Canine antibody against human and canine CTLA-4

Abstract

The present invention provides caninized anti-human CTLA-4 antibodies that have specific sequences and high binding affinity for canine CTLA-4. The present invention further relates to the use of these antibodies in the treatment of cancer in dogs and other companion animals.

Description

Technical Field

[0001] Cross-reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 874,287, filed on July 15, 2019, under 35 U.S.C. § 119(e), the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to antibodies against proteins involved in co-stimulatory or co-inhibitory signaling pathways (which include CTLA-4). More specifically, the present invention further relates to a canineized antibody against human CTLA-4, which has a specific sequence and a high binding affinity for canine CTLA-4. The present invention further relates to the use of the antibodies of the present invention in the treatment of canine cancer.

Background Art

[0003] The initiation or termination of an immune response is mediated through signaling pathways activated by complex interactions between a series of proteins expressed on the surfaces of many types of immune cells, particularly T lymphocytes and antigen-presenting cells (APCs). The co-stimulatory signaling pathway has been shown to result in the generation of an immune response and, most importantly, is mediated through the interaction of CD28 on the surface of T cells with members of the B7.1 (also known as CD80) and B7.2 (also known as CD86) family on the surface of APCs. B7.1 and B7.2 are thought to perform similar functions.

[0004] In contrast, co-inhibitory pathways have been shown to result in the inhibition or termination of the immune response and are mediated through the interaction between CTLA-4 on T cells and B7.1 / B7.2 proteins on APCs. Additional co-inhibitory signaling pathways have been shown to be mediated through the interaction between programmed cell death receptor 1 (PD-1) on T cells and programmed cell death receptor ligand 1 or 2 (PD-L1 / PD-L2) proteins on APCs. Furthermore, it has also been shown that the interaction between PD-L1 and B7.1 may also result in an inhibitory signal within T cells.

[0005] B7.1 and B7.2 are members of the immunoglobulin (Ig) superfamily [Sharpe and Freeman, Nature Reviews, 2:116-126 (2002)]. B7.1 is expressed on activated B cells, activated T cells, and macrophages and dendritic cells [Swanson and Hall, Eur J. Immunol., 23:295-298 (1993); Razi-Wolfe et al., PNAS, 89:4210-4214 (1992)]. B7.2 is constitutively expressed on dendritic cells, Langerhans cells, and B cells. Furthermore, B7.2 is expressed on monocytes and is upregulated after IFN-γ stimulation [Larsen et al., Immunol.,152:5208-5219 (1994); Inaba, J. Exp. Med. 180:1849-1860 (1994)].

[0006] B7.1 and B7.2 bind to CD28 and CTLA-4, resulting in different functional outcomes [Linsley et al., PNAS, 87:5031-5035 (1990); Linsley et al., J. Exp. Med., 173:721-730(1991); Azuma et al., Nature 366:76-79 (1993); Freeman et al., Science 262:909-912 (1993)]. The binding of B7.1 and B7.2 to CTLA-4 shows a much higher affinity than the binding of B7.1 and B7.2 to CD28 [van der Merwe, J. Exp. Med. 185:393-402 (1997)].

[0007] CD28 is a homodimeric glycoprotein that is a member of the Ig superfamily [Aruffo and Seed, PNAS, 84:8573-8577 (1987)]. The mature protein has a single extracellular variable domain consisting of 134 amino acid residues that contains the hexapeptide motif MYPPPY essential for counter-receptor binding [Riley and June, Blood, 105:13-21 (2005)]. The 41-amino acid cytoplasmic domain of CD28 contains four tyrosine residues that can be phosphorylated upon activation [Sharpe and Freeman, Nat. Rev. Immunol., 2:116-126 (2002)]. CD28 is expressed on the majority of CD4 + T cells and CD8 +It is expressed in approximately 50% of T cells [Gross et al., J. Immunol., 149:380-388 (1992); Riley and June, Blood, 105:13-21 (2005)]. After T cell receptor (TCR) ligation, B7.1 / B7.2 bound to CD28 provides important co-stimulatory signals to T cells, enabling T cell activation and subsequent generation of an immune response [Reiser et al., PNAS, 89:271-275 (1992); Jenkins et al., J. Immunol., 147:2461-2466 (1991)]. In the absence of CD28 signals, T cells have been shown to undergo apoptosis or become unresponsive [Jenkins et al., J. Exp. Med. 165:302-319 (1987); Jenkins et al., PNAS, 84:5409-5413 (1987); Schwartz, Science, 248:1349-1356 (1990)]. CD28-B7.1 / B7.2 binding can change the threshold level of TCR ligation required for activation (e.g., the amount of antigen-MHC complex), shorten the time required to stimulate naive cells, and enhance the magnitude of the T cell response [Soskic et al., Advances in Immunology, 124:96-123 (2014)].

[0008] CTLA-4 (CD152) is also a member of the Ig superfamily and consists of a single extracellular domain, a transmembrane domain, and a short cytoplasmic tail [Swanson, Immunology; 1010:169-177 (2000)]. Furthermore, CTLA-4 shares approximately 30% amino acid identity with CD28. CTLA-4 is not constitutively expressed in naive T cells but is rapidly upregulated immediately after CD28 ligation and T cell activation, and the expression level of CTLA-4 peaks at approximately 48-96 hours after the first T cell activation [Alegre et al., J. Immunol., 157:4762-4770 (1996); Freeman et al., J. Immunol., 149:3795-3801 (1992)]. CTLA-4 binds to both B7.1 and B7.2 with a much higher affinity than CD28 [van der Merwe et al., J. Exp. Med., 185:393-402 (1997)]. However, in contrast to the stimulatory effect of CD28 binding to B7.1 or B7.2, CTLA-4 functions as an inhibitory receptor essential for the downmodulation of the immune response [Walnus et al., Immunity, 1:405-413 (1994); Walnus, J. Exp. Med., 183:2541-2550 (1996); Krummel and Allison, J. Exp. Med., 183:2533-2540 (1996)]. The mechanism by which CTLA-4 mediates its immunosuppressive function is related to its ability to act as a competitive inhibitor of the interaction between CD28 and B7.1 / B7.2 [reviewed in "Swanson, Immunology, 1010:169-177 (2000)"]. The important role of CTLA-4 in immune downregulation has been demonstrated in CTLA-4-deficient mice.The CTLA-4-deficient mice die at 3 to 5 weeks of age due to the development of a lymphoproliferative disease characterized by T cell infiltration into multiple organs [Tivol et al., Immunity, 3:541-5417 (1995); Waterhouse et al., Science, 270:985-988 (1995)]. The results of CTLA-4 knockout also demonstrated that it depends on the interaction between CD28 and its ligands B7.1 and B7.2, as shown by the absence of disease in CTLA-4 / B7.1 / B7.2 triple knockout mice [Mandelbrot et al., J. Exp. Med., 189:435-440 (1999)]. This has also been confirmed by the protection against lymphoproliferation brought about by repeated administration of CTLA-4Ig to CTLA-4 knockout mice [Tivol et al., J Immunol., 158:5091-5094 (1997)].

[0009] Furthermore, it has been shown that blocking the effect of CTLA-4 with an antibody enhances T cell responses in vitro and in vivo and increases the anti-tumor immune response [Leach et al., Science, 271:1734-1736 (1996)]. Based on these findings, the development of CTLA-4 blockers, such as monoclonal antibodies, has been carried out to provide a therapeutic modality for treating cancer [Hodi et al., PNAS, 100(8):4712-4717 (2003); Phan GQ et al., PNAS,100(14):8372-8377 (2003); Attia, Journal of Clinical Oncology, 23(25):6043-6053 (2005); Comin-Anduix et al., Journal of Translational Medicine, 6:22-22 (2008); WO2000037504A2; U.S.8,017,114B2; WO2010097597A1; WO2012120125A1; and, Boutros et al., Nat Rev Clin Oncol., 13(8):473-486 (2016)].

[0010] PD-1 is a member of the CD28 / CTLA-4 family of immunoregulatory receptors. PD-1 is also a member of the Ig superfamily and contains an extracellular variable domain that binds to its ligand and a cytoplasmic tail that binds to signaling molecules [reviewed in "Zak et al., Cell Structure, 25:1163-1174 (2017)"]. The cytoplasmic tail of PD-1 contains two tyrosine-based signaling motifs [Zhang et al., Immunity 20:337-347 (2004)]. Expression of PD-1 is not seen in unstimulated T cells, B cells, or myeloid cells. However, PD-1 expression is upregulated in these cells after activation [Chemnitz et al., J. Immunol., 173:945-954 (2004); Petrvas et al., J. Exp. Med., 203:2281-2292 (2006)]. PD-1 is most closely related to CTLA-4 and shares approximately 24% amino acid identity [Jin et al., Current Topics in Microbiology and Immunology, 350:17-37 (2010)]. When PD-1 binds to PD-L1 and PD-L2, which are expressed on the surface of APCs, it reduces T cell activation. When either of these ligands binds to PD-1, antigen signaling via the T cell receptor (TCR) is negatively regulated. To date, only PD-L1 and PD-L2 have been shown to function as ligands for PD-1. Similar to the case of CTLA-4, PD-1 ligation appears to transmit a negative immunoregulatory signal. Ligation of PD-1 by PD-L1 or PD-L2 results in inhibition of proliferation and cytokine production via the TCR [Jin et al., Current Topics in Microbiology and Immunology, 350:17-37 (2010)].In contrast to CTLA-4-deficient animals, PD-1-deficient mice die much later in life and show signs of autoimmunity, although the severity of the observed effects is not as profound as that shown by CTLA-4-deficient animals [Nishimura et al., Immunity, 11(2):141-151 (1999); Nishimura et al., Science, 291(5502):319-322 (2001)]. The PD-1 signaling pathway is currently being intensively studied, and previous studies have suggested that the PD-L1 / PD-L2 / PD-1 interaction reduces signals downstream of TCR stimulation, which results in reduced cytokine secretion, dysfunction of T cell proliferation, and reduced production of cytotoxic molecules by T cells, thus being involved in the negative regulation of some immune responses [Freeman et al., J. Exp. Med., 192 (7):1027-1034 (2000)].

