Canine-derived antibodies against canine CTLA-4

Abstract

The present invention provides canine-derived murine antibodies against canine CTLA-4, which have a specific sequence and a high binding affinity for canine CTLA-4. The present invention further provides epitopes of canine CTLA-4 for use in canine-derived murine antibodies against canine CTLA-4. 5 The invention also relates to the use of these antibodies in the treatment of cancer in canine and other companion animals.

Description

[0001] This application is a divisional application of the invention patent application with application date of July 15, 2020, application number 202080050841.7, and invention name “Caninized antibodies against canine CTLA-4”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority under 35 USC §119(e) to provisional applications U.S. Serial No. 62 / 874,287 filed on July 15, 2019, U.S. Serial No. 62 / 926,047 filed on October 25, 2019, and U.S. Serial No. 63 / 048,873 filed on July 7, 2020, the entire contents of which are incorporated herein by reference. Field of the Invention

[0004] The present invention relates to antibodies against proteins of co-stimulatory or co-inhibitory signal transduction pathways, including CTLA-4. More particularly, the present invention further relates to caninized antibodies against canine CTLA-4, which have specific sequences and high binding affinity to canine CTLA-4. The present invention also relates to the use of the antibodies of the present invention in treating cancer in canines. Summary of the invention

[0005] The initiation or termination of an immune response is mediated via signaling pathways activated by a complex interaction between a group of proteins expressed on the surface of many immune cells, most notably T lymphocytes and antigen presenting cells (APCs). Co-stimulatory signaling pathways lead to the development of an immune response and have been shown to be most importantly mediated by 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) families on the surface of APCs. B7.1 and B7.2 are thought to perform similar functions.

[0006] In contrast, the co-inhibitory pathway results in the inhibition or termination of the immune response and has been shown to be mediated by the interaction between cytotoxic T lymphocyte-associated protein 4 (CTLA-4) on T cells and CD80 / CD86 proteins on APCs. An additional co-inhibitory signaling pathway has been shown to be mediated by 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. In addition, it has been shown that the interaction between PD-L1 and CD80 can also generate inhibitory signals in T cells.

[0007] CD80 and CD86 are members of the immunoglobulin (Ig) superfamily [Sharpe and Freeman, Nature Reviews, 2: 116-126 (2002)]. CD80 is expressed on activated B cells, activated T cells, as well as macrophages and dendritic cells [Swanson and Hall, Eur J. Immunol., 23: 295-298 (1993); Razi-Wolfe et al., PNAS, 89: 4210-4214 (1992)]. CD86 is constitutively expressed on dendritic cells, Langerhans cells and B cells. In addition, CD 86 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)].

[0008] CD80 and CD86 bind to CD28 and CTLA-4 with different functional consequences [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)]. CD80 and CD86 bind to CTLA-4 with much higher affinity than CD80 / CD86 binds to CD28 [van der Merwe, J. Exp. Med. 185:393-402 (1997)].

[0009] 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 of 134 amino acid residues, which contains the hexapeptide motif MYPPPY that is 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 most CD4+ T cells and about 50% of CD8+ 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, the binding of B7.1 / B7.2 to CD28 provides a critical co-stimulatory signal for T cells, allowing T cell activation and subsequent immune response development [Reiser et al., PNAS, 89: 271-275 (1992); Jenkins et al., J. Immunol., 147: 2461-2466 (1991)]. It has been shown that in the absence of CD28 signals, T cells undergo apoptosis or enter an unresponsive state [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), reduce the time required to stimulate naive cells and enhance the amplitude of T cell responses [Soskic et al., Advances in Immunology, 124: 96-123 (2014)].

[0010] 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)]. In addition, CTLA-4 shares about 30% amino acid identity with CD28. CTLA-4 is not constitutively expressed on naive T cells, although it is rapidly upregulated shortly after CD28 ligation and T cell activation, with the peak expression level of CTLA-4 being about 48-96 hours after naive T cell activation [Alegret 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 much higher affinity than CD28 [van der Merwe et al., J. Exp. Med., 185:393-402 (1997)]. However, in contrast to the stimulatory effects of CD28 binding to B7.1 or B7.2, CTLA-4 acts as an inhibitory receptor that is critical for downregulation of immune responses [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 CD80 / CD86 [reviewed in Swanson, Immunology, 1010: 169-177 (2000)]. The key role of CTLA-4 in immune downregulation was demonstrated in CTLA-4-deficient mice, which died at 3-5 weeks of age due to the development of a lymphoproliferative disease characterized by T cell infiltration of multiple organs [Tivol et al., Immunity, 3: 541-5417 (1995); Waterhouse et al., Science, 270: 985-988 (1995)]. It has also been demonstrated that the consequences of CTLA-4 knockout depend on the interaction of CD28 with its ligands CD80 and CD86, as shown by the lack of disease in CTLA-4 / CD80 / CD86 triple knockout mice [Mandelbrot et al., J. Exp. Med., 189:435-440 (1999)].This was also confirmed by the protection against lymphoproliferation obtained by repeated administration of CTLA-4Ig in CTLA-4 knockout mice [Tivol et al., J Immunol., 158:5091-5094 (1997)].

[0011] In addition, blocking the action of CTLA-4 with antibodies has been shown to enhance T cell responses in vitro and in vivo and increase anti-tumor immune responses [Leach et al., Science, 271: 1734-1736 (1996)]. Based on these findings, development of CTLA-4 blockers such as monoclonal antibodies is underway 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); WO2000037504 A2; US8,017,114 B2; WO2010097597A1; WO2012120125A1; and Boutros et al., Nat Rev Clin Oncol.,13(8):473-486(2016)].

[0012] PD-1 is a member of the CD28 / CTLA-4 immunomodulatory receptor family. PD-1 is also a member of the Ig superfamily and contains an extracellular variable domain that binds its ligand and a cytoplasmic tail that binds a signaling molecule [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)]. PD-1 expression is not found on unstimulated T cells, B cells, or bone marrow cells. However, after activation, PD-1 expression is upregulated on these cells [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, sharing approximately 24% amino acid identity [Jin et al., Current Topics in Microbiology and Immunology, 350: 17-37 (2010)]. When bound to PD-L1 and PD-L2 expressed on the surface of APCs, PD-1 attenuates T cell activation. Binding of any of these ligands to PD-1 negatively regulates antigen signaling via the T cell receptor (TCR). To date, only PD-L1 and PD-L2 have been found to act as ligands for PD-1. Like CTLA-4, PD-1 binding appears to transmit negative immunoregulatory signals. Ligation of PD-1 by PD-L1 or PD-L2 results in inhibition of TCR-mediated proliferation and cytokine production [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 effects observed is less severe than those displayed by CTLA-4-deficient animals [Nishimura et al., Immunity, 11(2):141–151 (1999); Nishimura et al., Science, 291(5502):319–322 (2001)].Although the PD-1 signaling pathway is currently under intensive investigation, studies to date indicate that the PD-L1 / PD-L2 / PD-1 interaction is involved in the negative regulation of some immune responses, as reduced signaling downstream of TCR stimulation leads to reduced cytokine secretion and impaired T cell proliferation as well as reduced production of cytotoxic molecules by T cells [Freeman et al., J. Exp. Med., 192(7):1027–1034 (2000)].

[0013] PD-L1 (CD274) is a type 1 membrane protein consisting of IgV-like and IgC-like extracellular domains, a hydrophobic transmembrane domain, and a short cytoplasmic tail (consisting of 30 amino acids) with unknown signal transduction properties. PD-L1 is considered to be a member of the B7 family and shares approximately 20% amino acid identity with B7 family members. PDL1 binds to its receptor PD-1 found on activated T cells, B cells, and myeloid cells. PD-L1 also binds to the co-stimulatory molecule CD80, but not CD86 [Butte et al., Immunology, 45(13):3567–3572(2008)]. The affinity of CD80 for PD-L1 is between its affinity for CD28 and CTLA-4. The related molecule PD-L2 has no affinity for CD80 or CD86, but shares PD-1 as a receptor. Engagement of PD-L1 with its receptor PD-1 on T cells delivers signals that inhibit TCR-mediated IL-2 production and T cell proliferation. Binding of PD-L1 to PD-1 also contributes to ligand-induced TCR downregulation during antigen presentation to naive T cells. In addition, binding of PD-L1 to CD80 on T cells leads to T cell apoptosis. The role of PD1 and PD-L1 as inhibitors of T cell activation has been demonstrated in many studies. Based on these findings, the development of PD-1 and PD-L1 blockers such as monoclonal antibodies is being conducted to provide a therapeutic modality for the treatment of cancer and infectious diseases.

[0014] Humanized monoclonal antibodies that block the binding and activity of canine PD-1, PDL1, and CTLA-4 have been developed and are currently available for treating human subjects diagnosed with one of several different types of cancer. Similarly, caninized monoclonal antibodies that block the binding and activity of canine PD-1 and PD L1 have also been reported [US9,944,704B2, US10,106,607B2, and US2018 / 0237535A1, the entire contents of which are incorporated herein by reference]. However, there have been no reports to date of caninized monoclonal antibodies that block the binding and activity of canine CTLA-4.

[0015] Citation of any reference herein should not be construed as an admission that such reference is available as "prior art" to the present application.

[0016] Brief description of the invention

[0017] The present invention relates to anti-canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibodies that bind to canine CTLA-4. In a specific embodiment, the antibody to canine CTLA-4 specifically binds to canine CTLA-4. In a more specific embodiment, the antibody to canine CTLA-4 also has the ability to block the binding of canine CTLA-4 to canine CD80. In other specific embodiments, the antibody to canine CTLA-4 also has the ability to block the binding of canine CTLA-4 to canine CD86. In still other specific embodiments, the antibody to canine CTLA-4 also has the ability to simultaneously block the binding of canine CTLA-4 to canine CD80 and block the binding of canine CTLA-4 to canine CD86.

[0018] In addition, the present invention relates to the complementarity determining regions (CDRs) comprised by these antibodies and the combination of these CDRs (e.g., obtained from murine anti-canine CTLA-4 antibodies) in a canine framework to form caninized anti-canine CTLA-4 antibodies. The present invention also relates to the use of such antibodies in the treatment of conditions such as cancer.

[0019] Thus, the present invention provides unique CDR sets from six (6) exemplary murine anti-canine CTLA-4 antibodies. The six exemplary murine anti-canine CTLA-4 antibodies have unique CDR sets, namely three light chain CDRs: CDR light chain 1 (CDRL1), CDR light chain 2 (CDRL2), and CDR light chain 3 (CDRL3) and three heavy chain CDRs: CDR heavy chain 1 (CDRH1), CDR heavy chain 2 (CDRH2), and CDR heavy chain 3 (CDRH3). As described in detail below, there is substantial sequence homology within each set of CDRs, and even some redundancy (e.g., see V in Table 1 below). L CDR-3' group). Therefore, the present invention not only provides the amino acid sequences of the six CDRs from six exemplary murine anti-canine CTLA-4 antibodies, but also provides conservatively modified variants of these CDRs, as well as variants that comprise (e.g., share) the same canonical structure and / or bind to one or more (e.g., 1, 2, 3, 4 or more) amino acid residues of canine CTLA-4 that are encompassed by an epitope of canine CTLA-4.

[0020] One aspect of the invention provides a mammalian antibody that binds to canine cytotoxic T lymphocyte-associated protein 4 (CTLA-4). In a specific embodiment, the mammalian antibody or antigen-binding fragment thereof of the invention is a murine antibody. In a preferred embodiment, the mammalian antibody (including the murine antibody of the invention) or antigen-binding fragment thereof of the invention is a caninized antibody or a caninized antigen-binding fragment thereof.

[0021] In a specific embodiment, the mammalian antibody specifically binds to canine CTLA-4. In a more specific embodiment, the mammalian antibody to canine CTLA-4 also has the ability to block the binding of canine CTLA-4 to canine CD80. In other specific embodiments, the mammalian antibody to canine CTLA-4 also has the ability to block the binding of canine CTLA-4 to canine CD86. In still other specific embodiments, the mammalian antibody to canine CTLA-4 has the ability to block both the binding of canine CTLA-4 to canine CD 80 and the binding of canine CTLA-4 to canine CD 86.

[0022] In certain embodiments, the mammalian antibody that binds to canine CTLA-4 is an isolated antibody. The invention also provides antigen-binding fragments of any of these mammalian antibodies that bind to canine CTLA-4. In a specific embodiment, the antibody comprises three light chain complementary determining regions: CDR light chain 1 (CDRL1), CDR light chain 2 (CDRL2), and CDR light chain 3 (CDRL3); and three heavy chain CDRs: CDR heavy chain 1 (CDRH1), CDR heavy chain 2 (CDRH2), and CDR heavy chain 3 (CDRH3).

[0023] In a specific embodiment, the mammalian antibody or antigen-binding fragment thereof comprises a CDRH3 comprising the amino acid sequence of SEQ ID NO: 90, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 90, or a variant of SEQ ID NO: 90 comprising canonical structural class 7. In a more specific embodiment, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRH2 comprising the amino acid sequence of SEQ ID NO: 88, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 88, or a variant of SEQ ID NO: 88 comprising canonical structural class 2A. In an even more specific embodiment, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 86, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 86, or a CDRH1 comprising a variant of SEQ ID NO: 86 comprising canonical structural class 1. In still more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRH3 comprising the amino acid sequence of SEQ ID NO: 96, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 96, or a variant of SEQ ID NO: 96 comprising canonical structural class 1. In still more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRH2 comprising the amino acid sequence of SEQ ID NO: 94, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 94, or a variant of SEQ ID NO: 94 comprising canonical structural class 1. In even more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 92, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 92, or a variant of SEQ ID NO: 92 comprising canonical structural class 4.

[0024] In alternative embodiments, the mammalian antibody or antigen-binding fragment thereof comprises a CDRH3 comprising the amino acid sequence of SEQ ID NO: 102, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 102, or a variant of SEQ ID NO: 102 comprising canonical structural class 9. In even more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRH2 comprising the amino acid sequence of SEQ ID NO: 100, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 100, or a variant of SEQ ID NO: 100 comprising canonical structural class 4. In even more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises: a CDRH1 comprising the amino acid sequence of SEQ ID NO: 98, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 98, or a CDRH1 comprising a variant of SEQ ID NO: 98 comprising canonical structural class 1. In still more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRL3 comprising the amino acid sequence of SEQ ID NO: 108, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 108, or a variant of SEQ ID NO: 108 comprising canonical structural class 1. In still more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRL2 comprising the amino acid sequence of SEQ ID NO: 106, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 106, or a variant of SEQ ID NO: 106 comprising canonical structural class 1. In even more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 104, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 104, or a variant of SEQ ID NO: 104 comprising canonical structural class 1.

[0025] In other alternative embodiments, the mammalian antibody or antigen-binding fragment thereof comprises a CDRH3 comprising the amino acid sequence of SEQ ID NO: 113, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 113, or a variant of SEQ ID NO: 113 comprising canonical structural class 7. In more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRH2 comprising the amino acid sequence of SEQ ID NO: 88, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 88, or a variant of SEQ ID NO: 88 comprising canonical structural class 2A. In even more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises: a CDRH1 comprising the amino acid sequence of SEQ ID NO: 86, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 86, or a CDRH1 comprising a variant of SEQ ID NO: 86 comprising canonical structural class 1. In still more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRL3 comprising the amino acid sequence of SEQ ID NO: 96, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 96, or a variant of SEQ ID NO: 96 comprising canonical structural class 1. In still more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRL2 comprising the amino acid sequence of SEQ ID NO: 94, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 94, or a variant of SEQ ID NO: 94 comprising canonical structural class 1. In even more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 117, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 117, or a variant of SEQ ID NO: 117 comprising canonical structural class 4.

[0026] In still other alternative embodiments, the mammalian antibody or antigen-binding fragment thereof comprises a CDRH3 comprising the amino acid sequence of SEQ ID NO: 115, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 115, or a variant of SEQ ID NO: 115 comprising canonical structural class 7. In more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRH2 comprising the amino acid sequence of SEQ ID NO: 88, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 88, or a variant of SEQ ID NO: 88 comprising canonical structural class 2A. In even more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises: a CDRH1 comprising the amino acid sequence of SEQ ID NO: 86, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 86, or a CDRH1 comprising a variant of SEQ ID NO: 86 comprising canonical structural class 1. In still more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRL3 comprising the amino acid sequence of SEQ ID NO: 96, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 96, or a variant of SEQ ID NO: 96 comprising canonical structural class 1. In still more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRL2 comprising the amino acid sequence of SEQ ID NO: 122, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 122, or a variant of SEQ ID NO: 122 comprising canonical structural class 1. In even more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 119, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 119, or a variant of SEQ ID NO: 119 comprising canonical structural class 4.

[0027] In still other alternative embodiments, the mammalian antibody or antigen-binding fragment thereof comprises a CDRH3 comprising the amino acid sequence of SEQ ID NO: 114, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 114, or a variant of SEQ ID NO: 114 comprising canonical structural class 7. In more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRH2 comprising the amino acid sequence of SEQ ID NO: 111, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 111, or a variant of SEQ ID NO: 111 comprising canonical structural class 2A. In even more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises: a CDRH1 comprising the amino acid sequence of SEQ ID NO: 109, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 109, or a CDRH1 comprising a variant of SEQ ID NO: 109 comprising canonical structural class 1. In still more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRL3 comprising the amino acid sequence of SEQ ID NO: 96, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 96, or a variant of SEQ ID NO: 96 comprising canonical structural class 1. In still more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRL2 comprising the amino acid sequence of SEQ ID NO: 121, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 121, or a variant of SEQ ID NO: 121 comprising canonical structural class 1. In even more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 118, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 118, or a variant of SEQ ID NO: 118 comprising canonical structural class 4.

[0028] In still other alternative embodiments, the mammalian antibody or antigen-binding fragment thereof comprises a CDRH3 comprising the amino acid sequence of SEQ ID NO: 116, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 116, or a variant of SEQ ID NO: 116 comprising canonical structural class 12. In more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRH2 comprising the amino acid sequence of SEQ ID NO: 112, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 112, or a variant of SEQ ID NO: 112 comprising canonical structural class 2A. In even more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises: a CDRH1 comprising the amino acid sequence of SEQ ID NO: 110, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 110, or a CDRH1 comprising a variant of SEQ ID NO: 110 comprising canonical structural class 1. In still more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRL3 comprising the amino acid sequence of SEQ ID NO: 124, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 124, or a variant of SEQ ID NO: 124 comprising canonical structural class 1. In still more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRL2 comprising the amino acid sequence of SEQ ID NO: 123, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 123, or a variant of SEQ ID NO: 123 comprising canonical structural class 1. In even more specific embodiments, the mammalian antibody or antigen-binding fragment thereof further comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 120, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 120, or a variant of SEQ ID NO: 120 comprising canonical structural class 2.

[0029] As described above, caninized antibodies or caninized antigen-binding fragments thereof against canine CTLA-4 are an important aspect of the present invention, and the present invention provides caninized mammalian antibodies of all such mammalian antibodies, including caninized murine antibodies. Therefore, the present invention also provides isolated caninized antibodies or antigen-binding fragments thereof that specifically bind to CTLA-4, which comprise a canine IgG heavy chain and a canine κ or λ light chain. In a specific embodiment of this type, the canine κ or λ light chain comprises three light chain complementary determining regions (CDRs): CDR light chain 1 (CDRL1), CDR light chain 2 (CDRL2), and CDR light chain 3 (CDRL3); and the canine IgG heavy chain comprises three heavy chain CDRs: CDR heavy chain 1 (CDRH1), CDR heavy chain 2 (CDRH2), and CDR heavy chain 3 (CDRH3), which are obtained from a murine anti-canine CTLA-4 antibody. Specific embodiments of caninized antibodies and antigen-binding 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 canine CD86.

[0030] The caninized antibody or caninized antigen-binding fragment thereof of the present invention may comprise an IgGD comprising a hinge region comprising the amino acid sequence of SEQ ID NO: 128. In a related embodiment, the hinge region comprises the amino acid sequence of SEQ ID NO: 129. In yet another related embodiment, the hinge region comprises the amino acid sequence of SEQ ID NO: 130. In yet another related embodiment, the hinge region comprises the amino acid sequence of SEQ ID NO: 131.

[0031] In alternative embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 62. In a specific embodiment of this type, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 61. In other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 64. In a specific embodiment of this type, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 63. In still other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 66. In a specific embodiment of this type, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 65. In a more specific embodiment, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 50. In a specific embodiment of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 49. In other specific embodiments, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 52. In a specific embodiment of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 51. In still other specific embodiments, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 54. In specific embodiments of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO:53.

