, subcutaneous, intraarterial, intra-articular, intrathecal, or inhalation routes.

[0101] The parvovirus antibodies described herein can be administered in an amount ranging from 0.01 mg / kg body weight to 100 mg / kg body weight per dose. In some embodiments, the parvovirus antibodies can be administered in an amount ranging from 0.5 mg / kg body weight to 50 mg / kg body weight per dose. In some embodiments, the parvovirus antibodies can be administered in an amount ranging from 0.1 mg / kg body weight to 10 mg / kg body weight per dose. In some embodiments, the parvovirus antibodies can be administered in an amount ranging from 0.1 mg / kg body weight to 100 mg / kg body weight per dose. In some embodiments, the parvovirus antibodies can be administered in an amount ranging from 1 mg / kg body weight to 10 mg / kg body weight per dose. In some embodiments, the parvovirus antibodies can be administered in an amount in the range of 0.5 mg / kg body weight to 100 mg / kg body weight, 1 mg / kg body weight to 100 mg / kg body weight, 5 mg / kg body weight to 100 mg / kg body weight, 10 mg / kg body weight to 100 mg / kg body weight, 20 mg / kg body weight to 100 mg / kg body weight, 50 mg / kg body weight to 100 mg / kg body weight, 1 mg / kg body weight to 10 mg / kg body weight, 5 mg / kg body weight to 10 mg / kg body weight, 0.5 mg / kg body weight to 10 mg / kg body weight, 0.01 mg / kg body weight to 0.5 mg / kg body weight, 0.01 mg / kg body weight to 0.1 mg / kg body weight, or 5 mg / kg body weight to 50 mg / kg body weight per dose. In some embodiments, the parvovirus antibodies can be administered in an amount of 0.5 mg / kg body weight.

[0102] The parvovirus antibodies or pharmaceutical compositions comprising the parvovirus antibodies can be administered as a single dose to a subject such as a human or a companion animal (e.g., a dog or a cat), either once or over a series of treatments. For example, the parvovirus antibodies or pharmaceutical compositions comprising the parvovirus antibodies can be administered at least once, more than once, at least twice, at least three times, at least four times, or at least five times.

[0103] In some embodiments, the dosage is administered once a week for at least two or three consecutive weeks, and in some embodiments, this treatment cycle is repeated more than once, with treatment-free periods of one or more weeks interspersed in some cases. In other embodiments, a therapeutically effective dosage is administered once a day for two to five consecutive days, and in some embodiments, this treatment cycle is repeated more than once, with treatment-free periods of one or more days or weeks interspersed in some cases.

[0104] In some embodiments, the dosage is administered to a subject such as a human or a companion animal (e.g., a dog or a cat) less than 1 week old, less than 2 weeks old, less than 3 weeks old, less than 4 weeks old, less than 5 weeks old, less than 6 weeks old, less than 6 weeks old, less than 7 weeks old, less than 8 weeks old, less than 9 weeks old, less than 10 weeks old, less than 11 weeks old, less than 12 weeks old, less than 6 months old, between 0 and 12 weeks old, between 0 and 10 weeks old, between 0 and 8 weeks old, between 0 and 6 weeks old, between 0 and 4 weeks old, between 0 and 2 weeks old, between 4 and 12 weeks old, between 6 and 12 weeks old, between 10 and 12 weeks old, between 4 weeks old and 6 months old, between 2 months old and 6 months old, between 4 months old and 6 months old, between 6 months old and 1 year old, over 13 weeks old, or over 1 year old.

[0105] It may be advantageous to deliver parvovirus antibodies or nucleic acids encoding parvovirus antibodies to prenatal and / or postnatal neonatal subjects to provide passive immunity against parvovirus infection. In some embodiments, the parvovirus antibodies are administered to a maternal subject during pregnancy or lactation, such as a human or a companion animal (e.g., a dog or a cat). In some embodiments, the parvovirus antibodies are administered to the placenta of a subject during pregnancy.

[0106] In some embodiments, a method of providing passive immunity to a neonatal subject against infection by canine or feline parvovirus comprises administering a therapeutically effective amount of a monoclonal antibody that binds to canine or feline parvovirus to a maternal subject during pregnancy or lactation. In some embodiments, the parvovirus antibodies are administered to the placenta of a subject during pregnancy. In some embodiments, the parvovirus antibodies are administered to a subject during lactation.

[0107] Provided herein are methods of using parvovirus antibodies, polypeptides, and polynucleotides for the detection, diagnosis, and monitoring of parvovirus infection. Also provided herein are methods of determining whether a subject will respond to parvovirus antibody therapy. In some embodiments, the methods include viral serum neutralization. In some embodiments, the methods include using a parvovirus antibody to detect whether a subject has cells that express parvovirus. In some embodiments, the detection method includes contacting a sample with an antibody, polypeptide, or polynucleotide and determining whether the level of binding differs from the level of a reference or comparison sample (such as a control). In some embodiments, the methods may be useful for determining whether an antibody or polypeptide described herein would be an appropriate therapeutic agent for a subject.

[0108] In some embodiments, the sample is a biological sample. The term "biological sample" means an amount of material from a living body or something that was previously a living body. In some embodiments, the biological sample is a swab containing cell debris, cells, or cell / tissue lysates. In some embodiments, the biological sample includes, but is not limited to, blood (e.g., whole blood), plasma, serum, urine, synovial fluid, lymphoid tissue, and epithelial cells.

[0109] In some embodiments, cells or cell / tissue lysates are contacted with a parvovirus antibody and the binding between the antibody and the cells is determined. If the test cells exhibit binding activity as compared to reference cells of the same tissue type, it may be shown that the subject would benefit from treatment with the parvovirus antibody. In some embodiments, the test cells are from a subject, such as a human or a companion animal (e.g., a dog or a cat) tissue.

[0110] A variety of methods known in the art for detecting specific antibody-antigen binding can be used. Exemplary immunoassays that can be performed include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). The indicator moiety, or label group, can be conjugated to the antibody of interest and is selected to match the requirements for the use of a variety of methods, which are often determined by the availability of assay equipment and the compatible immunoassay procedure. Suitable labels include, but are not limited to, radionuclides (e.g., 125 I, 131 I, 35 S, 3 H, or 32 P), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or b-galactosidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., Qdot TM nanoparticles supplied by Quantum Dot Corporation, Palo Alto, Calif.). General techniques used in performing the various immunoassays described above are known to those of skill in the art.

[0111] For diagnostic purposes, polypeptides containing antibodies can be labeled with detectable moieties including, but not limited to, radioisotopes, fluorescent labels, and various enzyme-substrate labels known in the art. Methods for conjugating labels to antibodies are known in the art. In some embodiments, parvovirus antibodies need not be labeled, and their presence can be detected using a second labeled antibody that binds to the first parvovirus antibody. In some embodiments, parvovirus antibodies can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987). Parvovirus antibodies and polypeptides can also be used in in vivo diagnostic assays such as in vivo imaging. Generally, an antibody or polypeptide is labeled with a radionuclide (e.g., 111 In, 99 Tc, 14 C, 131 I, 125 I, 3 H, or any other radionuclide label including those outlined herein) such that the location of the target cell or tissue can be identified using immunoscintigraphy. Antibodies may also be used as staining reagents in pathology using techniques well known in the art.

[0112] In some embodiments, the first antibody is used for diagnosis and the second antibody is used as a therapeutic agent. In some embodiments, the first antibody and the second antibody are different. In some embodiments, the first antibody and the second antibody can both bind to an antigen simultaneously by binding to different epitopes.

[0113] The following examples illustrate specific aspects of the disclosure and are not intended to limit the disclosure in any way.

Example

[0114] Example 1 Preparation of Chimeric Antibodies Mab A and Mab B Structural analyses of canine and feline parvoviruses complexed with antibody fragments from eight different neutralizing monoclonal antibodies were reported by Hafenstein S. et al., J Virol. 2009 Jun, 83(11):5556-66. Incomplete amino acid sequence information was provided for the rat Mab E and Mab F Fab fragments, which showed neutralization of canine parvovirus in in vitro assays. The incomplete amino acid sequences of the variable heavy and variable light chains of Mab E are SEQ ID NO:81 and SEQ ID NO:82, respectively. The incomplete amino acid sequences of the variable heavy and variable light chains of Mab F are SEQ ID NO:83 and SEQ ID NO:84, respectively.

[0115] Three-dimensional protein structure analysis was performed to construct redesigned first framework regions for both the heavy and light chains of Mab E and Mab F. The amino acid sequences of the heavy and light chains of the redesigned monoclonal antibodies named Mab A and Mab B are SEQ ID NO:20 (Mab A heavy chain), SEQ ID NO:22 (Mab A light chain), SEQ ID NO:62 (Mab B heavy chain), and SEQ ID NO:64 (Mab B light chain).

[0116] The DNA sequences encoding the chimeric antibodies were designed against fusions to the canine constant IgG-B heavy chains of Mab A VH (SEQ ID NO: 20) and Mab B VH (SEQ ID NO: 62) and the canine constant κ light chains of Mab A VL (SEQ ID NO: 22) and Mab B LC (SEQ ID NO: 64), and SEQ ID NO: 23 (chimeric A HC IgG-B), SEQ ID NO: 66 (chimeric B HC IgG-B), SEQ ID NO: 25 (chimeric A LC κ), and SEQ ID NO: 68 (chimeric B LC κ) were obtained. The heavy and light chain nucleotide sequences were chemically synthesized and inserted into expression vectors suitable for transfection into mammalian host cells. After transfecting and culturing cell pairs of the heavy and light chain vectors, the antibodies were affinity purified from the medium using CaptivA® Protein A affinity resin (Repligen). The purified chimeric canine Mab A and Mab B antibodies were confirmed by SDS-PAGE analysis (data not shown).

[0117] Chimeric antibodies can also be designed and prepared based on fusions to the canine constant IgG-A, IgG-C, and IgG-D heavy chains of Mab A VH (SEQ ID NO: 20) and Mab B VH (SEQ ID NO: 62).

[0118] Example 2 Chimeric Canine Antibodies Mab A and Mab B Activity Both purified chimeric Mabs A and B (formulated in PBS, pH 7.2 at 200 μg / mL) were used to evaluate anti-canine parvovirus activity using a hemagglutination inhibition (HI) assay. The assay was performed essentially as described by Carmichael et al., Am J of Vet Res. 1980 May, 41 (5):784-91 at the University of Wisconsin Companion Animal Vaccine and Immunodiagnostic Services Laboratory. Serial dilutions of samples of chimeric Mabs A and B incubated with CPV were prepared. Next, porcine red blood cells were added. Canine parvovirus induces the agglutination of porcine red blood cells. Both chimeric Mabs A and B prevented agglutination. Both antibodies resulted in a CPV-2b HI of 40960, while irrelevant canine IgG resulted in an HI of <20. Most vaccinated dogs are understood to have an antibody HI of around 1280.

[0119] Furthermore, the activity of the antibodies to prevent live CPV from infecting cells and destroying them was analyzed using a virus serum neutralization assay.

[0120] Example 3 Variant VH and VL sequences for enhanced expression of chimeric Mabs A and B Chimeric Mabs A and B were consistently poorly expressed in transient CHO S or stable CHO K1 cells. To increase the expression level, three-dimensional protein structure analysis was performed to identify VH and VL amino acid residues suitable for modification. Multiple constructs with various mutations in VH and VL were generated. The expression levels of antibodies having combinations of canine IgG-B with VH and VL variants were tested.