[0011] PD-L1 (CD274) is a type I membrane protein and is composed of an IgV-like extracellular domain, an IgC-like extracellular domain, a hydrophobic transmembrane domain, and a short cytoplasmic tail consisting of 30 amino acids with unknown signaling properties. PD-L1 is recognized as a member of the B7 family and shares approximately 20% amino acid identity with members of the B7 family. PD-L1 binds to the receptor PD-1 found on activated T cells, B cells, and myeloid cells. PD-L1 also binds to the costimulatory molecule B7.1 but does not bind to CD86 [Butte et al., Immunology, 45 (13):3567-3572 (2008)]. The affinity of B7.1 for PD-L1 is intermediate between its affinities for CD28 and CTLA-4. The related molecule PD-L2 has no affinity for either CD80 or CD86 but shares PD-1 as a receptor. When PD-L1 binds to its receptor PD-1 on T cells, signals are delivered that inhibit TCR-mediated IL-2 production and T cell proliferation. PD-L1 that binds to PD-1 also contributes to ligand-induced TCR downmodulation during antigen presentation to naive T cells. Furthermore, when PD-L1 binds to B7.1 on T cells, T cell apoptosis occurs. The roles of PD-1 and PD-L1 as inhibitors of T cell activation have been demonstrated in many studies. Based on these findings, the development of PD-1 and PD-L1 blockers, such as monoclonal antibodies, has been carried out to provide therapeutic modalities for treating cancer and infectious diseases.

[0012] Humanized monoclonal antibodies that block the binding and activity of canine PD-1, PD-L1, and CTLA-4 have been developed and are now available for use in the treatment of human subjects diagnosed with one of several different types of cancer. Similarly, canine monoclonal antibodies that block the binding and activity of canine PD-1 and PDL1 have also been reported [U.S. 9,944,704 B2, U.S. 10,106,607 B2, and U.S. 2018 / 0237535 A1; these are hereby incorporated by reference in their entirety]. However, to date, no canine monoclonal antibodies that block the binding and activity of canine CTLA-4 have been reported.

[0013] The citation of references in this specification should not be construed as an admission that any such reference is available as "prior art" to the present application.

Prior Art Documents

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Summary of the Invention

Problems to be Solved by the Invention

[0016] The present invention relates to a canine anti-human CTLA-4 antibody that has specific binding affinity for canine CTLA-4 and also has the ability to block the binding of canine CTLA-4 to canine CD80 and / or CD86. The present invention further relates to the use of such antibodies in the treatment of diseases such as cancer and / or diseases caused by infectious diseases.

[0017] Accordingly, the present invention provides an isolated canine antibody or an antigen-binding fragment of the canine antibody that contains a canine IgG heavy chain and a canine kappa or lambda light chain and specifically binds to CTLA-4. In certain embodiments of this type, the canine kappa or lambda light chain contains three light chain complementarity determining regions (CDRs) [i.e., CDR light 1 (CDRL1), CDR light 2 (CDRL2), and CDR light 3 (CDRL3)], and the canine IgG heavy chain contains three heavy chain CDRs [i.e., CDR heavy 1 (CDRH1), CDR heavy 2 (CDRH2), and CDR heavy 3 (CDRH3)], where all six of the CDRs are obtained from mammalian CTLA-4 antibodies. Certain embodiments of the canine antibody and fragments thereof of the present invention bind to canine CTLA-4 and / or block the binding of canine CTLA-4 to canine CD80 and / or CD86.

[0018] In certain embodiments, the CDRL1 of the canine antibody contains the amino acid sequence of SEQ ID NO: 54, CDRL2 contains the amino acid sequence containing SEQ ID NO: 56, and CDRL3 contains the amino acid sequence of SEQ ID NO: 58. In related embodiments, the CDRH1 of the canine antibody contains the amino acid sequence of SEQ ID NO: 48, CDRH2 contains the amino acid sequence containing SEQ ID NO: 50, and CDRH3 contains the amino acid sequence of SEQ ID NO: 52.

[0019] In certain embodiments, CDRL1 of the canine antibody is encoded by the nucleotide sequence of SEQ ID NO: 53, CDRL2 is encoded by the nucleotide sequence of SEQ ID NO: 55, and CDRL3 is encoded by the nucleotide sequence of SEQ ID NO: 57. In related embodiments, CDRH1 of the canine antibody is encoded by the nucleotide sequence of SEQ ID NO: 47, CDRH2 is encoded by the nucleotide sequence of SEQ ID NO: 49, and CDRH3 is encoded by the nucleotide sequence of SEQ ID NO: 51.

[0020] In more specific embodiments, CDRL1 of the canine antibody comprises the amino acid sequence of SEQ ID NO: 54, CDRL2 comprises the amino acid sequence comprising SEQ ID NO: 56, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 58. Additionally, CDRH1 of the canine antibody comprises the amino acid sequence of SEQ ID NO: 48, CDRH2 comprises the amino acid sequence comprising SEQ ID NO: 50, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 52.

[0021] In certain embodiments of the present invention, the heavy chain of the canine antibody comprises the amino acid sequence of SEQ ID NO: 34. In more specific embodiments, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 33. In related embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a conservative variant of the amino acid sequence of SEQ ID NO: 34. In still other embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a functionally conserved variant of the amino acid sequence of SEQ ID NO: 34.

[0022] In yet other embodiments, the heavy chain of the canineized antibody comprises the amino acid sequence of SEQ ID NO: 36. In a more specific embodiment, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 35. In related embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a conservative variant of the amino acid sequence of SEQ ID NO: 36. In yet other embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a functionally conserved variant of the amino acid sequence of SEQ ID NO: 36.

[0023] In yet other embodiments, the heavy chain of the canineized antibody comprises the amino acid sequence of SEQ ID NO: 38. In a more specific embodiment, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 37. In related embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a conservative variant of the amino acid sequence of SEQ ID NO: 38. In yet other embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a functionally conserved variant of the amino acid sequence of SEQ ID NO: 38.

[0024] In yet other embodiments, the heavy chain of the canineized antibody comprises the amino acid sequence of SEQ ID NO: 40. In a more specific embodiment, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 39. In related embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a conservative variant of the amino acid sequence of SEQ ID NO: 40. In yet other embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a functionally conserved variant of the amino acid sequence of SEQ ID NO: 40.

[0025] In certain embodiments of the present invention, the heavy chain of the canine antibody comprises the amino acid sequence of SEQ ID NO: 60. In more specific embodiments, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 59. In related embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a conservative variant of the amino acid sequence of SEQ ID NO: 60. In yet other embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a functionally conserved variant of the amino acid sequence of SEQ ID NO: 60.

[0026] In yet other embodiments, the heavy chain of the canine antibody comprises the amino acid sequence of SEQ ID NO: 62. In more specific embodiments, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 61. In related embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a conservative variant of the amino acid sequence of SEQ ID NO: 62. In yet other embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a functionally conserved variant of the amino acid sequence of SEQ ID NO: 62.

[0027] In yet other embodiments, the heavy chain of the canine antibody comprises the amino acid sequence of SEQ ID NO: 64. In more specific embodiments, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 63. In related embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a conservative variant of the amino acid sequence of SEQ ID NO: 64. In yet other embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a functionally conserved variant of the amino acid sequence of SEQ ID NO: 64.

[0028] In yet other embodiments, the heavy chain of the canine antibody comprises the amino acid sequence of SEQ ID NO: 66. In a more specific embodiment, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 65. In related embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a conservative variant of the amino acid sequence of SEQ ID NO: 66. In yet other embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a functionally conserved variant of the amino acid sequence of SEQ ID NO: 66.

[0029] In certain embodiments, the canine light chain of the canine antibody is a kappa chain. In an alternative embodiment, the canine light chain is a lambda chain. In certain embodiments, the kappa light chain comprises the amino acid sequence of SEQ ID NO: 42. In a more specific embodiment, the kappa light chain is encoded by the nucleotide sequence of SEQ ID NO: 41. In related embodiments, the kappa light chain comprises the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within a conservative variant of the amino acid sequence of SEQ ID NO: 42. In yet other embodiments, the kappa light chain comprises the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within a functionally conserved variant of the amino acid sequence of SEQ ID NO: 42.

[0030] In certain embodiments, the kappa light chain of the canine antibody comprises the amino acid sequence of SEQ ID NO: 44. In a more specific embodiment, the kappa light chain is encoded by the nucleotide sequence of SEQ ID NO: 43. In related embodiments, the kappa light chain comprises the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within a conservative variant of the amino acid sequence of SEQ ID NO: 44. In yet other embodiments, the kappa light chain comprises the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within a functionally conserved variant of the amino acid sequence of SEQ ID NO: 44.

[0031] In still other embodiments, the kappa light chain of the chimeric antibody comprises the amino acid sequence of SEQ ID NO: 46. In a more specific embodiment, the kappa light chain is encoded by the nucleotide sequence of SEQ ID NO: 45. In related embodiments, the kappa light chain comprises the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within a conservative variant of the amino acid sequence of SEQ ID NO: 46. In still other embodiments, the kappa light chain comprises the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within a functionally conserved variant of the amino acid sequence of SEQ ID NO: 46.

[0032] The present invention further provides a chimeric antibody comprising any light chain of the present invention and any heavy chain of the present invention. In a specific embodiment, the isolated chimeric antibody comprises a heavy chain comprising the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within the amino acid sequence of SEQ ID NO: 36, SEQ ID NO: 62, or a conservative variant of the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 62, and a kappa light chain comprising the amino acid sequence of SEQ ID NO: 46 or the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within a conservative variant of the amino acid sequence of SEQ ID NO: 46. In a more specific embodiment, the isolated chimeric antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and a kappa light chain comprising the amino acid sequence of SEQ ID NO: 46. In another specific embodiment, the isolated chimeric antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 62 and a kappa light chain comprising the amino acid sequence of SEQ ID NO: 46.

[0033] In other specific embodiments, the isolated canine antibody comprises a heavy chain comprising the amino acid sequences of SEQ ID NO: 40, SEQ ID NO: 66 or SEQ ID NO: 48, SEQ ID NO: 50 and SEQ ID NO: 52 in a conservative variant of the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 66, and a kappa light chain comprising the amino acid sequence of SEQ ID NO: 42 or the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56 and SEQ ID NO: 58 in a conservative variant of the amino acid sequence of SEQ ID NO: 42. In a more specific embodiment, the isolated canine antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 40 and a kappa light chain comprising the amino acid sequence of SEQ ID NO: 42. In other specific embodiments, the isolated canine antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 66 and a kappa light chain comprising the amino acid sequence of the sequence of SEQ ID NO: 42.