[0032] In alternative embodiments, the caninized antibody comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO:74. In specific embodiments of this type, the modified heavy chain is encoded by the nucleotide sequence of SEQ ID NO:73. In other embodiments, the caninized antibody comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO:76. In specific embodiments of this type, the modified heavy chain is encoded by the nucleotide sequence of SEQ ID NO:75. In still other embodiments, the caninized antibody comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO:78. In specific embodiments of this type, the modified heavy chain is encoded by the nucleotide sequence of SEQ ID NO:77. In more specific embodiments, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO:50. In specific embodiments of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO:49. In other specific embodiments, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO:52. In specific embodiments of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO:51. In still other specific embodiments, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 54. In specific embodiments of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO:53.

[0033] In specific embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 66 and a light chain comprising the amino acid sequence of SEQ ID NO: 52. In other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 66 and a light chain comprising the amino acid sequence of SEQ ID NO: 54.

[0034] In alternative embodiments, the caninized antibody comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 78 and a light chain comprising the amino acid sequence of SEQ ID NO: 52. In other embodiments, the caninized antibody comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 78 and a light chain comprising the amino acid sequence of SEQ ID NO: 54.

[0035] In other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 68. In a specific embodiment of this type, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 67. In other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 70. In a specific embodiment of this type, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 69. In still other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 72. In a specific embodiment of this type, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 71. In a more specific embodiment, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 56. In a specific embodiment of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 55. In other specific embodiments, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 58. In a specific embodiment of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 57. In still other specific embodiments, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 60. In specific embodiments of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO:59.

[0036] In alternative embodiments, the caninized antibody comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 80. In a specific embodiment of this type, the modified heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 79. In other embodiments, the caninized antibody comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 82. In a specific embodiment of this type, the modified heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 81. In still other embodiments, the caninized antibody comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 84. In a specific embodiment of this type, the modified heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 83. In a more specific embodiment, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 56. In a specific embodiment of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 55. In other specific embodiments, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 58. In a specific embodiment of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 57. In still other specific embodiments, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 60. In specific embodiments of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO:59.

[0037] In specific embodiments, the caninized antibody comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 72 and a light chain comprising the amino acid sequence of SEQ ID NO: 58. In other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 72 and a light chain comprising the amino acid sequence of SEQ ID NO: 60.

[0038] In alternative embodiments, the caninized antibody comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence of SEQ ID NO: 58. In other embodiments, the caninized antibody comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence of SEQ ID NO: 60.

[0039] The present invention further provides a method of reducing the amount of -12 M (e.g., 5 x 10 -13 M or less) with a dissociation constant (Kd) of 1×10-5 M to 1×10 -12 In a more specific embodiment, the mammalian antibody or antigen-binding fragment thereof binds to canine CTLA-4 with a dissociation constant of 1×10 -7 M to 1×10 -11 In still more specific embodiments, the mammalian antibody or antigen-binding fragment thereof binds to canine CTLA-4 with a dissociation constant of 1×10 -8 M to 1×10 -11 In still more specific embodiments, the mammalian antibody or antigen-binding fragment thereof binds to canine CTLA-4 with a dissociation constant of 1×10 -8 M to 1×10 -10 The dissociation constant of M binds to canine CTLA-4.

[0040] The present invention also provides a method with a concentration greater than 1×107M -1 s -1 In other embodiments, the mammalian antibody or antigen-binding fragment thereof binds to canine CTLA-4 at an on rate (kon) of 1×10 2 M -1 s -1 Up to 1×10 7 M -1 s -1 In a more specific embodiment, the mammalian antibody or antigen-binding fragment thereof binds to canine CTLA-4 at a binding rate of 1×10 3 M -1 s -1 Up to 1×10 6 M -1 s -1 In a more specific embodiment, the mammalian antibody or antigen-binding fragment thereof binds to canine CTLA-4 at a binding rate of 1×10 3 M -1 s -1 Up to 1×10 5 M -1 s -1 In still more specific embodiments, the mammalian antibody or antigen-binding fragment thereof binds to canine CTLA-4 at an on-rate of 1×10 4 M -1 s -1 Up to 1×10 5 M -1 s -1 The rate of binding to canine CTLA-4.

[0041] The present invention further provides a method of reducing the amount of -7 s-1 In other embodiments, the mammalian antibody or antigen-binding fragment thereof binds to canine CTLA-4 at an off rate (koff) of 1×10 -3 s -1 Up to 1×10 -8 s -1 In a more specific embodiment, the mammalian antibody or antigen-binding fragment thereof binds to canine CTLA-4 at an off-rate of 1×10 -4 s -1 Up to 1×10 -7 s -1 In still more specific embodiments, the mammalian antibody or antigen-binding fragment thereof binds to canine CTLA-4 at an off-rate of 1×10 -5 s -1 Up to 1×10 -7 s -1 The dissociation rate of canine CTLA-4 is 2.

[0042] In a specific embodiment, the mammalian antibodies (including chimeric antibodies) of the invention block the binding of canine CD80 and / or CD86 to canine CTLA-4. In a more specific embodiment, the antibodies are present at 1×10 -8 M to 1×10 -9 M or even lower concentrations of minimum EC50 blocking the binding of canine CD80 and / or CD86 to canine CTLA-4. In still more specific embodiments, EC 50 5×10 -9 M to 5×10 -13 M. Thus, in still more specific embodiments, EC 50 In 5×10 -9 M to 5×10 -11 M. Thus, in specific embodiments, the antibodies of the invention may exhibit one, two, three, four, or all of these properties, i.e., the above dissociation constant for canine CTLA-4, the above association rate for binding to canine CTLA-4, the above dissociation rate for dissociation from the antibody-canine CTLA-4 binding complex, or be effective in treating cancer in an animal subject.

[0043] The present invention further provides caninized mammal antibodies and antigen-binding fragments that cross-compete with the mammalian antibodies disclosed herein. In specific embodiments, the caninized mammal antibody cross-competes with an antibody comprising the 6 CDRs of 45A9 [see Table 1 below]. In related embodiments, the caninized mammal antibody cross-competes with an antibody comprising the 6 CDRs of 27G12 [see Table 1 below]. In still other related embodiments, the caninized mammal antibody cross-competes with an antibody comprising the 6 CDRs of 22A11 [see Table 1 below]. In still related embodiments, the caninized mammal antibody cross-competes with an antibody comprising the 6 CDRs of 110E3 [see Table 1 below]. In specific embodiments, the caninized mammal antibody cross-competes with an antibody comprising the 6 CDRs of 12B3 [see Tables 1 and 3 below]. In other specific embodiments, the caninized mammal antibody cross-competes with an antibody comprising the 6 CDRs of 39A11 [see Tables 1 and 3 below]. In specific embodiments, the assay is a standard binding assay. In one such embodiment, the caninized mammal antibody is cross-competed with an antibody comprising the 6 CDRs of 39A11. A standard binding assay is performed. In another such embodiment, the standard binding assay is performed using ELISA. In still another such embodiment, the standard binding assay is performed by flow cytometry.

[0044] As described above, the antibodies (and antigen-binding fragments thereof) of the present invention, including the aforementioned antibodies (and antigen-binding fragments thereof), can be monoclonal antibodies (and antigen-binding fragments thereof), mammalian antibodies (and antigen-binding fragments thereof) such as murine (mouse) antibodies (and antigen-binding fragments thereof), caninized antibodies (and antigen-binding fragments thereof), including caninized mouse antibodies (and antigen-binding fragments thereof). In certain embodiments, the antibodies (and antigen-binding fragments thereof) are isolated.

[0045] In a preferred embodiment, the caninized antibody or antigenic fragment thereof of the present invention binds to an epitope of the amino acid sequence of canine CTLA-4. In a specific embodiment, the caninized antibody interacts with one or more amino acid residues at positions T35, R38, T51, T53, Y90, K93, Y98, and Y102 of the amino acid sequence of SEQ ID NO: 138. In another embodiment, the caninized antibody interacts with one or more of the amino acid residues at positions 35T, R38, S42, K93, and Y102 of the amino acid sequence of SEQ ID NO: 138.

[0046] The present invention further provides caninized antibodies that bind to one or more epitopes or portions thereof of the amino acid sequences of SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136 and SEQ ID NO: 137. In a specific embodiment, the caninized antibody or antigenic fragment thereof of the present invention binds to an epitope or portion thereof encompassed by the amino acid sequence of SEQ ID NO: 132. In a more specific embodiment of this type, the epitope or portion thereof is encompassed by the amino acid sequence of SEQ ID NO: 134. In another embodiment of this type, the epitope or portion thereof is encompassed by the amino acid sequence of SEQ ID NO: 135. In certain embodiments, the epitope or portion thereof is encompassed by the amino acid sequence of SEQ ID NO: 133. In a more specific embodiment of this type, the epitope or portion thereof is encompassed by the amino acid sequence of SEQ ID NO: 136. In a related embodiment, the caninized antibody binds to one or more epitopes encompassed by the amino acid sequence of SEQ ID NO: 134 and / or SEQ ID NO: 136 and / or SEQ ID NO: 135, or a portion thereof.

[0047] The present invention also provides nucleic acids (including isolated and / or recombinant nucleic acids) encoding any light chain of the caninized antibodies of the present invention. Similarly, the present invention provides isolated nucleic acids (including isolated and / or recombinant nucleic acids) encoding any heavy chain of the caninized antibodies of the present invention.

[0048] The invention further provides expression vectors comprising one or more nucleic acids of the invention (including isolated nucleic acids). The invention also provides host cells comprising one or more expression vectors of the invention.

[0049] In a specific embodiment, the antibody is a recombinant antibody or an antigen-binding fragment thereof. In a related embodiment, the variable heavy chain domain and the variable light chain domain are connected by a flexible linker to form a single-chain antibody. In a specific embodiment, the antibody or antigen-binding fragment is a Fab fragment. In other embodiments, the antibody or antigen-binding fragment is a Fab' fragment. In still other embodiments, the antibody or antigen-binding fragment is a (Fab')2 fragment. In still other embodiments, the antibody or antigen-binding fragment is a double antibody. In a specific embodiment, the antibody or antigen-binding fragment is a domain antibody. In a specific embodiment, the antibody or antigen-binding fragment is a single domain antibody.

[0050] In a specific embodiment, the caninized murine anti-canine CTLA-4 antibody or antigen-binding fragment binds to CTLA-4 in an animal subject (e.g., a dog) being treated for cancer. In a more specific embodiment, administration of a caninized murine anti-canine CTLA-4 antibody or antigen-binding fragment of the invention is used to ameliorate one or more symptoms of cancer in an animal subject (e.g., a canine) being treated.

[0051] The present invention further provides an isolated nucleic acid encoding a caninized murine anti-canine CTLA-4 antibody or a portion thereof. In a related embodiment, such an antibody or antigen-binding fragment can be used to prepare a medicament for treating cancer in a canine subject. Alternatively or in combination, the present invention provides for use of any of the antibodies or antibody fragments of the present invention for diagnostic purposes. In a further embodiment, a kit comprising any of the caninized antibodies or antigen-binding fragments disclosed herein is provided.

[0052] The present invention further provides an isolated peptide that binds to the caninized antibody of the present invention, comprising 5 to 25 amino acid residues and being 90% identical or higher to the amino acid sequence of SEQ ID NO: 132. In a specific embodiment, the isolated peptide is identical to the amino acid sequence of SEQ ID NO: 132. In a more specific embodiment, the isolated peptide comprises 10 to 20 amino acid residues. In a related embodiment, the isolated peptide binds to the caninized antibody of the present invention, comprises 5 to 25 amino acid residues and being 90% identical or higher to the amino acid sequence of SEQ ID NO: 133. In a specific embodiment, the isolated peptide is identical to the amino acid sequence of SEQ ID NO: 133. In a more specific embodiment of this type, the isolated peptide comprises 10 to 20 amino acid residues.

[0053] In still other embodiments, the isolated peptide that binds to the caninized antibody of the present invention comprises an amino acid sequence that is 90% identical or higher to the amino acid sequence of SEQ ID NO: 134. In still other embodiments, the isolated peptide comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 134. In other embodiments, the isolated peptide that binds to the caninized antibody of the present invention comprises an amino acid sequence that is 90% identical or higher to the amino acid sequence of SEQ ID NO: 135. In other embodiments, the isolated peptide comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 135. In other embodiments, the isolated peptide that binds to the caninized antibody of the present invention comprises an amino acid sequence that is 90% identical or higher to the amino acid sequence of SEQ ID NO: 136. In still other embodiments, the isolated peptide comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 136.

[0054] The present invention further provides a fusion protein comprising such an isolated peptide that binds to a caninized antibody of the present invention. The present invention further provides a fusion protein comprising any of the above peptides. In a specific embodiment, the fusion protein comprises such an antigenic peptide and the Fc region of a non-canine mammal IgG antibody. In a more specific embodiment, the fusion protein comprises the Fc region of a non-canine mammal IgG antibody. In certain embodiments, the non-canine mammal IgG antibody is mouse IgG. In alternative embodiments, the non-canine mammal IgG antibody is human IgG. In other embodiments, the non-canine mammal IgG antibody is horse IgG. In still other embodiments, the non-canine mammal IgG antibody is porcine IgG. In still other embodiments, the non-canine mammal IgG antibody is bovine IgG.

[0055] In specific embodiments, the non-canine mammal IgG antibody is IgG1. In other embodiments, the non-canine mammal IgG antibody is IgG2a. In still other embodiments, the non-canine mammal IgG antibody is IgG3. In still other embodiments, the non-canine mammal IgG antibody is IgG4. In other embodiments, the fusion protein comprises any of the above-mentioned antigenic peptides and maltose binding protein. In still other embodiments, the fusion protein comprises any of the above-mentioned antigenic peptides and beta-galactosidase. In still other embodiments, the fusion protein comprises any of the aforementioned antigenic peptides and glutathione S transferase. In still other embodiments, the fusion protein comprises any of the aforementioned antigenic peptides and thioredoxin. In still other embodiments, the fusion protein comprises any of the aforementioned antigenic peptides and Gro E1. In still other embodiments, the fusion protein comprises any of the aforementioned antigenic peptides and NusA.

[0056] The present invention also provides nucleic acids (including isolated and / or recombinant nucleic acids) encoding one or more isolated immunogenic and / or antigenic peptides and / or fusion proteins of the present invention. The present invention further provides expression vectors comprising such isolated nucleic acids, and host cells comprising one or more expression vectors of the present invention.

[0057] The pharmaceutical composition may also include an antigenic peptide from canine CTLA-4 (including an isolated antigenic peptide), a fusion protein comprising an antigenic peptide from canine CTLA-4 of the present invention, a nucleic acid encoding an antigenic fragment and / or fusion protein of the present invention (including an isolated nucleic acid), an expression vector comprising such a nucleic acid, or any combination thereof, and a pharmaceutically acceptable carrier or diluent. In addition, the present invention includes a pharmaceutical composition comprising an anti-canine CTLA-4 antibody of the present invention (including a caninized mouse anti-canine CTLA-4 antibody) or an antigen-binding fragment thereof. Such a pharmaceutical composition can be used to treat cancer, infection or infectious diseases, as a vaccine adjuvant, and / or in a method for increasing the activity of immune cells, comprising administering a therapeutically effective amount of the pharmaceutical composition to a subject in need thereof.

[0058] In a specific embodiment, such a pharmaceutical composition further comprises an anti-canine PD-1 antibody (including a caninized mouse anti-canine PD-1 antibody) or an antigen-binding fragment thereof. In a more specific embodiment, the anti-canine PD-1 antibody is a caninized mouse anti-canine PD-1 antibody or an antigen-binding fragment of the caninized mouse anti-canine PD-1 antibody.

[0059] In a related embodiment, such a pharmaceutical composition further comprises an anti-canine PD-L1 antibody (including a caninized mouse anti-canine PD-L1 antibody) or an antigen-binding fragment thereof. In a specific embodiment, the anti-canine PD-L1 antibody is a caninized mouse anti-canine PD-1 antibody or an antigen-binding fragment of a caninized mouse anti-canine PD-1 antibody.

[0060] Thus, the present invention provides pharmaceutical compositions comprising one, two, three or more of the following: anti-canine PD-L1 antibodies, anti-canine PD-1 antibodies, anti-canine CTLA-4 antibodies, antigen-binding fragments of anti-canine PD-L1 antibodies, antigen-binding fragments of anti-canine PD-1 antibodies, or antigen-binding fragments of anti-canine CTLA-4 antibodies. In specific embodiments, such anti-canine protein (i.e., anti-canine PD-L1, PD-1 or CTLA-4) antibodies or antigen-binding fragments thereof are mouse anti-canine protein antibodies. In other embodiments, such anti-canine protein antibodies or antigen-binding fragments thereof are caninized anti-canine protein antibodies. In more specific embodiments, the anti-canine protein antibodies or antigen-binding fragments thereof are caninized mouse anti-canine protein antibodies.

[0061] In addition, the present invention provides a method for increasing the activity of immune cells, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a subject in need thereof. In certain embodiments, the method is used to treat cancer. In other embodiments, the method is used to treat infection or infectious diseases. In still other embodiments, the caninized antibody or antigen-binding fragment thereof of the present invention is used as a vaccine adjuvant. In a specific embodiment, a pharmaceutical composition comprising a caninized mouse anti-canine CTLA-4 antibody or an antigen-binding fragment thereof can be administered before, after, or simultaneously with a caninized mouse anti-canine PD-1 antibody or an antigen-binding fragment thereof and / or a caninized mouse anti-canine PD-L1 antibody or an antigen-binding fragment thereof.

[0062] These and other aspects of the invention will be better understood by reference to the following brief description and detailed description of the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The binding activity of six antibodies to canine CTLA-4 (cCTLA-4) was shown. Figure 1 Depicts a graph of the amount of individual canine CTLA-4 antibodies (in ng / mL) added to canine CTLA-4 in an ELISA (Ab log), which demonstrates the binding activity of the antibodies to cCTLA-4. The individual antibodies to canine CTLA-4 are represented as 27G12, 110E3, 12B3, 45A9, 39A11, and 22A11.

[0065] Figure 2 Depicted are antibodies that block the interaction of canine CD86 with CTLA-4. The figure depicts a graph of the amount (in ng / mL) of individual canine CTLA-4 antibodies added to cCTLA-4 to interfere with the binding of canine CTLA-4 to CD86 (Ab log). The antibodies against canine CTLA-4 are denoted as 39A11, 27G12, 45A9, 12B3, 110E3, and 22A11. As can be seen, the antibodies can block the interaction of canine CD86 with CTLA-4.

[0066] Figure 3 Depicted are antibodies that block the interaction of canine CD80 with CTLA-4. The figure depicts a graph of the amount (in ng / mL) of individual canine CTLA-4 antibodies added to cCTLA-4 to interfere with the binding of canine CTLA-4 to CD80 (Ab log). Individual antibodies against canine CTLA-4 are represented as 39A11, 27G12, 45A9, 12B3, 110E3, and 22A11. As can be seen, the antibodies can also block the interaction of canine CD80 with CTLA-4.

[0067] Figures 4A-4GAntibodies binding to CHO cells expressing canine CTLA-4 are depicted. Figure 4A is the isotype control (Iso-control), Figure 4B It is 39A11, Figure 4C It is 27G12, Figure 4D It is 12B3, Figure 4E It is 45A9. Figure 4F is 110E3, and Figure 4G It is 22A11. As can be seen, the antibody can bind to CHO cells expressing cCTLA-4.

[0068] Figure 5 Depicts a bar graph of three decreasing concentrations of a single canine CTLA-4 antibody added at 25 μg / ml, 50 μg / ml or 100 μg / ml (Ab) to quantify activation of canine PBMC cells to produce IFNγ in the presence of concanavalin A (CoA). The antibodies tested are on the abscissa, labeled CTLA-4 monoclonal antibodies (xCTLA-4mAb). As can be seen, the antibodies can activate canine PBMC cells to produce IFNγ.

[0069] Figure 6 A graph depicting the amount of CTLA-4 monoclonal antibody (xCTLA-4; Ab log ng / ml) that has the same reactivity with canine CTLA-4 as the parental antibody. The ELISA results show that both 12B3 and 39A11 were successfully caninized. Caninized c12B3L3H2 and L3H3 have similar reactivity with cCTLA-4 as the parental 12B3, and caninized C39A11L3H3 has similar reactivity with cCTLA-4 as the parental 39A11.

