Caninized antibodies against canine CTLA-4

Abstract

To provide anti-canine Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4) antibodies that bind canine CTLA-4.SOLUTION: The present invention provides caninized murine antibodies against canine CTLA-4 that have specific sequences and a high binding affinity for canine CTLA-4. The present invention further provides epitopes of canine CTLA-4 for caninized murine antibodies against canine CTLA-4.5. The invention also relates to use of these antibodies in the treatment of cancer in canines and other companion animals.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed under 35 U.S.C. § 119(e) on July 15, 2019. U.S. Provisional Patent Application No. 62 / 874,287, filed October 25, 2019 U.S. Provisional Patent Application No. 62 / 926,047 filed July 7, 2020 This application claims priority to U.S. Provisional Patent Application No. 63 / 048,873. The contents of U.S. Provisional Patent Application No. 62 / 926,047 and U.S. Provisional Patent Application No. 63 / 048,873 are incorporated herein by reference. No. 6,239,999, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to proteins involved in costimulatory or co-inhibitory signaling pathways (which may be CTL More particularly, the present invention further relates to antibodies against certain and having high binding affinity to canine CTLA-4. The present invention further relates to caninized antibodies against 4. The present invention further relates to the use of the antibodies of the present invention in the treatment of cancer in dogs. It also concerns the use of the body. [Background technology]

[0003] The initiation or termination of an immune response involves many types of immune cells, particularly T lymphocytes and antigen-presenting cells. Activated by complex interactions among a series of proteins expressed on the surface of the adipocyte (APC) Co-stimulatory signaling pathways are involved in the development of immune responses. Most importantly, CD28 on the surface of T cells and B7.1 on the surface of APCs (also known as CD80) and B7.2 (also known as CD86) It has been shown that this is mediated through interactions with the B7.1 and B7 .2 is believed to perform a similar function.

[0004] In contrast, the co-inhibitory pathway leads to the inhibition or termination of the immune response and involves the activation of cytotoxic T cells. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and CD80 / CD86 on APCs It has been shown that this is mediated through interactions between proteins. The primary signaling pathway is programmed death receptor 1 (PD-1) on T cells and programmed death receptor 1 (PD-2) on APCs. Interactions between the PD-L1 and PD-L2 proteins Furthermore, the interaction between PD-L1 and CD80 has been shown to be mediated by It has also been shown that its use can result in an inhibitory signal within T cells.

[0005] CD80 and CD86 are members of the immunoglobulin (Ig) superfamily. [Sharpe and Freeman, Nature Reviews, 2:11 6-126(2002)]. CD80 is expressed on activated B cells, activated T cells, and macrophages. Expressed in phages and dendritic cells [Swanson and Hall, Eur JI mmunol.,23:295-298(1993);Razi-Wolfe et a l., PNAS, 89:4210-4214 (1992)]. CD86 is expressed on dendritic cells, laminin, and dendritic cells. CD86 is constitutively expressed on Langerhans cells and B cells. In addition, CD86 is expressed on monocytes and and is upregulated after IFN-γ stimulation [Larsen et al .,Immunol.,152:5208-5219(1994);Inaba,JE xp.Med.180:1849-1860(1994)].

[0006] CD80 and CD86 bind to CD28 and CTLA-4 with different functional outcomes. Linsley et al., PNAS, 87:5031-5035 (199 0); Linsley et al., J. Exp. Med., 173:721-7 30(1991);Azuma et al.,Nature 366:76-79 ( 1993);Freeman et al.,Science 262:909-912 (1993)]. The binding of CD80 and CD86 to CTLA-4 is a CD80 / CD86 It shows a much higher affinity than the binding of CD28 to [van der Merwe, J. Exp. Med. 185:393-402(1997)].

[0007] CD28 is a homodimeric glycoprotein that is a member of the Ig superfamily. [Aruffo and Seed, PNAS, 84:8573-8577 (1987 The mature protein contains the hexapeptide motif MY, which is essential for counter-receptor binding. It has a single extracellular variable domain consisting of 134 amino acid residues containing PPPY [R iley and June, Blood, 105:13-21 (2005)]. CD2 The 41-amino acid cytoplasmic domain of 8 contains four tyrosine residues that can be phosphorylated upon activation. Including [Sharpe and Freeman, Nat. Rev. Immunol., 2:116-126(2002)]. CD28 is a + Most T cells and CD8 + T It is expressed in approximately 50% of cells [Gross et al., J. Immunol., 149 :380-388(1992);Riley and June,Blood,105: 13-21(2005)]. After T cell receptor (TCR) ligation, it binds to CD28 B7.1 / B7.2, which are involved in T cell proliferation, provide important costimulatory signals to T cells, promoting T cell activation. This allows for activation and subsequent development of an immune response [Reiser et al., PNAS ,89:271-275(1992);Jenkins et al.,J.Immun ol., 147:2461-2466 (1991)]. In the absence of CD28 signaling, , T cells have been shown to undergo apoptosis or become unresponsive [Jenk ins et al.,J.Exp.Med.165:302-319(1987);J enkins et al.,PNAS,84:5409-5413(1987);Sc hwartz, Science, 248:1349-1356 (1990)]. CD2 8-B7.1 / B7.2 binding determines the threshold level of TCR ligation required for activation (e.g. For example, the amount of antigen-MHC complexes (amount of antigen) can be varied to determine the time required to stimulate naive cells. shortening the IL-1 receptor and increasing the magnitude of the T cell response [Soskic et al., Ad vances in Immunology, 124:96-123(2014)].