[0034] The present invention further provides an isolated nucleic acid encoding any one of the light chains of the canine antibody of the present invention. Similarly, the present invention further provides an isolated nucleic acid encoding any one of the heavy chains of the canine antibody of the present invention. The present invention further provides an expression vector comprising one or more of the isolated nucleic acids of the present invention. The present invention further provides a host cell comprising one or more of the expression vectors of the present invention.

[0035] In certain embodiments, the antibody is a recombinant antibody or an antigen-binding fragment thereof. In related embodiments, the variable heavy chain domain and the variable light chain domain are linked by a flexible linker to form a single-chain antibody.

[0036] In certain embodiments, the antibody or antigen-binding fragment is a Fab fragment. In another embodiment, the antibody or antigen-binding fragment is a Fab’ fragment. In another embodiment, the antibody or antigen-binding fragment is a (Fab’)2 fragment. In yet another embodiment, the antibody or antigen-binding fragment is a diabody. In certain embodiments, the antibody or antigen-binding fragment is a domain antibody. In a more particular embodiment, the antibody or antigen-binding fragment is a camelized single domain antibody.

[0037] In certain embodiments, the camelized anti-human CTLA-4 antibody or antigen-binding fragment increases the immune response of the treated canine subject.

[0038] Accordingly, the invention further provides an isolated nucleic acid encoding a camelized anti-human CTLA-4 antibody or antigen-binding fragment disclosed herein. In related embodiments, such an antibody or antigen-binding fragment can be used in the preparation of a medicament for treating cancer in a canine subject. Alternatively or in combination, the invention provides the use of any of the antibodies or antibody fragments of the invention for diagnostic use. In further additional embodiments, kits are provided that contain any of the camelized antibodies or antigen-binding fragments disclosed herein.

[0039] In further additional embodiments, expression vectors are provided that contain an isolated nucleic acid encoding any of the camelized anti-human CTLA-4 antibodies or antigen-binding fragments of the invention. The invention further relates to host cells containing any of the expression vectors described herein. In certain embodiments, these nucleic acids, expression vectors or polypeptides of the invention are useful in methods of making antibodies.

[0040] The present invention further encompasses a pharmaceutical composition comprising the canine antibody of the present invention or an antigen-binding fragment thereof together with a pharmaceutically acceptable carrier or diluent. Further, the present invention provides a method for increasing the activity of immune cells, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of the present invention. In certain embodiments, the subject is a dog. In alternative embodiments, the subject is a cat. In yet other embodiments, the subject is a horse. In certain embodiments, the method is used for the treatment of cancer. In another embodiment, the method is used in the treatment of an infectious disease or disorder. In yet another embodiment, the canine antibody of the present invention or an antigen-binding fragment thereof is used as a vaccine adjuvant.

[0041] These and other aspects of the present invention will be better understood by reference to the following "Brief Description of the Drawings" and "Detailed Description".

Brief Description of the Drawings

[0042]

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DETAILED DESCRIPTION OF THE INVENTION

[0043] Abbreviations Throughout the detailed description and examples of the present invention, the following abbreviations are used.

Table 1

[0044] Definitions To make the present invention more readily understandable, specific technical terms and scientific terms are defined clearly below. Unless otherwise clearly defined elsewhere in this specification, all other technical terms and scientific terms used in this specification have the meanings generally understood by those skilled in the technical field to which the present invention pertains.

[0045] As used in this specification, including the appended "claims", the singular forms of words such as "a", "an", and "the" include their corresponding plural references unless the context clearly indicates otherwise.

[0046] "Activation" as applied to a cell or receptor means activation or treatment of the cell or receptor by a ligand, unless the context or otherwise explicitly indicates otherwise. "Ligand" includes natural ligands and synthetic ligands, such as cytokines, cytokine variants, analogs, mutant proteins, and binding compounds derived from antibodies. "Ligand" further includes small molecules, such as peptidomimetics of cytokines and peptidomimetics of antibodies. "Activation" can mean cell activation regulated by internal mechanisms and cell activation regulated by external factors or environmental factors.

[0047] The "activity" of a molecule can represent or indicate the following: the binding of the molecule to a ligand or receptor, catalytic activity; the ability to stimulate gene expression or cell signaling, differentiation or maturation; antigenic activity, regulation of the activity of other molecules, etc. The "activity" of a molecule can further indicate the following: activity in regulating or maintaining cell-cell interactions (e.g., adhesion), or activity in maintaining the structure of a cell (e.g., cell membrane or cytoskeleton). "Activity" can further mean the following: specific activity, e.g., [catalytic activity] / [mg protein] or [immunological activity] / [mg protein], concentration within a biological compartment, etc. "Activity" can also indicate the regulation of components of the innate or adaptive immune system.

[0048] "Administration" and "treatment", when applied to an animal (e.g., a canine subject), a cell, a tissue, an organ or a biological fluid, indicate the contact of an exogenous pharmaceutical, therapeutic, diagnostic or composition with the animal (e.g., a canine subject), the cell, the tissue, the organ or the biological fluid. Treatment of a cell includes the contact of a reagent with the cell and the contact of a reagent with a biological fluid where the biological fluid contacts the cell.

[0049] "Administration" and "treatment" further mean in vitro and ex vivo treatment of a cell, for example, by a reagent, a diagnostic, a binding compound, or by another cell. The term "subject" includes any organism, preferably includes an animal, more preferably includes a mammal (e.g., a dog, a cat or a horse), and most preferably includes a dog.

[0050] "Treat" or "treating" means administering a therapeutic agent (e.g., a composition comprising either an antibody or an antigen-binding fragment of the invention) internally or externally to a canine subject or patient, for example, having one or more disease symptoms for which the therapeutic agent has therapeutic activity or suspected of suffering from a disease. Typically, the therapeutic agent is administered in an amount effective to reduce and / or ameliorate such one or more disease symptoms in the subject or population being treated by inducing regression of the symptom(s) to a clinically measurable extent or preventing progression of the symptom(s). The amount of a therapeutic agent effective to reduce a particular disease symptom (also referred to as "therapeutically effective amount") can vary depending on factors such as the disease state, age and weight of the patient (e.g., canine, feline or equine), and the ability of the pharmaceutical composition to elicit the desired response in the subject. Whether a disease symptom has been reduced or ameliorated can be evaluated by any clinical measurement typically used by a veterinarian or another skilled healthcare provider to assess the severity or progression of that symptom. Embodiments of the invention (e.g., methods of treatment or articles of manufacture) may not be effective in reducing (one or more) target disease symptoms in all subjects, but should reduce (one or more) target disease symptoms in a statistically significant number of subjects as determined by any statistical test known in the art (e.g., Student's t-test, chi-square test, Mann-Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test).

[0051] "Treatment" refers to therapeutic treatment as well as research and diagnostic uses when applied to a human subject, veterinary subject (e.g., canine) or research subject. "Treatment" includes contacting an antibody or antigen-binding fragment of the invention with, for example, a canine or other animal subject (e.g., feline), cell, tissue, physiological compartment or physiological fluid when applied to a human subject, veterinary subject (e.g., canine) or research subject, or to a cell, tissue or organ.

[0052] The term "immune response" refers to, for example, the action of lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (e.g., antibodies, cytokines, and complements) produced by said cells or the liver, which results in selective damage to, destruction of, or removal from the mammalian body (e.g., the canine body) of cancer cells, cells or tissues infected with pathogens, or invading pathogens.

[0053] Canine anti-human CTLA-4 antibody The present invention provides an isolated canine anti-human CTLA-4 antibody or an antigen-binding fragment thereof that binds to canine CTLA-4, and the use of such an antibody or fragment thereof.

[0054] As used herein, a canine anti-human CTLA-4 antibody refers to a canine antibody that specifically binds to mammalian CTLA-4. An antibody that specifically binds to mammalian CTLA-4 (particularly, canine CTLA-4) is an antibody that exhibits preferential binding to mammalian CTLA-4 as compared to other antigens, although this specificity does not require absolute binding specificity. A canine anti-human CTLA-4 antibody is considered "specific" for canine CTLA-4 when its binding determines the presence of canine CTLA-4 in a biological sample obtained from a dog, or when it can alter the activity of canine CTLA-4 without unduly interfering with the activity of other unrelated canine proteins in the dog sample (e.g., without causing undesirable results such as false positives in a diagnostic situation or side effects in a therapeutic situation). The degree of specificity required for a canine anti-human CTLA-4 antibody may depend on the intended use of the antibody, and in any case, is defined by its suitability for use for the intended purpose. A binding compound derived from an antibody or an antigen-binding site of an antibody of the intended method binds to the antigen or its variant or mutant protein with an affinity that is at least 2-fold greater, preferably at least 10-fold greater, more preferably at least 20-fold greater, and most preferably at least 100-fold greater compared to its affinity for any other canine antigen. However, an isolated antibody that specifically binds to canine CTLA-4 may cross-react with other antigens, particularly closely related antigens such as feline CTLA-4, equine CTLA-4, and / or human CTLA-4.

[0055] As used herein, an antibody is said to specifically bind to a polypeptide containing the amino acid sequence of canine CTLA-4 if it binds to the polypeptide containing the sequence of canine CTLA-4 but does not bind at all to any such protein, even if there is another canine protein lacking the amino acid sequence of canine CTLA-4. For example, an antibody that specifically binds to a polypeptide containing canine CTLA-4 may bind to a FLAG®-tagged form of canine CTLA-4, but does not specifically bind to another FLAG®-tagged canine protein.

[0056] As used herein, unless otherwise indicated, "antibody fragment" or "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment (e.g., a fragment retaining one or more CDR regions) that retains the ability to specifically bind to an antigen (e.g., canine CTLA-4) bound by a full-length antibody. Examples of antigen-binding fragments include, but are not limited to, the following: Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; nanobodies, and multispecific antibodies formed from antibody fragments.