[0070] Figure 7 A-7B provides C12B3 on cCTLA-4 ( Figure 7 A) and C39A11( Figure 7 B) Binding epitopes. Two regions of the canine CTLA-4 protein are depicted, which have the amino acid sequences of SEQ ID NO: 132 and SEQ ID NO: 133, respectively (see Table 8 below). Both antibodies bind to the amino acid sequence SEQ ID NO: 136 containing the MYPPPY motif (SEQ ID NO: 137), and bind to the amino acid sequence of SEQ ID NO: 134. c12B3 also binds to the amino acid sequence of SEQ ID NO: 135. DETAILED DESCRIPTION OF THE INVENTION

[0072] abbreviation

[0073] Throughout the detailed description and examples of the present invention, the following abbreviations will be used:

[0074] ADCC Antibody-dependent cellular cytotoxicity

[0075] CDC Complement-dependent cytotoxicity

[0076] CDR Complementarity determining regions in immunoglobulin variable regions, defined using the Kabat numbering system

[0077] CHO Chinese Hamster Ovary

[0078] EC50 Concentration resulting in 50% efficacy or binding

[0079] ELISA enzyme-linked immunosorbent assay

[0080] FR Antibody Framework Region: The variable region of an immunoglobulin excluding the CDR region.

[0081] HRP Horseradish Peroxidase

[0082] IFN Interferon

[0083] IC50 concentration causing 50% inhibition

[0084] IgG Immunoglobulin G

[0085] Kabat Immunoglobulin alignment and numbering system pioneered by Elvin A. Kabat [Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)]

[0086] mAb Monoclonal Antibody (also called Mab or MAb)

[0087] MES 2-(N-morpholino)ethanesulfonic acid

[0088] MOA Mechanism of Action

[0089] NHS Normal Human Serum

[0090] PCR polymerase chain reaction

[0091] PK Pharmacokinetics

[0092] SEB Staphylococcal enterotoxin B

[0093] TT Tetanus toxoid

[0094] The V region is a segment of the IgG chain that is variable in sequence between different antibodies. It extends to Kabat residue 109 in the light chain and to Kabat residue 113 in the heavy chain.

[0095] V H Immunoglobulin heavy chain variable region

[0096] V L Immunoglobulin light chain variable region

[0097] VK immunoglobulin kappa light chain variable region

[0098] definition

[0099] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by ordinary technicians in the field to which the present invention belongs.

[0100] As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0101] "CTLA-4" is an abbreviation for "cytotoxic T lymphocyte-associated protein 4", also known as CD152 (cluster of differentiation 152), which is a protein receptor that acts as an immune checkpoint and downregulates immune responses. The amino acid sequence of canine CTLA-4 is SEQ ID NO: 126. The present invention further provides caninized murine antibodies against canine CTLA-4.

[0102] Unless the context indicates otherwise or makes it clear, "activation" as it applies to cells or receptors refers to activation or treatment of cells or receptors with a ligand. "Activation" can refer to cellular activation regulated by internal mechanisms as well as external or environmental factors.

[0103] "Ligand" encompasses natural and synthetic ligands, such as cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies."Ligand" also encompasses small molecules, such as peptide mimetics of cytokines and peptide mimetics of antibodies.

[0104] The "activity" of a molecule can describe or refer to the binding of a molecule to a ligand or receptor, catalytic activity; the ability to stimulate gene expression or cell signaling, differentiation or maturation; antigenic activity, regulation of other molecular activities, etc. The "activity" of a molecule can also refer to the activity of regulating or maintaining cell-to-cell interactions (e.g., adhesion), or the activity of maintaining cell structure (e.g., cell membrane or cytoskeleton). "Activity" can also mean specific activity, such as [catalytic activity] / [mg protein] or [immune activity] / [mg protein], concentration in a biological compartment, etc. "Activity" can refer to the regulation of components of the innate or adaptive immune system.

[0105] "Administering" and "treating" refer to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal (e.g., a canine subject), cell, tissue, organ, or biological fluid when applied to the animal (e.g., a canine subject), cell, tissue, organ, or biological fluid. Treatment of cells encompasses contact of an agent with a cell, as well as contact of an agent with a fluid, wherein the fluid is in contact with the cell.

[0106] "Administering" and "treating" also refer to in vitro and ex vivo manipulation of a cell, for example, by an agent, a diagnostic agent, a binding compound, or by another cell.

[0107] The term "subject" includes any organism, preferably an animal, more preferably a mammal (eg, a canine, a feline, or a human), most preferably a canine.

[0108] "Treat" or "treating" refers to the internal or external administration of a therapeutic agent, such as a composition containing any of the antibodies or antigen-binding fragments of the invention, to a canine subject or patient, e.g., having one or more symptoms of a disease or suspected of having a disease, which agent has therapeutic activity for the canine subject or patient.

[0109] Typically, the agent is administered in an amount that effectively alleviates and / or improves one or more disease symptoms in the treated subject or group, whether to induce the regression of such symptoms or inhibit the progression of such symptoms to any clinically measurable extent. The amount of the therapeutic agent that effectively alleviates any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to factors such as the disease state, age and weight of the patient (e.g., canine) and the ability of the pharmaceutical composition to induce a desired response in the subject. Any clinical measurement commonly used by a veterinarian or other skilled health care provider can be used to assess the severity or progression of the symptom to evaluate whether the disease symptom has been alleviated or improved. Although embodiments of the present invention (e.g., methods of treatment or articles) may not be effective in alleviating the target disease symptoms in each subject, it should alleviate the 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, according to the U-test of Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra-test and Wilcoxon-test).

[0110] "Treatment" as it applies to humans, veterinary (e.g., canines), or research subjects, refers to therapeutic treatment as well as research and diagnostic applications. When it applies to humans, veterinary (e.g., canines), or research subjects or cells, tissues, or organs, "treatment" encompasses contact of an antibody or antigen-binding fragment of the invention with, for example, a canine or other animal subject, a cell, tissue, a physiological compartment, or a physiological fluid.

[0111] Unless otherwise indicated, the term "canine" as used herein includes all domestic dogs, Canis lupus familiaris or Canis familiaris.

[0112] As used herein, the term "feline" refers to any member of the family Felidae. Members of this family include wild, zoo and domestic members, including domestic cats, purebred and / or hybrid companion cats, show cats, laboratory cats, cloned cats, and wild or untamed cats.

[0113] As used herein, the term "canine framework" refers to the amino acid sequence of the heavy and light chains of a canine antibody, excluding the hypervariable region residues defined herein as CDR residues. With regard to caninized antibodies, in most embodiments, the amino acid sequences of the native canine CDRs are replaced in both chains by the corresponding exogenous CDRs (e.g., those from a mouse antibody). Optionally, the heavy and / or light chains of the canine antibody may contain some exogenous non-CDR residues, for example, in order to retain the conformation of the exogenous CDRs within the canine antibody, and / or to modify the Fc function, as exemplified below.

[0114] Canine CTLA-4 has been found to comprise the amino acid sequence of SEQ ID NO: 126 (including the signal sequence). In a specific embodiment, canine CTLA-4 is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 125. The canine CTLA-4 sequence may differ, for example, by having conservative variations in non-conserved regions, but the canine CTLA-4 will have substantially the same biological function as the canine CTLA-4 comprised of the amino acid sequence of SEQ ID NO: 126.

[0115] As used herein, for example, "replacing an amino acid residue" with another amino acid residue in the amino acid sequence of an antibody is equivalent to "replacing an amino acid residue" with another amino acid residue, and indicates that a specific amino acid residue at a specific position in the amino acid sequence has been replaced (or substituted) by a different amino acid residue. Such a replacement can be specifically designed, i.e., purposefully replacing alanine with serine at a specific position in the amino acid sequence by, for example, recombinant DNA technology. Alternatively, for example, based on the ability of an antibody produced by a cell to bind to a given region on the antigen (e.g., a region containing an epitope or a portion thereof), and / or the ability to make the antibody contain a specific CDR that retains the same canonical structure as the CDR it replaces, a specific amino acid residue or residue string of an antibody can be replaced by one or more amino acid residues through a more natural selection process. Such a replacement / replacement can produce a "variant" CDR and / or a variant antibody.

[0116] The co-stimulatory signaling pathway leads to the development of the immune response and has been shown to be mediated by the interaction of CD28 with CD80 (also known as B7.1) and CD86 (also known as B7.2) on the surface of T cells. CTLA-4 binds to both CD80 and CD86 with a much higher affinity than CD28, thereby acting as an inhibitory receptor that is critical for the downregulation of the immune response. In fact, the mechanism by which CTLA-4 mediates its immunosuppressive function is related to its ability to act as a competitive inhibitor of the interaction of CD28 with CD80 and CD86. Therefore, the present invention describes the generation and characterization of monoclonal antibodies that block the binding of canine CD80 and canine CD 86 to CTLA-4, thereby allowing co-stimulatory signaling due to the binding of canine CD 28 to canine CD 80 and CD 86. Therefore, these antibodies can be used to treat cancer, as well as other diseases of companion animals as disclosed herein.

[0117] A specific canine CTLA-4 amino acid sequence will generally be at least 90% identical to a canine CTLA-4 comprising the amino acid sequence of SEQ ID NO: 126 (excluding the signal sequence). In certain cases, a canine CTLA-4 may be at least 95%, or even at least 96%, 97%, 98%, or 99% identical to a canine CTLA-4 comprising the amino acid sequence of SEQ ID NO: 126 (excluding the signal sequence). In certain embodiments, a canine CTLA-4 amino acid sequence will show no more than 10 amino acid differences from a canine CTLA-4 comprising the amino acid sequence of SEQ ID NO: 126 (excluding the signal sequence). In certain embodiments, a canine CTLA-4 amino acid sequence may show no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from a canine CTLA-4 comprising the amino acid sequence of SEQ ID NO: 126 (excluding the signal sequence). The percent identity may be determined as described below.

[0118] The term "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement), which results in selective damage, destruction, or elimination of cells or tissues of cancer cells, infectious pathogens, or invasive pathogens from the body of a mammal (e.g., a dog).

[0119] Anti-canine CTLA-4 antibody

[0120] The present invention provides isolated antibodies (particularly murine anti-canine CTLA-4 antibodies and caninized antibodies thereof) or antigen-binding fragments thereof that bind to canine CTLA-4, and uses of such antibodies or fragments thereof. In a specific embodiment, murine anti-canine CTLA-4 CDRs from murine anti-canine CTLA-4 antibodies are provided, which have been shown to both bind to canine CTLA-4 and block the binding of canine CTLA-4 to one or both of its ligands (canine CD86 or CD80). These CDRs can be inserted into a modified canine framework of a canine antibody to generate a caninized murine anti-canine CTLA-4 antibody.

[0121] As used herein, an "anti-canine CTLA-4 antibody" refers to an antibody that is raised against canine CTLA-4 (e.g., in a mammal such as a mouse or rabbit) and that specifically binds to canine CTLA-4. An antibody that "specifically binds to canine CTLA-4," an antibody that "particularly specifically binds to canine CTLA-4," or an antibody that "specifically binds to a polypeptide comprising the amino acid sequence of canine CTLA-4" is one that exhibits preferential binding to canine CTLA-4 compared to other canine antigens, but such specificity does not require absolute binding specificity. An anti-canine CTLA-4 antibody is considered "specific" for canine CTLA-4 if its binding determines the presence of canine CTLA-4 in a sample that is limited to canine proteins, or if it is able to alter the activity of canine CTLA-4 without unduly interfering with the activity of other molecules in a canine sample, e.g., without producing undesirable results, such as false positives in a diagnostic setting or side effects in a therapeutic setting. The degree of specificity required of an anti-canine CTLA-4 antibody may depend on the intended use of the antibody and is defined at least by its suitability for the intended purpose. The antibodies of the contemplated methods, or binding compounds derived from the antigen binding site of the antibodies, bind to their antigen, or variant or mutein thereof, with an affinity that is at least two-fold greater, preferably at least 10-fold greater, more preferably at least 20-fold greater, and most preferably at least 100-fold greater than to any other canine antigen.

[0122] As used herein, an antibody is said to specifically bind to a polypeptide comprising a given antigen sequence (in this case, a portion of the amino acid sequence of canine CTLA-4) if the antibody binds to a polypeptide comprising a portion of the amino acid sequence of canine CTLA-4 but does not bind to other canine proteins that lack that portion of the sequence of canine CTLA-4. For example, an antibody that specifically binds to a polypeptide comprising canine CTLA-4 may bind Tagged form of canine CTLA-4, but not bound to other Tagged canine protein. An antibody, or a binding compound derived from the antigen binding site of an antibody, binds to a canine antigen, or a variant or mutein thereof, "with specificity" when the affinity for the canine antigen, or a variant or mutein thereof, is at least 10-fold, more preferably at least 20-fold, even more preferably at least 100-fold greater than its affinity for any other canine antigen tested.

[0123] The term "antibody" as used herein refers to any form of antibody that exhibits the desired biological activity. Therefore, it is used in the broadest sense and specifically encompasses, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), canonical antibodies, fully canine antibodies, chimeric antibodies, and camelized single domain antibodies. A "parent antibody" is an antibody obtained by exposing the immune system to an antigen before modifying the antibody for its intended use (e.g., caninization of an antibody for use as a canine therapeutic antibody).

[0124] As used herein, unless otherwise indicated, "antibody fragment" or "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen bound by a full-length antibody, such as a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, 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 multiple antibody fragments.

[0125] "Fab fragment" consists of one light chain and the CH1 and variable region of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. "Fab fragment" can be the product of papain cleavage of an antibody.

[0126] The "fragment crystallizable" ("Fc") region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

[0127] A "Fab' fragment" contains a light chain and a portion or fragment of a heavy chain, which contains V H The Fab' fragments contain the CH1 and CH2 domains and the CH2 domains, which allow for the formation of an interchain disulfide bond between the two heavy chains of the two Fab' fragments to form a F(ab')2 molecule.

[0128] A "F(ab')2 fragment" contains two light chains and two heavy chains, wherein the heavy chain contains a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. Thus, a F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. A "F(ab')2 fragment" may be a product of pepsin cleavage of an antibody.

[0129] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0130] The term "single-chain Fv" or "scFv" antibody refers to an antibody comprising a V H and V L The Fv polypeptide is further comprised of a V domain and a V domain. H and V L A polypeptide linker between domains that enables the scFv to form the desired structure for antigen binding [see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113 Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); WO 88 / 01649; and US 4,946,778 and US 5,260,203].

[0131] As used herein, an anti-canine CTLA-4 antibody or antigen-binding fragment thereof that "blocks" or is "blocking" or is "blocking" the binding of canine CTLA-4 to its binding partner (ligand) (e.g., canine CD80 or canine CD86) is an antibody or antigen-binding fragment thereof that is detected in a standard binding assay (e.g., Anti-canine CTLA-4 antibodies or antigen-binding fragments thereof that (partially or completely) block the binding of canine CTLA-4 to canine CD86 and / or CD80 as determined by ELISA or flow cytometry. Such "blocking" is exemplified in Example 4 below using an ELISA-based blocking assay.

[0132] As used herein, the term "canonical structures" refers to the local conformations that each hypervariable region of an antibody's heavy and light chains can adopt within the framework they reside in. For each hypervariable region, there are a small number of canonical structures (usually represented by a simple integer, such as 1 or 2, etc.) that can be very accurately predicted from the amino acid sequence of the corresponding hypervariable region with great accuracy [particularly within the context of the amino acid sequence of the framework of its corresponding anti-canine CTLA-4 variable domain]. These canonical structures can determine whether modification of the amino acid sequence of a given CDR results in the retention or loss of the ability to bind its antigen-binding partner [see, Chothia and Lesk, Canonical Structures for the hypervariable regions of immunoglobulins, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Conformation of immunoglobulin hypervaribale regions, Nature, 34:877-883 (1989); and Al-Lazikani et al., Standard Conformations for the canonical structures of immunoglobulins, J. Mol. Biol. 273:927-948 (1997)].

[0133] "Domain antibodies" are immunologically functional immunoglobulin fragments that contain only the variable region of a heavy chain or the variable region of a light chain. H The two V domains of the bivalent domain antibody are covalently linked with a peptide linker to create a bivalent domain antibody. H The regions may target the same or different antigens.

[0134] A "bivalent antibody" contains two antigen binding sites. In some cases, the two binding sites have the same antigen specificity. However, a bivalent antibody can be bispecific (see below).

[0135] In certain embodiments, the monoclonal antibodies herein also include camelized single domain antibodies [see, e.g., Muyldermans et al., Trends Biochem. Sci. 26:230 (2001); Reichmann et al., J. Immunol. Methods 231:25 (1999); WO 94 / 04678; WO 94 / 25591; US6,005,079]. In one embodiment, the present invention provides a monoclonal antibody comprising two V domains with modified V domains.H A single domain antibody is formed by combining the domains of the antibody.

[0136] The term "diabodies" as used herein refers to small antibody fragments with two antigen-binding sites contained in the same polypeptide chain in combination with a light chain variable domain (V L ) connected to the heavy chain variable domain (V H )(V H -V L or V L -V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domain is forced to pair with the complementary domain of another chain and create two antigen-binding sites. [See EP 0 404 097 B1; WO 93 / 11161; and Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)]. For a review of engineered antibody variants [see generally Holliger and Hudson Nat. Biotechnol. 23:1126-1136 (2005)].

[0137] Typically, when activity is expressed on a molar basis, the antibodies or antigen-binding fragments of the invention retain at least 10% of their canine CTLA-4 binding activity (when compared to the parent antibody). Preferably, the antibodies or antigen-binding fragments of the invention retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the canine CTLA-4 binding affinity of the parent antibody. It is also contemplated that the antibodies or antigen-binding fragments of the invention may include conservative or non-conservative amino acid substitutions that do not substantially alter their biological activity (referred to as "conservative variants" or "function conservative variants" of the antibodies).

[0138] An "isolated antibody" refers to a purified state, and in this case refers to a molecule that is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium. In general, the term "isolated" is not intended to refer to the complete absence of such materials or the absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with the experimental or therapeutic use of the binding compounds as described herein.

[0139] As used herein, a "chimeric antibody" is an antibody having a variable domain from a first antibody and a constant domain from a second antibody, wherein the first and second antibodies are from different species. [US4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)]. Typically, the variable domains are obtained from an antibody ("parent antibody") from an experimental animal (e.g., a rodent), and the constant domain sequences are obtained from an animal subject antibody (e.g., a human or canine), such that the resulting chimeric antibody will be less likely to elicit an adverse immune response in a human or canine subject, respectively, than the parent (e.g., rodent) antibody.

[0140] As used herein, the term "caninized antibody" refers to an antibody form containing sequences from canine and non-canine (e.g., murine) antibodies. Typically, a caninized antibody will substantially contain all of at least one or more, typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-canine immunoglobulin (e.g., containing the 6 murine anti-canine CTLA-4 CDRs exemplified below), and all or substantially all of the framework (FR) regions (and typically all or substantially all of the remaining frameworks) are framework regions of canine immunoglobulin sequences. As exemplified herein, a caninized antibody comprises three heavy chain CDRs and three light chain CDRs from a murine anti-canine CTLA-4 antibody and a canine framework or a modified canine framework. The modified canine framework comprises one or more amino acid changes as exemplified herein, which further optimize the effectiveness of the caninized antibody, for example, to increase its binding to canine CTLA-4 and / or its ability to block canine CTLA-4 from binding to canine CD86 and / or CD80.

[0141] The term "full canine antibody" refers to an antibody that contains only canine immunoglobulin protein sequences. Full canine antibodies may contain rat carbohydrate chains if produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Similarly, "mouse antibodies" refer to antibodies that contain only mouse immunoglobulin sequences. Alternatively, full canine antibodies may contain rat carbohydrate chains if produced in rats, in rat cells, or in hybridomas derived from rat cells. Similarly, "rat antibodies" refer to antibodies that contain only rat immunoglobulin sequences.

[0142] There are four known IgG heavy chain subtypes of dog IgG, which are referred to as IgG-A, IgG-B, IgG-C and IgG-D. The two known light chain subtypes are referred to as λ and κ.

[0143] The variable regions of each light chain / heavy chain pair form an antibody binding site. Thus, typically, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are typically identical.

[0144] Typically, the variable domains of both the heavy and light chains contain three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FRs). The CDRs are typically aligned by the framework regions to enable binding to specific epitopes. Typically, from N-terminus to C-terminus, both the light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain is typically based on the definitions in: 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. al., J. Mol. Biol. 196:901-917 (1987) or Chothia, et al., Nature 342:878-883 (1989)].

[0145] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from the "complementarity determining region" or "CDR" (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 define the CDR regions of antibodies by sequence; see also Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) which define the CDR regions of antibodies by structure]. As used herein, the term "framework" or "FR" residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0146] In certain embodiments of the invention, canine or caninized antibodies to CTLA-4 optimally possess two properties in addition to binding and activating canine immune cells:

[0147] 1. Lack of effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and

[0148] 2. Ease of large-scale purification using industry standard techniques (e.g., Protein A chromatography-based techniques).

[0149] None of the naturally occurring canine IgG isotypes meet both of these 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, but also exhibits ADCC activity. In addition, neither IgG-C nor IgG-D can be purified on a Protein A column, although IgG-D does not exhibit ADCC activity. (IgG-C has considerable ADCC activity). One approach to addressing these issues is to provide a modified canine IgG-B antibody specific for CTLA-4 that lacks effector functions such as ADCC and can be easily purified using industry standard Protein A chromatography.