[0008] CTLA-4 (CD152) is also a member of the Ig superfamily and It consists of a single extracellular domain, a transmembrane domain, and a short cytoplasmic tail [Swanson, Immunology;1010:169-177(2000)]. Furthermore, CTLA- CTLA-4 shares approximately 30% amino acid identity with CD28. It is not constitutively expressed on T cells, but is rapidly expressed immediately after CD28 ligation and T cell activation. CTLA-4 is upregulated approximately 48-96 hours after initial T cell activation. The expression level of β-glucan is at its peak [Alegre et al., J. Immunol., 1 57:4762-4770(1996);Freeman et al., J. Immu Nol., 149:3795-3801(1992)]. CTLA-4 is more potent than CD28. It also binds to both B7.1 and B7.2 with extremely high affinity [van der Mer we et al., J.Exp.Med., 185:393-402 (1997) However, in contrast to the stimulatory effect of CD28-binding B7.1 or B7.2, CTL A-4 functions as an inhibitory receptor essential for down-modulation of immune responses. Walnus et al., Immunity, 1:405-413(1994); Walnus, J.Exp.Med.,183:2541-2550(1996);Kr ummel and Allison, J. Exp. Med., 183:2533-25 40 (1996)]. The mechanism by which CTLA-4 mediates its immunosuppressive function is through the interaction of CD28 and C This is related to its ability to act as a competitive inhibitor of the CD80 / CD86 interaction [ Swanson, Immunology, 1010:169-177 (2000). The important role of CTLA-4 in immune downregulation is reviewed in [1]. The division of the immune system has been demonstrated in CTLA-4-deficient mice, which show multiple Death occurs at 3-5 weeks of age due to the development of a lymphoproliferative disorder characterized by T cell infiltration in the organs of the cat. [Tivol et al.,Immunity,3:541-5417(1995); Waterhouse et al.,Science,270:985-988(19 95)]. The results of CTLA-4 knockout are consistent with the CTLA-4 / CD80 / CD86 triad. CD28 and its ligands are expressed in the ribosomal protein 1 (RI) and ribosomal protein 2 (RI), as shown by the absence of disease in ribosomal protein knockout mice. It has also been demonstrated that the expression of IL-1 is dependent on the interaction with the target genes CD80 and CD86 [Man elbrot et al., J. Exp. Med., 189:435-440(199 9)]. This suggests that repeated injection of CTLA-4Ig into CTLA-4 knockout mice This is also confirmed by the protection against lymphoproliferation afforded by administration of thrombin [T ivol et al.,J Immunol.,158:5091-5094(199 7)].

[0009] Furthermore, blocking the effects of CTLA-4 with antibodies significantly reduced T cell proliferation in vitro and in vivo. It has been shown that cellular responses are enhanced and antitumor immune responses are increased [Leach et al., Science, 271:1734-1736 (1996)]. Based on these findings, monoclonal antibodies have been developed to provide a therapeutic modality for treating cancer. The development of CTLA-4 blockers such as CTLA-4 inhibitors has been reported [Hodi et al., PNA S,100(8):4712-4717(2003);Phan GQ et al., PNAS,100(14):8372-8377(2003);Attia,Journ al of Clinical Oncology,23(25):6043-6053 (2005);Comin-Anduix et al., Journal of Tr. anslational medicine,6:22-22(2008); WO20 00037504A2;US8,017,114B2;WO2010097597 A1; WO2012120125A1; and Boutros et al., Nat Rev Clin Oncol.,13(8):473-486(2016)].

[0010] PD-1 is a member of the CD28 / CTLA-4 family of immunoregulatory receptors. PD-1 is also a member of the Ig superfamily and its ligands It contains an extracellular variable domain that binds to a signaling molecule and a cytoplasmic tail that binds to a signaling molecule. Zak et al.,Cell Structure,25:1163-1174(2 017). The cytoplasmic tail of PD-1 contains two tyrosine-based signaling motif [Zhang et al., Immunity 20 :337-347 (2004)]. PD-1 expression is expressed on unstimulated T cells, B cells, and However, PD-1 expression is not observed in blastocysts or myeloid cells. It is upregulated in cells [Chemnitz et al., J. Immunol. nol.,173:945-954(2004); Petrvas et al.,J Exp. Med., 203:2281-2292(2006)]. PD-1 inhibits CTL It is most closely related to A-4, sharing approximately 24% amino acid identity [Jin et al.,Current Topics in Microbiology an d Immunology, 350:17-37(2010)]. PD-1 is a Binding to PD-L1 and PD-L2 expressed on the surface of T cells reduces T cell activation. When any of these ligands bind to PD-1, it activates the T cell receptor (TCR). To date, only PD-L1 and PD-L2 are known to negatively regulate antigen signaling via PD-L1. It is known to function as a ligand for D-1, as in the case of CTLA-4. In addition, PD-1 ligation appears to transmit a negative immunoregulatory signal. Ligation of PD-1 by PD-L1 or PD-L2 inhibits TCR-mediated proliferation and cytokine production. This results in the inhibition of insulin production [Jin et al., Current Topics in n Microbiology and Immunology,350:17-37( In contrast to CTLA-4-deficient animals, PD-1-deficient mice show a lifelong Although the animals die later and show signs of autoimmunity, the severity of the effects observed is comparable to that of C This is not as severe as that seen in TLA-4-deficient animals [Nishimura et al., I mmunity, 11(2):141-151 (1999); et al., Science, 291(5502):319-322 (200 1)]. The PD-1 signaling pathway is currently under intensive investigation, but previous studies have PD-L1 / PD-L2 / PD-1 interaction reduces downstream signals of TCR stimulation. This results in reduced cytokine secretion and impaired T cell proliferation and T cell-mediated cytotoxicity. It is involved in the negative regulation of some immune responses, resulting in a reduction in the production of cytotoxic molecules. [Freeman et al., J. Exp. Med., 192(7) :1027-1034(2000)].

[0011] PD-L1 (CD274) is a type 1 membrane protein and has an IgV-like extracellular domain. IgC-like extracellular domains, hydrophobic transmembrane domains, and signaling properties unknown PD-L1 is a member of the B7 family of proteins. It is recognized as a B7 family member and shares approximately 20% amino acid identity with members of the B7 family. PD-L1 is a receptor found on activated T cells, B cells, and myeloid cells. PD-L1 also binds to the costimulatory molecule CD80, but not to CD86. No [Butte et al., Immunology, 45 (13):356 7-3572 (2008)]. The affinity of CD80 for PD-L1 is similar to that of CD28 and Its affinity is intermediate to that of CTLA-4. The related molecule PD-L2 is expressed by CD80 or CD8 6, but shares the same receptor as PD-1. When PD-1 binds to its receptor on T cells, it stimulates TCR-mediated IL-2 production and T cell proliferation. PD-L1 binds to PD-1 and delivers a signal that inhibits proliferation of naive T cells. It also contributes to ligand-induced TCR downmodulation during antigen presentation to cells. When PD-L1 binds to CD80 on T cells, apoptosis of the T cells occurs. The role of PD-1 and PD-L1 as inhibitors of cell activation has been demonstrated in many studies. Based on these findings, we hope to provide therapeutic modalities for treating cancer and infectious diseases. To combat this, PD-1 and PD-L1 blockers such as monoclonal antibodies have been developed. It was.