[0057] Typically, the canine antibody or antigen-binding fragment thereof of the present invention retains at least 10% of its activity (when compared to its corresponding parental antibody) when its canine CTLA-4 binding activity is expressed on a molar basis. Preferably, the antibody or antigen-binding fragment of the present invention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the canine CTLA-4 binding affinity of the parental antibody.

[0058] The present invention encompasses antibodies referred to as "conservative variants" of antibodies having a defined amino acid sequence. As used herein, "conservative variants" have one, two, three, or more conservative amino acid substitutions in their amino acid sequences as compared to the canineized antibodies of the present invention having the defined amino acid sequence. The present invention further encompasses antibodies referred to as "function conserved variants" of canineized antibodies having a defined amino acid sequence. As used herein, "function conserved variants" have one, two, three, or more non-conservative amino acid substitutions as compared to the amino acid sequence of the canineized antibody. The terms "conservative variants" and "function conserved variants" are used only with respect to changes in amino acid residues within the canine frame of the corresponding canineized antibodies of the present invention and are not used with respect to specific CDRs of the canineized antibody. Importantly, "conservative variants" and / or "function conserved variants" do not substantially alter the biological activity of the corresponding canineized antibodies of the present invention that contain the defined amino acid sequence.

[0059] "Isolated antibody" indicates a purified state and, in such context, means that the molecule is substantially free of other biological molecules (e.g., nucleic acids, proteins, lipids, carbohydrates) or other substances (e.g., cell debris and growth medium). Generally, the term "isolated" is not intended to indicate that such substances are completely absent or that water, buffer, or salt is absent, unless such substances or water, buffer, or salt are present in an amount that substantially interferes with the experimental or therapeutic use of the binding compounds described herein.

[0060] The variable regions of each light chain / heavy chain pair form the antigen-binding site of the antibody. Thus, generally, a complete antibody has two binding sites. Except in the case of bifunctional or bispecific antibodies, the two binding sites are generally identical.

[0061] Typically, both the variable domains of the heavy and light chains contain three hypervariable regions, also called complementarity-determining regions (CDRs), which are located within relatively conserved framework regions (FRs). The CDRs are usually adjacent to the framework regions, thereby enabling binding to a specific epitope. Generally, from the N-terminus to the C-terminus, both the light chain variable domain and the heavy chain variable domain contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments to each domain generally follow the following definitions: Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md., 5 th ed., NIH Publ. No. 91-3242 (1991); Kabat, Adv. Prot. Chem. 32:1-75 (1978); Kabat, et al., J. Biol. Chem. 252:6609-6616 (1977); Chothia, et al., J. Mol. Biol. 196:901-917 (1987) or Chothia, et al., Nature 342:878-883 (1989).

[0062] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are involved in antigen binding. The hypervariable regions include amino acid residues derived from "complementary determining regions" or "CDRs" (i.e., CDRL1, CDRL2, and CDRL3 in the light chain variable domain, and CDRH1, CDRH2, and CDRH3 in the heavy chain variable domain). [See "Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)", which defines the CDR regions of antibodies by sequence; see also "Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)", which defines the CDR regions of antibodies by structure].

[0063] As used herein, the term "framework" or "FR" residues refers to the variable domain residues other than the hypervariable region residues defined herein as CDR residues. The framework of a chimeric antibody represents a part of the framework of the non-human species.

[0064] As used herein, the term "canine" includes all domestic dogs, wolves (Canis lupus familiaris), or the genus Canis (Canis familiaris) unless otherwise indicated.

[0065] As used herein, the term "feline" refers to any member of the family Felidae. Domestic cats, purebred and / or hybrid companion cats, as well as wild or feral cats are all felines.

[0066] As used herein, the term "in-frame" refers to the amino acid sequences of the heavy and light chains of a canine antibody other than the hypervariable region residues defined as CDR residues herein. In connection with a caninized antibody, in most embodiments, the amino acid sequences of the native canine CDRs are replaced in both chains with the corresponding heterologous CDRs (e.g., CDRs from a murine antibody). Optionally, the heavy and / or light chains of the canine antibody can contain some heterologous non-CDR residues, for example, as discussed below, to preserve the conformation of the heterologous CDRs within the canine antibody and / or to modify the Fc function.

[0067] There are four known IgG heavy chain subtypes in canine IgG, which are designated IgG-A, IgG-B, IgG-C, and IgG-D. The two known light chain subtypes are designated lambda and kappa.

[0068] In addition to binding and activating canine immune cells, a canine antibody or caninized antibody against CTLA-4 can also be designed to have two additional attributes as follows: 1. Absence of effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC); and, 2. Can be easily purified on a large scale using industry standard techniques such as those based on protein A chromatography.

[0069] None of the naturally occurring canine IgG isotypes meet both criteria. For example, IgG-B can be purified using protein A, but has a high level of ADCC activity. On the other hand, IgG-A binds weakly to protein A and exhibits undesirable ADCC activity. Furthermore, IgG-D does not exhibit ADCC activity, but neither IgG-C nor IgG-D can be purified on a protein A column. (IgG-C exhibits a significant level of ADCC activity). The present invention overcomes this problem by providing mutant canine IgG-B antibodies that are specific for CTLA-4 [see U.S. 10,106,607 B2; which is hereby incorporated by reference in its entirety]. These antibodies all lack effector functions such as ADCC and can be easily purified using industry standard protein A chromatography.

[0070] As used herein, the term "caninized antibody" refers to an antibody that includes three heavy chain CDRs and three light chain CDRs derived from a non-canine source (e.g., an anti-human CTLA-4 antibody in a canine frame or a modified canine frame). A modified canine frame includes one or more amino acid changes. In certain embodiments, the modified canine frame further optimizes the efficacy of the caninized antibody, for example, by increasing its binding to canine CTLA-4 and / or increasing its ability to block the binding of canine CTLA-4 to canine CD80 and / or canine CD86.

[0071] "Identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptide sequences when optimally aligned. If a position in both of the two sequences being compared is occupied by the same base or amino acid monomer subunit, for example, if a position in each of two DNA molecules is occupied by adenine, then these molecules are identical at that position. The percentage of identity is obtained by dividing the number of identical positions shared by the two sequences by the total number of positions compared and multiplying by 100. For example, if 6 out of 10 positions in two sequences are identical or homologous when the two sequences are optimally aligned, then the two sequences are 60% identical. Generally, such comparisons are made when the two sequences are aligned so as to obtain the maximum percentage of identity.

[0072] As used herein, one amino acid sequence is 100% "identical" or exhibits 100% "identity" to a second amino acid sequence if the amino acid residues of both sequences are the same. Thus, an amino acid sequence is 50% "identical" to a second amino acid sequence if 50% of the amino acid residues of the two amino acid sequences are the same. Sequence comparisons are performed over contiguous blocks of amino acid residues contained within a given protein (e.g., a portion of the polypeptide being compared or a protein). In certain embodiments, selected deletions or insertions that can vary the correspondence between two amino acid sequences are taken into account.

[0073] "Isolated nucleic acid molecule" means a genomic, mRNA, cDNA, or DNA or RNA of synthetic origin, or some combination thereof, wherein the isolated polynucleotide is not associated with all or part of the polynucleotide found in nature, or is associated with a polynucleotide not linked in nature. For the purposes of this disclosure, it should be understood that a nucleic acid molecule "comprising" a particular nucleotide sequence does not include an intact chromosome. An isolated nucleic acid molecule "comprising" the designated nucleic acid sequence can include, in addition to the designated sequence, coding sequences for up to 10 or even up to 20 or more other proteins or portions or fragments thereof, or can include regulatory sequences operably linked to control the expression of the coding region of the described nucleic acid sequence, and / or can include vector sequences.

[0074] The phrase "control sequence" indicates a DNA sequence necessary to express a coding sequence operably linked in a particular host organism. Appropriate control sequences for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

[0075] A nucleic acid is "operably linked" when placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if expressed as a preprotein which participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence; or a ribosome binding site is operably linked to a coding sequence if positioned so as to promote translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking can be accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice. It will also be readily understood that when a nucleic acid sequence is provided herein, it may contain a termination codon. However, since the termination codon is exchangeable, inclusion of a particular termination codon in the sequence should not be regarded as a requirement for the necessary part of the sequence.

[0076] As used herein, the terms "cell", "cell line", and "cell culture" are used interchangeably and all such designations include progeny. Thus, the terms "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom without regard for the number of subcultures. It also is understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

[0077] As used herein, "germline sequence" refers to the sequence of an unrearranged immunoglobulin DNA sequence. Any suitable source of unrearranged immunoglobulin sequences can be used. Human germline sequences can be obtained, for example, from the JOINSOLVER® germline database on the website of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the United States National Institutes of Health. Mouse germline sequences can be obtained, for example, as described in Giudicelli et al. [Nucleic Acids Res. 33:D256-D261 (2005)].

[0078] Characteristics of Representative Canine anti-human CTLA-4 Antibodies The present invention provides an isolated canine anti-human CTLA-4 antibody and a method of using the antibody or an antigen-binding fragment thereof in the treatment of diseases (e.g., treatment of cancer in dogs). Examples of canine anti-human CTLA-4 antibodies that bind to canine CTLA-4 include, but are not limited to, antibodies that contain canine IgG-A, IgG-B, IgG-C, and IgG-D heavy chains and / or canine kappa light chains together with anti-human CTLA-4 CDRs. Accordingly, the present invention provides an isolated canine anti-human CTLA-4 antibody or an antigen-binding fragment thereof that binds to canine CTLA-4 and blocks the binding of canine CTLA-4 to canine CD80 and / or CD86.

[0079] "Conservatively modified variant" or "conservative substitution" refers to substituting an amino acid in a protein with another amino acid having similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation, and rigidity, etc.), such that as a result, the change can often be made without altering the biological activity of the protein. One of ordinary skill in the art generally recognizes that one amino acid substitution within a non-essential region of a polypeptide does not substantially modify biological activity [see, e.g., "Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p.224 (4th Ed.; 1987)"]. Additionally, substitution of amino acids that are structurally or functionally similar is less likely to disrupt biological activity. Various embodiments of the antibodies or antigen-binding fragments of the present invention include polypeptide chains having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more conservative amino acid substitutions within regions other than the polypeptide chain or CDR regions having the sequences disclosed herein (e.g., SEQ ID NO: 34, 36, 38, 40, 42, 44, 60, 62, 64 or 66). Exemplary conservative substitutions are set forth in Table A.