[0121] Enhanced expression variants were analyzed for their specific activities by an in vitro parvovirus HI assay. In these experiments, each antibody variant tested was formulated in PBS, pH 7.2 at 200 μg / mL. Both chimeric murine Mab A (SEQ ID NOs: 23 and 25) and chimeric murine Mab B (SEQ ID NOs: 66 and 68) showed a CPV-2b HI of 10240. The chimeric murine Mab A v2 (SEQ ID NOs: 24 and 25) and chimeric murine Mab B v2 (SEQ ID NOs: 67 and 69) of the enhanced expression variants maintained the same HI value of 10240, while other variants showed lower specific activities.

[0122] Example 4 Parvovirus infection assay activity (SVN) To determine the ability of chimeric murine Mab A v2 (SEQ ID NOs: 24 and 25) and chimeric murine Mab B v2 (SEQ ID NOs: 67 and 69) to prevent live parvovirus from infecting cells and destroying them, a serum virus neutralization (SVN) assay was performed. In this assay, Madin-Darby canine kidney (MDCK) cells were grown. Serial dilutions of the antibody were mixed with a fixed titer of parvovirus. The mixture was then incubated with the cells. After incubation, the cells were examined microscopically to identify the virus cytopathic effect (CPE). The reading was the final dilution of the antibody sample at which the parvovirus was prevented from causing a cytopathic effect.

[0123] (1) Kept at 4°C and (2) both chimeric murine Mab A v2 subjected to 7 freeze-thaw cycles at 200 μg / mL had the same activity: an SVN of 32000 against CPV-2b and 8000 against CPV2c.

[0124] Example 5 Stability of chimeric murine Mab A v2 and Mab B v2 The stability of chimeric murine Mab A v2 (SEQ ID NOs: 24 and 25) at 25 mg / mL and chimeric murine Mab B v2 (SEQ ID NOs: 67 and 69) at 20.5 mg / mL was tested. Chimeric murine Mab A v2 and chimeric murine Mab B v2 exhibit similar specific activities by HI assay at concentrations of approximately 25 mg / mL and approximately 20.5 mg / mL, respectively.

[0125] Both chimeric murine Mab A v2 and chimeric murine Mab B v2 produced and purified from CHOK1 stable pools can be easily concentrated to 25 mg / mL in PBS, pH 7.2, suggesting that there are no concerns about solubility for both antibodies.

[0126] Chimeric murine Mab A v2 and chimeric murine Mab B v2 were evaluated by size exclusion high performance liquid chromatography (HPLC) and were monomeric (>99%) at neutral pH and after 10 hours of storage at pH 4.1. In this analysis, monomer and aggregate of the antibody were separated by SEC-HPLC using an Agilent 1100 system, a Shodex KW803 column (8 mm × 300 mm) equipped with a KW-G guard column, and a mobile phase buffer of 2×PBS pH 7.2 (270 mM NaCl, 5.4 mM KCl, 8.6 mM Na2HPO4, 2.8 mM KPO4) at a constant flow rate of 0.5 mL / min. The column was calibrated using a Bio Rad marker (catalog number 151-1901) composed of thyroglobulin, γ-globulin, ovalbumin, myoglobin, and vitamin B12.

[0127] (1) The real-time stability of chimeric murine Mab A v2 at 25 mg / mL in PBS, pH 7.2 and (2) at various temperatures at 25 mg / mL in 50 mM sodium citrate pH 7, 150 mM NaCl was evaluated. The results of HPLC-SEC at 10 months are summarized in Table 3.

[0128] JPEG2025102776000015.jpg61169

[0129] The stability of the antibodies was also tested by subjecting them to three freeze-thaw cycles involving freezing at -70°C and thawing at room temperature. No loss of activity was observed for either antibody with respect to both the CPV-2b HI value and the CPV-2c HI value.

[0130] The stability of the antibodies was further tested by subjecting them to 7 freeze-thaw cycles involving freezing at -20°C and thawing at room temperature. Both antibodies maintained full activity when measured by the HI assay.

[0131] Differential scanning fluorimetry (DSF) was used to analyze the thermal stability of the two antibodies over a range of pH in different formulations. The melting temperature (Tm) of each antibody under different conditions was measured. Buffer and 12 μg of antibody were mixed with 1× Protein Thermal Shift Dye (Applied Biosystem, catalog number 4461146). A melting curve was performed using a StepOne Real-Time PCR system (Applied Biosystem, catalog number 4376357). The temperature was raised from 25°C to 99°C at a ramp rate of 1% according to the manufacturer's instructions. The data was analyzed using Protein Thermal Shift TM Software v1.0 (Applied Biosystem, catalog number 4466038), and the Tm was determined by calculating the highest value obtained by taking the first derivative of the protein melting curve. For each formulation tested containing PBS pH 7.2, chimeric Mab A v2 was significantly more stable than chimeric Mab B v2.

[0132] Example 6 VLP Binding of Chimeric Antibodies Mab A v2 and Mab B v2 The apparent affinities of chimeric murine Mab A v2 (SEQ ID NOs: 24 and 25) and chimeric murine Mab B v2 (SEQ ID NOs: 67 and 69) with parvovirus VLPs were measured. First, biotinylated chimeric murine Mab A v2 and chimeric murine Mab B v2 were bound to streptavidin sensor chips. Next, the binding of recombinant canine parvovirus CPV2b capsid (Advanced ImmunoChemical Inc Cat#8-CPV) to the Mab-binding sensor chips was evaluated.

[0133] Chimeric murine Mab A v2 and chimeric murine Mab B v2 antibodies each showed affinity for canine CPV 2b VLPs with kinetics potentially sufficient for therapeutic activity. Binding analysis was performed using an Octet biosensor as follows. Briefly, chimeric murine Mab A v2 (SEQ ID NOs: 24 and 25) and chimeric murine Mab B v2 (SEQ ID NOs: 67 and 69) were biotinylated by amine chemistry. Free unreacted biotin was removed by extensive dialysis. Biotinylated chimeric murine Mab A v2 and chimeric murine Mab B v2 were captured on streptavidin sensor chips. The association of either Mab A v2 or Mab B v2 with canine CPV 2b VLP (40 μg / mL) was monitored for 300 seconds. Dissociation was monitored for 300 seconds. Biotinylated irrelevant canine IgG-B was used as a negative control. ForteBio TM Data was fit to a 1:1 binding model using ForteBio data analysis software to determine kon, koff, and Kd. The buffer for dilution and all binding steps was 20 mM phosphate, 150 mM NaCl, pH 7.2. The Kd of chimeric murine Mab A v2 and VLP2b was 9.78×10 -11 M, and the Kd of chimeric murine Mab B v2 and VLP2b was 9.38×10 -12 M (see Figure 2).

[0134] Example 7 Caninization of Mab A v2 and Mab B v2 To reduce potential immunogenicity, particularly when repeated administration of antibody A or B is required, the VH and VL from Mab A v2 and B v2 were subjected to caninization. A number of caninized VH and VL were designed, expressed, and purified. The CPV2b HI assay was used to select the expressors with excellent specific activity. Caninized A HC v3 IgG-B (SEQ ID NO: 37) and caninized A LC κ (SEQ ID NO: 39), as well as caninized A HC v4 IgG-B (SEQ ID NO: 38) and caninized A LC κ (SEQ ID NO: 39), maintained sufficient activity compared to chimeric A HC IgG-B (SEQ ID NO: 23) and chimeric A LC κ (SEQ ID NO: 25), and also compared to chimeric A HC v2 IgG-B (SEQ ID NO: 24) and chimeric A LC κ (SEQ ID NO: 25). Caninized B HC v3 IgG-B (SEQ ID NO: 79) and caninized B LC v3 κ (SEQ ID NO: 80) maintained sufficient activity compared to chimeric B HC IgG-B (SEQ ID NO: 66) and chimeric B LC κ (SEQ ID NO: 68), and also compared to chimeric B HC v2 IgG-B (SEQ ID NO: 67) and chimeric B LC v2 κ (SEQ ID NO: 69).

[0135] Example 8 Feline chimeric Mab A and Mab B Chimeric canine Mab A v2 (SEQ ID NOs: 24 and 25) and chimeric canine Mab B v2 (SEQ ID NOs: 67 and 69) showed activity in the feline parvovirus HI assay. 200 μg / mL of chimeric canine Mab A v2 showed 262,144 feline parvovirus HI, and 200 μg / ml of chimeric canine Mab B v2 showed 131,072 feline parvovirus HI.

[0136] Therefore, Mab A and Mab B can be used for the prevention or treatment of feline panleukopenia virus (FPV). Furthermore, the chimeric feline Mab A variable heavy chain and feline IgG-1 were designed to pair with the chimeric feline Mab A variable light chain and feline kappa (SEQ ID NOs: 31 and 32). To remove potential heterogeneity, mutations were introduced to remove glycosylation sites, allowing chimeric feline Mab A-IgG1 to pair with nonglycosylated kappa (e.g., SEQ ID NOs: 31 and 33).

[0137] Furthermore, the chimeric feline Mab B variable heavy chain and feline IgG-1 were designed to pair with the chimeric feline Mab B variable light chain and feline kappa (SEQ ID NOs: 74 and 75). Mutations were introduced to remove glycosylation sites, allowing chimeric feline Mab A-IgG1 to pair with nonglycosylated kappa (e.g., SEQ ID NOs: 74 and 76).

[0138] Example 9 Safety and Pharmacodynamics in Puppies In vivo studies were performed using chimeric murine Mab A (SEQ ID NOs: 23 and 25) and chimeric murine Mab B (SEQ ID NOs: 66 and 68) monoclonal antibodies. The purpose of this study was to evaluate the safety, the effect of chimeric murine Mab A and chimeric murine Mab B on canine parvovirus-2 (CPV-2) titers, virus neutralization, and the response to a monovalent modified live vaccine in healthy, unvaccinated puppies raised for this purpose after treatment with the two antibodies. Refer to the summary of the study design in Table 4 below.

[0139] JPEG2025102776000016.jpg118170

[0140] After randomization, the puppies were assigned to two groups (3 Mab A (dog IDs BDW-8, DEW-8, and DFW-8): 3 Mab B (dog IDs CTW-8, CWW-8, and DHW-8)). On study day 0, each antibody was administered to the puppies as a single intravenous dose (5 mg / kg) via the shaved lateral saphenous vein. Initially, puppies were administered the monovalent CPV vaccine Nobivac 1-Pv (Merck Animal Health, Kenilworth, NJ) at a titer of 80 or less. The second dose was administered 21 days after the first dose. A third dose was administered if the puppies did not seroconvert approximately 21 days after the second dose.

[0141] The puppies were monitored for a total of 100 days using hemagglutination inhibition (HI) and virus neutralization (VN). HI quantifies the amount of antibody in a serum sample, and VN is utilized to determine the ability of an antibody to prevent live virus from infecting and destroying cells. The puppies were also followed to determine if they seroconverted after CPV vaccination.

[0142] All puppies were verified to have an HI titer of less than 20 on day 0 before intravenous administration of Mab A or B antibody. The HI titer on day 1 after administration of Mab A or B was between 2560 and 5120. The titer was monitored for 100 days to document antibody degradation and evaluate serum half-life.

[0143] On day 37 when the titer of individual puppies began to decline to 1:40, vaccination was initiated to evaluate the neutralization of CPV-2 of the vaccination. Mab A or B antibody has been documented to block immunization at various levels in puppies. The puppies were given subsequent doses of the vaccine as scheduled and the response was verified. Seroconversion of the vaccination ultimately occurred in 5 out of 6 puppies. The HI assay titers of the animals in both groups are reflected in Table 5 below, and Figures 3A (Mab A group), 3B (Mab B group), and 3C (a combination of both groups).