[0012] Humanized monoclonal antibodies that block the binding and activity of canine PD-1, PD-L1, and CTLA-4 A monoclonal antibody has been developed and is now being used to treat one of several different types of cancer. Canine PD-1 and PDL1 can be used in the treatment of human subjects. Caninized monoclonal antibodies that block the binding and activity of have also been reported [US9, 944,704B2, US10,106,607B2 and US2018 / 023 7535A1; the contents of which are incorporated herein by reference in their entireties]. However, to date, no canine monoclonal antibody has been shown to block the binding and activity of canine CTLA-4. No antibodies have been reported.

[0013] Any citation of a reference herein is to be construed as an indication that such reference is incorporated by reference into this application. This disclosure is not to be construed as an admission that any of the subject matter is available as "prior art" under any provision of this patent. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] WO2000037504A2 [Patent Document 2] US8,017,114B2 [Patent Document 3] WO2010097597A1 [Patent Document 4] WO2012120125A1 [Patent Document 5] US9,944,704B2 [Patent Document 6] US10,106,607B2 [Patent Document 7] US2018 / 0237535A1 [Non-patent literature]

[0015] [Non-Patent Document 1] Sharpe and Freeman, Nature Reviews,2:116-126(2002) [Non-licensed document 2] Swanson and Hall,Eur J. Immunol.,23:295-298(1993) [Non-licensed document 3] Razi-Wolfe et al.,PNAS,89:4210-4214(1992)

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Summary of the Invention

[0016] The present invention relates to an anti-canine cytotoxic T lymphocyte-associated protein 4 (CTLA-4) that binds to canine CTLA-4. In a specific embodiment, the antibody against canine CTLA-4 is In a more particular embodiment, the antibody specifically binds to canine CTLA-4. The antibody against this antibody also has the ability to block the binding of canine CTLA-4 to canine CD80. In another specific embodiment, the antibody to canine CTLA-4 further comprises canine CTLA-4. It also has the ability to block the binding of CTLA-4 to canine CD86. In this embodiment, the antibody to canine CTLA-4 is a combination of canine CTLA-4 and canine CD80. and blocking the binding of canine CTLA-4 to canine CD86. It has the ability to do both.

[0017] Furthermore, the present invention relates to the complementarity determining regions (CDRs) formed by these antibodies, and , and the canine CDRs in frame to form a caninized anti-canine CTLA-4 antibody. The present invention also relates to combinations (e.g., derived from murine anti-canine CTLA-4 antibodies). It also relates to the use of such antibodies in the treatment of conditions such as cancer.

[0018] Thus, the present invention provides CDRs derived from six exemplified murine anti-canine CTLA-4 antibodies. The six exemplified murine anti-canine CTLA-4 antibodies provide a unique set of CDRs. a unique set of three light chain CDRs [CDR light 1 (CDRL1), CDR light 2 (CDR light 3)]. CDR light 3 (CDRL2) and CDR light 3 (CDRL3)] and three heavy chain CDRs [CDR heavy 1 (CDR H1), CDR duplex 2 (CDRH2) and CDR duplex 3 (CDRH3). As detailed in, there is substantial sequence homology within each group of CDRs, and certain There is even a degree of redundancy (see, for example, the set of VL CDR-3s in Table 1 below). Thus, the present invention provides six exemplified murine anti-canine CTLA-4 antibodies. The present invention not only provides the amino acid sequences of the six CDRs, but also the conservation of these CDRs. Substantially modified variants, as well as variants that include (e.g., share) the same canonical structure. and / or one or more canine CTLA-4 epitopes Variants that bind (eg, 1, 2, 3, 4 or more) amino acid residues are also provided.

[0019] One aspect of the present invention is a method for producing a canine cytotoxic T lymphocyte-associated protein (CTLA-4)-binding protein. In certain embodiments, the mammalian antibody of the present invention or its antigen is provided. The binding fragment is a murine antibody. In a preferred embodiment, the mammalian antibody of the present invention (This includes murine antibodies of the present invention) or antigen-binding fragments thereof, such as caninized antibodies. or a caninized antigen-binding fragment thereof.

[0020] In certain embodiments, the mammalian antibody specifically binds to canine CTLA-4. In certain embodiments, the mammalian antibody to canine CTLA-4 is further selected from the group consisting of canine CTLA- In another specific embodiment, the antibody has the ability to block the binding of IgG4 to canine CD80. The mammalian antibody against canine CTLA-4 further binds canine CTLA-4 and canine CD86. In yet another specific embodiment, the canine CTLA- Mammalian antibodies against -4 block the binding of canine CTLA-4 to canine CD80 and blocking the binding of canine CTLA-4 to canine CD86. is doing.

[0021] In certain embodiments, the mammalian antibody that binds canine CTLA-4 is an isolated antibody. The present invention further provides antigen binding assays of these mammalian antibodies that bind to canine CTLA-4. In certain embodiments, the antibody fragments comprise three light chain complementarity determining regions (C CDR light 1 (CDRL1), CDR light 2 (CDRL2) and CDR light 3 (CDR L3)] and three heavy chain CDRs [CDR heavy 1 (CDRH1), CDR heavy 2 (CDRH2) and CDR heavy 3 (CDRH3)].