Table 2

[0080] Functionally conserved variants of the antibodies of the present invention are also contemplated by the present invention. "Functionally conserved variant" as used herein refers to an antibody or fragment in which one or more amino acid residues have been changed without altering desired properties such as antigen affinity and / or specificity. Such variants include, but are not limited to, those in which a particular amino acid has been replaced with an amino acid having similar properties such as the conservative amino acid substitutions of Table A.

[0081] Nucleic Acids The present invention further encompasses nucleic acids encoding the immunoglobulin chains of the anti-human CTLA-4 antibody and antigen-binding fragments thereof disclosed herein. For example, the present invention encompasses all of the novel nucleic acids described in the following table.

[0082] Furthermore, when comparison is performed by the BLAST algorithm, nucleic acids encoding immunoglobulin polypeptides comprising an amino acid sequence of an in-frame of the antibody provided herein that is at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical, and most preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) are also encompassed by the present invention, wherein the parameters of the algorithm are selected to give the maximum match between the respective sequences over the full length of each reference sequence. The present invention further provides nucleic acids encoding an in-frame of an immunoglobulin polypeptide comprising an amino acid sequence that is at least about 70% similar, preferably at least about 80% similar, more preferably at least about 90% similar, and most preferably at least about 95% similar (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to any of the reference amino acid sequences when comparison is performed by the BLAST algorithm, wherein the parameters of the algorithm are selected to give the maximum match between the respective sequences over the full length of each reference sequence and are similarly encompassed by the present invention.

[0083] Sequence identity indicates the degree to which the amino acids of two polypeptides are identical at positions where the two sequences are optimally aligned. Sequence similarity includes identical residues and non-identical amino acids that are biochemically related. Biochemically related amino acids that share similar properties and may be exchangeable are discussed above.

[0084] The following references pertain to the BLAST algorithms often used for sequence analysis: BLAST ALGORITHMS: Altschul, S.F., et al., J. Mol. Biol. 215:403-410 (1990); Gish, W., et al., Nature Genet. 3:266-272 (1993); Madden, T.L., et al., Meth. Enzymol. 266:131-141(1996); Altschul, S.F., et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang, J., et al., Genome Res. 7:649-656 (1997); Wootton, J.C., et al., Comput. Chem. 17:149-163 (1993); Hancock, J.M. et al., Comput. Appl. Biosci. 10:67-70 (1994); ALIGNMENT SCORING SYSTEMS: Dayhoff, M.O., et al., “A model of evolutionary change in proteins.” in Atlas of Protein Sequence and Structure, vol. 5, suppl. 3. M.O. Dayhoff (ed.), pp. 345-352, (1978); Natl. Biomed. Res. Found., Washington, DC; Schwartz, R.M., et al., “Matrices for detecting distant relationships.” in Atlas of Protein Sequence and Structure, vol. 5, suppl. 3.“ (1978), M.O. Dayhoff (ed.), pp. 353-358 (1978), Natl. Biomed. Res. Found., Washington, DC; Altschul, S.F., J. Mol. Biol. 219:555-565 (1991); States, D.J., et al., Methods 3:66-70(1991); Henikoff, S., et al., Proc. Natl. Acad. Sci. USA 89:10915-10919 (1992); Altschul, S.F., et al., J. Mol. Evol. 36:290-300 (1993); ALIGNMENT STATISTICS: Karlin, S., et al., Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990); Karlin, S., et al., Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); Dembo, A., et al., Ann. Prob. 22:2022-2039 (1994); and Altschul, S.F. “Evaluating the statistical significance of multiple distinct local alignments.” in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), pp. 1-14, Plenum, New York (1997).

[0085] The present invention further provides an expression vector containing the isolated nucleic acid of the present invention, wherein the nucleic acid is operably linked to a control sequence recognized by a host cell when the host cell is transfected with the vector. Furthermore, a host cell containing the expression vector of the present invention, and a method for producing the antibody or antigen-binding fragment thereof disclosed herein are also provided, wherein the method includes culturing a host cell carrying an expression vector encoding the antibody or antigen-binding fragment in a medium and isolating the antigen or antigen-binding fragment thereof from the host cell or the medium.

[0086] Epitope Binding and Binding Affinity The present invention further provides a canine anti-human CTLA-4 antibody comprising the amino acid sequence of SEQ ID NO: 36 and / or SEQ ID NO: 46, and an antibody or an antigen-binding fragment thereof that binds to the same epitope on canine CTLA-4. The canine anti-human CTLA-4 antibody or its antigen-binding fragment can inhibit the binding of canine CTLA-4 to canine CD80 and / or CD86.

[0087] As described in the following examples, the canine anti-human CTLA-4 antibody can be recombinantly produced. Mammalian cell lines available as hosts for the expression of the antibodies or fragments disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, simian kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and many other cell lines. Mammalian host cells include cells of humans, mice, rats, dogs, monkeys, pigs, goats, cows, horses, and hamsters. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other cell lines that can be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. When introducing a recombinant expression vector encoding the heavy chain or its antigen-binding portion or fragment, the light chain and / or its antigen-binding fragment into a mammalian host cell, the antibody is produced by culturing the host cell for a period sufficient to allow expression of the antibody in the host cell, or, more preferably, for a period sufficient to allow secretion of the antibody into the medium in which the host cell grows.

[0088] The antibody can be recovered from its culture medium using standard protein purification methods. Furthermore, the expression of the antibody of the present invention (or other components derived therefrom) from the production cell line can be enhanced using many known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common approach for enhancing expression under specific conditions. The GS system is considered in whole or in part in relation to European Patent No. 0216846, European Patent No. 0256055, European Patent No. 0323997, and European Patent Application No. 89303964.4.

[0089] Generally, glycoproteins produced in a particular cell line or transgenic animal have a glycosylation pattern characteristic of the glycoproteins produced in that cell line or transgenic animal. Thus, the specific glycosylation pattern of an antibody depends on the particular cell line or transgenic animal used to produce the antibody. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein constitute the present invention, regardless of the glycosylation pattern that the antibody may have. Similarly, in certain embodiments, antibodies having a glycosylation pattern that contains only non-fucosylated N-glycans may be advantageous because these antibodies have been shown to typically exhibit greater potency than their fucosylated counterparts both in vitro and in vivo [see, for example: Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Patent No. 6,946,292 and U.S. Patent No. 7,214,775].

[0090] The present invention further encompasses antibody fragments of the canine anti-human CTLA-4 antibody disclosed herein. Such antibody fragments include, for example, F(ab)2 fragments that can be generated by enzymatic cleavage of IgG with pepsin. Fab fragments can be generated, for example, by reducing F(ab)2 with dithiothreitol or mercaptoethylamine. The Fab fragment is a V H -C H1 chain added to the V L -C L chain. The F(ab)2 fragment is two Fab fragments added by two disulfide bridges. The Fab portion of the F(ab)2 molecule contains a portion of the F c region where a disulfide bridge is located in between. The F V fragment is the V L region or the V H region.

[0091] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region, such as a canine constant region, such as IgG-A, IgG-B, IgG-C, and IgG-D canine heavy chain constant regions or variants thereof. In another embodiment, the antibody or antigen-binding fragment comprises a light chain constant region, such as a canine light chain constant region, such as lambda or kappa canine light chain regions or variants thereof. By way of example, and not limitation, the canine heavy chain constant region can be derived from IgG-B [see, e.g., modified IgG-B; U.S. 10,106,607 B2, the content of which is incorporated herein by reference in its entirety], and the canine light chain constant region can be derived from kappa.

[0092] Antibody Engineering The canine anti-human CTLA-4 antibody of the present invention was engineered, for example, to include modifications to the canine framework residues within the variable domain of the parental (i.e., canine) monoclonal antibody to improve the properties of the antibody.

[0093] Experimental and Diagnostic Uses The canine anti-human CTLA-4 antibody or antigen-binding fragment thereof of the present invention may further be useful in diagnostic assays for canine CTLA-4 protein, for example, in the detection of its expression in certain tumor cells, tissues or sera. Such diagnostic methods may be useful in the diagnosis of various diseases (especially certain cancers in dogs).

[0094] For example, such a method includes the following steps: (a) Coating a substrate (e.g., the surface of a microtiter plate well, e.g., the surface of a plastic plate) with the canine anti-human CTLA-4 antibody or antigen-binding fragment thereof; (b) Applying a sample to be tested for the presence of canine CTLA-4 to the substrate; (c) Washing the plate to remove unbound substances in the sample; (d) Applying a detectable labeled antibody (e.g., an enzyme-conjugated antibody) that is also specific for the CTLA-4 antigen; (e) Washing the substrate to remove unbound labeled antibody; (f) When the labeled antibody is enzyme-conjugated, applying a chemical substance that is converted by the enzyme into a fluorescent signal; and, (g) Detecting the presence of the labeled antibody.

[0095] In a further embodiment, the labeled antibody is labeled with a peroxidase that reacts with ABTS [e.g., 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] or 3,3',5,5'-tetramethylbenzidine to produce a detectable color change. Alternatively, the labeled antibody is labeled with a detectable radioisotope (e.g., 3 H) that can be detected by a scintillation counter in the presence of a scintillant. The canine anti-human CTLA-4 antibody of the present invention can be used in Western blotting or immunoprotein blotting.

[0096] Such methods form part of the present invention and, for example, include the following: (i) contacting a membrane or another solid substrate to be tested for the presence of bound canine CTLA-4 or a fragment thereof with the canine anti-human CTLA-4 antibody of the present invention or an antigen-binding fragment thereof. Such a membrane can be in the form of a nitrocellulose or vinyl-based [e.g., polyvinylidene fluoride (PVDF)] membrane onto which a protein whose presence of canine CTLA-4 in a non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or an SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel is to be tested (e.g., after electrophoretic separation in the gel) has been transferred. Prior to contacting the membrane with the canine anti-human CTLA-4 antibody or an antigen-binding fragment thereof, the membrane is optionally blocked, e.g., with non-fat dry milk, so that non-specific protein-binding sites on the membrane bind; (ii) washing the membrane one or more times to remove unbound canine anti-human CTLA-4 antibody or an antigen-binding fragment thereof and other unbound substances; and (iii) detecting the bound canine anti-human CTLA-4 antibody or an antigen-binding fragment thereof.