[0144] To obtain fast distribution and slow decay, a biphasic decay kinetics was used with HI as a function of time. The half-life of chimeric Mab A was 10.1 days (Figure 4A), and that of chimeric Mab B was 8.6 days (Figure 4B). The serum VN assay mirrored the HI titers (see Table 6 below). The half-life was approximately consistent with maternally derived antibodies. (Carmichael, LE, Joubert, JC and Pollock, RVH; “A Modified Live Canine Parvovirus Vaccine. II. Immune Response;” Cornell Vet, Volume 73, Pages 13-29; 1983).

[0145] This first pharmacodynamic study demonstrated that intravenously administered chimeric Mab A (SEQ ID NOs: 23 and 25) and chimeric Mab B (SEQ ID NOs: 66 and 68) resulted in initial antibody titers between 2560 and 5120 within 24 hours after administration. Additionally, VN closely follows the HI titers. The approximate half-life of the antibodies is between 7 and 10 days. The duration of immunity (i.e., the duration during which the HI titer was maintained above 80) ranged from 30 to 42 days after a single administration of chimeric Mab A or chimeric Mab B. Interference with vaccination may occur until the antibodies are sufficiently degraded so that they no longer neutralize the vaccine-inoculated CPV-2. Both chimeric Mab A and chimeric Mab B had high titers, good tolerability, and no adverse events or injection site reactions were reported.

[0146] The first CPV vaccination did not result in seroconversion in 5 out of 6 puppies due to residual chimeric Mab A or B antibodies that potentially neutralize the vaccine. Blocking of vaccine virus replication by both canine Mab A and chimeric Mab B was the first in vivo correlation with in vitro neutralization data. Subsequently, 3 puppies seroconverted after the second vaccination, and 1 puppy seroconverted after the third vaccination.

[0147] Regarding puppies CWW-8, the seroconversion on day 56 did not correlate with the time frame of vaccination administered on day 37. In this study, it is suspected that vaccine virus excreted from other puppies exposed CWW-8 and caused seroconversion when the titer dropped below 20.

[0148] JPEG2025102776000017.jpg234157

[0149] JPEG2025102776000018.jpg249141

[0150] Example 10 Prevention and Treatment In this in vivo study, MabA v2 antibody was utilized. The purpose of this study was to verify the dose of live CPV-2 to be used for exposure to dogs in future important studies of MabA v2, and to provide proof of concept of MabA v2 for both two indications: as a prophylactic treatment to prevent CPV-2 infection and as a therapeutic treatment for dogs infected with CPV-2. Refer to the summary of the study design in Table 7 below.

[0151] JPEG2025102776000019.jpg103170

[0152] Twelve dogs were randomized and assigned to one of six groups, each containing both male and female. Dogs in groups 1 - 3 were given only live CPV-2 exposure to verify the doses required to generate the morbidity and mortality rates required in three different virus logs. Dogs in groups 4 and 5 were administered Mab A v2 as a prophylactic treatment prior to inoculation with live CPV-2b. Dogs in group 6 were administered Mab A v2 after exposure, and the occurrence of CPV-2 infection was confirmed by fecal cage-side ELISA SNAP test. Refer to Table 8 (below) for the CPV-2 inoculation exposure schedule.

[0153] JPEG2025102776000020.jpg61170

[0154] According to Table 9 below, in Groups 4 and 5, MabA v2 was administered on Day 1 of the study.

[0155] JPEG2025102776000021.jpg26170

[0156] After each puppy tested positive in the cage-side CPV-2 ELISA test to confirm CPV-2 in feces for parvovirus infection, in Group 6, Mab A v2 was administered on Day 7 of the study. See Table 10 below.

[0157] JPEG2025102776000022.jpg26170

[0158] This study provided the first pharmacodynamic data on subcutaneously administered MabA v2. The dose administered intravenously to dogs in Group 4 resulted in an immediate increase in HI titer as predicted. The dose administered subcutaneously to dogs in Group 5 resulted in equivalent titers over 24 hours. In Table 11 below, it is possible to see how intravenous and subcutaneous administrations differed in terms of the timing at which the HI titer peak was achieved after MabA v2 administration.

[0159] As shown in Table 12 below, administration of MabA v2 resulted in circulating antibody titers in dogs within 24 hours (IV) and 48 hours (SC) after administration. Facing a high-load pathogenic exposure, protective passive antibody titers were maintained for 16 days.

[0160] JPEG2025102776000023.jpg255130

[0161] JPEG2025102776000024.jpg255133

[0162] On Day 1, MabA v2 was administered intravenously or subcutaneously to Groups 4 and 5, respectively. On Day 4, pathogenic CPV-2 was administered intranasally to all 12 dogs in all 6 groups, at the inoculation doses shown in Table 8. The dogs were monitored every 6 hours for 14 days in total to record general health observations (GHO), and clinical scores (using a validated system, according to Mohr et al., 2003) were documented every 12 hours, and frequent blood sampling was performed for hematological tests, HI, and VN, as well as fecal hemagglutination assay (HA). In addition, a cage-side CPV-2 ELISA test was performed daily on all dogs after inoculation until a positive determination was made to evaluate when parvovirus infection was present. Table 13 summarizes the clinical signs of CPV-2 observed during the study.

[0163] JPEG2025102776000025.jpg85170

[0164] The dogs in Groups 3 and 6 (10 6 dogs that were exposed to live CPV-2 and not pre-treated with MabA v2) showed positive results in the cage-side CPV-2 ELISA test on Day 3 or 4 after inoculation. Both dogs in Group 3 (a de facto control group) developed severe classical clinical signs of parvovirus infection, including depression, vomiting, hemorrhagic diarrhea, and progressive dehydration, became moribund, and were euthanized by Day 7 after inoculation. Both dogs in Group 6 showed positive results in the cage-side CPV-2 ELISA test 3 days after inoculation with live CPV-2. After confirming that the cage-side CPV-2 ELISA test was positive, the dogs were subsequently treated with 5 mg / kg of intravenous Mab A v2. The dogs did not receive additional adjunctive or supportive care.

[0165] The dogs in Group 3 continued to deteriorate and ultimately became moribund and were humanely euthanized, while the dogs in Group 6 survived and showed complete clinical recovery during the study period. The dogs in Group 6 also reverted to negative in the cage-side CPV-2 ELISA test, indicating that the dogs had stopped releasing virus particles.

[0166] All dogs in Groups 4 and 5 that were administered Mab A v2 as a preventive measure not only survived but also maintained their health (e.g., they never tested positive for CPV-2 ELISA at the cage side and never showed clinical signs of parvovirus infection).

[0167] Anatomical and histopathological diagnoses of Groups 3 - 6 were performed after humane euthanasia at the scheduled end of the study, unless euthanasia was carried out earlier for humane reasons. Groups 4 and 5 showed no significant pathological changes. Group 6 showed lymphocytic depletion of the thymus, but the other organs examined were normal. Group 3 showed lymphoid depletion of all lymphoid organs and moderate to severe small intestinal villous atrophy / necrosis with mild to severe crypt necrosis indicating CPV-2 infection.

[0168] In this proof-of-concept study, it was demonstrated that Mab A v2 is promising as a preventive and therapeutic intervention. All dogs in the preventive group (n = 4) did not develop parvovirus infection and remained clinically normal. All dogs in the treatment group (n = 2) that were confirmed to be infected with parvovirus survived and experienced complete clinical recovery after being administered Mab A v2 without any other adjunctive or supportive treatment. This study also verified that subcutaneous administration of Mab A v2 for prevention is a feasible option. Finally, this formulation was well-tolerated with no adverse events or injection site reactions. In this proof-of-concept study, 100% of the puppies were protected from CPV-2 infection by pre-exposure prophylactic administration of Mab A v2. In addition, 100% of the puppies that received therapeutic administration of Mab A v2 after CPV infection confirmation survived and recovered.

[0169] Example 11 Central efficacy study of therapeutic treatment This pivotal study evaluates the efficacy of Mab A v2 as a therapeutic agent for canine parvovirus (CPV) disease. Successful completion of this study and demonstration of efficacy could support the claim of the label that "this formulation has been shown to be effective in the treatment of dogs 13 weeks of age and older against canine parvovirus (CPV) disease."

[0170] This GCP, randomized, blinded, placebo-controlled trial is 14 days in length after a minimum 7-day acclimation period. A total of 28 naive, CPV-2 antibody-negative dogs (less than or equal to 13 weeks of age at Day 0, any gender, body weight greater than or equal to 1.5 kg) are randomized to the study in a 3:1 ratio into two groups (n = 21 dogs in Group 1 and n = 7 dogs in Group 2) as shown in Table 14.

[0171] During acclimation, dogs are acclimated to feeding, housing, and handling procedures while being administered anthelmintics and antibiotics to eliminate common helminth and coccidia infections.

[0172] The day of CPV-2b exposure is considered Day 0. On Day 0, all dogs in Groups 1 and 2 are administered pathogenic CPV-2b by intranasal (IN) (approximate volume of 1 mL; approximately 0.5 mL per nostril) dose of 6 TCID 50 . On the same day that CPV is detected in the feces (cage-side CPV SNAP test is positive), each dog is administered Mab A v2 or Control Product (CP; phosphate-buffered saline (PBS)) by intravenous (IV) injection into an accessible vein (e.g., lateral saphenous) catheter using a Luer-lock syringe. Mab A v2 is administered as a single dose of 5 mg / kg based on body weight. The pH of CP is 7.2 ± 0.2. The doses of Mab A v2 and CP are calculated as follows: {(body weight in kg) × (5 mg / kg)} ÷ {25 mg / mL} = volume in mL to be injected

[0173] At the scheduled time outlined in Table 15, the health status of each dog will be closely monitored for 14 days using physical examination (PE), general health observation (GHO), measurement of rectal temperature (RT), in-cage CPV SNP testing, serum HI, fecal hemagglutination assay (HA), serum biochemistry testing, and hematology testing.

[0174] The primary efficacy variable is the prevention of death due to CPV infection in dogs in Group 1 versus dogs in Group 2.

[0175] Please refer to the treatment groups in Table 14 below.

[0176] JPEG2025102776000026.jpg43170

[0177] Randomization In this study, two-stage randomization may be required. The first stage is to determine which 28 dogs will be enrolled in the study. Next, the dogs will be randomized at a ratio of 3:1 for Groups 1 and 2, respectively, with a block size of 4 based on littermates if possible. Randomization is based on random numbers generated by the PLAN procedure in SAS (version 9.4 and later, SAS Institute, Cary NC) using the randomization selection method with a seed number. Upon arrival at the test facility, the dogs will be placed in two-room cages in the order of arrival, and dogs from the same litter / block will be kept in the same room if possible. Dogs may be secondarily exposed to the virus excreted from infected dogs. To maintain a similar level of secondary virus exposure in both treatment groups and to maintain blinding for the test facility personnel, both treatment groups will be included in each room. After selecting 28 dogs, their cages will be numbered sequentially from 1 to 28. Dogs from the same litter will have adjacent cage numbers, and 4 dogs from the same litter will take the earlier numbers (e.g., 1 - 4, 5 - 8, etc.) for each room. One room should contain cage numbers 1 - 12, and the other room should contain cage numbers 13 - 28. The dogs will be enrolled in the order of the cage numbers on the randomization list.

[0178] Blinding in the study The principal investigator of the clinical trial and all other study site personnel (except the dispenser and the one designated to restrain the dog for dosing) are unaware of the actual treatment group assignment on Day 0. Only the dispenser and the designated personnel to restrain the dog during dosing have access to the actual treatment group assignment (Mab A v2 or CP) of each dog. The dispenser and the dog restrainer / holder do not perform any study evaluations.