[0022] In certain embodiments, the mammalian antibody or antigen-binding fragment thereof has the sequence of SEQ ID NO: 90 Amino acid sequence, conservatively modified variants or canonical of the amino acid sequence of SEQ ID NO: 90 and CDRH3 comprising variants of SEQ ID NO: 90 that comprise structural class 7. More specific embodiments In one embodiment, the mammalian antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO: 88: Conservatively modified variants of the amino acid sequence of SEQ ID NO: 88 or canonical structure class 2A Further particular embodiments include CDRH2 comprising a variant of SEQ ID NO: 88 comprising: In the case of a mammalian antibody or antigen-binding fragment thereof, the mammalian antibody or antigen-binding fragment thereof also comprises the amino acid sequence of SEQ ID NO: 86. CDRH1 comprising the sequence, conservatively modified variants or variants of the amino acid sequence of SEQ ID NO: 86 Further included are CDRH1s comprising variants of SEQ ID NO: 86 that contain nonical structural class 1. In a more particular embodiment, the mammalian antibody or antigen-binding fragment thereof is similarly the amino acid sequence of SEQ ID NO: 96, conservatively modified variants of the amino acid sequence of SEQ ID NO: 96, or further comprises a CDRL3 comprising a variant of SEQ ID NO: 96 that comprises canonical structure class 1. In an even more particular embodiment, the mammalian antibody or antigen-binding fragment thereof has SEQ ID NO: the amino acid sequence of SEQ ID NO: 94, conservatively modified variants or variants of the amino acid sequence of SEQ ID NO: 94 and further comprising a CDRL2 comprising a variant of SEQ ID NO: 94 that comprises nonical structural class 1. In a more particular embodiment, the mammalian antibody or antigen-binding fragment thereof is similarly the amino acid sequence of SEQ ID NO: 92, conservatively modified variants of the amino acid sequence of SEQ ID NO: 92, or further includes a CDRL1 comprising a variant of SEQ ID NO: 92 that comprises canonical structure class 4.

[0023] In an alternative embodiment, the mammalian antibody or antigen-binding fragment thereof has the sequence of SEQ ID NO:1. 102, conservatively modified variants of the amino acid sequence of SEQ ID NO: 102 or and CDRH3 comprising variants of SEQ ID NO: 102 that contain nonical structural class 9. In a typical embodiment, the mammalian antibody or antigen-binding fragment thereof comprises the antigen of SEQ ID NO: 100. amino acid sequence, conservatively modified variants or canonical versions of the amino acid sequence of SEQ ID NO: 100 Further included are CDRH2s comprising variants of SEQ ID NO: 100 that comprise structural class 4. In certain embodiments, the mammalian antibody or antigen-binding fragment thereof also has the sequence of SEQ ID NO: CDRH1 comprising the amino acid sequence of SEQ ID NO: 98, a conservatively modified version of the amino acid sequence of SEQ ID NO: 98 and CDRH1 comprising a variant of SEQ ID NO: 98 containing canonical structure class 1. In even more particular embodiments, a mammalian antibody or antigen-binding fragment thereof. Similarly, the amino acid sequence of SEQ ID NO: 108, the amino acid sequence of SEQ ID NO: 108, CD containing modified variants or variants of SEQ ID NO: 108 containing canonical structure class 1 In an even more particular embodiment, the mammalian antibody or antigen-binding fragment thereof further comprises RL3. The fragment also has the amino acid sequence of SEQ ID NO: 106, the amino acid sequence of SEQ ID NO: 106 Conservatively modified variants of or variants of SEQ ID NO: 106 containing canonical structure class 1 In an even more particular embodiment, the mammalian antibody or its The antigen-binding fragment may also have the amino acid sequence of SEQ ID NO: 104, SEQ ID NO: 10 containing conservatively modified variants of amino acid sequence or canonical structure class 1 Further included is a CDRL1 comprising a variant of 4.

[0024] In another alternative embodiment, the mammalian antibody or antigen-binding fragment thereof has SEQ ID NO: the amino acid sequence of SEQ ID NO: 113, conservatively modified variants of the amino acid sequence of SEQ ID NO: 113, or contains a CDRH3 comprising a variant of SEQ ID NO: 113 that contains canonical structure class 7. In a specific embodiment, the mammalian antibody or antigen-binding fragment thereof has the sequence of SEQ ID NO: 88 Amino acid sequence, conservatively modified variants or canonical of the amino acid sequence of SEQ ID NO: 88 Further included are CDRH2s comprising variants of SEQ ID NO: 88 that comprise structural class 2A. In certain embodiments, the mammalian antibody or antigen-binding fragment thereof also has the sequence of SEQ ID NO: CDRH1 comprising the amino acid sequence of SEQ ID NO: 86, a conservatively modified version of the amino acid sequence of SEQ ID NO: 86 and CDRH1 comprising a variant of SEQ ID NO: 86 containing canonical structural class 1. In even more particular embodiments, a mammalian antibody or antigen-binding fragment thereof. Similarly, the amino acid sequence of SEQ ID NO: 96, conservative modifications of the amino acid sequence of SEQ ID NO: 96 CDRL3 comprising a variant of SEQ ID NO: 96 containing a modified variant or a variant of SEQ ID NO: 96 containing canonical structure class 1 In an even more particular embodiment, the mammalian antibody or antigen-binding fragment thereof The amino acid sequence of SEQ ID NO: 94 is also a conservatively derived amino acid sequence of SEQ ID NO: 94. CDRs containing modified variants or variants of SEQ ID NO: 94 containing canonical structure class 1 In an even more particular embodiment, the mammalian antibody or antigen-binding fragment thereof further comprises L2. The fragment is similarly the amino acid sequence of SEQ ID NO: 117, Conservatively modified variants or variants of SEQ ID NO: 117 containing canonical structure class 4

[0032] The present invention further includes a CDRL1 comprising:

[0025] In yet another alternative embodiment, the mammalian antibody or antigen-binding fragment thereof comprises: Amino acid sequence of SEQ ID NO: 115, conservatively modified variants of the amino acid sequence of SEQ ID NO: 115 CDRH3 containing variants of SEQ ID NO: 115 containing variants or canonical structure class 7 In a more particular embodiment, the mammalian antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 88, conservatively modified variants of the amino acid sequence of SEQ ID NO: 88 or The present invention further includes a CDRH2 comprising a variant of SEQ ID NO: 88 that comprises a neuronal structural class 2A. In a more particular embodiment, the mammalian antibody or antigen-binding fragment thereof is similarly CDRH1 comprising the amino acid sequence of SEQ ID NO: 86, a conservatively modified version of the amino acid sequence of SEQ ID NO: 86 CDRH containing a modified variant or variant of SEQ ID NO: 86 containing canonical structure class 1 In an even more particular embodiment, the mammalian antibody or antigen-binding fragment thereof The amino acid sequence of SEQ ID NO: 96, a conservative amino acid sequence of SEQ ID NO: 96, or a variant of SEQ ID NO: 96 containing canonical structure class 1. In an even more particular embodiment, the mammalian antibody or antigen-binding fragment thereof further comprises RL3. The fragment also has the amino acid sequence of SEQ ID NO: 122, the amino acid sequence of SEQ ID NO: 122 Conservatively modified variants of SEQ ID NO: 122 or variants of SEQ ID NO: 122 containing canonical structure class 1 In an even more particular embodiment, the mammalian antibody or its The antigen-binding fragment may also have the amino acid sequence of SEQ ID NO: 119, SEQ ID NO: 11 containing conservatively modified variants of amino acid sequence or canonical structure class 4 Further included is a CDRL1 comprising 9 variants.