[0097] Detection of the bound antibody or antigen-binding fragment can be carried out by binding the antibody or antigen-binding fragment to a secondary antibody (anti-immunoglobulin antibody) labeled detectably and then detecting the presence of the secondary antibody.

[0098] The anti-human CTLA-4 canine antibodies and antigen-binding fragments thereof disclosed herein can further be used in immunohistochemistry. Such methods form part of the present invention and include, for example: (1) contacting cells to be tested for the presence of canine CTLA-4 with the anti-human CTLA-4 canine antibody or antigen-binding fragment thereof of the present invention; and (2) detecting the antibody or fragment on or within the surface of the cells. If the antibody or antigen-binding fragment itself is detectably labeled, it can be detected directly. Alternatively, a secondary antibody detectably labeled can be bound to the antibody or antigen-binding fragment and detected.

[0099] The specific anti-human CTLA-4 canine antibodies and antigen-binding fragments thereof disclosed herein can further be used in in vivo tumor imaging. Such methods include injecting a radio-labeled anti-human CTLA-4 canine antibody or antigen-binding fragment thereof into the body of a dog to be tested for the presence of a tumor associated with the expression of canine CTLA-4, followed by subjecting the body of the patient to nuclear imaging to detect the presence of the labeled antibody or antigen-binding fragment, for example, at locations containing a high concentration of the antibody or antigen-binding fragment bound to the tumor.

[0100] Imaging techniques include SPECT imaging (single photon emission computed tomography) or PET imaging (positron emission tomography), etc. Labels include, for example, the following: for example, iodine-123 ( 123 I) and technetium-99m ( 99m Tc), or, for example, in combination with PET imaging, 11 C, 13 N, 15 O or 18F, or indium-111 [see, e.g., "Gordon et al., International Rev. Neurobiol. 67:385-440 (2005)"].

[0101] Pharmaceutical Compositions and Administration To prepare a pharmaceutical composition or a sterile composition of a canine anti-human CTLA-4 antibody or an antigen-binding fragment thereof, the canine anti-human CTLA-4 antibody or an antigen-binding fragment thereof is mixed with a pharmaceutically acceptable carrier or excipient. [See, e.g., the following: Remington’s Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984)].

[0102] Formulations of therapeutic and diagnostic agents can be prepared by mixing them, for example, in the form of lyophilized powders, slurries, aqueous solutions or suspensions, with acceptable carriers, excipients or stabilizers [see, for example: Hardman, et al. (2001) Goodman and Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY]. In one embodiment, the anti-CTLA-4 antibody of the invention is diluted to an appropriate concentration in a sodium acetate solution (pH 5-6), and NaCl or sucrose is added for tonicity. To enhance stability, additional agents such as polysorbate 20 or polysorbate 80 can be added.

[0103] The toxicity and therapeutic efficacy of the antibody composition administered alone or in combination with another agent are, for example, LD 50 (the dose lethal to 50% of the population) and ED 50Can be confirmed by standard pharmaceutical procedures in cell cultures or experimental animals to determine a therapeutically effective dose (in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (LD 50 / ED 50 ). In certain embodiments, antibodies with a high therapeutic index are desirable. The data obtained from these cell culture assays and animal tests can be used to set a dosage range for use in dogs. The dosage of such a compound preferably lies within a range of circulating concentrations that includes an ED 50 with little or no toxicity. The dosage can vary within this range depending on the dosage form and route of administration used.

[0104] The methods of administration can vary. Suitable routes of administration include, among others, the following: oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, dermal, transdermal, or intraarterial.

[0105] In certain embodiments, the canine anti-human CTLA-4 antibody or antigen-binding fragment thereof can be administered by an invasive route such as injection. In further embodiments of the invention, the canine anti-human CTLA-4 antibody or antigen-binding fragment thereof or a pharmaceutical composition thereof is administered intravenously, subcutaneously, intramuscularly, intraarterially, intratumorally, or by inhalation, aerosol delivery. Administration by non-invasive routes (e.g., orally; e.g., in pills, capsules or tablets) is also within the scope of the invention.

[0106] The composition can be administered using medical devices known in the art. For example, the pharmaceutical composition of the present invention can be administered by injection with a hypodermic needle (which includes, for example, a prefilled syringe or an autoinjector). The pharmaceutical composition disclosed herein can further be administered using a needleless subcutaneous injection device (for example, the devices disclosed in U.S. Patents: No. 6,620,135; No. 6,096,002; No. 5,399,163; No. 5,383,851; No. 5,312,335; No. 5,064,413; No. 4,941,880; No. 4,790,824 or No. 4,596,556).

[0107] The pharmaceutical composition disclosed herein can also be administered by infusion. Examples of well-known implants and modules for administering pharmaceutical compositions include, among others: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for administering a drug at a controlled rate; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering a drug at an accurate infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow rate implantable infusion device for sustained drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments. Many other such implants, delivery systems and modules are well known to those skilled in the art.

[0108] Alternatively, for example, in many cases, the chimeric anti-human CTLA-4 antibody can be administered locally rather than systemically by directly injecting the chimeric anti-human CTLA-4 antibody into the interior of a joint of a pathogen-induced lesion or arthritis characterized by immunopathology, for example, in a depot formulation or a sustained release formulation. Further, for example, the chimeric anti-human CTLA-4 antibody can be administered in a targeted drug delivery system that targets a joint of a pathogen-induced lesion or arthritis characterized by immunopathology, for example, encapsulated in liposomes coated with a tissue-specific antibody. The liposomes target the diseased tissue and are selectively taken up by the diseased tissue.

[0109] The dosing regimen depends on several factors including the serum or tissue turnover rate of the therapeutic antibody, the level of the symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of the target cells in the biological matrix. Preferably, the dosing regimen delivers a sufficient amount of the therapeutic antibody to effect improvement at the target disease site while minimizing unwanted side effects simultaneously. Thus, the amount of the biological agent delivered depends in part on the particular therapeutic antibody and the severity of the condition being treated. It is possible to obtain guidance in the selection of an appropriate dose of a therapeutic antibody [see, for example: Wawrzynczak Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK (1996); Kresina (ed.) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY (1991); Bach (ed.) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY (1993); Baert, et al. New Engl. J. Med. 348:601-608 (2003); Milgrom et al. New Engl. J. Med. 341:1966-1973 (1999); Slamon et al. New Engl. J. Med. 344:783-792 (2001); Beniaminovitz et al. New Engl. J. Med. 342:613-619 (2000); Ghosh et al. New Engl. J. Med. 348:24-32 (2003); Lipsky et al. New Engl. J. Med. 343:1594-1602 (2000)].

[0110] The determination of an appropriate dosage is made by a veterinarian, for example, using parameters or factors that are known or presumed in the art to affect the treatment. Generally, the administration starts at a somewhat lower amount than the optimal dosage and is then increased gradually until the desired or optimal effect is achieved as compared to negative side effects. Important diagnostic criteria include diagnostic criteria for symptoms, for example, diagnostic criteria for symptoms such as inflammation or the level of inflammatory cytokines produced.

[0111] The antibodies or antigen-binding fragments thereof disclosed herein can be provided by continuous infusion or, for example, by doses administered once a day, 1 to 7 times a week, once a week, once every two weeks, once a month, once every two months, once every three months, once every six months, once a year, etc. The doses can be provided, for example, intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. The total dose for one week is generally at least 0.05 μg / kg body weight, more generally at least 0.2 μg / kg, 0.5 μg / kg, 1 μg / kg, 10 μg / kg, 100 μg / kg, 0.25 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 5.0 mg / mL, 10 mg / kg, 25 mg / kg, 50 mg / kg or more [see, for example: Yang, et al. New Engl. J. Med. 349:427-434 (2003); Herold, et al. New Engl. J. Med. 346:1692-1698 (2002); Liu, et al. J. Neurol. Neurosurg. Psych. 67:451-456 (1999); Portielji, et al. Cancer Immunol. Immunother. 52:133-144 (2003)]. The doses can also be provided to achieve a predetermined target concentration (e.g., 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg / mL or more) of the canine anti-human CTLA-4 antibody in the serum of the subject. In another embodiment, the canine anti-human CTLA-4 antibody of the invention is administered subcutaneously or intravenously at 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg / subject based on once a week, once every two weeks, "once every four weeks", once a month, once every two months or once every three months.

[0112] As used herein, "inhibiting" or "treating" or "treatment" encompasses delaying the onset of symptoms associated with a disorder and / or reducing the severity of symptoms of such a disorder. These terms further encompass ameliorating existing uncontrolled or undesirable symptoms, preventing additional symptoms, and ameliorating or preventing the cause underlying such symptoms. Accordingly, these terms mean that a beneficial result has been provided to a vertebrate subject having or at risk of developing such a disorder, disease or symptom.

[0113] As used herein, the terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to an amount of an anti-human CTLA-4 antibody of the invention or an antigen-binding fragment thereof that is effective, alone or in combination with additional therapeutic agents, when administered to a cell, tissue or subject, to produce a measurable improvement in one or more symptoms of a disease or condition or in the progression of such a disease or condition. A therapeutically effective dose further refers to an amount of a binding compound sufficient to effect at least a partial amelioration of symptoms, e.g., treatment, cure, prevention or amelioration of a related medical condition or an increase in the rate of treatment, cure, prevention or amelioration of such a condition. When applied to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to the total amount of the active ingredients that, when administered together, whether continuously or simultaneously, produce a therapeutic effect. An effective amount of a therapeutic agent produces an improvement of at least 10%, usually at least 20%, preferably at least about 30%, more preferably at least 40%, most preferably at least 50% of a diagnostic criterion or parameter. An effective amount can also produce an improvement in subjective criteria, when such are used to evaluate the severity of a disease.

[0114] Other Combinatorial Therapies As described above, the canine anti-human CTLA-4 antibody or antigen-binding fragment thereof can be co-administered with one or more other therapeutic agents (e.g., chemotherapeutic agents). The antibody can be conjugated to the other therapeutic agent (as an immune complex), or can be administered separately from the other therapeutic agent. In the latter case (separate administration), the antibody can be administered before, after, or simultaneously with the other therapeutic agent, or can be co-administered with another known therapy.