[0150] In an alternative embodiment of the invention, the canine IgG-B or IgG-C antibody specific for CTLA-4 is intentionally not modified to remove / substantially reduce effector function such as ADCC, and thus retains effector function such as ADCC.

[0151] "Homology" refers to the sequence similarity between two polynucleotide sequences or two polypeptide sequences when they are optimally aligned. When one position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if one position in each of the two DNA molecules is occupied by adenine, the molecules are homologous at that position. The homology percentage is the number of homologous positions shared by the two sequences divided by the total number of positions compared × 100. For example, when the sequences are optimally aligned, if 6 of the 10 positions of the two sequences are matched or homologous, the two sequences are 60% homologous. Typically, the comparison is made when the two sequences are aligned to give the maximum homology percentage.

[0152] An "isolated nucleic acid molecule" refers to a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, that is unrelated to all or part of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature. For the purposes of this disclosure, it is understood that a "nucleic acid molecule comprising a specific nucleotide sequence" does not encompass a complete chromosome. An isolated nucleic acid molecule "comprising" a specified nucleic acid sequence may include, in addition to the specified sequence, coding sequences for up to 10 or even up to 20 or more other proteins, or portions or fragments thereof, or may include operably linked regulatory sequences that control the expression of the coding region of the enumerated nucleic acid sequence, and / or may include vector sequences.

[0153] The phrase "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes include, for example, a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

[0154] A nucleic acid is "operably linked" when it is functionally associated with another nucleic acid sequence. For example, if the DNA of a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, it is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a sequence, it is operably linked to a coding sequence; or if a ribosome binding site is positioned to facilitate translation, it is operably linked to a coding sequence. Typically, "operably linked" means that the DNA sequences being linked are continuous, and in the case of a secretory leader, continuous and in reading phase. However, enhancers do not have to be continuous. The connection is accomplished by connecting at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0155] As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all of these names include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It should also be understood that not all progeny will have exactly the same DNA content, due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. Where different names are contemplated, this will be clear from the context.

[0156] As used herein, "germline sequence" refers to the sequence of an unrearranged immunoglobulin DNA sequence. Any suitable source of unrearranged immunoglobulin sequences may be used. Human germline sequences can be obtained, for example, from 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)].

[0157] Properties of mouse anti-canine CTLA-4 and caninized mouse anti-canine CTLA-4 antibodies

[0158] The present invention provides isolated mouse anti-canine CTLA-4 antibodies and caninized antibodies thereof, and methods of using the antibodies or antigen-binding fragments thereof in treating diseases (e.g., treating cancer in canines). In canines, there are four IgG heavy chains, referred to as A, B, C, and D. These heavy chains represent four different subclasses of canine IgG, referred to as IgGA, IgGB, IgGC, and IgGD. Each of the two heavy chains consists of a variable domain (V H The CH-1 domain is connected to the CH-2 domain via an amino acid sequence called a "hinge" or alternatively a "hinge region".

[0159] The DNA and amino acid sequences of these four heavy chains were first identified by Tang et al. [Vet. Immunol. Immunopathol. 80: 259-270 (2001)]. The amino acid and DNA sequences of these heavy chains can also be obtained from the GenBank database. For example, the amino acid sequence of the IgGA heavy chain has accession number AAL35301.1, IgGB has accession number AAL35302.1, IgGC has accession number AAL35303.1, and IgGD has accession number (AAL35304.1). Canine antibodies also contain two types of light chains, κ and λ. The DNA and amino acid sequences of these light chains can be obtained from the GenBank database. For example, the κ light chain amino acid sequence has accession number ABY 57289.1, and the λ light chain has accession number ABY 55569.1.

[0160] In the present invention, the amino acid sequence of each of the four canine IgG Fc fragments is based on the identified boundaries of the CH1 and CH2 domains as determined by Tang et al. (supra). Caninized mouse anti-canine CTLA-4 antibodies that bind to canine CTLA-4 include, but are not limited to, antibodies comprising canine IgG-A, IgG-B, IgG-C, and IgG-D heavy chains and / or canine kappa light chains and mouse anti-canine CTLA-4 CDRs. Thus, the present invention provides isolated mouse anti-canine CTLA-4 and / or caninized mouse anti-canine CTLA-4 antibodies or antigen-binding fragments thereof that bind to canine CTLA-4 and block the binding of canine CTLA-4 to canine CD86 and / or canine CD80.

[0161] The present invention further provides full-length canine heavy chains, which can be matched with corresponding light chains to prepare caninized antibodies. Therefore, the present invention also provides caninized mouse anti-canine antigen antibodies (including isolated caninized mouse anti-canine PD-1 antibodies) and methods of using the antibodies or antigen-binding fragments thereof in treating diseases (e.g., treating cancer in canines).

[0162] The present invention also provides caninized mouse anti-canine-CTLA-4 antibodies comprising a canine fragment crystallizable region (cFc region), wherein the cFc has been genetically modified to enhance, reduce or eliminate one or more effector functions. In one aspect of the invention, the genetically modified cFc reduces or eliminates one or more effector functions. In another aspect of the invention, the genetically modified cFc enhances one or more effector functions. In certain embodiments, the genetically modified cFc region is a genetically modified canine IgGb Fc region. In another such embodiment, the genetically modified cFc region is a genetically modified canine IgGC Fc region. In a specific embodiment, the effector function is an antibody-dependent cellular toxicity (ADCC) that is enhanced, reduced or eliminated. In another embodiment, the effector function is a complement-dependent cytotoxicity (CDC) that is enhanced, reduced or eliminated. In still another embodiment, the cFc region has been genetically modified to enhance, reduce or eliminate both ADCC and CDC.

[0163] To generate variants of canine IgG lacking effector function, a number of mutant canine IgGb heavy chains were generated. These variants may include one or more of the following single or combined substitutions in the Fc portion of the heavy chain amino acid sequence: P4A, D31A, N63A, G64P, T65A, A93G, and P95A. The variant heavy chains (i.e., containing such amino acid substitutions) were cloned into expression plasmids and transfected into HEK 293 cells along with a plasmid containing the gene encoding the light chain. The intact antibodies expressed and purified from HEK 293 cells were evaluated for expression with Fc γBinding of RI and C1q to assess their potential to mediate immune effector functions [see US 10,106,607 B2, the entire contents of which are incorporated herein by reference].

[0164] The present invention also provides a modified canine IgGD comprising a hinge region from:

[0165] IgGA: fnecrctdtppcpvpep, SEQ ID NO: 128;

[0166] IgGB: pkrengrvprppdcpkcpapem, SEQ ID NO: 129; or

[0167] IgGC:akececkcncnncpcpgcgl, SEQ ID NO:130.

[0168] Alternatively, the IgGD hinge region can be genetically modified by replacing serine residues with proline residues, i.e. SEQ ID NO: 131 (proline residues (P) are underlined, and bold replaces naturally occurring serine residues). Such modifications can produce canine IgGDs that lack fab arm exchange. Modified canine IgGDs can be constructed using standard methods of recombinant DNA technology [e.g., Maniatis et al., Molecular Cloning, A Laboratory Manual (1982)]. To construct these variants, nucleic acids encoding the amino acid sequence of canine IgGD can be modified so that they encode modified IgGDs. The modified nucleic acid sequence is then cloned into an expression plasmid for protein expression.

[0169] The antibody or antigen-binding fragment thereof that binds to canine CTLA-4 can comprise 1, 2, 3, 4, 5 or 6 complementarity determining regions (CDRs) of a murine anti-canine antibody as described herein. 1, 2, 3, 4, 5 or 6 CDRs can be independently selected from the CDR sequences of those provided below. In a further embodiment, the isolated antibody or antigen-binding fragment thereof that binds to canine CTLA-4 comprises a canine antibody kappa or lambda light chain containing a murine light chain CDR-1, CDR-2 and / or CDR-3 and a canine antibody heavy chain IgG containing a murine heavy chain CDR-1, CDR-2 and / or CDR-3.

[0170] In other embodiments, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to canine CTLA-4 and has a canine antibody kappa or lambda light chain comprising a given set of three CDRs having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequences of SEQ ID NOs: 92, 94 and 96 for VLCDR-1, VLCDR-2 and VLCDR-3, respectively, and a canine antibody heavy chain IgG comprising a given set of three CDRs having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequences of SEQ ID NOs: 86, 88 and 90 for VLCDR-1, VLCDR-2 and VLCDR-3, respectively; or a canine antibody kappa or lambda light chain comprising a given set of three CDRs having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequences of SEQ ID NOs: 86, 88 and 90 for VLCDR-1, VLCDR-2 and VLCDR-3, respectively. NO:104, 106 and 108 having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a given group of three CDRs, and a canine antibody heavy chain IgG comprising a different group of CDRs having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequences of SEQ ID NO:98, 100 and 102 for VLCDR-1, VLCDR-2 and VLCDR-3, respectively; or a canine antibody kappa or lambda light chain comprising a given group of three CDRs having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequences of SEQ ID NO:117, 94 and 96 for VLCDR-1, VLCDR-2 and VLCDR-3, respectively, and a canine antibody heavy chain IgG comprising a different group of CDRs having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequences of SEQ ID NO:98, 100 and 102 for VLCDR-1, VLCDR-2 and VLCDR-3, respectively; ID NO:86, 88 and 113 having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity; or a canine antibody kappa or lambda light chain comprising a given set of three CDRs having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequences SEQ ID NO:119, 122 and 96 for VLCDR-1, VLCDR-2 and VLCDR-3, respectively, and a canine antibody heavy chain IgG comprising a given set of three CDRs having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequences SEQ ID NO:86, 88 and 115 for VLCDR-1, VLCDR-2 and VLCDR-3, respectively; while still showing the desired binding and functional properties.In another embodiment, an antibody or antigen-binding fragment of the invention comprises a canine framework comprising an IgG heavy chain sequence in combination with a kappa or lambda light chain having the above three light chain CDRs and one or more of the three heavy chain CDR groups having 0, 1, 2, 3, 4 or 5 conservative or non-conservative amino acid substitutions, while still exhibiting the desired binding and functional properties.

[0171] Sequence identity refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when the two sequences are optimally aligned. As used herein, one amino acid sequence is 100% "identical" to a second amino acid sequence when the amino acid residues of the two sequences are identical. Thus, one amino acid sequence is 50% "identical" to a second amino acid sequence when 50% of the amino acid residues of the two amino acid sequences are identical. Sequence alignment is performed in a continuous stretch of amino acid residues comprised in a given protein, e.g., to compare portions of a protein or polypeptide. In a specific embodiment, selected deletions or insertions are contemplated that would otherwise alter the correspondence between the two amino acid sequences.

[0172] Sequence similarity includes identical residues and non-identical biochemically related amino acids. Biochemically related amino acids that share similar properties and are interchangeable are discussed.

[0173] "Conservatively modified variants" or "conservative substitutions" refer to amino acids in a protein that are replaced by other amino acids with similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein. Those skilled in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially change the biological activity [see, e.g., Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.; 1987)]. In addition, substitutions of amino acids that are structurally or functionally similar are less likely to destroy biological activity. Exemplary conservative substitutions are listed directly in Table A below.

[0174] Table A

[0175] Exemplary conservative amino acid substitutions

[0176] Original residue Conservative substitution Ala(A) Gly; Ser Arg(R) Lys; His Asn(N) Gln; His Asp(D) Glu; Asn Cys(C) Ser; Ala Gln(Q) Asn Glu(E) Asp; Gln Gly(G) Ala His(H) Asn; Gln Ile(I) Leu; Val Leu(L) Ile; Val Lys(K) Arg; His Met(M) Leu; Ile; Tyr Phe(F) Tyr;Met;Leu Pro(P) Ala; Gly Ser(S) Thr Thr(T) Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Phe Val(V) Ile; Leu

[0177] The present invention also contemplates functional conservative variants of the antibodies of the present invention. As used herein, "functional conservative variants" refer to antibodies or fragments in which one or more amino acid residues have been altered without changing desired properties (such as antigen affinity and / or specificity). Such variants include, but are not limited to, replacing amino acids with amino acids having similar properties, such as conservative amino acid substitutions of Table A above.

[0178] Nucleic Acids

[0179] The invention also includes nucleic acids encoding the immunoglobulin chains of the murine anti-canine CTLA-4 and / or caninized murine anti-canine CTLA-4 antibodies and antigen-binding fragments thereof disclosed herein (see Examples below).

[0180] The present invention also includes nucleic acids encoding immunoglobulin polypeptides, which, when compared by the BLAST algorithm (wherein the parameters of the algorithm are selected to give the maximum match between the corresponding sequences over the entire length of the corresponding reference sequences), comprise an amino acid sequence that is at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical, and most preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the amino acid sequence of the caninized antibody provided herein. The present invention further provides nucleic acids encoding immunoglobulin polypeptides, which, when compared with the BLAST algorithm (wherein the parameters of the algorithm are selected to give the maximum match between the corresponding sequences over the entire length of the corresponding reference sequences), comprise 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 reference amino acid sequence, and are also included in the present invention.

[0181] As used herein, using C, MacVector (MacVector, Inc. Cary, NC27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996) and Clustal W algorithm (with default parameters for comparison and default parameters for identity), nucleotide and amino acid sequence identity percentages can be determined. These commercially available programs can also be used to determine sequence similarity using the same or similar default parameters. Alternatively, Advanced Blast searches under default filtering conditions can be used, for example, using GCG (Genetics Computer Group, Program Manual for the GCG Package, 7th Edition, Madison, Wisconsin) pileup program using default parameters.

[0182] The following references relate to the BLAST algorithm that is frequently used for sequence analysis: BLAST ALGORITHMS: Altschul, SF, et al., J. Mol. Biol. 215:403-410 (1990); Gish, W., et al., Nature Genet. 3:266-272 (1993); Madden, TL, et al., Meth. Enzymol. 266:131-141 (1996); Altschul, SF, et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang, J., et al., Genome Res. 7:649-656 (1997); Wootton, JC, et al., Comput. Chem. 17:149-163 (1993); Hancock, J. Met. al., Comput.Appl.Biosci.10:67-70(1994); ALIGNMENT SCORINGSYSTEMS: Dayhoff, MO, et al., "A model of evolutionary change in proteins." in Atlas of Protein Sequence and Structure, Volume 5, Supplement 3, MODayhoff (ed.), pp.345-352, (1978); Natl.Biomed.Res.Found., Washington, DC; Schwartz, RM, et al., "Matrices for detecting distant relationships." in Atlas of Protein Sequenceand Structure, vol.5, suppl.3." (1978), MODayhoff (ed.), pp.353-358 (1978), Natl.Biomed.Res.Found., Washington, DC; Altschul, SF, J.Mol.Biol.219:555-565 (1991); States, DJ, et al., Methods 3:66-70(1991); Henikoff, S., et al., Proc. Natl. Acad. Sci. USA 89: 10915-10919 (1992); Altschul, SF, et al., J. Mol. Evol.36:290-300(1993);ALIGNMENT STATISTICS:Karlin,S.,et al.,Proc.Natl.Acad.Sci.USA 87:2264-2268(1990);Karlin,S.,et al.,Proc.Natl.Acad.Sci.USA 90:5873-5877(1993); Dembo, A., et al., Ann. Prob. 22: 2022-2039 (1994); and Altschul, SF "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). .

[0183] The present invention also provides an expression vector comprising a nucleic acid of the present invention, wherein the nucleic acid is operably linked to a control sequence that is recognized by a host cell when the host cell is transfected with the vector. Also provided are a host cell comprising the expression vector of the present invention and a method for producing an antibody or antigen-binding fragment thereof disclosed herein, the method comprising: culturing a host cell carrying an expression vector encoding an antibody or antigen-binding fragment in a culture medium, and isolating the antigen or antigen-binding fragment thereof from the host cell or culture medium.

[0184] Caninized mouse anti-canine CTLA-4 antibodies can be recombinantly produced by methods known in the art. Mammalian cell lines that can be used as hosts for expressing 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 Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, 3T3 cells, HEK-293 cells, and many other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other cell lines that can be used are insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. When recombinant expression vectors encoding heavy chains or antigen-binding portions or fragments thereof, light chains and / or antigen-binding fragments thereof are introduced into mammalian host cells, antibodies are produced by culturing the host cells for a period of time sufficient to allow expression of the antibodies in the host cells or, more preferably, secretion of the antibodies into the culture medium in which the host cells are grown.

[0185] The antibody can be recovered from the culture medium using standard protein purification methods. In addition, many known techniques can be used to enhance the expression of the antibodies of the invention (or other parts thereof) from production cell lines. For example, the glutamine synthetase gene expression system (GS system) is a common method for enhancing expression under certain conditions. The GS system is discussed in whole or in part in conjunction with European Patent Nos. 0 216 846, 0 256 055 and 0 323 997 and European Patent Application No. 89303964.4.

[0186] In general, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern specific to the glycoprotein produced in the cell line or transgenic animal. Therefore, the specific glycosylation pattern of an antibody will depend on the specific 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 with a glycosylation pattern comprising only non-fucosylated N-glycans may be advantageous because these antibodies have been shown to generally show more effective efficacy than their fucosylated counterparts both in vitro and in vivo [see, e.g., Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Pat. Nos. 6,946,292 and 7,214,775].

[0187] The present invention also includes antibody fragments of the mouse anti-canine CTLA-4 antibodies disclosed herein. The antibody fragments include F(ab)2 fragments, which can be produced by, for example, enzymatic cleavage of IgG by pepsin. Fab fragments can be produced by, for example, reducing F(ab)2 with dithiothreitol or mercaptoethylamine. Fab fragments can be attached to V by disulfide bonds. H -C H1 Chain V L -C L The F(ab)2 fragment is two Fab fragments attached by two disulfide bonds. The Fab portion of the F(ab)2 molecule includes a portion of the Fc region between which the disulfide bridge is located. The Fv fragment is a V L or V H area.

[0188] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region, e.g., a canine constant region, such as an IgGA, IgGB, IgGC, and IgGD canine heavy chain constant region or a variant thereof. In another embodiment, the antibody or antigen-binding fragment comprises a light chain constant region, e.g., a canine light chain constant region, such as a lambda or kappa canine light chain region or a variant thereof. By way of example, and not limitation, the canine heavy chain constant region may be from IgG-B, and the canine light chain constant region may be from kappa.

[0189] Antibody Engineering

[0190] The caninized murine anti-canine CTLA-4 antibodies of the invention can be engineered to include modifications to the canine framework and / or canine framework residues within the variable domain of the parent (ie, canine) monoclonal antibody, e.g., to improve the properties of the antibody.

[0191] Epitope Binding and Binding Affinity

[0192] The present invention further provides antibodies or antigen-binding fragments thereof that bind to the same amino acid residues of the canine CTLA-4 epitope as the murine anti-canine CTLA-4 antibodies disclosed herein. In a specific embodiment, the murine anti-canine CTLA-4 or antigen-binding fragment thereof is also capable of inhibiting / blocking the binding of canine CTLA-4 to canine CD86 and / or CD80. In a related embodiment, the caninized murine anti-canine CTLA-4 antibody or antigen-binding fragment thereof is also capable of inhibiting / blocking the binding of canine CTLA-4 to canine CD86 and / or CD80.

[0193] Experimental and diagnostic uses

[0194] The murine anti-canine CTLA-4 and / or caninized murine anti-canine CTLA-4 antibodies or antigen-binding fragments thereof of the invention can also be used in diagnostic assays for canine CTLA-4 protein, e.g., to detect its expression associated and / or related to cancer.

[0195] For example, this method includes the following steps:

[0196] (a) coating a substrate (e.g., the surface of a microtiter plate well, e.g., a plastic plate) with a murine anti-canine CTLA-4 antibody or an antigen-binding fragment thereof;

[0197] (b) applying to the matrix a sample to be tested for the presence of canine CTLA-4;

[0198] (c) washing the plate to remove unbound substances in the sample;

[0199] (d) applying a detectably labeled antibody (e.g., an enzyme-linked antibody) that is also specific for the CTLA-4 antigen;

[0200] (e) washing the matrix to remove unbound, labeled antibodies;

[0201] (f) if the labeled antibody is enzyme-linked, applying a chemical that is converted into a fluorescent signal by the enzyme; and

[0202] (g) detecting the presence of the labeled antibody.

[0203] 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 (TMB) to produce a detectable color change. Alternatively, the labeled antibody is labeled with a detectable radioisotope (e.g., 3H) that can be detected with a scintillation counter in the presence of a scintillator. The mouse anti-canine PD-1 antibodies of the present invention can be used in a Western blot or immuno-western blot procedure.