[0179] Specifications A total of at least 30 healthy, unvaccinated, CPV-2 seronegative (HI < 20) beagle dogs bred for the purpose are procured. Upon arrival at the study site, the dogs are confirmed to be CPV negative by a CPV SNAP test. Twenty-eight dogs are included in the study registration, and two additional dogs are purchased to ensure that the number of dogs is met after screening and acclimation. Ideally, the 30 dogs consist of 7 litters of 4 puppies each and 2 dogs from another litter (partial litter).

[0180] Central efficacy determination The central efficacy serial is used to set the release efficacy of subsequent serials, and each serial must be at least as potent as the central efficacy serial when compared to the potency assay in order to be considered sufficient for release. A reference monitoring assay is used to test the central efficacy serial in order to set the master reference for the potency assay. The test is conducted on Days 0 ± 7 of dosing for the first effective dose administered to the test dogs.

[0181] [Observation]

[0182] Physical examination (PE) The physical examination is performed on all dogs by the study site veterinarian at least on Days -7, -1, the Mab A v2 / CP dosing day, 13, and on unscheduled days as needed. The physical examination includes a comprehensive evaluation of all body tissues, including hydration status and BW, and is recorded.

[0183] Food consumption Once a day, in conjunction with general health observations (GHO), a qualitative assessment of food consumption is performed. All abnormalities in the observed food consumption are recorded.

[0184] General health observations (GHO) and measurement of rectal temperature (RT) GHO is performed at least once a day during the acclimation period from day - 7 to day - 1 by a veterinarian or their designee at the test facility. GHO is performed every 12 hours (±1 hour) from day 0 to day 12. Starting on day 0, in combination with each GHO, RT is also measured using a calibrated thermometer on the Fahrenheit scale.

[0185] Specific GHO information is collected regarding these four parameters. · Attitude - Recorded as normal, mild to moderate depression, severe depression, or prostration or moribund state · Appetite - Recorded as normal, eats a small amount spontaneously, has no interest in food, or not offered · Vomiting - Recorded as none, mild (once every 12 hours), moderate (2 - 5 times every 12 hours), or severe (6 or more times every 12 hours) · Feces - Recorded as well - formed or none, soft or pasty feces, watery non - bloody diarrhea, or watery bloody diarrhea GHO may include other findings in addition to these four parameters. These parameters are documented for each dog during each observation period.

[0186] Cage - side CPV SNAP test The cage - side CPV ELISA test is performed using the IDEXX CPV SNAP test according to the manufacturer's instructions. Upon arrival from the vendor on day - 7, a preliminary CPV SNAP test is performed on all dogs to confirm that the dogs do not have an active CPV infection. Subsequent CPV SNAP tests are evaluated at a consistent time in the morning from day - 1 until the end of the test. If a dog is not determined to be positive in the CPV SNAP test and thus does not receive Mab A v2 / CP, that dog is excluded from the efficacy analysis.

[0187] Serum CPV-2 Hemagglutination (HI) Test Whole blood samples are collected from all dogs via the jugular, cephalic, or lateral saphenous vein into serum separator tubes (approx. 1.5 mL), processed into serum on days -7, 0, 3, 4, 6, 8, 10, and 13, and the day 0 samples are collected prior to exposure to pathogenic CPV-2b. Samples are also collected prior to unscheduled euthanasia. Samples are inventory managed and shipped on dry ice to the study physician in the laboratory at the earliest opportunity. Samples are stored frozen (-60°C or colder) until shipment. Data is recorded.

[0188] Fecal CPV-2 Hemagglutination Assay (HA) Test Bulk fecal samples are collected from each dog from day 0 until the end of the study. Bulk feces are collected from each dog into 50 mL tubes, individually frozen, used to verify CPV SNAP test results, and stored for fecal HA testing to meet one of four CPV-2b infection criteria. Fecal samples are homogenized prior to HA testing to maximize result accuracy and sample size. Procedures for fecal collection, storage, and transport follow the instructions of the study physician in the laboratory. Data is recorded.

[0189] CPV-2b Infection Criteria The criteria for CPV infection are in accordance with 9 CFR Section 113.317, (c), (3), (i). This regulation defines four criteria for active CPV infection. A valid CPV-2b exposure should cause three of the four criteria, or result in death in at least 4 out of 7 CP dogs. According to the definition in 9 CFR Section 113.317, the four criteria for parvovirus are as follows: · Body temperature ≥ 103.4°F · Lymphopenia ≥ 50% of pre-exposure normal values · Diarrhea, mucus, and / or blood in feces · Viral hemagglutinin level ≥ 1:64 in a 1:5 dilution of feces (or a test of equivalent sensitivity) These four criteria are monitored daily in all dogs via GHO every 12 hours, including rectal temperature (RT), hematological examinations at specified intervals to monitor lymphocyte counts, and daily CPV SNAP tests verified via fecal HA. After exposure, any dog in a group that dies due to CPV infection is considered to have died from CPV infection. Dogs that are considered moribund and must be euthanized under the rescue provisions are also considered to have died from CPV infection.

[0190] Clinical Pathology (Hematology and Clinical Biochemistry) - On day 7, baseline complete blood count (CBC) and biochemical aspects are evaluated to confirm that the dog is healthy and has no existing medical conditions. - On days 1, 0, 3, 4, 5, 6, 7, 8, 10, and 13, blood samples are taken for CBC to monitor lymphocyte counts. Samples should be collected and recorded at a certain time every morning. Approximately 0.5 mL of blood is collected for hematological examinations, and for clinical biochemical examinations, approximately 1 mL of blood is collected via any accessible vein (e.g., jugular vein, cephalic vein, or saphenous vein). All samples are analyzed to evaluate CBC data in real-time and determine whether lymphopenia, one of the four clinical signs consistent with active CPV based on 9 CFR 113.317, has occurred.

[0191] [Efficacy Outcome]

[0192] The primary outcome, the primary efficacy variable, is death induced by CPV. Mortality rates for the Mab A v2 and CP groups are calculated. Using the Cochran-Mantel-Haenszel method (SAS, SAS Institute, Cary NC, FREQ procedure version 9.4 and later), with room as the stratifying variable, the prevention fraction (PF) and the lower limit of the 95% confidence interval are calculated to compare the Mab A v2 group to the CP group. If the PF cannot be generated for the data (e.g., 100% Mab A v2 survival) and the results are homogeneous across rooms, the same analysis is done without using room as the stratifying variable. If the lower limit of the 95% confidence interval of the PF is >0 and the mortality rate in the CP group is higher than that in the Mab A v2 group, the primary efficacy endpoint is met. Mortality in Mab A v2 / CP dogs is defined as death due to CPV infection or as dogs that are moribund and must be euthanized according to the rescue clause. In the event a dog is not determined positive in the CPV SNAP test and thus not administered Mab A v2 / CP, that dog is excluded from the efficacy analysis.

[0193] Rescue clause If an individual dog is deemed moribund by the veterinarian at the test facility, they can exclude that dog from the study and choose euthanasia according to the rescue clause. Dogs excluded from the study for humane reasons are included in the efficacy assessment as "mortality" unless the reason for euthanasia is not related to CPV.

[0194] The schedule of events is provided in Table 15 below.

[0195] JPEG2025102776000027.jpg255164

[0196] Next is the canine parvovirus exposure vaccination procedure: 1. Remove food approximately 12 hours before the exposure material is administered. Water remains available. 2. Remove the exposure material from the ultra-low temperature (-80°C) freezer and thaw at room temperature approximately 1 hour before use. 3. To ensure a backup dose in case of handling errors, thaw five extra aliquots. 4. Keep the exposed material on wet ice until use. 5. Prepare 1 mL of the exposed material aliquots at a time with a syringe dose. 6. Administer 0.5 mL to each nostril using a syringe with a nasal cannula (without a needle). 7. Restraint is by hand; no sedative is required. 8. The handler holds the dog's head up with the nose slightly raised during dosing. 9. Leave a few seconds between each nostril until the dog swallows and is comfortable. 10. Anticipate that some of the exposed material may be discharged from the nostrils. 11. Once dosing is complete, record the dog ID, date, and time in the animal exposure record. 12. Return food to all dogs immediately after dosing. 13. Return the unused exposed material to an -80°C freezer and label it with "Retained Aliquot, Date". 14. Retain the aliquot until the end of the study and return it to the study physician in the laboratory.

[0197] Next are the bulk fecal collection procedures: 1. Select the most abnormal individual fecal pile (or piece of pile). 2. Use a separate wooden tongue depressor to collect up to the maximum number of grams of feces from each individual dog - collect once per day per dog. Diarrheal feces may need to be collected using a syringe. 3. Place the feces in a 50 mL conical plastic centrifuge tube. The tube should be less than half full. 4. Label with the dog ID and date. 5. Group by date and treatment group and store in a plastic freezer bag. 6. Freeze at -80°C until shipped to the study physician in the laboratory. 7. At the end of the collection period, all collected feces are transported with dry ice and delivered to the study physician in the laboratory over night.

[0198] Example 12 Study of Central Efficacy of Prevention The objective of this pivotal study is to evaluate the efficacy of Mab A v2 as a prophylactic treatment for preventing clinical signs of canine parvovirus (CPV) infection.

[0199] Successful completion of this study and demonstration of efficacy could support the claim on the label that "this preparation has been shown to be effective for passive immunization of healthy dogs 13 weeks of age and older against canine parvovirus (CPV) disease."

[0200] This GCP, randomized, blinded, placebo-controlled trial is 14 days in length following at least a 7-day acclimation period. A total of 25 naive, CPV-2 seronegative (CPV hemagglutination inhibition (HI) titer <20) dogs are randomized into two groups (n = 20 dogs in group 1 and n = 5 dogs in group 2) as shown in Table 16. Dogs are less than 13 weeks of age on day 0, of either sex, weigh at least 1.5 kg upon arrival, are in good health, and have no clinical signs of illness at the start of the study.

[0201] During acclimation, dogs are acclimated to feeding, housing, and handling procedures while being administered anthelmintics and antibiotics to eliminate common worm and coccidia infections.

[0202] On day 0, all dogs are administered the assigned Mab A v2 (group 1) or control preparation [CP; group 2; phosphate buffered saline (PBS)] by subcutaneous (SC) injection. Mab A v2 is administered as a single dose of 5 mg / kg based on body weight. The pH of CP is 7.2 ± 0.2 and it is administered in an equal volume to group 1. The doses of Mab A v2 and CP are calculated as follows: {(body weight in kg) × (5 mg / kg)} ÷ {25 mg / mL} = volume in mL to be injected

[0203] Local and systemic safety data are collected from day 0 (post-treatment) through day 2. On day 3, all dogs from both groups 1 and 2 are administered pathogenic CPV-2b intranasally (IN) (a volume equal to approximately 0.5 mL per nostril) at a dose of about 1×10 6 TCID 50 / mL.

[0204] At the scheduled times outlined in Table 17, the health status of individual dogs is closely monitored for 14 days by physical examination (PE), measurement of rectal temperature (RT), cage-side CPV-2 ELISA IDEXX SNAP test (CPV SNAP test), serum hemagglutination inhibition assay (HI), fecal hemagglutination assay (HA), and hematology tests.