[0026] In yet another alternative embodiment, the mammalian antibody or antigen-binding fragment thereof comprises: Amino acid sequence of SEQ ID NO: 114, conservatively modified variants of the amino acid sequence of SEQ ID NO: 114 CDRH3 containing variants of SEQ ID NO: 114 containing variants or canonical structure class 7 In a more particular embodiment, the mammalian antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 111, conservatively modified variants of the amino acid sequence of SEQ ID NO: 111, or and further comprising a CDRH2 comprising a variant of SEQ ID NO: 111 that comprises canonical structure class 2A. In even more particular embodiments, the mammalian antibody or antigen-binding fragment thereof is CDRH1 comprising the amino acid sequence of SEQ ID NO: 109, Conservatively modified variants or variants of SEQ ID NO: 109 containing canonical structure class 1 In an even more particular embodiment, the mammalian antibody or its anti- The original binding fragment may also have the amino acid sequence of SEQ ID NO: 96, the amino acid sequence of SEQ ID NO: 96, Conservatively modified variants of sequences or variations of SEQ ID NO: 96 containing canonical structure class 1 In an even more particular embodiment, the mammalian antibody or its The antigen-binding fragment of SEQ ID NO: 121 may also be a fragment of SEQ ID NO: 122. SEQ ID NO: 1, including conservatively modified variants of the amino acid sequence or canonical structure class 1 In a still more particular embodiment, the mammal further comprises a CDRL2 comprising 21 variants. The antibody or antigen-binding fragment thereof may also comprise the amino acid sequence of SEQ ID NO: 118, SEQ ID NO: Conservatively modified variants of the amino acid sequence of No. 118 or canonical structure class 4 Further included is a CDRL1 comprising a variant of SEQ ID NO:118.

[0027] In yet another alternative embodiment, the mammalian antibody or antigen-binding fragment thereof comprises: Amino acid sequence of SEQ ID NO: 116, conservatively modified variants of the amino acid sequence of SEQ ID NO: 116 A CDRH3 comprising a variant of SEQ ID NO: 116 containing a variant or canonical structural class 12. In more particular embodiments, the mammalian antibody or antigen-binding fragment thereof comprises SEQ ID NO: the amino acid sequence of SEQ ID NO: 112, conservatively modified variants of the amino acid sequence of SEQ ID NO: 112, or The present invention further includes a CDRH2 comprising a variant of SEQ ID NO: 112 that contains canonical structure class 2A. In even more particular embodiments, the mammalian antibody or antigen-binding fragment thereof is Similarly, CDRH1 comprising the amino acid sequence of SEQ ID NO: 110, Conservatively modified variants of or variants of SEQ ID NO: 110 containing canonical structure class 1 In an even more particular embodiment, the mammalian antibody or its The antigen-binding fragment may also have the amino acid sequence of SEQ ID NO: 124, SEQ ID NO: 12 containing conservatively modified variants of amino acid sequence or canonical structure class 1 In an even more particular embodiment, the mammalian antibody further comprises a CDRL3 comprising a variant of The antibody or antigen-binding fragment thereof may also have the amino acid sequence of SEQ ID NO: 123, SEQ ID NO: Conservatively modified variants of the 123 amino acid sequence or sequences containing canonical structure class 1 In an even more particular embodiment, the CDRL2 comprises the variant of sequence number 123. The mammalian antibody or antigen-binding fragment thereof may also comprise the amino acid sequence of SEQ ID NO: 120. , conservatively modified variants or canonical structure classes of the amino acid sequence of SEQ ID NO: 120 2.

[0028] As described above, caninized antibodies against canine CTLA-4 or caninized antigens thereof Binding fragments are an important aspect of the present invention, and the present invention provides all such Caninized mammalian antibodies (including caninized mouse antibodies) are provided. Therefore, the present invention further provides a CT comprising a canine IgG heavy chain and a canine kappa or lambda light chain. The present invention provides an isolated caninized antibody or antigen-binding fragment thereof that specifically binds to LA-4. In certain embodiments of this type, the canine kappa or lambda light chains are provided as three light chain phases. Complementarity determining regions (CDRs) [CDR light 1 (CDRL1), CDR light 2 (CDRL2) and CDR light and the canine IgG heavy chain contains the murine anti-canine CTLA- Three heavy chain CDRs (CDR heavy 1 (CDRH1), CDR heavy 2 (CDR heavy 3)) obtained from four antibodies The caninized antibodies of the present invention and their antigen-binding domains comprise CDR heavy chains (CDR heavy chains H2 and H3) and CDR heavy chains 3 (CDRH3). Particular embodiments of the fragments bind to canine CTLA-4 and / or canine CTL Blocks the binding of A-4 to canine CD80 and / or canine CD86.

[0029] The caninized antibody or caninized antigen-binding fragment thereof of the present invention can be caninized with the amino acid sequence of SEQ ID NO: 128. In a related embodiment, the antibody can include an IgGD comprising a hinge region comprising a nucleotide sequence. In yet another related embodiment, the range region comprises the amino acid sequence of SEQ ID NO: 129. 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.

[0030] In an alternative embodiment, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 62. In a particular embodiment of this type, the heavy chain is represented by the nucleotide sequence of SEQ ID NO: 61. In another embodiment, the caninized antibody comprises the amino acid sequence of SEQ ID NO: 64. In particular embodiments of this type, the heavy chain comprises the nucleotide sequence of SEQ ID NO: 63. In yet another embodiment, the caninized antibody is encoded by the sequence of SEQ ID NO: 66. In a particular embodiment of this type, the heavy chain comprises the amino acid sequence of SEQ ID NO: 65. In a more particular embodiment, the caninized antibody is encoded by the nucleotide sequence of: and further comprising a light chain comprising the amino acid sequence of SEQ ID NO: 50. In particular embodiments of this type The light chain is encoded by the nucleotide sequence of SEQ ID NO: 49. In this embodiment, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 52. In a specific embodiment of the present invention, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 51. In yet another specific embodiment, the caninized antibody comprises the amino acid sequence of SEQ ID NO: 54. In particular embodiments of this type, the light chain further comprises a light chain comprising the nucleotide sequence of SEQ ID NO: 53. It is encoded by the octide sequence.