[0204] Such procedures form part of the present invention and include, for example:

[0205] (i) contacting a membrane or other solid matrix to which the canine CTLA-4 or fragment thereof to be tested for binding is present with the caninized murine anti-canine CTLA-4 antibody or antigen-binding fragment thereof of the invention. Such a membrane can take the form of a nitrocellulose or vinyl [e.g., polyvinylidene fluoride (PVDF)] based membrane to which the protein to be tested for the presence of canine CTLA-4 has been transferred in a non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel (e.g., after electrophoretic separation in the gel). Prior to contacting the membrane with the caninized murine anti-canine CTLA-4 antibody or antigen-binding fragment thereof, the membrane is optionally blocked with, for example, skim milk powder, to bind nonspecific protein binding sites on the membrane.

[0206] (ii) washing the membrane one or more times to remove unbound caninized murine anti-canine CTLA-4 antibody or antigen-binding fragment thereof and other unbound substances; and

[0207] (iii) detecting bound caninized murine anti-canine CTLA-4 antibody or antigen-binding fragment thereof.

[0208] The bound antibody or antigen-binding fragment can be detected by binding the antibody or antigen-binding fragment to a detectably labeled second antibody (anti-immunoglobulin antibody) and then detecting the presence of the second antibody.

[0209] The mouse anti-canine CTLA-4 antibodies, caninized mouse anti-canine CTLA-4 antibodies, and / or antigen-binding fragments thereof disclosed herein may also be used for 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, for example, a mouse anti-canine CTLA-4 antibody or antigen-binding fragment thereof of the present invention; and (2) detecting the antibody or fragment on or in the cell. If the antibody or antigen-binding fragment itself is detectably labeled, direct detection may be possible. Alternatively, the antibody or antigen-binding fragment may be bound by a detectably labeled second antibody that is detected.

[0210] Imaging techniques include SPECT imaging (single photon emission computed tomography) or PET imaging (positron emission tomography). Labels include, for example, iodine-123 (123I) and technetium-99m (99mTc), for example in combination with SPECT imaging, or 11 C. 13 N. 15 O or 18 F, for example in conjunction with PET imaging, or indium-111 [see, e.g., Gordon et al., International Rev. Neurobiol. 67:385-440 (2005)].

[0211] Cross-blocking antibodies

[0212] In addition, the anti-canine CTLA-4 antibodies or antigen-binding fragments thereof of the invention include any antibodies or antigen-binding fragments thereof that bind to the same epitope in canine CTLA-4 that the antibodies and fragments discussed herein bind to, as well as any antibodies or antigen-binding fragments thereof that cross-block (partially or completely) or are cross-blocked (partially or completely) by the antibodies or fragments discussed herein for canine CTLA-4 binding; and any variants thereof.

[0213] The cross-blocking antibodies and antigen-binding fragments thereof discussed herein can be determined based on their performance in standard binding assays (e.g., ELISA, as exemplified below, or flow cytometry) to identify their ability to cross-compete with the antibodies disclosed herein (based on the CDRs provided in Example 5 below), i.e., 45A9, 27G12, 22A11, 110E3; and more specifically, 12B3 and / or 39A11. For example, a standard ELISA assay can be used in which recombinant canine CTLA-4 protein is immobilized on a plate, one of the antibodies is fluorescently labeled, and the ability of the unlabeled antibody to compete away the binding of the labeled antibody is assessed. Additionally or alternatively, a The ability of the antibodies to cross-compete is assessed by assays. The ability of the test antibodies to inhibit, for example, the binding of 27G12, 45A9, 110E3 and / or 22A11; and even more particularly, the ability of 12B3 and / or 39A11 to canine CTLA-4 antibodies is demonstrated to indicate that the tested antibodies can compete with 27G12, 45A9, 110E3 and / or 22A11 and / or 12B3 and / or 39A11 for binding to canine CTLA-4 and thus, in some cases, can bind to the same epitope on canine CTLA-4 as 27G12, 45A9, 110E3 and / or 22A11 and / or 12B3 and / or 39A11. As described above, antibodies and fragments that bind to the same epitope as any of the anti-canine CTLA-4 antibodies or fragments of the invention also form part of the invention.

[0214] Pharmaceutical compositions and administration

[0215] To prepare a pharmaceutical or sterile composition of a caninized murine anti-canine CTLA-4 antibody or antigen-binding fragment thereof, it can be mixed with a pharmaceutically acceptable carrier or excipient [see, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984)].

[0216] Therapeutic and diagnostic agents can be prepared by mixing with acceptable carriers, excipients or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions [see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: 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 at pH 5-6, and NaCl or sucrose is added to improve tonicity. Additional agents such as polysorbate 20 or polysorbate 80 may be added to enhance stability.

[0217] Toxicity and therapeutic efficacy of an antibody composition administered alone or in combination with another agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as for determining LD 50 (a dose lethal to 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index (LD 50 / ED 50 ). In certain aspects, antibodies that exhibit high therapeutic indices are desirable. The data obtained from these cell culture assays and animal studies can be used to formulate a dosage range for canines. The dosage of such compounds is preferably within the range that includes the ED 50The dosage may vary within this range depending upon the dosage form employed and the route of administration employed.

[0218] The mode of administration may vary. Suitable routes of administration include oral, rectal, transmucosal, enteral, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, directly intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal or intraarterial. In a specific embodiment, the caninized mouse anti-canine CTLA-4 antibody or its antigen-binding fragment can be administered by an invasive route, such as by injection. In a further embodiment of the present invention, the caninized mouse anti-canine CTLA-4 antibody or its antigen-binding fragment or its pharmaceutical composition is administered intravenously, subcutaneously, intramuscularly, intraarterially or by inhalation, aerosol delivery. Administration by non-invasive routes (e.g., oral, such as in pills, capsules or tablets) is also within the scope of the present invention.

[0219] The composition can be administered with medical devices known in the art. For example, the pharmaceutical composition of the present invention can be administered by injection with a hypodermic needle (including, for example, a prefilled syringe or an automatic injector). The pharmaceutical compositions disclosed herein can also be administered with a needle-free hypodermic injection device; such as the devices disclosed in U.S. Patent Nos.: 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.

[0220] The pharmaceutical composition disclosed herein can also be administered by infusion. Examples of well-known implants and module forms for administering pharmaceutical compositions include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses an implantable infusion device with a variable flow rate for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with a multi-chamber compartment. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0221] Alternatively, the murine anti-canine or caninized murine anti-canine CTLA-4 antibody can be administered in a local rather than systemic manner, for example, by direct injection of the antibody into an arthritic joint or pathogen-induced lesion characterized by immunopathology, often in a depot or sustained release formulation. Moreover, the antibody can be administered in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody that targets, for example, an arthritic joint or pathogen-induced lesion characterized by immunopathology. The liposome will selectively target and be taken up by the diseased tissue.

[0222] The administration regimen depends on several factors, including serum or tissue turnover of the therapeutic antibody, symptom levels, immunogenicity of the therapeutic antibody, and accessibility of target cells in the biomatrix. Preferably, the administration regimen delivers enough therapeutic antibodies to achieve improvement in the target disease state while minimizing undesirable side effects. Therefore, the amount of the biologic delivered depends in part on the specific therapeutic antibody and the severity of the condition being treated. Guidance for selecting appropriate doses of therapeutic antibodies is available [see, e.g., Wawrzynczak Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK (1996); Kresina (ed.) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY (1991); Bach (ed.) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY (1993); Baert, et al. New Engl. J. Med. 348:601-608 (2003); Milgrom et al. New Engl. J. Med. 341:1966-1973 (1999); Slamon et al. New Engl. J. Med. 344:783-792 (2001); Beniaminovitz et al. New Engl. J. Med. 345:783-792 (2002); et al. New Engl. J. Med. 342: 613-619 (2000); Ghosh et al. New Engl. J. Med. 348: 24-32 (2003); Lipsky et al.

[0223] Determination of the appropriate dosage is made by a veterinarian, for example using parameters or factors known in the art or suspected to affect treatment. Typically, the dosage is started with an amount slightly less than the optimal dosage and increased in small increments thereafter until the desired or optimal effect is obtained relative to any negative side effects. Important diagnostic measures include those of symptoms such as tumor size.

[0224] The antibodies or antigen-binding fragments thereof disclosed herein can be provided by continuous infusion or by dosages administered, for example, daily, 1-7 times per week, weekly, biweekly, monthly, bimonthly, quarterly, semi-annually, yearly, etc. The dosage can be provided, for example, intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. The total weekly dose is usually at least 0.05 μg / kg body weight, more usually 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, e.g., Yang, et al. New Engl. J. Med. 349:427-434 (2003); Herold, et al. New Engl. J. Med. 346:1692-1698 (2002); Liu, et al. J. Neurol. Neurosurg. Psych. 67:451-456 (1999); Portielji, et al. Cancer Immunol. Immunother. 52: 133-144 (2003)]. Doses can also be provided to achieve a predetermined target concentration of caninized murine anti-canine CTLA-4 antibody in the subject's serum, e.g., 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg / ml or more. In other embodiments, the caninized murine anti-canine CTLA-4 antibody of the invention is administered subcutaneously or intravenously at 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg / subject weekly, biweekly, "every 4 weeks", monthly, bimonthly or quarterly.

[0225] Antigenic peptides recognized by anti-canine CTLA-4 mAbs (e.g., peptides comprising epitopes from CTLA-4 or portions thereof) can also be used as vaccines to elicit antibodies that block the binding of canine CTLA-4 to canine CD80 and / or CD86. Such vaccines can be used as therapeutic vaccines for diseases such as cancer. In order to use these antigenic peptides as vaccines, one or more of these peptides can be chemically coupled or coupled to another carrier protein by recombinant DNA technology to enhance the immunogenicity of these peptides and elicit peptide-specific antibodies. The techniques for coupling peptides to carrier proteins are known to those skilled in the art. Peptide vaccines can be used to vaccinate animals by IM, S / C, oral, spray or in ovo routes. Peptide vaccines can be used as subunit proteins expressed from bacteria, viruses, yeast or baculovirus systems. Alternatively, such peptide vaccines can be delivered after administration of a variety of viral or bacterial vectors expressing such peptide vaccines, as can be implemented by methods known to those skilled in the art. Peptide vaccines can be administered at a dose of 1-1000 μg and can optionally contain adjuvants and acceptable pharmaceutical carriers.

[0226] As used herein, "inhibit" or "treat" or "treatment" includes a delay in the development of symptoms associated with a disorder and / or a reduction in the severity of symptoms of such a disorder. The term also includes ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of these symptoms. Thus, the term means that a beneficial result has been conferred on a vertebrate subject (e.g., a canine) having a disorder, disease, or symptom or having the potential to develop such a disorder, disease, or symptom.

[0227] As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of the caninized murine anti-canine CTLA-4 antibody or antigen-binding fragment thereof of the invention, which, when administered to a cell, tissue, or subject alone or in combination with another therapeutic agent, is effective to cause a measurable improvement in one or more symptoms of a disease or condition, or the progression of such a disease or condition. A therapeutically effective dose further refers to an amount of a binding compound sufficient to result in at least a partial improvement in symptoms, such as treatment, healing, prevention, or amelioration of a related medical condition, or an increase in the rate of treatment, healing, prevention, or amelioration of such a condition. When applied to a single 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 combined amount of the active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent will result in an improvement in a diagnostic measure or parameter of at least 10%; typically at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably at least 50%. In cases where subjective measurements are used to assess disease severity, an effective amount may also result in an improvement in a subjective measurement.

[0228] Other combination therapies

[0229] As previously described, the caninized mouse anti-canine CTLA-4 antibody or antigen-binding fragment thereof and / or antigenic peptide of the present invention can be co-administered with one or more other therapeutic agents (e.g., inhibitors discussed in the next paragraph) and / or caninized mouse anti-canine PD-1 antibody [see, e.g., US9,944,704B2 and US10,106,107B2, the entire contents of both of which are incorporated herein by reference] and / or caninized mouse anti-canine PD-L1 antibody [see, e.g., US20180237535A1, the entire contents of which are incorporated herein by reference]. The antibody can be linked to the agent (as an immune complex), and / or can be administered separately from the agent or other antibodies. In the latter case (separate administration), the antibody can be administered before, after, or simultaneously with the agent, or can be co-administered with other known therapies.

[0230] Reagent test kit

[0231] Kits are further provided that include one or more components, including but not limited to an antibody or antigen-binding fragment that specifically binds to CTLA-4 as discussed herein (e.g., a caninized murine anti-canine CTLA-4 antibody or antigen-binding fragment thereof) in combination with one or more additional components (including a caninized murine anti-canine PD-1 antibody and / or a caninized murine anti-canine PD-L1 antibody). The combination composition as described immediately above can be formulated as a pure composition or combined in a pharmaceutical composition with a pharmaceutically acceptable carrier.

[0232] In one embodiment, the kit includes a binding composition of the invention (e.g., caninized murine anti-canine CTLA-4 or a pharmaceutical composition thereof in one container (e.g., in a sterile glass or plastic vial), caninized murine anti-canine PD-1 antibody and / or caninized murine anti-canine PD-L1 antibody or a pharmaceutical composition thereof in another container (e.g., in a sterile glass or plastic vial)).

[0233] If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit may also include a device for performing such administration. For example, the kit may include one or more hypodermic needles or other injection devices as described above. The kit may also include a package insert including information about the pharmaceutical composition and dosage form in the kit. Typically, such information helps pet owners and veterinarians to effectively and safely use the encapsulated pharmaceutical composition and dosage form. For example, the following information about the combination of the present invention may be provided in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdose, appropriate dosage and administration, supply specifications, appropriate storage conditions, references, manufacturer / distributor information, and patent information.

[0234] For convenience, the antibodies or specific binding agents disclosed herein can be provided in a kit, i.e., a predetermined amount of reagents and a packaged combination of instructions for diagnosis or detection assays. When one or more antibodies are labeled with enzymes, the kit will include substrates and cofactors required for the enzyme (e.g., substrate precursors that can detect chromophores or fluorophores are provided). In addition, other additives may be included, such as stabilizers, buffers (e.g., blocking buffer or lysis buffer), etc. The relative amounts of various reagents can vary widely, to provide a concentration of the reagent in solution that substantially optimizes the sensitivity of the assay. In particular, the reagent can be provided as a dry powder, typically lyophilized, including an excipient, which will provide a reagent solution with an appropriate concentration when dissolved. Example

[0235] Example 1

[0236] Generation of mouse monoclonal antibodies and corresponding mouse-canine chimeric antibodies against canine CTLA-4

[0237] Mouse monoclonal antibodies were generated using mouse hybridoma technology using canine CTLA-4 (cCTLA-4) recombinant protein as an immunogen. Positive hybridoma clones were selected based on antibody reactivity with cCTLA-4 and blocking of the interaction of canine CD86 or CD80 with cCTLA-4 (blocking activity) by ELISA and FACS assays. Selected hybridoma clones were subjected to V by rapid amplification of cDNA ends (RACE). H and V LSequencing of antibody fragments of the sequences. The six monoclonal antibodies selected are denoted as: 12B3, 27G12, 39A11, 45A9, 110E3 and 22A11. The amino acid sequences of the six antibodies are SEQ ID NOs: 2, 4, 6, 8, 10 and 12 for the heavy chain variable region, and SEQ ID NOs: 14, 16, 18, 20, 22 and 24 for the light chain variable region. The CDRs are underlined in the sequences provided below [see also Table 1 below]. The corresponding nucleotide sequences encoding the above amino acid sequences are listed as SEQ ID NOs: 1, 3, 5, 7, 9 and 11 for the heavy chain variable region, and SEQ ID NOs: 13, 15, 17, 19, 21 and 23 for the light chain variable region. The nucleotide sequences of the heavy chain variable regions were fused to the nucleotide sequences of the modified canine constant heavy chain (CH1-hinge-CH2-CH3) to generate chimeric mouse-canine heavy chain nucleotide sequences designated as SEQ ID NOs: 25, 27, 29, 31, 33, and 35, respectively. The variable regions are shown in bold. The nucleotide sequences of the light chain variable regions were fused to the nucleotide sequences of the canine constant kappa light chain domain to generate chimeric mouse-canine light chain nucleotide sequences designated as SEQ ID NOs: 37, 39, 41, 43, 45, and 47, respectively. The variable regions are shown in bold. The amino acid sequences encoded by the chimeric mouse-canine heavy chain nucleotide sequences were designated as SEQ ID NOs: 26, 28, 30, 32, 34, and 36. The amino acid sequences encoded by the chimeric mouse-canine light chain nucleotide sequences were designated as SEQ ID NOs: 38, 40, 42, 44, 46, and 48. The variable regions are shown in bold, and the CDRs are underlined. 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 HEK 293 cells, and the expressed antibodies were purified from the HEK 293 cell supernatant using protein A.

[0238] Example 2

[0239] Amino acid sequences of mouse CDRs

[0240] The CDRs from mouse anti-canine CTLA-4 monoclonal antibodies are listed in Table 1 below.

[0241] Table 1

[0242] Amino acid sequences of mouse CDRs

[0243]

[0244]

[0245] The individual canonical structural assignments for the six CDRs for each of the six antibodies are provided in Table 2 below.

[0246] Table 2

[0247] Canonical structure of mouse CDRs

[0248] Antibody L1 L2 L3 H1 H2 H3 12B3 4 1 1 1 2A 7 27G12 4 1 1 1 2A 7 39A11 1 1 1 1 4 9 45A9 4 1 1 1 2A 7 22A11 2 1 1 1 2A 12 110E3 4 1 1 1 2A 7

[0249] Example 3

[0250] Reactivity of chimeric antibodies with canine CTLA-4

[0251] Chimeric antibodies generally have the same reactivity as their parental mouse antibodies. To confirm the reactivity of the six antibodies with cCTLA-4, a mouse-canine chimeric antibody was generated and tested for reactivity with cCTLA-4 by ELISA as follows:

[0252] 1. Coat 200 ng / well of cCTLA-4 in immunoplates and incubate the plates overnight at 4°C.

[0253] 2. Wash the plate three times with PBS containing 0.05% Tween 20 (PBST).

[0254] 3. Block the plates with 0.5% BSA in PBS for 45-60 minutes at room temperature.

[0255] 4. Wash the plate 3 times with PBST.

[0256] 5. Dilute the antibody three-fold in each column or row of the dilution plate.

[0257] 6. Transfer the diluted antibody to each column or row of the plate and incubate the plate for 45-60 minutes at room temperature.

[0258] 7. Wash the plate 3 times with PBST.

[0259] 8. Add 1:2000 dilution of horseradish peroxidase-labeled anti-canine IgG Fc to each well of the plate and incubate the plate for 45-60 minutes at room temperature.

[0260] 9. Wash the plate 3 times with PBST.

[0261] 10. Add TMB substrate to each well of the plate and incubate the plate for 10 to 15 minutes at room temperature to allow color development.

[0262] 11. Add 100 μL of 1.5 M phosphoric acid to each well to stop the reaction.

[0263] 12. Read the plate at 450 nm using a reference wavelength of 540 nm.

[0264] ELISA results showed that the chimeric antibody could bind to cCTLA-4 [see Figure 1 ].

[0265] Example 4

[0266] Blocking activity of chimeric antibodies against canine CD86 or CD80 and the interaction with canine CTLA-4

[0267] To investigate the blocking activity of the chimeric antibodies, an ELISA-based blocking assay was performed as follows:

[0268] 1. Coat 200 ng / well of cCTLA-4 in immunoplates and incubate the plates overnight at 4°C.

[0269] 2. Wash the plate three times with PBS containing 0.05% Tween 20 (PBST).

[0270] 3. Block the plates with 0.5% BSA in PBS for 45-60 minutes at room temperature.

[0271] 4. Wash the plate 3 times with PBST.

[0272] 5. Dilute the antibody in each column or row of the dilution plate three-fold, and then add 100 ng / well of biotinylated CD86 or CD80. Next, mix with the antibody.

[0273] 6. Transfer the mixture to each column or row of the immunoplate and incubate the plate for 45-60 minutes at room temperature.

[0274] 7. Wash the plate 3 times with PBST.

[0275] 8. Add 1:2000 dilution of horseradish peroxidase-conjugated streptavidin to each well of the plate and incubate the plate for 45-60 minutes at room temperature.

[0276] 9. Wash the plate 3 times with PBST.

[0277] 10. Add TMB substrate to each well of the plate and incubate the plate for 10 to 15 minutes at room temperature to allow color development.

[0278] 11. Add 100 μL of 1.5 M phosphoric acid to each well to stop the reaction.

[0279] 12. Read the plate at 450 nm using a reference wavelength of 540 nm.

[0280] Chimeric antibodies were found to block cCTLA-4 and CD86[ Figure 2 ] and CD80[ Figure 3 ] interaction.