[0205] The primary efficacy variable is the prevention of CPV infection in dogs in group 1 as defined by the criteria of 9 CFR section 113.317, (c), (3), (i): · Body temperature ≧103.4°F · Lymphopenia of 50% or more of normal values prior to CPV-2b exposure · Diarrhea, mucus, and / or blood in the feces · Viral hemagglutinin at a level of 1:64 or greater in a 1:5 dilution of feces (or a test of equivalent sensitivity)

[0206] Efficacy is demonstrated if at least 80% of the dogs in group 2 (CP) have at least 3 of the 4 criteria outlined in 9 CFR section 113.317, (c), (3), (i), and at least 19 dogs treated with Mab A v2 remain alive and exhibit ≤1 of the 4 possible clinical signs.

[0207] Refer to the treatment groups in Table 16 below.

[0208] JPEG2025102776000028.jpg30170

[0209] Randomization In this study, two-stage randomization may be required. The first stage is to determine which 25 dogs are enrolled in the study. Next, the dogs are randomized with a block size of 5 at a ratio of 4:1 for groups 1 and 2, respectively. Randomization is based on random numbers generated by the PLAN procedure in SAS (version 9.4 or later, SAS Institute, Cary NC) using the randomization selection method with a seed number. Upon arrival at the test facility, the dogs are placed in two-room cages in the order of arrival, and dogs from the same litter / block are kept in the same room if possible. Dogs may be secondarily exposed to the virus excreted from infected dogs. To maintain a similar level of secondary virus exposure in both treatment groups and to maintain blinding for the test facility personnel, both treatment groups are included in each room. After selecting 25 dogs, their cages are numbered sequentially from 1 to 25. Dogs from the same litter have adjacent cage numbers, and 5 litters take early numbers (e.g., 1-5, 6-10, etc.) for each room. One room should contain cage numbers 1-10, and the other room should contain cage numbers 11-25. The dogs are enrolled in the order of the cage numbers on the randomization list.

[0210] Study Blinding The principal investigator of the clinical trial and all other test facility personnel (except for the dispenser and one designated to restrain the dog for dosing) do not have knowledge of the actual treatment group assignment on day 0. Only the dispenser and the designated personnel for restraining the dog during dosing have access to the actual treatment group assignment (Mab A v2 or CP) of each dog. The dispenser and the dog restrainer / holder do not perform any study evaluations.

[0211] At least 27 unvaccinated, CPV-2 seronegative (HI < 20), healthy beagle dogs, reared with the aim of the total specifications, are procured. Upon arrival at the test facility, the dogs are confirmed to be CPV negative by CPV SNAP test. Although 25 dogs are included in the study registration, 2 more dogs are purchased to ensure that the number of dogs is met after screening and acclimatization. Ideally, 27 dogs are composed of 5 litters of 5 puppies each and 2 dogs from another litter (partial litter).

[0212] [Observation]

[0213] Physical examination (PE) Physical examination is performed on all dogs by a veterinarian at the test facility on days -7, 0, 3, 13, and on unscheduled days as necessary. Physical examination includes a comprehensive evaluation of all body tissues, including hydration status and BW.

[0214] Food consumption A qualitative evaluation of food consumption is performed once a day in conjunction with general health observation (GHO). All abnormalities in the observed food consumption are recorded.

[0215] Injection site observation (ISO) ISO is performed once a day on days 1 and 2, 4 hours ± 1 hour after administration of Mab A v2 / CP for clinical signs of local inflammation (e.g., erythema, heat, swelling). The reaction size is recorded. All abnormalities observed are recorded.

[0216] General health observation (GHO) and measurement of rectal temperature (RT) GHO is performed once a day by a veterinarian at the test facility or their designee during the acclimatization period from day -7 to day -1. GHO is performed every 12 hours (± 1 hour) or unscheduled from day 0 to day 12. Starting on day 0, in combination with each GHO, rectal temperature (RT) is also measured using a calibrated thermometer on the Fahrenheit scale. General health observation includes, but is not limited to, observation of general physical appearance, abnormalities in food or water consumption, and / or occurrence of vomiting or diarrhea. The status of all dogs, including those without abnormal signs, is documented.

[0217] Cage-side CPV ELISA (CPV SNAP test) The cage-side CPV ELISA test is performed using the IDEXX CPV SNAP test according to the manufacturer's instructions. Upon arrival from the vendor on day -7, a preliminary CPV SNAP test is performed on all dogs to confirm the absence of active CPV infection in the dogs. Subsequent CPV SNAP tests are evaluated at a consistent time in the morning on days 0 and 3 (before inoculation with pathogenic CPV-2b), and then once daily until the end of the test. CPV SNAP test data are recorded.

[0218] Serum CPV-2 hemagglutination (HI) test Whole blood samples are collected from all dogs into serum separator tubes (approx. 1.5 mL) via the jugular, cephalic, or lateral saphenous veins, processed into serum at a consistent time in the morning on days -7, 0, 2, 3, 4, 6, 8, 10, and 13, and the samples on day 3 are collected before inoculation with pathogenic CPV-2b. Samples are also collected prior to unscheduled euthanasia. Samples are inventory managed and shipped to the study physician in the laboratory on dry ice at the earliest opportunity. Samples are stored frozen (-60°C or below) until shipment. Data are recorded.

[0219] Fecal CPV-2 hemagglutination assay (HA) test Bulk fecal samples are collected from each dog at a consistent time in the morning on days 0 and 3 until the end of the study. Bulk feces are collected into 50 mL tubes from each dog, frozen individually, stored for the fecal HA test to verify CPV SNAP test results and to meet one of four CPV-2b infection criteria. Fecal samples are homogenized prior to the HA test to maximize result accuracy and sample size. Procedures for fecal collection, storage, and transport follow the instructions of the study physician in the laboratory. Data are recorded.

[0220] CPV-2b Infection Criteria The criteria for CPV infection comply with 9 CFR Section 113.317, (c), (3), (i). This regulation defines four criteria for active CPV infection. An effective CPV-2b exposure must meet three of the four criteria in at least 80% of the exposed control dogs. According to the definition in 9 CFR Section 113.317, the four criteria for parvovirus are as follows: · Body temperature ≥ 103.4°F · Lymphopenia of more than 50% of the pre-exposure normal value · Diarrhea, mucus and / or blood in feces · Viral hemagglutinin at a level of 1:64 or higher in a 1:5 dilution of feces (or a test of equivalent sensitivity)

[0221] These four criteria are monitored daily in all dogs via GHO every 12 hours, including rectal temperature (RT), hematological examinations at specified intervals to monitor lymphocyte counts, and daily CPV SNAP tests verified via fecal HA.

[0222] Clinical Pathology (Hematology and Clinical Biochemistry) - On day 7, evaluate the baseline complete blood count (CBC) and biochemical aspects to confirm that the dog is healthy and has no existing medical conditions. On days 0, 3, 5, 6, 7, 8, 9, 10, and 13, blood is collected for CBC and blood lymphocytes are monitored. Samples are collected and recorded at a fixed time every morning. Approximately 0.5 mL of blood is collected for hematological examination, and for clinical biochemical examination, approximately 1 mL of blood is collected via any accessible vein (e.g., jugular vein, cephalic vein, or lateral saphenous vein). Analyze all samples to evaluate CBC data in real time and determine whether lymphopenia, one of the four clinical signs consistent with active CPV based on 9 CFR 113.317, has occurred.

[0223] [Efficacy Outcome]

[0224] The primary outcome The primary efficacy variable is prevention of CPV infection as defined by the criteria of 9 CFR Section 113.317, (c), (3), (i): · Body temperature ≥ 103.4°F · Lymphopenia of ≥ 50% of pre - exposure normal values (average of pre - exposure days - 7, 0, and 3) before CPV - 2b exposure · Diarrhea, mucus, and / or blood in feces · Viral hemagglutinin at a level of ≥ 1:64 in a 1:5 dilution of feces (or a test of equivalent sensitivity) Efficacy is established when 80% of the control dogs have 3 out of 4 criteria outlined in 9 CFR 113.317 and at least 19 Mab A v2 - treated dogs survive and exhibit ≤ 1 of 4 possible clinical signs.

[0225] Secondary outcome Secondary outcomes such as CPV SNAP test results, serum HI titers, or other parameters may be evaluated.

[0226] Safety outcome The dogs in this pivotal study are applicable to the required number of dogs treated in the Mab A v2 pivotal field safety study. Safety assessment is done by collation of all local and systemic adverse events (AEs) from day 0 (post - treatment) through day 3. Specifically, dogs are monitored for injection - site reactions such as erythema, heat, and swelling (by daily ISO) and systemic reactions including anaphylaxis after administration of Mab A v2. AE data are not statistically analyzed but are summarized and tabulated for the final report.

[0227] Serious AE A serious AE is any AE that results in death, is life - threatening, causes persistent or significant disability / incapacity, or causes congenital anomaly or congenital defect. The principal investigator of the clinical trial notifies the clinical development manager within 24 hours of the occurrence of a serious AE.

[0228] The schedule of events is provided in Table 17 below.

[0229] JPEG2025102776000029.jpg255162JPEG2025102776000030.jpg255113

[0230] Next is the canine parvovirus exposure vaccination procedure: 1. Remove food approximately 12 hours before the exposure material is administered. Water can still be used. 2. Remove the exposure material from the ultra-low temperature (-80°C) freezer and thaw it at room temperature about 1 hour before use. 3. Thaw five extra aliquots to ensure a backup dose in case of handling errors. 4. Keep the exposure material on wet ice until use. 5. Prepare 1 mL of the aliquot exposure material at a time with a syringe per dose. 6. Using a syringe with a nasal cannula (without a needle), administer 0.5 mL to each nostril. 7. Restraint is by hand; no sedatives are required. 8. The handler holds the dog's head up with the dog's nose slightly raised during dosing. 9. Leave a few seconds between each nostril until the dog swallows and is comfortable. 10. Anticipate that some exposure material may be discharged from the nostrils. 11. Once dosing is complete, record the dog ID, date, and time in the animal exposure record. 12. Return food to all dogs immediately after dosing. 13. Return the unused exposure material to the -80°C freezer and label it "Retained aliquot, date". 14. Retain the aliquot until the end of the study and return it to the study physician in the laboratory.

[0231] Next is the bulk feces collection procedure: 1. Select the most abnormal-appearing individual fecal pile (or piece of a pile). 2. Using a separate wooden tongue depressor, collect up to the maximum number of grams of feces from each individual dog - collect once per day per dog. Diarrheic feces may need to be collected using a syringe. 3. Place the feces in a 50 mL conical plastic centrifuge tube. The tube should be less than half full. 4. Label with dog ID and date. 5. Group by date and treatment group and store in plastic freezer bags. 6. Freeze at -80 °C until shipped to the study physician in the laboratory. 7. At the end of the collection period, transport all collected feces on dry ice and have it reach the study physician in the laboratory overnight.

[0232] Example 13 Decrease in half-life and lack of antibody interference This in vivo study monitors the in vivo degradation of Mab A v2 and evaluates the duration for which the hemagglutination inhibition (HI) titer levels remain sufficient to neutralize the vaccine strain of CPV. The following objectives are addressed: · Characterize the decrease in the half-life of Mab A v2 after subcutaneous (SC) administration · Determine the time frame after administration when Mab A v2 ceases to inhibit active immunity against parvovirus (lack of antibody interference, or LOAI)

[0233] Serological monitoring of the HI titer after administration of Mab A v2 can characterize the degradation half-life. LOAI is determined by the active immune response against parvovirus after vaccination in at least 80% of the dogs within each group. The active response is defined as a four-fold increase in titer (seroconversion) above baseline with an antibody titer of 1:80 dilution or greater as determined by HI. This level of antibody response provides immunity against canine parvovirus as shown by the challenge in the immunological study (Pollock and Carmichael, JAVMA 1982 Jan 1;180(1):37-42).