[0115] Kits Furthermore, provided is a kit comprising one or more components that specifically bind to CTLA-4 (including, but not limited to, the antibodies or antigen-binding fragments discussed herein) (e.g., the canine anti-human CTLA-4 antibody or antigen-binding fragment thereof of the present invention) together with one or more additional components (including, but not limited to, the pharmaceutically acceptable carriers and / or chemotherapeutic agents discussed herein). The binding composition and / or the chemotherapeutic agent can be formulated as a pure composition or in combination with a pharmaceutically acceptable carrier in a pharmaceutical composition.

[0116] In one embodiment, the kit comprises the binding composition of the present invention (e.g., the canine anti-human CTLA4 antibody or its pharmaceutical composition containing the amino acid sequences of SEQ ID NO: 36 and SEQ ID NO: 46) in one container (e.g., a sterile glass or plastic vial) and a pharmaceutical composition and / or chemotherapeutic agent in another container (e.g., a sterile glass or plastic vial).

[0117] In another embodiment, the kit comprises the combination of the present invention comprising a binding composition component (e.g., the canine anti-human CTLA4-antibody containing the amino acid sequences of SEQ ID NO: 36 and SEQ ID NO: 46) together with a pharmaceutically acceptable carrier, optionally in combination with one or more therapeutic agent components formulated together (optionally in a pharmaceutical composition), in a single common container.

[0118] If the kit contains a pharmaceutical composition for parenteral administration to a subject, the kit can include an apparatus for performing such administration. For example, the kit can include one or more hypodermic needles or other injection devices discussed above. The kit can further include a package insert containing information regarding the pharmaceutical composition and dosage form in the kit. Generally, such information is useful for the pet owner and veterinarian to use the enclosed pharmaceutical composition and dosage form effectively and safely. For example, the following information regarding the combination of the present invention can be provided in the package insert: pharmacokinetics, pharmacodynamics, clinical trials, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdose, appropriate dosage and administration, supply method, appropriate storage conditions, references, manufacturer / distributor information, and patent information.

[0119] For convenience, the antibodies or specific binding agents disclosed herein can be provided in a kit, i.e., a packaged combination of a predetermined amount of reagent together with instructions for performing a diagnostic or detection assay. If the antibody is labeled with an enzyme, the kit contains a substrate and cofactors required by the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives, such as stabilizers, buffers (e.g., blocking buffer or lysis buffer), etc. are also included. The relative amounts of the various reagents can be varied widely to provide concentrations of the reagents in solution that substantially optimize the sensitivity of the assay. In particular, the reagent can be provided as a usually lyophilized, dry powder containing an excipient that provides a reagent solution having an appropriate concentration when dissolved.

Examples

[0120] Example 1 Construction of Anti-CTLA-4 Chimeric Antibodies Prior art monoclonal antibodies raised against human CTLA-4, which are known to both bind to human CTLA-4 and block the binding of human CTLA-4 to human CD86, were also thought to potentially bind to canine CTLA-4 and perhaps block canine CD86 and canine CTLA-4. To test this possibility, a known nucleotide sequence corresponding to the heavy chain variable region of the anti-human CTLA-4 monoclonal antibody 3B10 (disclosed in WO2012120125) was fused to the nucleotide sequence of a modified canine constant heavy chain (CH1-hinge-CH2-3) to generate a chimeric mouse-canine heavy chain nucleotide sequence shown as SEQ ID NO: 1. A second known nucleotide sequence encoding the amino acid sequence corresponding to the light chain variable region of anti-human CTLA-4 was fused to the nucleotide sequence of the canine constant kappa light chain domain to generate a chimeric mouse-canine light chain nucleotide sequence shown as SEQ ID NO: 3. The protein encoded by the chimeric mouse-canine heavy chain nucleotide sequence contains the amino acid sequence of SEQ ID NO: 2; and the protein encoded by the chimeric mouse-canine light chain nucleotide sequence contains the amino acid sequence of SEQ ID NO: 4. The chimeric human-canine heavy and light chains were cloned into separate expression plasmids using standard molecular biology techniques. Plasmids containing the heavy and light chain genes were transfected into HEK293 cells, and the expressed antibody was purified from the HEK293 cell supernatant using Protein A.

[0121] Similarly, the chimeric antibody 8H5 containing variable domains previously disclosed in WO2012120125 was also constructed as exemplified by antibodies formed by combinations of heavy and light chains containing the DNA sequences and amino acid sequences described as SEQ ID NO: 5 to SEQ ID NO: 8, respectively. Furthermore, the chimeric antibodies 411, 418, and 611 containing variable domains disclosed in WO2000037504 were also constructed as exemplified by antibodies formed by combinations of heavy and light chains containing the nucleic acid sequences and amino acid sequences described as SEQ ID NO: 9 to SEQ ID NO: 20, respectively. The chimeric antibodies 10D1 and 1E2 containing variable domains disclosed in U.S.8,017,114B2 were also constructed as exemplified by antibodies formed by combinations of heavy and light chains containing the nucleotide sequences and amino acid sequences described as SEQ ID NO: 21 to SEQ ID NO: 28, respectively. Furthermore, the chimeric antibody 3B3 containing variable domains disclosed in WO2010097597 was also constructed as exemplified by antibodies formed by combinations of heavy and light chains containing the nucleotide sequences and amino acid sequences described as SEQ ID NO: 29 to SEQ ID NO: 32, respectively. The amino acid sequence identification numbers of the chimeric antibodies are shown in Table 1 below.

Table 3

[0122] Example 2 Reactivity of Anti-CTLA-4 Chimeric Antibodies The chimeric antibody of Example 1 was expressed in Expi293 cells [Expi293 expression system obtained from THERMO FISHER SCINETIFIC (registered trademark)] and purified by a Protein A column. Then, the chimeric antibody was tested for reactivity with canine CTLA-4 by ELISA as follows: 1. Coated 200 ng / well of CTLA-4 on an immunoplate and incubated the plate overnight at 4°C; 2. Washed the plate three times with PBS containing 0.05% Tween 20 (PBST); 3. The plate was blocked with 0.5% BSA in PBS at room temperature for 45 - 60 minutes; 4. The plate was washed three times with PBST; 5. In each column or row of the dilution plate, the antibody was diluted three - fold; 6. The diluted antibody was transferred to each column or row of the immunoplate, and the plate was incubated at room temperature for 45 - 60 minutes; 7. The plate was washed three times with PBST; 8. 1:2000 - diluted horseradish peroxidase - labeled anti - dog IgG Fc was added to each well of the plate, and the plate was incubated at room temperature for 45 - 60 minutes; 9. The plate was washed three times with PBST; 10. TMB substrate was added to each well of the plate, and the plate was incubated at room temperature for 10 - 15 minutes to develop color; 11. 100 μL of 1.5 M phosphoric acid was added to each well to stop the reaction; 12. The plate was read at 450 nm using a reference wavelength of 540 nm.

[0123] The results of the ELISA are shown in Figure 1, and it shows that the selected antibody was able to bind to dog CTLA - 4. Furthermore, the 3B10 chimeric antibody was found to be the best binder among this group of chimeric antibodies.

[0124] Example 3 Construction of Canine Anti-Human CTLA-4 Monoclonal Antibody 3B10 Since it was confirmed that the 3B10 chimeric antibody had the strongest binding affinity for canine CTLA-4 (see Figure 1), a set of six CDRs of this chimeric antibody was selected and added to the canine framework. To carry out the process of caninization, DNA sequences encoding the heavy and light chains of canine IgG were identified [see U.S. 10,106,607 B2; the content of which is hereby incorporated by reference in its entirety]. The DNA sequences and protein sequences of the canine heavy and light chains are known in the art and can be obtained by searching the NCBI gene and protein databases. There are four known IgG subtypes of canine IgG, which are designated IgG-A, IgG-B, IgG-C, and IgG-D. Canine antibodies have two types of light chains, called kappa and lambda.

Table 4

Table 5

[0125] Although not restricted to a particular approach, the overall process of generating caninized heavy and light chains that can be mixed in various combinations to produce caninized anti-human CTLA-4 mAb may include the following scheme: (i) Identify the CDRs of the H and L chains of the desired anti-CTLA-4 mAb. Reverse translate the amino acid sequences of those CDRs into appropriate DNA sequences; (ii) Identify the appropriate DNA sequences for the H and L chains of canine IgG (e.g., the modified heavy chain and light kappa chain of IgG-B described in U.S. 10,106,607 B2); (iii) Identify the DNA sequences encoding the endogenous CDRs of the canine IgG H and L chain DNAs of the above sequences; (iv) Replace the DNA sequence encoding the endogenous canine H-chain and L-chain CDRs with the DNA sequence encoding the desired anti-human CTLA-4 CDRs. When using the modified canine IgG-B, the modifications at D31A and N63A numbered in SEQ ID NO: 69 were performed in canine IgG-B as previously described in U.S. 10,106,607 B2 (which is incorporated herein by reference in its entirety). Additionally, optionally, the DNA encoding some canine framework amino acid residues was replaced with the DNA encoding selected amino acid residues from the desired anti-human CTLA-4 mAb canine region. This process is referred to as back-mutation. Two sets of four mutants for each heavy chain and one set of three mutants for the light chain variable region, described in Tables 4 and 5, were developed using each mutant of the set containing different back-mutation sites; (v) Synthesize the DNA from step (iv) and clone it into an appropriate expression plasmid; (vi) Transfect HEK293 cells with the plasmid containing the desired humanized H-chain and L-chain; (vii) Purify the expressed humanized antibody from the HEK293 supernatant; (viii) Test the purified humanized antibody for binding to canine CTLA-4.

[0126] A set of humanized H-chain and L-chain sequences was developed. The sequence numbers are described in Tables 4 and 5 below. [Table 6] [Table 7]

[0127] The present invention provides a canineized antibody formed by any combination of the canineized heavy and light chains described in Tables 4 and 5 above. Subsequently, the antibody was expressed in Expi293 cells and purified. The ELISA results shown in Figure 2 indicate that 3B10 was successfully canineized, and that 3B10L3H2, 3B10L3H3, and 3B10L3H4 all have similar tight binding activity to the parental mouse-canine chimeric antibody of 3B10.