[0281] Example 5

[0282] FACS assay for testing the binding activity of chimeric antibodies to CHO-cCTLA-4

[0283] A CHO-K1 cell line that stably expresses cCTLA-4 was generated. The cells were used to test antibody binding and blocking activity in a FACS flow assay. To test the cCTLA-4 binding activity of the chimeric antibody, a FACS assay was performed as follows:

[0284] 1. Grow CHO-K1-CCTLA-4 cells in culture medium in T-75 flasks.

[0285] When cell confluence reached 90%, cells were passaged.

[0286] Culture medium: F12K (Gibco, catalog number 21127-022), 10% FBS (Gibco, catalog number 10099-141), and 4 μg / ml puromycin (Gibco, catalog number A1113803).

[0287] 2. Detach the cells by trypsin-EDTA solution, resuspend the cells in culture medium, and count the live cells with a viability of more than 95%.

[0288] 3. Centrifuge the cells, aspirate the supernatant, and resuspend the cells in FACS buffer (ThermoFisher Scientific, catalog number BDB554656) to 1×10 7 cells / ml.

[0289] 4. Add the antibody to 100 μL of cells and incubate at room temperature for 30 minutes with gentle shaking.

[0290] 5. Wash the cells 3 x 250 μL of FACS buffer and resuspend the cells in 100 μL of FACS buffer.

[0291] 6. Stain the cells with FITC-conjugated anti-canine IgG and incubate at room temperature for 30 minutes with gentle shaking.

[0292] 7. Wash the cells with 3 x 250 μL of FACS buffer and resuspend the cells in 500 μL of FACS buffer.

[0293] 8. Read 10,000 cells by flow cytometry.

[0294] FACS results showed that the chimeric antibody could bind to CHO-CCTLA-4 cells [see Figures 4A-4G ].

[0295] Example 6

[0296] Interferon gamma (IFNγ) production by canine PBMC activated by chimeric antibodies Isolation of canine peripheral blood mononuclear cells

[0297] 1. Collect ~20 ml of whole blood in EDTA or sodium heparin tubes.

[0298] 2. Transfer blood to a 50 mL polystyrene tube and dilute 50:50 with HBSS (Thermo Fisher Scientific Cat. No. 21022CM).

[0299] 3. Add 15 ml of Ficoll-Plaque Plus to 4 x 50 ml of SepMate TM Then, slowly add ~10 ml of 50:50 diluted blood and add to each Sepmate containing Ficoll. TM One side of the tube.

[0300] 4. Centrifuge the tubes at 1200 x g for 20 minutes.

[0301] 5. Collect cells from the gradient interface and transfer the cells to a 50 mL polypropylene tube. Add HBSS to the 40-45 ml mark and centrifuge the cells at 800 x g for 10 minutes.

[0302] 6. Discard the supernatant, resuspend the cells in 40-45 ml of HBSS and centrifuge the tube again at 800 x g for 10 minutes.

[0303] 7. Discard the supernatant and resuspend the cells from each tube with 2 ml of canine lymphocyte culture medium (RPMI medium, Lonza, catalog number 12-167Q). Pool cells from the same animal.

[0304] 8. Take a small aliquot of the cell suspension, mix it with 0.04% Trypan blue and count the number of cells.

[0305] 9. Store the cell suspension at 2-7°C until use, but no longer than 24 hours before use.

[0306] Cell proliferation assay of canine peripheral blood mononuclear cells

[0307] 1. Dilute the antibody in canine lymphocyte culture medium to give a final concentration of 40 μg / mL (make 160 μg / mL) and sterilize using a 0.2 μm syringe filter. Place the two-fold dilution of the antibody on a sterile dilution plate and set it aside.

[0308] 2. Dilute the cells to 2.5 × 10 in canine lymphocyte culture medium. 6 cells / mL and dispensed at 100 μL / well throughout a 96-well tissue culture plate.

[0309] 3. Dilute Con A in canine lymphocyte culture medium to a final concentration of 250 ng / mL (make 1000 ng / mL), sterilize using a 0.2 μm syringe filter, and add 50 μL to all wells. (Con A was not added to the column of eight wells for the cell only control and to each well for the cell only + mAb control).

[0310] 4. Add 100 μL of canine lymphocyte culture medium / well to the cell-only wells and add 50 μL of culture medium to the column containing Con A control wells (Con A+ cells without mAb treatment).

[0311] 5. Add 50 μL of diluted mAb to duplicate wells.

[0312] 6. Incubate the plate at 36 ± 2 °C, 4.0-6.0% CO2 in a humidified incubator for 68 to 124 hours.

[0313] IFNγ ELISA

[0314] 1. After 68-124 hours of incubation, centrifuge the plate at 800 x g for 10 minutes.

[0315] 2. Collect the supernatant from each well and pool the replicates. These samples can be frozen at ≤ -50°C for later use or tested immediately.

[0316] 3. If necessary, dilute the supernatant sample appropriately and perform IFN-γ ELISA according to the instructions of the Canine IFN-γ Quantikine ELISA Kit [R&D Systems, catalog number CAIF00].

[0317] The results confirmed that the selected antibodies (including 12B3) can activate canine T cells to produce IFN [see below Figure 5 ].

[0318] Example 7

[0319] Construction of canine anti-cCTLA-4 monoclonal antibodies 12B3 and 39A11

[0320] Due to their strong binding affinity to cCTLA-4 and their blocking activity to cCTLA-4 and its ligands CD86 and CD80, the mouse antibodies 12B3 and 39A11 were selected for the preparation of the initial caninized antibodies. In order to perform the caninization process, the DNA sequences encoding the heavy and light chains of canine IgG were determined. The DNA and protein sequences of canine heavy and light chains are known in the art and can be obtained by searching the NCBI gene and protein database. There are four known IgG subtypes of canine IgG, which are referred to as IgGA, IgGB, IgGC and IgGD. Like human IgG1, canine IgGB has strong effector functions. In order to knock out the effector functions of IgGB, a modified IgGB (IgGBm) was constructed, which removes the natural ADCC and CDC functions [see US10,106,107B2, the entire contents of which are incorporated herein by reference]. There are two types of light chains in canine antibodies, called κ and λ. Without being bound by any particular method, the overall process of generating caninized heavy and light chains that can be mixed in various combinations to generate caninized anti-canine CTLA-4 mAbs may include the following protocol:

[0321] i) Identify the CDRs of the H and L chains of the selected antibody. Translate the amino acid sequence of the CDRs back into the appropriate DNA sequence.

[0322] ii) Identification of suitable DNA sequences for the H and L chains of canine IgG (eg the heavy and light kappa chains of IgGB).

[0323] iii) Identify the DNA sequences encoding the endogenous CDRs of canine IgG H and L chain DNAs of the above sequences.

[0324] iv) Replace the DNA sequences encoding the endogenous canine H and L chain CDRs with DNA sequences encoding the CDRs of the selected antibody. In addition, the DNA encoding some canine framework amino acid residues is optionally replaced with DNA encoding selected amino acid residues from the framework region of the selected antibody.

[0325] v) Synthesizing the DNA from step (iv) and cloning it into a suitable expression plasmid.

[0326] vi) The synthesized plasmid was transfected into HEK 293 cells.

[0327] vii) Purification of expressed caninized antibodies from HEK 293 supernatant.

[0328] viii) Purified caninized antibodies were tested for binding to canine CTLA-4.

[0329] The nucleotide and amino acid sequences of the CDRs of 12B3 and 39A11 are listed in Table 3 below.

[0330] Table 3

[0331] Nucleotide and amino acid sequences for CDRs of caninized antibodies

[0332]

[0333]

[0334] A set of caninized light and heavy chain sequences were constructed. Their sequence identification numbers are provided in Tables 4-6 below.

[0335] Table 4

[0336] SEQ ID numbers of caninized light chains of 12B3 and 39A11

[0337]

[0338] 1. Underline the CDRs; 2. The variable regions are indicated in bold in the sequences that follow.

[0339] Table 5

[0340] SEQ ID NO: 12B3 and 39A11 with wild-type IgGb (native) caninized heavy chain

[0341]

[0342] 1. Underline the CDRs; 2. The variable regions are indicated in bold in the sequences that follow.

[0343] Table 6

[0344] SEQ ID NO: 12B3 and 39A11 with caninized heavy chain of IgGBm (modified IgGB)

[0345]

[0346] 1. The CDRs are underlined; 2. The variable regions are in bold in the subsequent sequences.

[0347] Table 7

[0348] Related prior art series

[0349]

[0350] The present invention provides caninized antibodies of 12B3 and 39A11 formed from a combination of caninized heavy and light chains of each of the antibodies listed in the table above; such antibodies demonstrate particularly strong binding to cCTLA-4. Figure 6 As shown, the ELISA results indicate that both 12B3 and 39A11 were successfully caninized. Caninized C12B3L3H2 and L3H3 have similar reactivity with cCTLA-4 as the parental 12B3; caninized c39A11L3H3 has similar reactivity with cCTLA-4 as the parental 39A11. The chimeras of 12B3 and 39A11 represent their parental antibodies.

[0351] Nucleotide (NA) and amino acid (AA) sequences

[0352] SEQ ID NO: 1: NA sequence of the heavy chain variable region of mouse monoclonal antibody 12B3

[0353] cagatccagttggtgcagtctggacctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacactggagagcca acatatgctgatgacttcaagggacggtttgccttctctttggaaacctctgccagcactgcctatttgcagatcaacaacctcaaaaatgaggacatggctacatatttctgtgcaagacggtcaatttattacccgtactggggccaaggcaccactctcacagtctcctca

[0354] SEQ ID NO: 2: AA sequence of the heavy chain variable region of mouse monoclonal antibody 12B3

[0355]

[0356] SEQ ID NO: 3: NA sequence of the heavy chain variable region of mouse monoclonal antibody 27G12

[0357] cagatccagttggtacagtctggacctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaacctatggagtgagctgggtgaaacaggctccaggaaagggtttaaggtggatgggctggataaacacctactctggaatgcca acatatgttgatgacttcaagggacggtttgccttctctctttggaaacctctgccagcactgcctttttgcagatcaacaacctcaaaaatgaggacacggctatatatttctgtgcaagacggggtatctcctttgactactggggccaaggcaccactctcacagtctcctca

[0358] SEQ ID NO:4: AA sequence of the heavy chain variable region of mouse monoclonal antibody 27G12

[0359]

[0360] SEQ ID NO:5: NA sequence of the heavy chain variable region of mouse monoclonal antibody 39A11

[0361] gaggtgaagctggtggagtctggaggaggcttggtacagcctgggggttccctgagtctctcctgtgcaacttctggattcaccttcagtgattactacatgagctgggtccgccagtctccggggaaggcacttgagtggatgggttttattagaaacaaagctaatggttacacaaca gagtacagcgcatctctgaagggtcggttcaccatctccagagataattcccaaagcatcctctatcttcaaatgaatgtcctgagagctgaggacagtgccacttattactgtgtaagatttgggttaatgtactactttgactactggggccaaggcaccactctcacagtctcctca

[0362] SEQ ID NO:6: AA sequence of the heavy chain variable region of mouse monoclonal antibody 39A11

[0363]

[0364] SEQ ID NO:7: NA sequence of the heavy chain variable region of mouse monoclonal antibody 45A9

[0365] cagatccagttggtgcagtctggacctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacactggagagcca acatatgctgatgacttcaagggacggtttgccttctctttggaaacctctgccagcactgcctatttgcagatcaacaacctcaaaaatgaggacacggctacatatttctgtgcaagaagggggacctactataggccctggggccaaggcaccactctcacagtctcctca

[0366] SEQ ID NO:8: AA sequence of the heavy chain variable region of mouse monoclonal antibody 45A9

[0367]

[0368] SEQ ID NO:9: NA sequence of the heavy chain variable region of mouse monoclonal antibody 110E3

[0369] cagatccagttggtgcagtctggacctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttctggatataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacactggagagcca acatatgctgatgacttcaagggacgggttgccttctctttggaaacctctgccagcactgcctttttgcagatcaacaacctcaaaaatgaggacacggctacatatttctgtgcaaggcggggggtacgactggactactggggccaaggcaccactctcacagtctcctca

[0370] SEQ ID NO: 10: AA sequence of the heavy chain variable region of mouse monoclonal antibody 110E3

[0371]

[0372] SEQ ID NO: 11: NA sequence of the heavy chain variable region of mouse monoclonal antibody 22A11

[0373] caggtccaactgcagcagcctgggactgaactggtgaagcctggggcttcagtgaagctgtcctgcaaggcctctggctataccttcaccagctactggatgcactgggtgaagcagaggcctggacaaggccttgagtggattggaaatatcaatcctagcaatggtggtactaggttca atgagaagttcaagaacaaggccacactgactgaagacaaatcctccagcacagcctacatgcagctcagtagcctgacatctgaggactctgcggtctattattgtgcaagatcgaactacggtagtggctgggcctggtttgcttactggggccaagggactctggtcactgtctctgca

[0374] SEQ ID NO: 12: AA sequence of the heavy chain variable region of mouse monoclonal antibody 22A11

[0375]

[0376] SEQ ID NO: 13: NA sequence of the light chain variable region of mouse monoclonal antibody 12B3

[0377] gatgttttgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctagtcagagcattgtatatagtaatggaaacacctatttagaatggtacctgcagaaaccaggccagtctccaaagctcctgatctacaaagtt tccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagttattactgctttcaaggttcacatgttccgtggacgttcggtggaggcaccaagctggaaatcaaa

[0378] SEQ ID NO: 14: Mouse monoclonal antibody 12B3 light chain variable region AA sequence

[0379]

[0380] SEQ ID NO: 15: NA sequence of the light chain variable region of mouse monoclonal antibody 27G12

[0381] Gatgttttgatgacccagactccactctccctgcctgtcagtcttggagatcacgcctccatctcttgcaaatctagtcagagcattgtatatattaatggaaacacctatttagaatggtacctgcagaagccaggccagtctccaaagctcctgatctacaaaagtt tccaaacgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagttattactgctttcaaggttcacatgttccgtggacgttcggtggaggcaccaagctggaaatcaaa

[0382] SEQ ID NO: 16: Mouse monoclonal antibody 27G12 light chain variable region AA sequence

[0383]

[0384] SEQ ID NO: 17: NA sequence of the light chain variable region of mouse monoclonal antibody 39A11

[0385] gaaaatgtgctcatccagtctccagcaatcatgtctgcttctccaggggaaaaggtcaccatgacctgcagggccagctcaagtgtaagttccagttacttgcactggtaccagcagaagtcaggtgcctcccccaaactctggatttttagcacatccaac ttggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttattctctcacaatcaacagtgtggaggctgaagatgctgccacttattactgccagcagtacagtggtctcccactcacgttcggaggggggaccaagctggaaataaaa

[0386] SEQ ID NO: 18: Mouse monoclonal antibody 39A11 light chain variable region AA sequence

[0387]

[0388]

[0389] SEQ ID NO: 19: NA sequence of the light chain variable region of mouse monoclonal antibody 45A9

[0390] gatgttttgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctagtcagagtattgtatatagtcatggaaacacctatttagaatggtacctgcagaaaccaggccagtctccaaaggtcctgatctacaaaagtt tccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagttattactgctttcaaggttcacatgttccgtggacgttcggtggaggcaccaagctggaaatcaaa

[0391] SEQ ID NO: 20: Mouse monoclonal antibody 45A9 light chain variable region AA sequence

[0392]

[0393] SEQ ID NO:21: NA sequence of the light chain variable region of mouse monoclonal antibody 110E3

[0394] gatgttttgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctagtcagagcattgtatatattagtggaagcacctatttagaatggtatctgcagaaaccaggccagtctccaaagctcctgatctacaaaagtt tccagtcgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagttattactgctttcaaggttcacatgttccgtggacgttcggtggaggcaccaagctggaaatcaaa

[0395] SEQ ID NO: 22: Mouse monoclonal antibody 110E3 light chain variable region AA sequence

[0396]

[0397] SEQ ID NO: 23: NA sequence of the light chain variable region of mouse monoclonal antibody 22A11

[0398] gacatccagatgaaccagtctccatccagtctgtctgcatcccttggagacacaattaccatcacttgccatgccagtcagaacattaatgtttggttaagctggtaccagcagaaaccaggaaatattcctaaacttttgatctataagtcttccaact tgcacacaggcgtcccatcaaggtttagtggcagtggatctggaacaggtttcacattaaccatcagcagcctgcagcctgaagacattgccacttactactgtcaacagggtcaaagttatccgtggacgttcggtggaggcaccaagctggaaatcaaa

[0399] SEQ ID NO: 24: Mouse monoclonal antibody 22A11 light chain variable region AA sequence

[0400]

[0401] SEQ ID NO:25: Mouse-canine chimeric antibody 12B3 heavy chain NA sequence

[0402]

[0403]

[0404] SEQ ID NO:26: Mouse-canine chimeric antibody 12B3 heavy chain AA sequence

[0405]

[0406] SEQ ID NO:27: Mouse-canine chimeric antibody 27G12 heavy chain NA sequence

[0407]

[0408]

[0409] SEQ ID NO:28: Mouse-canine chimeric antibody 27G12 heavy chain AA sequence

[0410]

[0411] SEQ ID NO: 29: Mouse-canine chimeric antibody 39A11 heavy chain NA sequence

[0412]

[0413]

[0414] SEQ ID NO:30: Mouse-canine chimeric antibody 39A11 heavy chain AA sequence

[0415]

[0416] SEQ ID NO:31: Mouse-canine chimeric antibody 45A9 heavy chain NA sequence

[0417]

[0418] SEQ ID NO:32: Mouse-canine chimeric antibody 45A9 heavy chain AA sequence

[0419]

[0420]

[0421] SEQ ID NO:33: Mouse-canine chimeric antibody 110E3 heavy chain NA sequence

[0422]

[0423] SEQ ID NO:34: Mouse-canine chimeric antibody 110E3 heavy chain AA sequence

[0424]

[0425] SEQ ID NO:35: Mouse-canine chimeric antibody 22A11 heavy chain NA sequence

[0426]

[0427]

[0428] SEQ ID NO:36: Mouse-canine chimeric antibody 22A11 heavy chain AA sequence

[0429]

[0430] SEQ ID NO:37: Mouse-canine chimeric antibody 12B3 light chain NA sequence

[0431]

[0432]

[0433] SEQ ID NO:38: Mouse-canine chimeric antibody 12B3 light chain AA sequence

[0434]

[0435] SEQ ID NO:39: Mouse-canine chimeric antibody 27G12 light chain NA sequence

[0436]

[0437] SEQ ID NO:40: Mouse-canine chimeric antibody 27G12 light chain AA sequence

[0438]

[0439] SEQ ID NO:41: Mouse-canine chimeric antibody 39A11 light chain NA sequence

[0440]

[0441] SEQ ID NO:42: Mouse-canine chimeric antibody 39A11 light chain AA sequence

[0442]

[0443] SEQ ID NO:43: Mouse-canine chimeric antibody 45A9 light chain NA sequence

[0444]

[0445]

[0446] SEQ ID NO:44: Mouse-canine chimeric antibody 45A9 light chain AA sequence

[0447]

[0448] SEQ ID NO:45: Mouse-canine chimeric antibody 110E3 light chain NA sequence

[0449]

[0450] SEQ ID NO:46: Mouse-canine chimeric antibody 110E3 light chain AA sequence

[0451]

[0452] SEQ ID NO:47: Mouse-canine chimeric monoclonal antibody 22A11 light chain NA sequence

[0453]

[0454] SEQ ID NO:48: Mouse-canine chimeric antibody 22A11 light chain AA sequence

[0455]

[0456] SEQ ID NO:49: Caninized 12B3 light chain NA sequence (V L 1)

[0457]

[0458]

[0459] SEQ ID NO:50: Caninized 12B3 light chain AA sequence (VL1)

[0460]

[0461] SEQ ID NO:51: Caninized 12B3 light chain NA sequence (VL2)

[0462]

[0463] SEQ ID NO:52: Caninized 12B3 light chain AA sequence (VL2)

[0464]

[0465] SEQ ID NO:53: Caninized 12B3 light chain NA sequence (VL3)

[0466]

[0467] SEQ ID NO:54: Caninized 12B3 light chain AA sequence (VL3)

[0468]

[0469] SEQ ID NO:55: Caninized 39A11 light chain NA sequence (VL1)

[0470]

[0471]

[0472] SEQ ID NO:56: Caninized 39A11 light chain AA sequence (VL1)

[0473]

[0474] SEQ ID NO:57: Caninized 39A11 light chain NA sequence (VL2)

[0475]

[0476] SEQ ID NO:58: Caninized 39A11 light chain AA sequence (VL2)

[0477]

[0478] SEQ ID NO:59: Caninized 39A11 light chain NA sequence (VL3)

[0479]

[0480] SEQ ID NO:60: Caninized 39A11 light chain AA sequence (VL3)

[0481]

[0482] SEQ ID NO:61: Caninized 12B3 heavy chain NA sequence with IgGB (VH1)

[0483]

[0484]

[0485] SEQ ID NO:62: Caninized 12B3 heavy chain AA sequence with IgGB (VH1)

[0486]

[0487] SEQ ID NO:63: Caninized 12B3 heavy chain NA sequence with IgGB (VH2)

[0488]

[0489]

[0490] SEQ ID NO:64: Caninized 12B3 heavy chain AA sequence with IgGB (VH2)

[0491]

[0492]

[0493] SEQ ID NO:65: Caninized 12B3 heavy chain NA sequence with IgGB (VH3)

[0494]

[0495] SEQ ID NO:66: Caninized 12B3 heavy chain AA sequence (VH3) with IgGB

[0496]

[0497]

[0498] SEQ ID NO:67: Caninized 39A11 heavy chain NA sequence (VH1) with IgGB

[0499]

[0500]

[0501] SEQ ID NO:68: Caninized 39A11 heavy chain AA sequence with IgGB (VH1)

[0502]

[0503] SEQ ID NO:69: Caninized 39A11 heavy chain NA sequence with IgGB (VH2)

[0504]

[0505]

[0506] SEQ ID NO:70: Caninized 39A11 heavy chain AA sequence with IgGB (VH2)

[0507]

[0508] SEQ ID NO:71: Caninized 39A11 heavy chain NA sequence (VH3) with IgGB

[0509]

[0510]

[0511] SEQ ID NO:72: Caninized 39A11 heavy chain AA sequence with IgGB (VH3)

[0512]

[0513]

[0514] SEQ ID NO:73: Caninized 12B3 heavy chain NA sequence (VH1) with IgGBm

[0515]

[0516] SEQ ID NO:74: Caninized 12B3 heavy chain AA sequence (VH1) with IgGBm

[0517]

[0518]

[0519] SEQ ID NO:75: Caninized 12B3 heavy chain NA sequence (VH2) with IgGBm

[0520]

[0521]

[0522] SEQ ID NO:76: Caninized 12B3 heavy chain AA sequence (VH2) with IgGBm

[0523]

[0524] SEQ ID NO: 77. Caninized 12B3 heavy chain NA sequence (VH3) with IgGBm

[0525]

[0526]

[0527] SEQ ID NO:78: Caninized 12B3 heavy chain AA sequence (VH3) with IgGBm

[0528]

[0529] SEQ ID NO:79: Caninized 39A11 heavy chain NA sequence (VH1) with IgGBm

[0530]

[0531]

[0532] SEQ ID NO:80: Caninized 39A11 heavy chain AA sequence (VH1) with IgGBm

[0533]

[0534] SEQ ID NO:81: Caninized 39A11 heavy chain NA sequence (VH2) with IgGBm

[0535]

[0536] SEQ ID NO:82: Caninized 39A11 heavy chain AA sequence (VH2) with IgGBm

[0537]

[0538]

[0539] SEQ ID NO:83: Caninized 39A11 heavy chain NA sequence (VH3) with IgGBm

[0540]

[0541] SEQ ID NO:84: Caninized 39A11 heavy chain AA sequence (VH3) with IgGBm

[0542]

[0543] SEQ ID NO:125: Canine CTLA-4 NA sequence (NCBI reference sequence: NP_001003106).