[0234] Second, safety data is collected after treatment and prior to CPV immunization and can be used to contribute to the total number of dogs required to meet the minimum requirement of 300 dogs by USDA-CVB to satisfy Veterinary Services Memorandum (VSM) No. 800.204.

[0235] Satisfactory completion of this study and demonstration of when LOAI for CPV immunization occurs can support the two label claims stating: · “This formulation has been shown to passively immunize healthy dogs 13 weeks of age and older for the prevention of canine parvovirus (CPV) disease.” · “This formulation has been shown to be effective in the treatment of canine parvovirus (CPV) disease in dogs 13 weeks of age and older.”

[0236] In Example 9 above, which described the previous study KB-030-PK-301, Mab A v2 neutralized vaccine CPV that closely resembled maternally derived antibodies. The current study can provide information regarding the timing of CPV vaccination after administration of Mab A v2 as a prophylactic treatment.

[0237] This is a randomized, unblinded, open-label GCP study conducted in healthy CPV seronegative beagle dogs. The length of the study is up to 113 days with a 7-day acclimation period. During acclimation, dogs are acclimated to feeding, housing, and handling procedures; and are administered anthelmintics and antibiotics to rule out common worm and coccidia infections.

[0238] On Day 0, 5 mg / kg of Mab A v2 is administered SC to the interscapular region of Groups 1 and 2. Groups 3 and 4 receive no Mab A v2. From Day 0 to Day 41, General Health Observations (GHO), Physical Examinations (PE), and Injection Site Observations (ISO) are collected at various time points and dogs are monitored for local and systemic reactions to Mab A v2.

[0239] On day 42, one of the pre - assigned monovalent CPV vaccines is administered to groups 1 and 3, and another pre - assigned monovalent CPV vaccine is administered to groups 2 and 4. After collecting serum for HI titers, on day 42, one of the pre - assigned monovalent CPV vaccines (either Vaccine 1 or Vaccine 2) is administered to each dog. If seroconversion of the vaccine has not been demonstrated after the previous vaccination, a booster vaccination ("second CPV vaccine") is given on day 63, three weeks after the first vaccination. If seroconversion of the vaccine has not been demonstrated after the second CPV vaccine, a third booster of the pre - assigned monovalent CPV vaccine ("third booster") is administered on day 84. The end of the study for an individual dog occurs when an HI result demonstrating seroconversion is received. If some dogs did not initiate an immune response to the vaccination after the HI titers on day 91, a fourth and final monovalent CPV booster ("fourth booster") is administered on day 105. Serum hemagglutination inhibition (HI) titers are measured at various time points to monitor the degradation of Mab A v2 and when a LOAI to the CPV vaccination occurs.

[0240] From day 43 until the end of the study, GHO and PE are collected at various time points to monitor the general health status of the dogs.

[0241] The two monovalent CPV vaccines pre - assigned and administered in the study are as follows: [Vaccine 1] Registered name: Nobivac® Canine 1 - PV Vaccine: Canine parvovirus (modified live) Dosage form: Sterile injection Test dose: 1 mL (1 dose) Route of administration: Subcutaneous Manufacturer: Merck Animal Health Packaging: Individual vials per manufacturer's package Storage conditions: 2 - 7 °C Available units: 1 vial [Vaccine 2] Registered Name: Recombitek (Registered Trademark) Vaccine: Canine Parvovirus (Modified Live) Dosage Form: Sterile Injection Test Dosage: 1 mL (1 dose) Route of Administration: Subcutaneous Manufacturer: Merial Packaging: Individual vials per manufacturer's package Storage Conditions: 2 - 7 °C Available Unit: 1 vial

[0242] Refer to Treatment Group in Table 18 below.

[0243] JPEG2025102776000031.jpg54170

[0244] Randomization Dogs (13 weeks of age or younger, any gender, body weight 1.5 kg or more on Day 0) are randomized to Groups 1 to 4 at a ratio of 2:2:1:1 on Day 0 or earlier using a randomization list provided by the sponsor's research programmer. Treatments are randomly assigned within each block of 6 dogs. Randomization is based on random numbers generated by the PLAN procedure of SAS (SAS Institute, Cary NC, version 9.4 or later) using a randomization selection method with a seed number.

[0245] [Observation]

[0246] Physical Examination (PE) PE is performed on all days including -7 days, 0 days, the end of the study, and on unscheduled days as necessary. Physical examination includes a comprehensive evaluation of all body tissues including hydration status and body weight (BW).

[0247] Injection Site Observation (ISO) - On Day 3, the injection site is shaved for the preparation of the dosing on Day 0. Before dosing, the shaved injection site is evaluated and recorded as being free of inflammation or shaving wounds. Starting from Day 0, the injection site is evaluated once a day until Day 14 for local injection site reactions including, but not limited to, erythema, heat, and swelling (e.g., nodules). The size of the reaction is recorded. The observed injection site reactions are documented on the injection site observation form and recorded as adverse events (AE).

[0248] General Health Observation (GHO) GHO is performed once a day by the study physician or designee from Day -7 until the end of the study. General health observation includes, but is not limited to, observation of general physical appearance, abnormalities in food or water consumption, and / or the occurrence of vomiting or diarrhea. The status of all dogs, including those without abnormal signs, is documented on the general health observation form for each dog during each observation period. At -7, Day 0, and at the end of the study when PE is performed, PE replaces GHO.

[0249] Serum CPV-2 Hemagglutination (HI) Test - Whole blood samples are collected from all dogs (approximately 1.5 mL) via the jugular vein, cephalic vein, or lateral saphenous vein on Days -7, 0, 1, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105, 112, placed in serum separator tubes, and processed into serum. If seroconversion is demonstrated by HI, subsequent scheduled sampling is aborted and the individual dog terminates the study. Samples are stored frozen (60 to -80 °C) until sent to the analytical laboratory at the earliest opportunity.

[0250] Clinical Pathology - On Day -7, baseline complete blood count (CBC) and biochemical profiles are evaluated to confirm that the dog is healthy and free of existing medical conditions. Approximately 0.5 mL of blood is collected for hematology testing and approximately 1 mL is collected from the jugular vein, cephalic vein, or lateral saphenous vein for clinical biochemistry testing.

[0251] Adverse Event (AE) An AE is defined as any finding in dogs that occurs after the use of an animal formulation, whether undesirable, unexpected, unintended, and regardless of whether it is considered related to the formulation. When an experimental treatment is initiated (day 0 when Mab A v2 is administered), abnormal GHO, PE, or ISO is considered an AE.

[0252] Primary Endpoint The primary endpoint for each group is the number of days from day 0 until more than 80% of the dogs in the group show CPV seroconversion to monovalent CPV vaccination via serum HI titer. Seroconversion is determined by serum HI titer and is defined as a four-fold increase in titer relative to baseline, accompanied by achievement of an antibody titer of 1:80 dilution or higher. The number of dogs per group that showed an immune response to the vaccine and the study day on which this occurred are not statistically analyzed but are summarized and tabulated for the final report.

[0253] Refer to the schedule of events in Table 19 below.

[0254] JPEG2025102776000032.jpg255169JPEG2025102776000033.jpg32170

[0255] Example 14 Multicenter Central Safety Study in Healthy Dogs This in vivo field safety study evaluates the local and systemic tolerability of MabA v2. Individual dogs are observed for inflammation at the injection site (including but not limited to erythema, heat, and / or swelling) and systemic reactions (e.g., anaphylaxis).

[0256] Mab A v2 is administered to at least 300 dogs by either the subcutaneous (SC) (n = 150) or intravenous (IV) (n = 150) route. The safety data collected during this study is intended to meet the requirements of the central field safety of 300 dogs at the United States Department of Agriculture's Center for Veterinary Biologics (USDA-CVB).

[0257] The satisfactory completion of this study and demonstration of safety can support the following two label claims: · "This formulation has been shown to be effective for passive immunization of healthy dogs 10 weeks of age and older for the prevention of canine parvovirus (CPV) disease." · "This formulation has been shown to be effective for the treatment of canine parvovirus (CPV) disease in dogs 10 weeks of age and older."

[0258] This is a randomized, non-blinded GCP field safety study conducted in dogs bred for young, healthy purposes. This study is 16 days in length at each study site. At least 100 minimum-age dogs (≤10 weeks of age) of either sex and at least 200 dogs older than the minimum age (>10 weeks of age) of either sex are enrolled in the study.

[0259] Exclusion Criteria - Dogs: · Currently enrolled in another study. · Enrolled in another study within the past 30 days. · Administered a chimeric biologic agent up to 90 days prior to Day - 2. · Not cooperative with study procedures. · Not reasonably expected to survive during the study period. · Have an existing dermatologic condition that could confound ISO. · Have a current medical history suggesting a significant co - morbidity (e.g., polyuria, polydipsia, polyphagia, vomiting or diarrhea, unexplained weight loss, etc.).

[0260] The minimum - age dogs are placed at one site (Ridglan Farms (Mount Horeb, WI)), and the remaining dogs are divided among three test facilities in three geographic regions. To diversify genetics as much as possible, at least three test facilities and unique breeding colonies in three separate geographic regions are used. At each site, the dogs are randomized 1:1:1:1 into Groups 1, 2, 3, and 4 according to Table 20.

[0261] On day 0, all dogs are administered Mab A v2 either by SC or IV. In this study, two different pre-license serials (PLS) of Mab A v2 are administered. Within each age group and treatment group, approximately half of the dogs are randomly administered PLS No. 1 and the other half are randomly administered PLS No. 2.

[0262] The dose volume is calculated in mL by the EDC system based on the body weight (BW) on day 0. The dose is calculated by the EDC system as follows: {(body weight in kg) × (5 mg / kg)} · {TBD mg / mL} = volume in mL to be injected

[0263] The subcutaneous dose is administered subcutaneously in the interscapular region. The intravenous administration is administered to the radial cutaneous vein.

[0264] At the scheduled times described in Table 20, the health of individual dogs is closely monitored via physical examination (PE), injection site observation (ISO), and general health observation (GHO).

[0265] The safety assessment is by collation of all adverse events (AE). Specifically, dogs are monitored for injection site reactions (via daily ISO) such as erythema, heat or swelling, and for systemic reactions including anaphylaxis, after administration of Mab A v2. The AE data are not statistically analyzed but are summarized and presented in a table.

[0266] Refer to the treatment groups in Table 20 below.

[0267] JPEG2025102776000034.jpg61170

[0268] Since it is difficult to find test facilities that are raising dogs of the appropriate age randomly, approximately 100 dogs of the lowest age are procured from one location. The remaining approximately 200 dogs are divided among test facilities in at least three geographical regions and divided as evenly as possible among the test facilities. Randomization is stratified into blocks of 4 dogs by test facility site. Within each site, the dogs are randomized in a 1:1:1:1 ratio for groups 1 (PLS #1, SC), 2 (PLS #2, SC), 3 (PLS #1, IV), and group 4 (PLS #2, IV). Randomization is based on random numbers generated by the SAS PLAN procedure (SAS Institute, Cary NC, version 9.4 and later) using the randomization selection method with seed numbers.

[0269] Acclimation This study is conducted at test facilities where the dogs have been previously acclimated to diet, housing, handling procedures, etc. Therefore, a 2-day acclimation period is implemented to confirm that the dogs meet the registration criteria and to shave the injection site on day -2.