[0128] Example 4 Blocking Activity of Chimeric and Canine Anti-Human CTLA-4 Monoclonal Antibody 3B10 against the Interaction between Canine CD86 and CTLA-4 To confirm that the canineized 3B10 antibody maintains the same neutralizing activity as the parental antibody against canine CTLA-4, the canineized antibody was tested by ELISA for its blocking activity against the interaction between canine-CD-80 and canine CTLA-4 as follows: 1. Coated CTLA-4 at 200 ng / well on an immunoplate and incubated the plate overnight at 4°C; 2. Washed the plate three times with PBS containing 0.05% Tween 20 (PBST); 3. Blocked the plate with 0.5% BSA in PBS for 45 - 60 minutes at room temperature; 4. Washed the plate three times with PBST; 5. In each column or row of the dilution plate, the antibody was diluted three-fold, then biotinylated CD-86 at 100 ng / well was added and mixed with the antibody; 6. Transferred the mixture of the diluted antibody and CD-86 to each column or row of the immunoplate and incubated the plate for 45 - 60 minutes at room temperature; 7. Washed the plate three times with PBST; 8. Added streptavidin conjugated with horseradish peroxidase diluted 1:2000 to each well of the plate and incubated the plate for 45 - 60 minutes at room temperature; 9. Washed the plate three times with PBST; 10. TMB substrate was added to each well of the plate, and the plate was incubated at room temperature for 10 - 15 minutes to develop color; 11. 100 μL of 1.5 M phosphoric acid was added to each well to stop the reaction; 12. The plate was read at 450 nm using a reference wavelength of 540 nm.

[0129] The plot of the ELISA plate results shown in Figure 3 indicates that the canine 3B10 mutant has blocking activity similar to that of the parental 3B10.

[0130] Example 5 IFNγ Production by Canine PBMC Activated with Chimeric Antibodies Introduction: Interferon - gamma (IFN - γ; also known as type II interferon) is mainly produced by activated T lymphocytes and possibly by natural killer cells. This property has been widely applied as an indicator of T - cell activation by quantitatively testing IFN - γ production in ELISA - based assays. To identify functional antibodies against CTLA - 4 (anti - CTLA - 4 antibodies), the selected antibodies were tested for their activity to stimulate IFN - γ production in canine peripheral blood mononuclear cells using the following protocol.

[0131] Experiments and Results Isolation of Canine Peripheral Blood Mononuclear Cells 1. Approximately 20 mL of whole blood was collected into an EDTA or sodium heparin tube; 2. The blood was transferred to a 50 mL polystyrene tube and diluted 50:50 with Hank's balanced salt solution (HBSS); 3. 15 mL of Ficoll - PlaquePlus was added to 4 × 50 mL SepMate TM tubes. Then, approximately 10 mL of the 50:50 - diluted blood was slowly added to the side of each SepMate TM tube containing Ficoll; 4. The tube was centrifuged at 1200×g for 20 minutes; 5. Cells were collected from the gradient interface and transferred to 50 mL polypropylene tubes. HBSS was added to the 40 - 45 mL mark, and the cells were centrifuged at 800×g for 10 minutes; 6. The supernatant was discarded, the cells were resuspended in 40 - 45 mL HBSS, and the tube was centrifuged again at 800×g for 10 minutes; 7. The supernatant was discarded, and the cells from each tube were resuspended in 2 mL of canine lymphocyte medium (cells were pooled from the same animal); 8. A small aliquot of the cell suspension was taken, mixed with 0.04% trypan blue, and the cell count was determined; 9. The cell suspension was stored at 2 - 7°C until use [however, it was not stored at 2 - 7°C starting 24 hours before use].

[0132] Cell Proliferation Assay for Canine Peripheral Blood Mononuclear Cells 1. The antibody was diluted in canine lymphocyte medium [RPMI medium (catalog number 12 - 167Q) purchased from LONZA, or equivalent] to a final concentration of 40 μg / mL (prepared at 160 μg / mL) and sterilized using a 0.2 μm syringe filter. The antibody was serially diluted 2 - fold in a sterile dilution plate and left; 2. The cells were diluted in canine lymphocyte medium to 2.5×10 6 cells / mL, and 100 μL per well was dispensed into all wells of a 96 - well tissue culture plate; 3. Con A was diluted in canine lymphocyte medium to a final concentration of 250 ng / mL (prepared at 1000 ng / mL), sterilized using a 0.2 μm syringe filter, and 50 μL was added to all wells. (Note: Con A was not added to one row of 8 wells for the cell - only control and the wells for the cell + mAb - only control); 4. 100 μL of canine lymphocyte medium per well was added to the cell - only wells, and 50 μL of medium was added to the row containing the Con A control wells (Con A + cells without mAb treatment); 5. 50 μL of the diluted mAb was added to the replicate wells; 6. The plates were incubated in a humidified incubator at 36 ± 2 °C and 4.0 - 6.0% CO2 for 68 - 124 hours.

[0133] IFNγ ELISA 1. After 68 - 124 hours of incubation, the plates were centrifuged at 800 × g for 10 minutes; 2. Supernatants were collected from each well and pool replicate. These samples can be frozen at -50 °C or below for later use or can be tested immediately; 3. If necessary, the supernatant samples were appropriately diluted and IFN-γ ELISA was performed according to the instructions of the Canine IFN-γ Quantikine TM ELISA kit [R&D Systems catalog number CAIF00].

[0134] The results shown in Figure 4 indicate that the chimeric antibodies tested (especially 3B10, 411, and 611) significantly activated canine T cells to produce IFN-γ.

[0135] Example 6 Epitope Mapping of Canine 3B10 Antibody The binding epitopes of the canineized antibody 3B10L3H2 on canine CTLA-4 (NCBI reference sequence: NP_001003106; accession number 68) were mapped by chemical cross-linking and mass spectrometry. The results indicate that the antibody binds to two separate linear regions on the antigen CTLA-4 containing the amino acids R33, R38, S42, T45, R83, T87, Y90, K93, and Y98 (see Figure 5). Figure 5 shows that the amino acid residues related to the non-adjacent epitopes of CTLA-4 to which the canineized antibody 3B10L3H2 binds are composed of two linear parts (amino acid residues 30 - 50 and amino acid residues 80 - 100, respectively).

Table 8

[0136] In all of the above nucleotide sequences, the variable regions are shown in bold, in the corresponding amino acid sequences, the sequences related to the variable regions are shown in bold, and the CDRs are underlined and shown in bold.

Claims

**Claim 1**: An isolated canine antibody or antigen-binding fragment thereof that specifically binds to canine cytotoxic T lymphocyte antigen 4 (CTLA-4) and comprises a canine IgG heavy chain and a canine kappa light chain; wherein the canine kappa light chain comprises three light chain complementarity determining regions (CDRs) [CDR light 1 (CDRL1), CDR light 2 (CDRL2), and CDR light 3 (CDRL3)]; and the canine IgG heavy chain comprises three heavy chain CDRs [CDR heavy 1 (CDRH1), CDR heavy 2 (CDRH2), and CDR heavy 3 (CDRH3)]. (a) wherein CDRL1 comprises the amino acid sequence of SEQ ID NO: 54; (b) wherein CDRL2 comprises the amino acid sequence of SEQ ID NO: 56; (c) wherein CDRL3 comprises the amino acid sequence of SEQ ID NO: 58; (d) wherein CDRH1 comprises the amino acid sequence of SEQ ID NO: 48; (e) wherein CDRH2 comprises the amino acid sequence of SEQ ID NO: 50; and (f) wherein CDRH3 comprises the amino acid sequence of SEQ ID NO: 52; wherein the isolated canine antibody or antigen-binding fragment thereof inhibits the binding of canine CTLA-4 to canine CD80, inhibits the binding of canine CTLA-4 to canine CD86, or inhibits the binding of canine CTLA-4 to both canine CD80 and canine CD86 The isolated canine antibody or antigen-binding fragment thereof. **Claim 2** The isolated canine antibody or antigen-binding fragment thereof according to claim 1, wherein the canine IgG heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, and SEQ ID NO:

66. **Claim 3** The isolated canine antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the kappa light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO:

46. Claim 4 The isolated canine antibody or antigen-binding fragment thereof according to claim 1, wherein the canine IgG heavy chain comprises the amino acid sequence of SEQ ID NO: 36 and the canine kappa light chain comprises the amino acid sequence of SEQ ID NO:

46. Claim 5 The isolated canine antibody or antigen-binding fragment thereof according to claim 1, wherein the canine IgG heavy chain comprises the amino acid sequence of SEQ ID NO: 62 and the canine kappa light chain comprises the amino acid sequence of SEQ ID NO:

46. Claim 6 An isolated nucleic acid encoding the canine kappa light chain of the canine antibody or antigen-binding fragment thereof according to claim 1, 2, 3, 4 or 5. Claim 7 An isolated nucleic acid encoding the canine IgG heavy chain of the canine antibody or antigen-binding fragment thereof according to claim 1, 2, 3, 4 or 5. Claim 8 An expression vector comprising the isolated nucleic acid according to claim 6 or 7. Claim 9 A host cell comprising one or more expression vectors according to claim 8. Claim 10 A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to claim 1, 2, 3, 4 or 5 and a pharmaceutically acceptable carrier or diluent. Claim 11 A method of increasing the activity of immune cells, comprising administering to a non-human subject in need thereof a therapeutically effective amount of the pharmaceutical composition according to claim 10. Claim 12 The method as claimed in claim 11, wherein the method is (i) for treating cancer; (ii) for treating an infectious disease or an infectious disorder; or (iii) as a vaccine adjuvant. used. Claim 13 Use of the pharmaceutical composition according to claim 10 in the manufacture of a medicament for increasing the activity of immune cells in a non-human subject in need of increasing the activity of immune cells.

14. Use of a pharmaceutical composition in the manufacture of a medicament according to claim 13, wherein the medicament is (i) for treating cancer; (ii) for treating an infectious disease or an infectious disorder; or (iii) as a vaccine adjuvant; used, the use.

15. A method for generating a canine antibody or an antigen-binding fragment thereof that specifically binds to CTLA-4, comprising a. culturing the host cell according to claim 9 in a medium under conditions in which the nucleic acid is expressed, thereby generating a polypeptide comprising a light chain and a heavy chain variable region; and b. recovering the polypeptide from the host cell or the medium; the method comprising.

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