[0544] atggcgggctttggctttcgccgccatggcgcgcagccggatctggcgagccgcacctggccgtgcaccgcgctgtttagcctgctgtttattccggtgtttagcaaaggcatgcatgtggcgcagccggcggtggtgctggcgagcagccgcggcgtggcgagctttgtgtgcgaatatggcagcagcggcaacgcggcggaagtgcgcgtgaccgtgctgcgccaggcgggcagccagatgaccgaagtgtgcgcggcgacctataccgtggaagatgaactggcgtttctggatgatagcacctgcaccggcaccagcagcggcaacaaagtgaacctgaccattcagggcctgcgcgcgatggataccggcctgtatatttgcaaagtggaactgatgtatccgccgccgtattatgtgggcatgggcaacggcacccagatttatgtgattgatccggaaccgtgcccggatagcgattttctgctgtggattctggcggcggtgagcagcggcctgtttttttatagctttctgattaccgcggtgagcctgagcaaaatgctgaaaaaacgcagcccgctgaccaccggcgtgtatgtgaaaatgccgccgaccgaaccggaatgcgaaaaacagtttcagccgtattttattccgattaac

[0545] SEQ ID NO:126: Canine CTLA-4 AA sequence (NCBI reference sequence: NP_001003106) SEQ ID NO:138, mature sequence (i.e., minus the signal sequence), bold:

[0546]

[0547]

[0548] SEQ ID NO: 127: Genetically modified canine IgG B cFc region (from US10,106,107B2)

[0549] LGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARG QAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK

[0550] Example 8

[0551] Epitope Mapping of Caninized Anti-cCTLA-4 Monoclonal Antibodies 12B3 and 39A11

[0552] The interaction of an antibody with its cognate protein antigen is mediated by the binding of specific amino acids (paratopes) of the antibody to specific amino acids (epitopes) of the target antigen. An epitope is an antigenic determinant that causes a specific response by an immunoglobulin. An epitope consists of a group of amino acids on the surface of an antigen. A protein of interest may contain several epitopes that are recognized by different antibodies. Epitopes recognized by antibodies are classified as either linear epitopes or conformational epitopes. A linear epitope is formed by a continuous stretch of amino acids in a protein, whereas a conformational epitope consists of amino acids that are discontinuous (e.g., far apart) in the primary amino acid sequence but come together after three-dimensional protein folding.

[0553] Epitope mapping refers to the process of identifying the amino acid sequence (i.e., epitope) recognized by an antibody on its target antigen. Identifying the epitope recognized by a monoclonal antibody (mAb) on a target antigen has important applications. For example, it can help develop new therapeutic agents, diagnostic agents, and vaccines. Epitope mapping can also help select optimized therapeutic mAbs and help elucidate their mechanisms of action. Epitope information on canine CTLA-4 can also clarify unique epitopes and define the protective or pathogenic effects of vaccines. Epitope identification can also lead to the development of subunit vaccines based on chemical or genetic coupling of identified peptide epitopes to carrier proteins or other immunostimulants.

[0554] Epitope mapping can be performed using polyclonal or monoclonal antibodies, and epitope identification is performed using several methods, depending on the suspected nature of the epitope (i.e., linear versus conformational). Positioning linear epitopes is more direct and relatively easier to perform. For this purpose, commercial services for linear epitope mapping often use peptide scanning. In this case, a group of overlapping short peptide sequences of the target protein are chemically synthesized and tested for their ability to bind to the antibody of interest. This strategy is fast, high-throughput, and relatively cheap to perform. On the other hand, the positioning of discontinuous epitopes is technically more challenging and requires more specialized techniques, such as X-ray co-crystal analysis of monoclonal antibodies together with their target proteins, hydrogen-deuterium (H / D) exchange, mass spectrometry coupled to enzymatic digestion, and several other methods known to those skilled in the art.

[0555] Mapping the Canine CTLA-4 Receptor Alpha Epitope Using Mass Spectrometry:

[0556] To determine the epitopes of caninized 12B3 (exemplified by 12B3L2H3) and 39A11 (exemplified by 39A11L3H3) on canine CTLA-4, each complex of cCTLA-4 / c12B3L2H3 and cCTLA-4 / c39A11L2H3 was incubated with a deuterated cross-linker and subjected to multiple enzymatic cleavages. After enrichment of the cross-linked peptides, the samples were analyzed by high-resolution mass spectrometry (NLC-LTQ-OrbitrapMS), and the resulting data were analyzed using Xquest and Stavrox software.

[0557] The analysis showed that c12B3L2H3 interacts with amino acid residues 35, 38, 51, 53, 90, 93, 98 and 102 on cCTLA-4 comprising the amino acid sequence SEQ ID NO: 138 ( Figure 7 A); c39A11L2H3 interacts with amino acid residues 35, 38, 42, 93, and 102 on cCTLA-4 comprising the amino acid sequence SEQ ID NO: 138 ( Figure 7 B) Two specific regions of the canine CTLA-4 protein are depicted in Figure 7A and 7B: amino acid sequences SEQ ID NO: 132 and SEQ ID NO: 133, respectively (see Table 8 below). It is noteworthy that both antibodies bind to SEQ ID NO: 134 and SEQ ID NO: 136 on cCTLA-4, which contain the MYPPPY motif (SEQ ID NO: 137). The MYPPPY motif forms a loop binding with CD80 and CD86, which is a conserved motif of CTLA-4 across species (accross species). C12B3 also appears to bind to an additional region on canine CTLA-4 containing the amino acid sequence SEQ ID NO: 135. Combined with the results of Example 4, the epitope mapping results further confirm that C12B3 and C39A11 are both functional antibodies with the ability to block the interaction of canine CTLA-4 with its ligands CD80 and CD86. In addition, caninized antibodies that bind to epitopes in SEQID NO: 134 and SEQ ID NO: 136 are also part of the present invention.

[0558] Table 8

[0559] The amino acid region of canine CTLA-4 to which THE 12B3 and C39A1 bind

[0560] SEQ ID NO:132:AEVRVTVLRQAGSQMTEVCAATYTVEDELAF

[0561] SEQ ID NO:133:YICKVELMYPPPYYVGMGNGT

[0562] SEQ ID NO:134:TVLRQAGS

[0563] SEQ ID NO:135:ATYTV

[0564] SEQ ID NO:136:YICKVELMYPPPY

[0565] SEQ ID NO:137:MYPPPY.

[0566] Preferred embodiment:

[0567] 1. An isolated mammalian antibody or antigen-binding fragment that binds to canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and blocks the binding of canine CTLA-4 to canine CD80, blocks the binding of canine CTLA-4 to canine CD86, or blocks both the binding of canine CTLA-4 to canine CD80 and the binding of canine CTLA-4 to canine CD86;

[0568] wherein the antibody comprises a set of six complementarity determining regions (CDRs), three of which are light chain CDRs: CDR light chain 1 (CDRL1), CDR light chain 2 (CDRL2), and CDR light chain 3 (CDRL3); and three of which are heavy chain CDRs: CDR heavy chain 1 (CDRH1), CDR heavy chain 2 (CDRH2), and CDR heavy chain 3 (CDRH3);

[0569] wherein the group of six CDRs is selected from group (i), group (ii), group (iii), group (iv), group (v) and group (vi);

[0570] Where for group (i):

[0571] CDRL1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:92, a conservatively modified variant of SEQ ID NO:92, and a variant of SEQ ID NO:92 comprising canonical structural class 4;

[0572] CDRL2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:94, a conservatively modified variant of SEQ ID NO:94, and a variant of SEQ ID NO:94 comprising canonical structural class 1;

[0573] CDRL3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:96, conservatively modified variants of SEQ ID NO:96, and variants of SEQ ID NO:96 comprising canonical structural class 1;

[0574] CDRH1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:86, conservatively modified variants of SEQ ID NO:86, and variants of SEQ ID NO:86 comprising canonical structural class 1;

[0575] The CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:88, conservatively modified variants of SEQ ID NO:88, and variants of SEQ ID NO:88 comprising canonical structural class 2A; and

[0576] The CDRH3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:90, conservatively modified variants of SEQ ID NO:90, and variants of SEQ ID NO:90 comprising canonical structural class 7; wherein for group (ii),

[0577] CDRL1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 104, a conservatively modified variant of SEQ ID NO: 104, and a variant of SEQ ID NO: 104 comprising canonical structural class 1;

[0578] CDRL2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 106, a conservatively modified variant of SEQ ID NO: 106, and a variant of SEQ ID NO: 106 comprising canonical structural class 1;

[0579] CDRL3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 108, a conservatively modified variant of SEQ ID NO: 108, and a variant of SEQ ID NO: 108 comprising canonical structural class 1;

[0580] CDRH1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:98, conservatively modified variants of SEQ ID NO:98, and variants of SEQ ID NO:98 comprising canonical structural class 1;

[0581] CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 100, conservatively modified variants of SEQ ID NO: 100, and a variant of SEQ ID NO: 100 comprising canonical structural class 4; and

[0582] CDRH3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 102, a conservatively modified variant of SEQ ID NO: 102, and a variant of SEQ ID NO: 102 comprising canonical structural class 9;

[0583] For group (iii),

[0584] CDRL1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 117, a conservatively modified variant of SEQ ID NO: 117, and a variant of SEQ ID NO: 117 comprising canonical structural class 4;

[0585] CDRL2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:94, a conservatively modified variant of SEQ ID NO:94, and a variant of SEQ ID NO:94 comprising canonical structural class 1;

[0586] CDRL3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:96, conservatively modified variants of SEQ ID NO:96, and variants of SEQ ID NO:96 comprising canonical structural class 1;

[0587] CDRH1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:86, conservatively modified variants of SEQ ID NO:86, and variants of SEQ ID NO:86 comprising canonical structural class 1;

[0588] The CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:88, conservatively modified variants of SEQ ID NO:88, and variants of SEQ ID NO:88 comprising canonical structural class 2A; and

[0589] CDRH3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 113, a conservatively modified variant of SEQ ID NO: 113, and a variant of SEQ ID NO: 113 comprising canonical structural class 7;

[0590] For group (iv),

[0591] CDRL1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 119, a conservatively modified variant of SEQ ID NO: 119, and a variant of SEQ ID NO: 119 comprising canonical structural class 4;

[0592] CDRL2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 122, a conservatively modified variant of SEQ ID NO: 122, and a variant of SEQ ID NO: 122 comprising canonical structural class 1;

[0593] CDRL3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:96, conservatively modified variants of SEQ ID NO:96, and variants of SEQ ID NO:96 comprising canonical structural class 1;

[0594] CDRH1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:86, conservatively modified variants of SEQ ID NO:86, and variants of SEQ ID NO:86 comprising canonical structural class 1;

[0595] The CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:88, conservatively modified variants of SEQ ID NO:88, and variants of SEQ ID NO:88 comprising canonical structural class 2A; and

[0596] CDRH3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 115, a conservatively modified variant of SEQ ID NO: 115, and a variant of SEQ ID NO: 115 comprising canonical structural class 7;

[0597] where for group (v),

[0598] CDRL1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 118, a conservatively modified variant of SEQ ID NO: 118, and a variant of SEQ ID NO: 118 comprising canonical structural class 4;

[0599] CDRL2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 121, a conservatively modified variant of SEQ ID NO: 121, and a variant of SEQ ID NO: 121 comprising canonical structural class 1;

[0600] CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:96, a conservatively modified variant of SEQ ID NO:96, and a variant of SEQ ID NO:96 comprising canonical structural class 1; CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:109, a conservatively modified variant of SEQ ID NO:109, and a variant of SEQ ID NO:109 comprising canonical structural class 1;

[0601] CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 111, a conservatively modified variant of SEQ ID NO: 111, and a variant of SEQ ID NO: 111 comprising canonical structural class 2A; and

[0602] The CDRH3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 114, a conservatively modified variant of SEQ ID NO: 114, and a variant of SEQ ID NO: 114 comprising canonical structural class 7; and

[0603] For group (vi),

[0604] CDRL1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 120, a conservatively modified variant of SEQ ID NO: 120, and a variant of SEQ ID NO: 120 comprising canonical structural class 2; and

[0605] CDRL2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 123, a conservatively modified variant of SEQ ID NO: 123, and a variant of SEQ ID NO: 123 comprising canonical structural class 1;

[0606] CDRL3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 124, a conservatively modified variant of SEQ ID NO: 124, and a variant of SEQ ID NO: 124 comprising canonical structural class 1;

[0607] CDRH1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 110, a conservatively modified variant of SEQ ID NO: 110, and a variant of SEQ ID NO: 110 comprising canonical structural class 1;

[0608] The CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 112, conservatively modified variants of SEQ ID NO: 112, and variants of SEQ ID NO: 112 comprising canonical structural class 2A; and

[0609] The CDRH3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 116, a conservatively modified variant of SEQ ID NO: 116, and a variant of SEQ ID NO: 116 comprising canonical structural class 12.

[0610] 2. The isolated mammalian antibody or antigen-binding fragment thereof of item 1, wherein

[0611] (a) CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:92, a conservatively modified variant of SEQ ID NO:92, and a variant of SEQ ID NO:92 comprising canonical structural class 4;

[0612] (b) CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:94, a conservatively modified variant of SEQ ID NO:94, and a variant of SEQ ID NO:94 comprising canonical structural class 1;

[0613] (c) CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:96, a conservatively modified variant of SEQ ID NO:96, and a variant of SEQ ID NO:96 comprising canonical structural class 1;

[0614] (d) the CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:86, a conservatively modified variant of SEQ ID NO:86, and a variant of SEQ ID NO:86 comprising canonical structural class 1;

[0615] (e) the CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:88, a conservatively modified variant of SEQ ID NO:88, and a variant of SEQ ID NO:88 comprising canonical structural class 2A; and

[0616] (f) the CDRH3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:90, a conservatively modified variant of SEQ ID NO:90, and a variant of SEQ ID NO:90 comprising canonical structural class 7.

[0617] 3. The isolated mammalian antibody or antigen-binding fragment thereof of item 1, wherein

[0618] (a) CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 104, a conservatively modified variant of SEQ ID NO: 104, and a variant of SEQ ID NO: 104 comprising canonical structural class 1;

[0619] (b) CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 106, a conservatively modified variant of SEQ ID NO: 106, and a variant of SEQ ID NO: 106 comprising canonical structural class 1;

[0620] (c) CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 108, a conservatively modified variant of SEQ ID NO: 108, and a variant of SEQ ID NO: 108 comprising canonical structural class 1;

[0621] (d) the CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:98, conservatively modified variants of SEQ ID NO:98, and variants of SEQ ID NO:98 comprising canonical structural class 1;

[0622] (e) the CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 100, a conservatively modified variant of SEQ ID NO: 100, and a variant of SEQ ID NO: 100 comprising canonical structural class 4; and

[0623] (f) the CDRH3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 102, a conservatively modified variant of SEQ ID NO: 102, and a variant of SEQ ID NO: 102 comprising canonical structural class 9.

[0624] 4. The isolated mammalian antibody or antigen-binding fragment thereof of item 1, wherein

[0625] (a) CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 117, a conservatively modified variant of SEQ ID NO: 117, and a variant of SEQ ID NO: 117 comprising canonical structural class 4;

[0626] (b) CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:94, a conservatively modified variant of SEQ ID NO:94, and a variant of SEQ ID NO:94 comprising canonical structural class 1;

[0627] (c) CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:96, a conservatively modified variant of SEQ ID NO:96, and a variant of SEQ ID NO:96 comprising canonical structural class 1;

[0628] (d) the CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:86, a conservatively modified variant of SEQ ID NO:86, and a variant of SEQ ID NO:86 comprising canonical structural class 1;

[0629] (e) the CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:88, a conservatively modified variant of SEQ ID NO:88, and a variant of SEQ ID NO:88 comprising canonical structural class 2A; and

[0630] (f) the CDRH3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 113, a conservatively modified variant of SEQ ID NO: 113, and a variant of SEQ ID NO: 113 comprising canonical structural class 7.

[0631] 5. The isolated mammalian antibody or antigen-binding fragment thereof of item 1, wherein

[0632] (a) CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 119, a conservatively modified variant of SEQ ID NO: 119, and a variant of SEQ ID NO: 119 comprising canonical structural class 4;

[0633] (b) CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 122, a conservatively modified variant of SEQ ID NO: 122, and a variant of SEQ ID NO: 122 comprising canonical structural class 1;

[0634] (c) CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:96, a conservatively modified variant of SEQ ID NO:96, and a variant of SEQ ID NO:96 comprising canonical structural class 1;

[0635] (d) the CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:86, a conservatively modified variant of SEQ ID NO:86, and a variant of SEQ ID NO:86 comprising canonical structural class 1;

[0636] (e) the CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:88, a conservatively modified variant of SEQ ID NO:88, and a variant of SEQ ID NO:88 comprising canonical structural class 2A; and

[0637] (f) the CDRH3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 115, a conservatively modified variant of SEQ ID NO: 115, and a variant of SEQ ID NO: 115 comprising canonical structural class 7.

[0638] 6. The isolated mammalian antibody or antigen-binding fragment thereof of item 1, wherein

[0639] (a) CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 118, a conservatively modified variant of SEQ ID NO: 118, and a variant of SEQ ID NO: 118 comprising canonical structural class 4;

[0640] (b) CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 121, a conservatively modified variant of SEQ ID NO: 121, and a variant of SEQ ID NO: 121 comprising canonical structural class 1;

[0641] (c) CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:96, a conservatively modified variant of SEQ ID NO:96, and a variant of SEQ ID NO:96 comprising canonical structural class 1;

[0642] (d) the CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 109, a conservatively modified variant of SEQ ID NO: 109, and a variant of SEQ ID NO: 109 comprising canonical structural class 1;

[0643] (e) the CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 111, a conservatively modified variant of SEQ ID NO: 111, and a variant of SEQ ID NO: 111 comprising canonical structural class 2A; and

[0644] (f) the CDRH3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 114, a conservatively modified variant of SEQ ID NO: 114, and a variant of SEQ ID NO: 114 comprising canonical structural class 7.