[0031] In an alternative embodiment, the caninized antibody comprises a modified mAb comprising the amino acid sequence of SEQ ID NO: 74. In particular embodiments of this type, the modified heavy chain comprises a heavy chain having the nucleic acid sequence of SEQ ID NO: 73. In another embodiment, the caninized antibody is encoded by the nucleotide sequence SEQ ID NO:7. In a particular embodiment of this type, the modified heavy chain comprises an amino acid sequence of The modified heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 75. In embodiments, the caninized antibody comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO:78. In a particular embodiment of this type, the modified heavy chain has the nucleotide sequence of SEQ ID NO: 77 In a more specific embodiment, the caninized antibody is encoded by the amino acid sequence of SEQ ID NO: 50. In particular embodiments of this type, the light chain further comprises a light chain comprising the amino acid sequence of SEQ ID NO: In another specific embodiment, the caninized antibody is encoded by the nucleotide sequence of SEQ ID NO:49. The antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 52. In one embodiment, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 51. In certain embodiments, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 54. In particular embodiments of this type, the light chain is represented by the nucleotide sequence of SEQ ID NO: 53. It is coded as follows.

[0032] In certain embodiments, the caninized antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO: 66 and the sequence In another embodiment, the caninized antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 52. It 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.

[0033] In an alternative embodiment, the caninized antibody comprises a modified mAb comprising the amino acid sequence of SEQ ID NO: 78. and a light chain comprising the amino acid sequence of SEQ ID NO: 52. The antibody comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO:78 and a modified heavy chain comprising the amino acid sequence of SEQ ID NO:54. The light chain contains a sequence.

[0034] In another embodiment, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 68. In a particular embodiment of this type, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 67. In another embodiment, the caninized antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO: 70. In a particular embodiment of this type, the heavy chain comprises the nucleotide sequence of SEQ ID NO: 69. In yet another embodiment, the caninized antibody is encoded by the amino acid sequence of SEQ ID NO: 72. In a particular embodiment of this type, the heavy chain comprises a heavy chain comprising the nucleic acid sequence of SEQ ID NO: 71. In a more particular embodiment, the caninized antibody is encoded by the sequence In a particular embodiment of this type, the light chain further comprises an amino acid sequence of 56. The strand is encoded by the nucleotide sequence of SEQ ID NO: 55. In another particular embodiment, The caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 58. In certain embodiments, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 57. In yet another specific embodiment, the caninized antibody comprises the amino acid sequence of SEQ ID NO: 60. In a particular embodiment of this type, the light chain further comprises the nucleotide sequence of SEQ ID NO: 59. It is coded by a code sequence.

[0035] In an alternative embodiment, the caninized antibody comprises a modified mAb comprising the amino acid sequence of SEQ ID NO: 80. In particular embodiments of this type, the modified heavy chain comprises a heavy chain having the nucleic acid sequence of SEQ ID NO: 79. In another embodiment, the caninized antibody is encoded by the nucleotide sequence SEQ ID NO: 8 In particular embodiments of this type, the modified heavy chain comprises an amino acid sequence of The modified heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 81. In embodiments, the caninized antibody comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO:84. In a particular embodiment of this type, the modified heavy chain has the nucleotide sequence of SEQ ID NO: 83 In a more specific embodiment, the caninized antibody is encoded by the amino acid sequence of SEQ ID NO: 56. In particular embodiments of this type, the light chain further comprises a light chain comprising the amino acid sequence of SEQ ID NO: In another specific embodiment, the caninized antibody is encoded by the nucleotide sequence of SEQ ID NO: 55. The antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 58. In one embodiment, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 57. In certain embodiments, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 60. In particular embodiments of this type, the light chain is represented by the nucleotide sequence of SEQ ID NO: 59. It is coded as follows.

[0036] In certain embodiments, the caninized antibody comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 72. In another embodiment, the caninized antibody comprises a nucleotide sequence of SEQ ID NO: 58 and a light chain comprising the amino acid sequence of SEQ ID NO: 58. 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 include.

[0037] In an alternative embodiment, the caninized antibody comprises a modified mAb comprising the amino acid sequence of SEQ ID NO: 84. and a light chain comprising the amino acid sequence of SEQ ID NO: 58. The antibody comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO:84 and the amino acid sequence of SEQ ID NO:60. The light chain contains a sequence.

[0038] The present invention further provides a method for producing a 1×10 -12 Less than M (e.g., 5 × 10 -13 Dissociation determination of (M or less) A mammalian antibody or antigen-binding fragment thereof that binds to canine CTLA-4 with a Kd. In another embodiment, the mammalian antibody or antigen-binding fragment thereof is 1×10 -5 M~1×10 -12 It binds to canine CTLA-4 with a dissociation constant of M. In some embodiments, the mammalian antibody or antigen-binding fragment thereof is in a concentration of 1×10 -7 M~1×10 - 11 In an even more particular embodiment, mammalian CTLA-4 binds to canine CTLA-4 with a dissociation constant of M. The animal antibody or its antigen-binding fragment is 1 × 10 -8 M~1×10 -11 Dissociation constant of M In an even more particular embodiment, the mammalian antibody or its of antigen-binding fragments is 1 × 10 -8 M~1×10 -10 Canine CTL with M dissociation constant Binds to A-4.

[0039] The present invention further provides a method for producing a 1×10 7 M -1 s -1 Larger on-speed (k on ) for canine CT Mammalian antibodies or antigen-binding fragments thereof that bind to LA-4 are provided. In some embodiments, the mammalian antibody or antigen-binding fragment thereof is 1×10 2 M -1 s -1 ~1× 10 7 M -1 s -1 In a more particular embodiment, , mammalian antibodies or antigen-binding fragments thereof, 1 × 10 3 M -1 s -1 ~1×10 6 M -1 s -1 In an even more particular embodiment, , mammalian antibodies or antigen-binding fragments thereof, 1 × 10 3 M -1 s -1 ~1×10 5 M -1 s -1 In an even more particular embodiment, , mammalian antibodies or antigen-binding fragments thereof, 1 × 10 4 M -1 s -1 ~1×10 5 M -1 s -1 Binds to canine CTLA-4 with an on-rate of .