[0270] Physical Examination (PE) PE is performed by on-site veterinarians on day -2, day 0 (before dosing), day 7, and day 14. PE includes subjective evaluations of the general appearance and attitude, ears, eyes, oral cavity, mucous membranes, respiratory, cardiovascular, gastrointestinal, neurological, musculoskeletal, integumentary, and urogenital systems. Specific PE abnormalities are described using VeDDRA's low-level terms. Abnormalities recorded after day 0 (excluding pre-existing conditions and those that deteriorate) are considered AEs.

[0271] Injection Site Observation (ISO) On day -2, the injection site is shaved in preparation for dosing on day 0. Before the procedure, the shaved injection site is evaluated and recorded as having no inflammation or clipper injury. Starting on day 0 and up to day 14, the position of the injection site is evaluated daily for local injection site reactions including, but not limited to, erythema, heat, swelling (e.g., lump). The size of the reaction is recorded. Any observed injection site reaction is recorded as an AE.

[0272] General Health Observation (GHO) GHO is conducted once daily from the start of the study until the end in Hue, with one exception. On days -2, 0, 7, and 14 when PE is performed, PE replaces GHO. General health observation includes, but is not limited to, observation of general physical appearance, abnormalities in food or water consumption, and / or occurrence of vomiting or diarrhea. Specific observation results are described using low-level terms of VeDDRA. The status of all dogs, including those without abnormal signs, is documented.

[0273] Body weight On day 0, the BW (recorded in kilograms (kg)) is measured and recorded using a calibrated weighing scale. The dog can be weighed after being fed or fasted. The body weight is documented.

[0274] Safety analysis population The safety analysis population consists of all dogs administered Mab A v2.

[0275] Safety outcome Safety assessment is performed by collating all AEs. Specifically, dogs are monitored for injection site reactions such as erythema, heat, or swelling (by daily ISO) and systemic reactions including anaphylaxis after administration of Mab A v2. AE data are not statistically analyzed but are summarized and presented in a table.

[0276] Adverse event (AE) An AE is defined as any finding in a dog that occurs after the use of an animal formulation, whether undesirable, unexpected, unintended, and regardless of whether it is considered related to the formulation. All AEs are documented and recorded using low-level VEDDRA terms.

[0277] Serious AE A fatal or life-threatening AE, or an AE that requires medical intervention (e.g., such that the study physician determines that the dog's health condition warrants euthanasia or medical treatment).

[0278] Non-serious AE An AE that is not severe enough to require medical intervention or exclusion of the dog from the study.

[0279] Refer to the schedule of the events in Table 21 below.

[0280] JPEG2025102776000035.jpg86170

[0281] Example 15 Identification of Variant Canine IgG-B Fc Polypeptides for Enhanced ADCC Activity Structural models of canine CD16 and canine IgG-B Fc were prepared using the structural coordinates of the human IgG1 Fc / CD16 complex (1e4k1.pdb) as a template. See Sondermann P., “The 3.2-A Crystal Structure of the Human IgG1 Fc Fragment-Fc gammaRIII complex,” Nature 406(6793):267-73 (2000). Amino acid residues that are likely to interact between wild-type canine IgG-B Fc (SEQ ID NO: 91) and canine CD16 (SEQ ID NO: 90) were identified. Both Ser10 and Ile103 of canine IgG-B Fc SEQ ID NO: 91 are close to Lys148 of canine CD16 SEQ ID NO: 90. To obtain electrostatic interactions, Ser10 and / or Ile103 of canine IgG-B Fc SEQ ID NO: 90 can be substituted with the acidic amino acids Asp or Glu to introduce a salt bridge to K148 of canine CD16 SEQ ID NO: 90. The distances between Ser10Asp and Lys148; Ser10Glu and Lys148; Ile103Asp and Lys148; and Ile103Glu and Lys148 are each less than 4 Å (Figure 5). Exemplary variant canine IgG-B Fc polypeptide sequences for enhanced ADCC activity include SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99. Such variant canine IgG-B Fc polypeptides can be used in place of wild-type canine IgG-B Fc to enhance the killing of parvovirus-infected cells.

[0282] References: 1. S. Nandi, Manoj Kumar. Canine Parvovirus: Current Perspective. Indian J. Virol. 2010; 21(1):31-44。

[0283] 2. Carla Miranda, Gertrude Thompson. Canine parvovirus: the worldwide occurrence of antigenic variants. Journal of General Virology. 2016; 97, 2043-2057。

[0284] 3. Melissa Kennedy, Adesola Odunayo. Canine Parvovirus. Clinician’s Brief. 2017。

[0285] 4. Brindhalakshmi B, Mukhopadhyay HK, Antony PX, Thanislass J, Vijayalakshmi P, Mangadevi N. Isolation and molecular characterization of canine and feline parvovirus strains - an updated review. Journal of Dairy, Veterinary & Animal Research. 2016; 3(5):164-169。

[0286] 5. Emilee Venn, Karolina Preisner, Pedro Boscan, David Twedt, Lauren A. Sullivan. Evaluation of an outpatient protocol in the treatment of canine parvoviral enteritis. Journal of Veterinary Emergency and Critical Care. 2017; 27(1):52-65。

Claims

1. An isolated antibody that binds to canine parvovirus and / or feline parvovirus, wherein (a) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, and (iv) an HC-FR1 sequence of SEQ ID NO: 7 or SEQ ID NO: 8; or (b) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 42, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43, (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 44, and (iv) an HC-FR1 sequence of SEQ ID NO: 45 or SEQ ID NO: 46 is included.

2. An isolated antibody that binds to canine parvovirus and / or feline parvovirus, wherein (a) (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and (iv) an LC-FR1 sequence of SEQ ID NO: 16; or (b) (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54, and (iv) an LC-FR1 sequence of SEQ ID NO: 55 or SEQ ID NO: 56 is included.

3. The isolated antibody according to claim 1, wherein the antibody of (a) comprises an HC-FR4 sequence of SEQ ID NO:

12.

4. The isolated antibody according to claim 1, wherein the antibody of (b) comprises an HC-FR2 sequence of SEQ ID NO:

48.

5. The isolated antibody according to claim 1 or claim 4, wherein the antibody of (b) comprises an HC-FR3 sequence of SEQ ID NO:

50.

6. The isolated antibody according to claim 2, wherein the antibody of (b) comprises an HC-FR3 sequence of SEQ ID NO:

59.

7. The isolated antibody according to claim 2 or claim 6, wherein the antibody of (b) comprises an HC-F4 sequence of SEQ ID NO:

61.

8. An isolated antibody that binds to canine parvovirus and / or feline parvovirus, wherein a) a heavy chain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, or b) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 42; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 44, a heavy chain An antibody, which is a caninized or felinized antibody comprising the same. **Claim 9** An isolated antibody that binds to canine parvovirus and / or feline parvovirus, a) (i) A light chain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, or b) (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54, a light chain An antibody, which is a caninized or felinized antibody comprising the same. **Claim 10** An isolated antibody that binds to canine parvovirus and / or feline parvovirus, a) (i) A heavy chain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, and b) (i) A light chain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15 An antibody, which is a caninized or felinized antibody comprising the same. **Claim 11** An isolated antibody that binds to canine parvovirus and / or feline parvovirus, a) (i) A heavy chain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 42; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 44, and b) (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54 An antibody, which is a caninized or felinized antibody comprising the same. **Claim 12** The antibody according to any one of claims 1 to 11, wherein the antibody is a chimeric antibody. **Claim 13** The antibody according to any one of claims 1 to 12, wherein the antibody comprises a canine or feline constant heavy chain region or a canine or feline constant light chain region. **Claim 14** The antibody is (a) a canine heavy chain constant region selected from IgG-A, IgG-B, IgG-C, and IgG-D constant regions; or (b) a feline heavy chain constant region selected from IgG1, IgG2a, and IgG2b constant regions The antibody according to any one of claims 1 to 13, comprising [

15. ] The antibody according to any one of claims 1 to 14, wherein the antibody comprises a wild-type or variant IgG Fc having complement binding activity. [

16. ] The antibody according to any one of claims 1 to 15, wherein the antibody comprises a wild-type or variant IgG Fc having antibody-dependent cell-mediated cytotoxicity (ADCC) activity. [

17. ] The antibody according to any one of claims 1 to 16, wherein the antibody comprises a wild-type or variant IgG Fc having antibody-dependent cell phagocytosis (ADCP) activity. [

18. ] The antibody is a) aspartic acid or glutamic acid at a position corresponding to position 10 of SEQ ID NO: 91; b) aspartic acid or glutamic acid at position 10 of SEQ ID NO: 91; c) aspartic acid or glutamic acid at a position corresponding to position 103 of SEQ ID NO: 91; d) aspartic acid or glutamic acid at position 103 of SEQ ID NO: 91; e) aspartic acid or glutamic acid at a position corresponding to position 10 and / or 103 of SEQ ID NO: 91; f) aspartic acid or glutamic acid at position 10 and / or 103 of SEQ ID NO: 91; or g) the amino acid sequence of SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, or SEQ ID NO: 99 The antibody according to any one of claims 1 to 17, comprising [

19. ] The antibody according to any one of claims 1 to 18, wherein the antibody comprises a canine κ light chain constant region or a feline κ light chain constant region. [

20. ] The antibody according to any one of claims 1 to 19, wherein the antibody comprises a feline κ light chain constant region having no one or more N-glycosylation sites. [

21. ] The antibody according to any one of claims 1 to 20, wherein the antibody binds to an epitope comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO:

3. [

22. ] The antibody binds to canine parvovirus and / or feline parvovirus with a dissociation constant (Kd) of less than 5×10 －６ M, less than 1×10 －６ M, less than 5×10 －７ M, less than 1×10 －７ M, less than 5×10 －８ M, less than 1×10 －８ M, less than 5×10 －９ M, less than 1×10 －９ M, less than 5×10 －１０ M, less than 1×10 －１０ M, less than 5×10 －１１ M, less than 1×10 －１１ M, less than 5×10 －１２ M, less than 1×10 －１２ M, less than 5×10 －１３ M, or less than 1×10 －１３ M, and is an antibody according to any one of claims 1 to 21. [

23. ] The antibody according to any one of claims 1 to 22, wherein the antibody binds to canine parvovirus or feline parvovirus as determined by immunoblot analysis and / or biolayer interferometry. [

24. ] The antibody according to any one of claims 1 to 23, wherein the antibody at a concentration of 200 μg / mL has a hemagglutination inhibition value of at least 8000, at least 16000, or at least 32000.

25. The antibody according to any one of claims 1 to 24, wherein the antibody is a monoclonal antibody.

26. The antibody according to any one of claims 1 to 25, comprising one or more of: (a) the (HC-FR1) sequence of SEQ ID NO: 7, 8, 45, or 46; (b) the HC-FR2 sequence of SEQ ID NO: 9, 47, or 48; (c) the HC-FR3 sequence of SEQ ID NO: 10, 49, or 50; (d) the HC-FR4 sequence of SEQ ID NO: 11, 12, or 51; (e) the variable region light chain framework 1 (LC-FR1) sequence of SEQ ID NO: 16, 55, or 56; (f) the LC-FR2 sequence of SEQ ID NO: 17 or 57; (g) the LC-FR3 sequence of SEQ ID NO: 18, 58, or 59; or (h) the LC-FR4 sequence of SEQ ID NO: 19, 60, or 61.

27. The antibody, comprises: (a) the variable heavy chain sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 40, SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO: 88; and / or (b) the variable light chain sequence of SEQ ID NO: 22, SEQ ID NO: 87, SEQ ID NO: 41, SEQ ID NO: 64, SEQ ID NO: 65, or SEQ ID NO: 89 according to any one of claims 1 to 26.