[0645] 7. The isolated mammalian antibody or antigen-binding fragment thereof of item 1, wherein

[0646] (a) CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 120, a conservatively modified variant of SEQ ID NO: 120, and a variant of SEQ ID NO: 120 comprising canonical structural class 2;

[0647] (b) CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 123, a conservatively modified variant of SEQ ID NO: 123, and a variant of SEQ ID NO: 123 comprising canonical structural class 1;

[0648] (c) CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 124, a conservatively modified variant of SEQ ID NO: 124, and a variant of SEQ ID NO: 124 comprising canonical structural class 1;

[0649] (d) CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 110, a conservatively modified variant of SEQ ID NO: 110, and a variant of SEQ ID NO: 110 comprising canonical structural class 1;

[0650] (e) the CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 112, a conservatively modified variant of SEQ ID NO: 112, and a variant of SEQ ID NO: 112 comprising canonical structural class 2A; and

[0651] (f) the CDRH3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 116, a conservatively modified variant of SEQ ID NO: 116, and a variant of SEQ ID NO: 116 comprising canonical structural class 12.

[0652] 8. The isolated mammalian antibody or antigen-binding fragment thereof of item 1, 2, 3, 4, 5, 6 or 7, wherein the mammalian antibody is a murine antibody.

[0653] 9. The isolated mammalian antibody or antigen-binding fragment thereof of item 1, 2, 3, 4, 5, 6 or 7, or the murine antibody of item 8, which is a caninized antibody or a caninized antigen-binding fragment thereof.

[0654] 10. The caninized antibody or caninized antigen-binding fragment thereof according to claim 9, which comprises a hinge region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 and SEQ ID NO: 131.

[0655] 11. The isolated mammalian antibody or antigen-binding fragment thereof according to item 2, which is a caninized antibody or a caninized antigen-binding fragment thereof.

[0656] 12. The caninized antibody or antigen-binding fragment thereof of item 11, which comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:62, SEQ ID NO:64 and SEQ ID NO:66, a modified heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:74, SEQ ID NO:76 and SEQ ID NO:78, or a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:50, SEQ ID NO:52 and SEQID NO:54, or a combination of the heavy chain or the modified heavy chain and the light chain.

[0657] 13. The caninized antibody or antigen-binding fragment thereof of item 12, comprising a heavy chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63 and SEQ ID NO:65, a modified heavy chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:75 and SEQ ID NO:77, and a light chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NO:51 and SEQ ID NO:53, or a combination of the heavy chain or the modified heavy chain and the light chain.

[0658] 14. The caninized antibody or antigen-binding fragment thereof of item 12, which comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 66.

[0659] 15. The caninized antibody or antigen-binding fragment thereof of item 12, which comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 78.

[0660] 16. The caninized antibody or antigen-binding fragment thereof according to item 14 or 15, further comprising a light chain comprising the amino acid sequence of SEQ ID NO:52.

[0661] 17. The caninized antibody or antigen-binding fragment thereof according to item 14 or 15, further comprising a light chain comprising the amino acid sequence of SEQ ID NO:54.

[0662] 18. The isolated mammalian antibody or antigen-binding fragment thereof according to item 3, which is a caninized antibody or a caninized antigen-binding fragment thereof.

[0663] 19. The caninized antibody or antigen-binding fragment thereof of item 18, which comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 68, SEQ ID NO: 70 and SEQ ID NO: 72, a modified heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 80, SEQ ID NO: 82 and SEQ ID NO: 84, and a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 56, SEQ ID NO: 58 and SEQ ID NO: 60, or a combination of the heavy chain or the modified heavy chain and the light chain.

[0664] 20. The caninized antibody or antigen-binding fragment thereof of item 19, comprising a heavy chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 67, SEQ ID NO: 69 and SEQ ID NO: 71, a modified heavy chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 79, SEQ ID NO: 81 and SEQ ID NO: 83, and a light chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 55, SEQ ID NO: 57 and SEQ ID NO: 59, or a combination of the heavy chain or the modified heavy chain and the light chain.

[0665] 21. The caninized antibody or antigen-binding fragment thereof of item 19, which comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 72.

[0666] 22. The caninized antibody or antigen-binding fragment thereof of item 19, which comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 84.

[0667] 23. The caninized antibody or antigen-binding fragment thereof of item 21 or 22, further comprising a light chain comprising the amino acid sequence of SEQ ID NO:58.

[0668] 24. The caninized antibody or antigen-binding fragment thereof according to item 21 or 22, further comprising a light chain comprising the amino acid sequence of SEQ ID NO: 60.

[0669] 25. The caninized antibody or antigen-binding fragment thereof according to any one of items 9 to 24, wherein the caninized antibody or antigen-binding fragment thereof exhibits one, two, three, four or all five of the following properties:

[0670] (i) Take 1×10 -5 M to 1×10 -12 The dissociation constant (Kd) of M binds to canine CTLA-4;

[0671] (ii) 1×10 2 M -1 s-1 Up to 1×107M -1 s -1 The binding rate (kon) binds to canine CTLA-4; (iii) at 1×10 -3 s - 1 to 1×10 -8 s -1 The dissociation rate (koff) binds to canine CTLA-4;

[0672] (iv) blocking the binding of canine CTLA-4 to canine CD80; and

[0673] (v) Blocking the binding of canine CTLA-4 to canine CD86.

[0674] 26. A caninized monoclonal antibody or antigen-binding fragment thereof that cross-competes with any one of items 9-25 for binding to canine CTLA-4; wherein the caninized monoclonal antibody and antigen-binding fragment thereof binds to canine CTLA-4 and blocks the binding of canine CTLA-4 to canine CD80 and / or canine CD86.

[0675] 27. The caninized antibody of any one of items 9 to 26, wherein the caninized antibody binds to any one or more amino acid sequences selected from SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136 and SEQ ID NO: 137.

[0676] 28. The caninized antibody of item 27, which binds to any one or more amino acid sequences selected from SEQ ID NO: 134, SEQ ID NO: 135 and SEQ ID NO: 136.

[0677] 29. The caninized antibody of claim 28, which binds to the amino acid sequences of SEQ ID NO: 134 and SEQ ID NO: 136.

[0678] 30. The caninized antibody of claim 29, which further binds to SEQ ID NO: 135.

[0679] 31. An isolated nucleic acid encoding the heavy chain of the caninized antibody or antigen-binding fragment thereof according to any one of items 9 to 30.

[0680] 32. An isolated nucleic acid encoding the light chain of the caninized antibody or antigen-binding fragment thereof according to any one of items 9 to 30.

[0681] 33. An expression vector comprising the isolated nucleic acid of item 31 and / or item 32.

[0682] 34. A host cell comprising the expression vector of item 33.

[0683] 35. A pharmaceutical composition comprising the caninized antibody of any one of items 9 to 30 and a pharmaceutically acceptable carrier or diluent.

[0684] 36. A method for increasing the activity of immune cells, comprising administering a therapeutically effective amount of the pharmaceutical composition of item 35 to a subject in need thereof.

[0685] 37. The method of item 36, wherein the method is used for:

[0686] (i) Treatment of cancer;

[0687] (ii) treatment of infection or infectious disease;

[0688] (iii) as a vaccine adjuvant; or (iv) any combination thereof.

[0689] 38. An isolated peptide comprising 5 to 25 amino acid residues that are 90% identical or higher to an amino acid sequence selected from SEQ ID NO: 132 and SEQ ID NO: 133, and binds to the caninized antibody of any one of items 9-26.

[0690] 39. The isolated peptide of item 38, wherein the amino acid sequence is 90% identical or higher to an amino acid sequence selected from the group consisting of SEQ ID NO: 134, SEQ ID NO: 135 and SEQ ID NO: 136.

[0691] 40. The isolated peptide of item 39, wherein the amino acid sequence is identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 134, SEQ ID NO: 135 and SEQ ID NO: 136.

[0692] 41. A fusion protein comprising the peptide according to item 40.

[0693] 42. The fusion protein of item 41, which further comprises the Fc region of non-canine mammal IgG.

[0694] 43. An isolated nucleic acid encoding the isolated peptide of item 38, 39 or 40, or the fusion protein of item 41 or item 42, or any combination thereof.

[0695] 44. An expression vector comprising the isolated nucleic acid of item 43.

[0696] 45. A host cell comprising the expression vector of item 44.

Claims

1. An isolated mammalian antibody or antigen-binding fragment that binds to canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and blocks the binding of canine CTLA-4 to canine CD80, blocks the binding of canine CTLA-4 to canine CD86, or blocks both the binding of canine CTLA-4 to canine CD80 and the binding of canine CTLA-4 to canine CD86; wherein the antibody comprises a set of six complementarity determining regions (CDRs), three of which are light chain CDRs: CDR light chain 1 (CDRL1), CDR light chain 2 (CDRL2), and CDR light chain 3 (CDRL3); and three of which are heavy chain CDRs: CDR heavy chain 1 (CDRH1), CDR heavy chain 2 (CDRH2), and CDR heavy chain 3 (CDRH3); wherein the group of six CDRs is selected from group (i), group (ii), group (iii), group (iv), group (v) and group (vi); Where for group (i): CDRL1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:92, a conservatively modified variant of SEQ ID NO:92, and a variant of SEQ ID NO:92 comprising canonical structural class 4; CDRL2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:94, a conservatively modified variant of SEQ ID NO:94, and a variant of SEQ ID NO:94 comprising canonical structural class 1; CDRL3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:96, conservatively modified variants of SEQ ID NO:96, and variants of SEQ ID NO:96 comprising canonical structural class 1; CDRH1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:86, conservatively modified variants of SEQ ID NO:86, and variants of SEQ ID NO:86 comprising canonical structural class 1; The CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:88, conservatively modified variants of SEQ ID NO:88, and variants of SEQ ID NO:88 comprising canonical structural class 2A; and CDRH3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:90, a conservatively modified variant of SEQ ID NO:90, and a variant of SEQ ID NO:90 comprising canonical structural class 7; For group (ii), CDRL1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 104, a conservatively modified variant of SEQ ID NO: 104, and a variant of SEQ ID NO: 104 comprising canonical structural class 1; CDRL2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 106, a conservatively modified variant of SEQ ID NO: 106, and a variant of SEQ ID NO: 106 comprising canonical structural class 1; CDRL3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 108, a conservatively modified variant of SEQ ID NO: 108, and a variant of SEQ ID NO: 108 comprising canonical structural class 1; CDRH1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:98, conservatively modified variants of SEQ ID NO:98, and variants of SEQ ID NO:98 comprising canonical structural class 1; CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 100, conservatively modified variants of SEQ ID NO: 100, and a variant of SEQ ID NO: 100 comprising canonical structural class 4; and CDRH3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 102, a conservatively modified variant of SEQ ID NO: 102, and a variant of SEQ ID NO: 102 comprising canonical structural class 9; For group (iii), CDRL1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 117, a conservatively modified variant of SEQ ID NO: 117, and a variant of SEQ ID NO: 117 comprising canonical structural class 4; CDRL2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:94, a conservatively modified variant of SEQ ID NO:94, and a variant of SEQ ID NO:94 comprising canonical structural class 1; CDRL3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:96, a conservatively modified variant of SEQ ID NO:96, and a variant of SEQ ID NO:96 comprising canonical structural class 1; CDRH1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:86, conservatively modified variants of SEQ ID NO:86, and variants of SEQ ID NO:86 comprising canonical structural class 1; The CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:88, conservatively modified variants of SEQ ID NO:88, and variants of SEQ ID NO:88 comprising canonical structural class 2A; and CDRH3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 113, a conservatively modified variant of SEQ ID NO: 113, and a variant of SEQ ID NO: 113 comprising canonical structural class 7; For group (iv), CDRL1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 119, a conservatively modified variant of SEQ ID NO: 119, and a variant of SEQ ID NO: 119 comprising canonical structural class 4; CDRL2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 122, a conservatively modified variant of SEQ ID NO: 122, and a variant of SEQ ID NO: 122 comprising canonical structural class 1; CDRL3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:96, conservatively modified variants of SEQ ID NO:96, and variants of SEQ ID NO:96 comprising canonical structural class 1; CDRH1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:86, conservatively modified variants of SEQ ID NO:86, and variants of SEQ ID NO:86 comprising canonical structural class 1; The CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:88, conservatively modified variants of SEQ ID NO:88, and variants of SEQ ID NO:88 comprising canonical structural class 2A; and CDRH3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 115, a conservatively modified variant of SEQ ID NO: 115, and a variant of SEQ ID NO: 115 comprising canonical structural class 7; where for group (v), CDRL1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 118, a conservatively modified variant of SEQ ID NO: 118, and a variant of SEQ ID NO: 118 comprising canonical structural class 4; CDRL2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 121, a conservatively modified variant of SEQ ID NO: 121, and a variant of SEQ ID NO: 121 comprising canonical structural class 1; CDRL3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:96, conservatively modified variants of SEQ ID NO:96, and variants of SEQ ID NO:96 comprising canonical structural class 1; CDRH1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 109, conservatively modified variants of SEQ ID NO: 109, and variants of SEQ ID NO: 109 comprising canonical structural class 1; CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 111, a conservatively modified variant of SEQ ID NO: 111, and a variant of SEQ ID NO: 111 comprising canonical structural class 2A; and The CDRH3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 114, a conservatively modified variant of SEQ ID NO: 114, and a variant of SEQ ID NO: 114 comprising canonical structural class 7; and For group (vi), CDRL1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 120, a conservatively modified variant of SEQ ID NO: 120, and a variant of SEQ ID NO: 120 comprising canonical structural class 2; and CDRL2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 123, a conservatively modified variant of SEQ ID NO: 123, and a variant of SEQ ID NO: 123 comprising canonical structural class 1; CDRL3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 124, a conservatively modified variant of SEQ ID NO: 124, and a variant of SEQ ID NO: 124 comprising canonical structural class 1; CDRH1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 110, a conservatively modified variant of SEQ ID NO: 110, and a variant of SEQ ID NO: 110 comprising canonical structural class 1; The CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 112, conservatively modified variants of SEQ ID NO: 112, and variants of SEQ ID NO: 112 comprising canonical structural class 2A; and The CDRH3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 116, a conservatively modified variant of SEQ ID NO: 116, and a variant of SEQ ID NO: 116 comprising canonical structural class 12.

2. The isolated mammalian antibody or antigen-binding fragment thereof of claim 1, wherein (a) CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:92, a conservatively modified variant of SEQ ID NO:92, and a variant of SEQ ID NO:92 comprising canonical structural class 4; (b) CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:94, a conservatively modified variant of SEQ ID NO:94, and a variant of SEQ ID NO:94 comprising canonical structural class 1; (c) CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:96, a conservatively modified variant of SEQ ID NO:96, and a variant of SEQ ID NO:96 comprising canonical structural class 1; (d) the CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:86, a conservatively modified variant of SEQ ID NO:86, and a variant of SEQ ID NO:86 comprising canonical structural class 1; (e) the CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:88, a conservatively modified variant of SEQ ID NO:88, and a variant of SEQ ID NO:88 comprising canonical structural class 2A; and (f) the CDRH3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:90, a conservatively modified variant of SEQ ID NO:90, and a variant of SEQ ID NO:90 comprising canonical structural class 7.

3. The isolated mammalian antibody or antigen-binding fragment thereof of claim 1, wherein (a) CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 104, a conservatively modified variant of SEQ ID NO: 104, and a variant of SEQ ID NO: 104 comprising canonical structural class 1; (b) CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 106, a conservatively modified variant of SEQ ID NO: 106, and a variant of SEQ ID NO: 106 comprising canonical structural class 1; (c) CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 108, a conservatively modified variant of SEQ ID NO: 108, and a variant of SEQ ID NO: 108 comprising canonical structural class 1; (d) the CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:98, conservatively modified variants of SEQ ID NO:98, and variants of SEQ ID NO:98 comprising canonical structural class 1; (e) the CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 100, a conservatively modified variant of SEQ ID NO: 100, and a variant of SEQ ID NO: 100 comprising canonical structural class 4; and (f) the CDRH3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 102, a conservatively modified variant of SEQ ID NO: 102, and a variant of SEQ ID NO: 102 comprising canonical structural class 9.

4. The isolated mammalian antibody or antigen-binding fragment thereof of claim 1, wherein (a) CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 117, a conservatively modified variant of SEQ ID NO: 117, and a variant of SEQ ID NO: 117 comprising canonical structural class 4; (b) CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:94, a conservatively modified variant of SEQ ID NO:94, and a variant of SEQ ID NO:94 comprising canonical structural class 1; (c) CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:96, a conservatively modified variant of SEQ ID NO:96, and a variant of SEQ ID NO:96 comprising canonical structural class 1; (d) the CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:86, a conservatively modified variant of SEQ ID NO:86, and a variant of SEQ ID NO:86 comprising canonical structural class 1; (e) the CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:88, a conservatively modified variant of SEQ ID NO:88, and a variant of SEQ ID NO:88 comprising canonical structural class 2A; and (f) the CDRH3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 113, a conservatively modified variant of SEQ ID NO: 113, and a variant of SEQ ID NO: 113 comprising canonical structural class 7.

5. The isolated mammalian antibody or antigen-binding fragment thereof of claim 1, wherein (a) CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 119, a conservatively modified variant of SEQ ID NO: 119, and a variant of SEQ ID NO: 119 comprising canonical structural class 4; (b) CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 122, a conservatively modified variant of SEQ ID NO: 122, and a variant of SEQ ID NO: 122 comprising canonical structural class 1; (c) CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:96, a conservatively modified variant of SEQ ID NO:96, and a variant of SEQ ID NO:96 comprising canonical structural class 1; (d) the CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:86, a conservatively modified variant of SEQ ID NO:86, and a variant of SEQ ID NO:86 comprising canonical structural class 1; (e) the CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:88, a conservatively modified variant of SEQ ID NO:88, and a variant of SEQ ID NO:88 comprising canonical structural class 2A; and (f) the CDRH3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 115, a conservatively modified variant of SEQ ID NO: 115, and a variant of SEQ ID NO: 115 comprising canonical structural class 7.

6. The isolated mammalian antibody or antigen-binding fragment thereof of claim 1, wherein (a) CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 118, a conservatively modified variant of SEQ ID NO: 118, and a variant of SEQ ID NO: 118 comprising canonical structural class 4; (b) CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 121, a conservatively modified variant of SEQ ID NO: 121, and a variant of SEQ ID NO: 121 comprising canonical structural class 1; (c) CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:96, a conservatively modified variant of SEQ ID NO:96, and a variant of SEQ ID NO:96 comprising canonical structural class 1; (d) the CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 109, a conservatively modified variant of SEQ ID NO: 109, and a variant of SEQ ID NO: 109 comprising canonical structural class 1; (e) the CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 111, a conservatively modified variant of SEQ ID NO: 111, and a variant of SEQ ID NO: 111 comprising canonical structural class 2A; and (f) the CDRH3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 114, a conservatively modified variant of SEQ ID NO: 114, and a variant of SEQ ID NO: 114 comprising canonical structural class 7.

7. The isolated mammalian antibody or antigen-binding fragment thereof of claim 1, wherein (a) CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 120, a conservatively modified variant of SEQ ID NO: 120, and a variant of SEQ ID NO: 120 comprising canonical structural class 2; (b) CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 123, a conservatively modified variant of SEQ ID NO: 123, and a variant of SEQ ID NO: 123 comprising canonical structural class 1; (c) CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 124, a conservatively modified variant of SEQ ID NO: 124, and a variant of SEQ ID NO: 124 comprising canonical structural class 1; (d) CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 110, a conservatively modified variant of SEQ ID NO: 110, and a variant of SEQ ID NO: 110 comprising canonical structural class 1; (e) the CDRH2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 112, a conservatively modified variant of SEQ ID NO: 112, and a variant of SEQ ID NO: 112 comprising canonical structural class 2A; and (f) the CDRH3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 116, a conservatively modified variant of SEQ ID NO: 116, and a variant of SEQ ID NO: 116 comprising canonical structural class 12.

8. The isolated mammalian antibody or antigen-binding fragment thereof of claim 1, 2, 3, 4, 5, 6 or 7, wherein the mammalian antibody is a murine antibody.

9. The isolated mammalian antibody or antigen-binding fragment thereof of claim 1, 2, 3, 4, 5, 6 or 7, or the murine antibody of claim 8, which is a caninized antibody or a caninized antigen-binding fragment thereof.

10. The caninized antibody or caninized antigen-binding fragment thereof of claim 9, comprising a hinge region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 and SEQ ID NO: 131.

Images