[0040] The present invention further provides a method for producing a 1×10-7 s -1 slower off-rate (k off ) and canine CTL A mammalian antibody or antigen-binding fragment thereof that binds to A-4 is provided. In the case of a mammalian antibody or antigen-binding fragment thereof, -3 s -1 ~1×10 - 8 s -1 In a more particular embodiment, the mammalian CTLA-4 The antibody or antigen-binding fragment thereof is 1×10 -4 s -1 ~1×10 -7 s -1 Off In an even more particular embodiment, the mammalian antibody or The antigen-binding fragment is 1 × 10 -5 s -1 ~1×10 -7 s -1 Off speed Binds to CTLA-4.

[0041] In certain embodiments, the mammalian antibodies of the present invention (including chimeric antibodies) Blocks the binding of canine CD80 and / or canine CD86 to canine CTLA-4. In certain embodiments, the antibody is at a concentration of 1 x 10 -8 M~1×10 -9 Minimum of M or lower concentration Blocks the binding of canine CD80 and / or canine CD86 to canine CTLA-4 at EC50 In an even more particular embodiment, the EC50 is 5 x 10 -9 M~5×10 -13 M In an even more particular embodiment, the EC50 is 5 x 10 -9 M~5×10 -11 M. Thus, in certain embodiments, antibodies of the invention comprise one, two, three, four or All of these properties, i.e., the dissociation constant with canine CTLA-4, the binding affinity with canine CTLA-4, the on-rate for association with the canine CTLA-4 complex and the off-rate for dissociation from the anti-canine CTLA-4 binding complex. or can be shown to effectively treat cancer in animal subjects.

[0042] The present invention further provides caninized antibodies that cross-compete with the mammalian antibodies disclosed herein. Mammalian antibodies and antigen-binding fragments are provided. In certain embodiments, caninized mammalian antibodies and antigen-binding fragments are provided. The antibody cross-competes with an antibody containing the six CDRs of 45A9 [see Table 1 below]. In a related embodiment, the caninized mammalian antibody is an antibody comprising the six CDRs of 27G12. In yet another related embodiment, the canine The engineered mammalian antibodies cross-compete with antibodies containing the six CDRs of 22A11 [see Table 1 below]. In yet another related embodiment, the caninized mammalian antibody is Cross-competes with antibodies containing six CDRs [see Table 1 below]. The caninized mammalian antibodies cross-compete with antibodies containing the six CDRs of 12B3 [see table below]. 1 and Table 3]. In another specific embodiment, the caninized mammalian antibody is 39A 11 cross-competes with antibodies containing six CDRs [see Tables 1 and 3 below]. In one embodiment, the assay is a standard binding assay. Standard binding assays are performed using BIACore®. In such embodiments, a standard binding assay is performed using an ELISA. In another such embodiment, the standard binding assay is performed by flow cytometry. It will be implemented.

[0043] As indicated above, the present invention includes the antibody (and antigen-binding fragment thereof). The antibodies (and antigen-binding fragments thereof) of the invention are monoclonal antibodies (and antigen-binding fragments thereof). mammalian antibodies (and antigen-binding fragments thereof), e.g., murine ( murine (mouse) antibodies (and antigen-binding fragments thereof), canine caninized antibodies (and antigen-binding fragments thereof), e.g., caninized murine antibodies (and antigen-binding fragments thereof); In certain embodiments, antibodies (and their antigen-binding fragments) may be used. The fragment is isolated.

[0044] In a preferred embodiment, the caninized antibody or antigenic fragment thereof of the present invention is canine CT In certain embodiments, the caninized antibody binds to an epitope of the amino acid sequence of LA-4. Positions T35, R38, T51, T53, Y90, K93 of the amino acid sequence of SEQ ID NO: 138 , Y98 and Y102. In one embodiment, the caninized antibody comprises a nucleotide sequence at positions 35T, R38, S42 of the amino acid sequence of SEQ ID NO: 138. , K93 and Y102.

[0045] The present invention further relates to SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 13 5. One or more epitopes of the amino acid sequences of SEQ ID NO: 136 and SEQ ID NO: 137 or parts thereof In certain embodiments, caninized antibodies of the invention or their derivatives are provided. The antigenic fragment may be an epitope or fragments comprising the amino acid sequence of SEQ ID NO: 132. In more particular embodiments of this type, the epitope or a portion thereof The portion is composed of the amino acid sequence of SEQ ID NO: 134. Another embodiment of this type In one embodiment, the epitope or a portion thereof comprises the amino acid sequence of SEQ ID NO: 135. In certain embodiments, the epitope or a portion thereof is selected from the group consisting of amino acids 133, 134, 136, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, In a more particular embodiment of this type, the epitope or A portion of which is comprised of the amino acid sequence of SEQ ID NO: 136. In some embodiments, the caninized antibody is SEQ ID NO: 134 and / or SEQ ID NO: 136 and / or SEQ ID NO: 1 It binds to one or more epitopes or parts thereof that are formed by a sequence of 35 amino acids.

[0046] The present invention further provides a nucleic acid encoding any one of the light chains of the caninized antibodies of the present invention. provides nucleic acids (which include isolated and / or recombinant nucleic acids). The present invention provides an isolated nucleic acid encoding any one of the heavy chains of the caninized antibodies of the present invention. The present invention provides nucleic acids (which include isolated and / or recombinant nucleic acids).

[0047] The present invention further provides one or more of the nucleic acids of the present invention (which includes isolated nucleic acids). The present invention further provides an expression vector comprising one or more expression vectors of the present invention. A host cell comprising the vector is provided.

[0048] In certain embodiments, the antibody is a recombinant antibody or antigen-binding fragment thereof. In certain embodiments, the variable heavy and variable light domains are linked by a flexible linker. In certain embodiments, the antibody or antigen-binding fragment comprises: In another embodiment, the antibody or antigen-binding fragment is a Fab fragment. In yet another embodiment, the antibody or antigen-binding fragment is In yet another embodiment, the antibody or antigen-binding fragment is a Fab' fragment. In certain embodiments, the antibody or antigen-binding fragment is a diabody. In certain embodiments, the antibody or antigen-binding fragment is a single domain antibody. It is an antibody.