28. The antibody, comprises: (a) the variable heavy chain sequence of SEQ ID NO: 20 and the variable light chain sequence of SEQ ID NO: 22; (b) the variable heavy chain sequence of SEQ ID NO: 21 and the variable light chain sequence of SEQ ID NO: 22; (c) the variable heavy chain sequence of SEQ ID NO: 85 and the variable light chain sequence of SEQ ID NO: 87; (d) the variable heavy chain sequence of SEQ ID NO: 86 and the variable light chain sequence of SEQ ID NO: 87; (e) the variable heavy chain sequence of SEQ ID NO: 40 and the variable light chain sequence of SEQ ID NO: 41; (f) the variable heavy chain sequence of SEQ ID NO: 62 and the variable light chain sequence of SEQ ID NO: 64 or SEQ ID NO: 65; (g) the variable heavy chain sequence of SEQ ID NO: 63 and the variable light chain sequence of SEQ ID NO: 64 or SEQ ID NO: 65; or (h) the variable heavy chain sequence of SEQ ID NO: 88 and the variable light chain sequence of SEQ ID NO: 89 according to any one of claims 1 to 27.

29. The antibody, (a)(i) The heavy chain sequence of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 79; and / or (ii) the light chain sequence of SEQ ID NO: 25, SEQ ID NO: 39, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 80; or (b)(i) The heavy chain sequence of SEQ ID NO: 31, SEQ ID NO: 40, or SEQ ID NO: 74; and / or (ii) the light chain sequence of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 41, SEQ ID NO: 75, or SEQ ID NO: 76 The antibody according to any one of claims 1 to 28, comprising the same.

30. The antibody is (a) The heavy chain sequence of SEQ ID NO: 23 or SEQ ID NO: 24 and the light chain sequence of SEQ ID NO: 25; (b) The heavy chain sequence of SEQ ID NO: 31 and the light chain sequence of SEQ ID NO: 32 or SEQ ID NO: 33; (c) The heavy chain sequence of SEQ ID NO: 37 or SEQ ID NO: 38 and the light chain sequence of SEQ ID NO: 39; (d) The heavy chain sequence of SEQ ID NO: 66 or SEQ ID NO: 67 and the light chain sequence of SEQ ID NO: 68 or SEQ ID NO: 69; (e) The heavy chain sequence of SEQ ID NO: 66 and the light chain sequence of SEQ ID NO: 68; (f) The heavy chain sequence of SEQ ID NO: 67 and the light chain sequence of SEQ ID NO: 69; (g) The heavy chain sequence of SEQ ID NO: 74 and the light chain sequence of SEQ ID NO: 75 or SEQ ID NO: 76; or (h) The heavy chain sequence of SEQ ID NO: 79 and the light chain sequence of SEQ ID NO: 80 The antibody according to any one of claims 1 to 29, comprising the same.

31. The antibody according to any one of claims 1 to 30, wherein the antibody comprises the heavy chain sequence of SEQ ID NO: 24 and the light chain sequence of SEQ ID NO:

25.

32. An isolated nucleic acid encoding the antibody according to any one of claims 1 to 31.

33. A host cell comprising the nucleic acid according to claim 31.

34. A method for producing an antibody, comprising culturing the host cell according to claim 32 and isolating the antibody.

35. A pharmaceutical composition comprising one or more antibodies according to any one of claims 1 to 30 and a pharmaceutically acceptable carrier.

36. The pharmaceutical composition according to claim 34, wherein the pharmaceutically acceptable carrier is phosphate buffered saline.

37. A method for providing passive immunity to a subject against infection with canine or feline panleukopenia virus, comprising administering to the subject a therapeutically effective amount of a monoclonal antibody that binds to canine or feline panleukopenia virus.

38. The method according to claim 37, wherein the monoclonal antibody is administered before exposure to canine or feline parvovirus.

39. The method according to claim 37 or claim 38, wherein the monoclonal antibody is administered after exposure to canine or feline parvovirus.

40. The method according to any one of claims 37 to 39, wherein the monoclonal antibody is administered after infection with canine or feline parvovirus.

41. The method according to any one of claims 37 to 40, wherein the monoclonal antibody is administered after the subject exhibits at least one symptom selected from fever, vomiting, diarrhea, lymphopenia, and sepsis.

42. The method according to any one of claims 37 to 41, wherein the monoclonal antibody is administered after canine or feline parvovirus is detected in feces, as determined, for example, by a positive cage-side SNAP test.

43. The method according to any one of claims 37 to 42, wherein the subject has previously been administered a parvovirus vaccine.

44. The method according to any one of claims 37 to 42, wherein the subject has not previously been administered a parvovirus vaccine.

45. The method according to any one of claims 37 to 44, wherein the subject is not protected at birth due to lack of maternally-derived antibodies against canine or feline parvovirus or failure of passive transfer of antibodies against canine or feline parvovirus.

46. The method according to any one of claims 37 to 45, wherein the subject is a kitten or puppy, the subject's mother does not produce milk, or the subject is unable to produce antibodies against parvovirus.

47. The method according to any one of claims 37 to 46, wherein the subject lives in an environment contaminated with canine or feline parvovirus.

48. A method of treating canine or feline parvovirus infection in a subject, comprising administering to the subject a therapeutically effective amount of a monoclonal antibody that binds to canine or feline parvovirus.

49. The method according to claim 48, wherein the monoclonal antibody is administered after the subject exhibits at least one symptom selected from fever, vomiting, diarrhea, lymphopenia, and sepsis.

50. The method according to claim 48 or claim 49, wherein the monoclonal antibody is administered after canine or feline parvovirus is detected in feces, as determined, for example, by a positive cage-side SNAP test.

51. The method according to any one of claims 48 to 50, wherein the subject has previously been administered a parvovirus vaccine.

52. The method according to any one of claims 48 to 50, wherein the subject has not previously been administered a parvovirus vaccine.

53. The method according to any one of claims 48 to 52, wherein the subject lives in an environment contaminated with canine or feline parvovirus.

54. The method according to any one of claims 48 to 53, wherein the subject is a dog or a cat.

55. The method according to any one of claims 48 to 53, wherein the subject is a human.

56. The method according to any one of claims 37 to 55, comprising administering to the subject a therapeutically effective amount of a monoclonal antibody that binds to an epitope comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO:

3.

57. The method comprises (a) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 42; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43; and (iii) a heavy chain comprising CDR-H3 comprising the amino acid sequence of SEQ ID NO: 44; and (b) (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53; and (iii) a light chain comprising CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54 The method according to any one of claims 37 to 56, comprising administering to the subject a therapeutically effective amount of a monoclonal antibody comprising the same.

58. The method according to any one of claims 37 to 57, comprising administering to the subject a therapeutically effective amount of the antibody according to any one of claims 1 to 31 or the pharmaceutical composition according to claim 35 or claim 36.

59. The method according to any one of claims 37 to 58, wherein the antibody or pharmaceutical composition is administered parenterally.

60. The method according to any one of claims 37 to 59, wherein the antibody or pharmaceutical composition is administered by intramuscular, intraperitoneal, intrathecal, subcutaneous, intraarterial, intrasynovial, intrathecal, intravenous, or inhalation routes.

61. The method according to any one of claims 37 to 60, wherein the antibody or pharmaceutical composition is administered intravenously.

62. The method according to any one of claims 37 to 60, wherein the antibody or pharmaceutical composition is administered subcutaneously.

63. The method according to any one of claims 37 to 62, wherein the subject is less than 1 week old, less than 2 weeks old, less than 3 weeks old, less than 4 weeks old, less than 5 weeks old, less than 6 weeks old, less than 6 weeks old, less than 7 weeks old, less than 8 weeks old, less than 9 weeks old, less than 10 weeks old, less than 11 weeks old, less than 12 weeks old, less than 6 months old, from 0 to 12 weeks old, from 0 to 10 weeks old, from 0 to 8 weeks old, from 0 to 6 weeks old, from 0 to 4 weeks old, from 0 to 2 weeks old, from 4 to 12 weeks old, from 6 to 12 weeks old, from 10 to 12 weeks old, from 4 weeks to 6 months old, from 2 months to 6 months old, from 4 months to 6 months old, from 6 months to 1 year old, more than 13 weeks old, or more than 1 year old.

64. The method according to any one of claims 37 to 63, wherein the subject is 13 weeks old or older.

65. The method according to any one of claims 37 to 64, wherein the antibody is administered in an amount in the range of 0.01 mg / kg body weight to 100 mg / kg body weight per dose.

66. The method according to any one of claims 37 to 65, wherein the antibody is administered in an amount of 5 mg / kg body weight per dose.

67. The method according to any one of claims 37 to 66, wherein the antibody or pharmaceutical composition is administered as a single dose.

68. The method according to any one of claims 37 to 67, wherein the antibody or pharmaceutical composition is administered repeatedly, such as once a week for at least 2 weeks or 3 weeks continuously.

69. The method according to any one of claims 37 to 68, wherein the method comprises administering to the subject a therapeutically effective amount of two or more different antibodies according to any one of claims 1 to 31, the two or more different antibodies being administered simultaneously or sequentially, and in some cases, the administration of the two or more different antibodies being separated by more than one day.

70. The method according to any one of claims 37 to 69, wherein it is determined by a hemagglutination inhibition assay before administration of the antibody or pharmaceutical composition that the subject has a hemagglutination inhibition titer of less than 20.

71. The method according to any one of claims 37 to 70, wherein it is determined by a hemagglutination inhibition assay before administration of the antibody or pharmaceutical composition that the subject is negative for parvovirus titer.

72. The method according to any one of claims 37 to 71, wherein after administration of the antibody or pharmaceutical composition, the subject survives an infection by canine or feline parvovirus.

73. A method for reducing parvovirus infection of cells, comprising exposing the cells to an antibody according to any one of claims 1 to 31 or a pharmaceutical composition according to claim 35 or claim 36 under conditions that permit binding of the antibody to the parvovirus.

74. The method according to claim 73, wherein the cells are exposed to the antibody or the pharmaceutical composition in vitro.

75. The method according to claim 73 or claim 74, wherein the cells are mammalian cells, human cells, canine cells, or feline cells.

76. A method for detecting parvovirus infection in a sample from a subject, comprising contacting the sample with an antibody according to any one of claims 1 to 31 or a pharmaceutical composition according to claim 35 or claim 36 under conditions that permit binding of the antibody to the parvovirus, and detecting whether a complex is formed between the antibody and parvovirus in the sample.

77. The method according to claim 76, wherein the sample is a biological sample obtained from a dog, a cat, or a human.

78. a) Aspartic acid or glutamic acid at a position corresponding to position 10 of SEQ ID NO: 91; b) Aspartic acid or glutamic acid at position 10 of SEQ ID NO: 91; c) Aspartic acid or glutamic acid at a position corresponding to position 103 of SEQ ID NO: 91; d) Aspartic acid or glutamic acid at position 103 of SEQ ID NO: 91; e) Aspartic acid or glutamic acid at a position corresponding to position 10 and / or 103 of SEQ ID NO: 91; or f) Aspartic acid or glutamic acid at position 10 and / or 103 of SEQ ID NO: 91 A variant IgG Fc polypeptide comprising the same.

79. A polypeptide comprising the variant IgG Fc polypeptide according to claim 78.

80. A polypeptide comprising the amino acid sequence of SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, or SEQ ID NO:

99.

81. An isolated nucleic acid encoding the polypeptide according to any one of claims 78 to 80.

82. A host cell comprising the nucleic acid according to claim 81.

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