[0049] In certain embodiments, a caninized murine anti-canine CTLA-4 antibody or antigen-binding fragment binds to CTLA-4 in animal subjects (eg, dogs) undergoing treatment for cancer. In a more specific embodiment, the caninized murine anti-canine CTLA-4 antibody or antigen-binding fragment of the invention is Administration of the fragment inhibits one or more of the cancers in the animal subject (e.g., dog) being treated. Helps improve symptoms.

[0050] The present invention further provides a method for producing a caninized murine anti-canine CTLA-4 antibody or portion thereof. In a related embodiment, such an antibody or antigen-binding fragment is provided. The ment can be used in the preparation of a medicament for treating cancer in a canine subject. Alternatively or in combination, the present invention provides an antibody or antibody fragment of the invention for diagnostic use. In yet further embodiments, the present invention provides the use of any of the compounds disclosed herein. Kits containing either the caninized antibodies or antigen-binding fragments are provided.

[0051] The present invention further provides a caninized antibody of the present invention, which binds to the caninized antibody of the present invention, comprises 5 to 25 amino acid residues, and An isolated peptide is provided that is 90% or more identical to the amino acid sequence of SEQ ID NO: 132. In certain embodiments, the isolated peptide is identical to the amino acid sequence of SEQ ID NO: 132. In a more particular embodiment, the isolated peptide is a peptide of 10 to 20 amino acid residues. In a related embodiment, the isolated peptide binds to a caninized antibody of the invention and comprises: Contains 5 to 25 amino acid residues and is 90% or more identical to the amino acid sequence of SEQ ID NO: 133 In certain embodiments, the isolated peptide has the amino acid sequence of SEQ ID NO: 133. In a more particular embodiment of this type, the isolated peptide is 10 to 20 It contains amino acid residues.

[0052] In yet another embodiment, an isolated peptide that binds to a caninized antibody of the invention has the sequence The amino acid sequence of SEQ ID NO:134 is 90% or more identical to the amino acid sequence of SEQ ID NO:134. In one embodiment, the isolated peptide comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 134. In another embodiment, an isolated peptide that binds to a caninized antibody of the invention comprises the sequence: It includes an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 135. In embodiments, the isolated peptide has an amino acid sequence identical to the amino acid sequence of SEQ ID NO:135. In another embodiment, an isolated peptide that binds to a caninized antibody of the invention comprises the amino acid sequence comprises an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 136. In another embodiment, the isolated peptide has an amino acid sequence identical to the amino acid sequence of SEQ ID NO:136. Contains the amino acid sequence.

[0053] The present invention further includes such isolated peptides that bind to the caninized antibodies of the present invention. The present invention further provides a fusion protein comprising any of the above peptides. In certain embodiments, the fusion protein provides such an antigenic peptide. In a more particular embodiment, the fusion protein comprises the Fc region of a canine and a non-canine mammalian IgG antibody. The protein comprises the Fc region of a non-canine mammalian IgG antibody. The mammalian IgG antibody is a mouse IgG. In an alternative embodiment, the non-canine mammalian IgG antibody is a mouse IgG. In another embodiment, the non-canine mammalian IgG antibody is a horse IgG antibody. In yet another embodiment, the non-canine mammalian IgG antibody is a porcine IgG. In yet another embodiment, the non-canine mammalian IgG antibody is bovine IgG.

[0054] In a particular embodiment, the non-canine mammalian IgG antibody is an IgG1. In yet another embodiment, the non-canine mammalian IgG antibody is IgG2a. In yet another embodiment, the mammalian IgG antibody is an IgG3. In another embodiment, the fusion protein comprises the antigenic peptide G. and a maltose binding protein. The protein comprises any of the above antigenic peptides and beta-galactosidase. In this embodiment, the fusion protein comprises any of the antigenic peptides described above and glutathione S In yet another embodiment, the fusion protein comprises an antigenic -transferase. In yet another embodiment, the fusion protein comprises either a nucleotide sequence or a peptide sequence and thioredoxin. In yet another embodiment, the antigenic peptide comprises any of the above antigenic peptides and Gro EL. The fusion protein comprises any of the above antigenic peptides and NusA.

[0055] The present invention further provides one or more isolated immunogenic and / or antigenic peptides of the invention. and / or nucleic acids encoding the fusion proteins (which may be isolated and / or recombinant). The present invention further provides a method for producing a nucleic acid comprising the steps of: Expression vectors and host cells comprising one or more expression vectors of the invention are provided.

[0056] The pharmaceutical composition further comprises an antigenic peptide from canine CTLA-4, which is isolated and fusion proteins comprising antigenic peptides from canine CTLA-4 of the present invention. The present invention also provides a method for the preparation of a fusion protein, an antigenic fragment, and / or a fusion protein of the present invention. Nucleic acids (which includes isolated nucleic acids), expression vectors containing such nucleic acids, or includes any combination thereof, as well as a pharmaceutically acceptable carrier or diluent. Furthermore, the present invention provides the anti-canine CTLA-4 antibody of the present invention (which can be a canine clone). Pharmaceutical compositions containing mouse anti-canine CTLA-4 antibodies or antigen-binding fragments thereof Such pharmaceutical compositions include those for treating cancer, infections or infectious diseases. and can be used as a vaccine adjuvant. and / or can be used in a method for increasing immune cell activity, The method comprises administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition.

[0057] In certain embodiments, such pharmaceutical compositions further comprise an anti-canine PD-1 antibody, which , including caninized murine anti-canine PD-1 antibodies) or antigen-binding fragments thereof. In a more specific embodiment, the anti-canine PD-1 antibody is a caninized murine anti-canine PD-1 antibody or Antigen-binding fragment of caninized murine anti-canine PD-1 antibody.

[0058] In a related embodiment, such pharmaceutical compositions further comprise an anti-canine PD-L1 antibody ( This includes caninized murine anti-canine PD-L1 antibodies) or antigen-binding fragments thereof. In certain embodiments, the anti-canine PD-L1 antibody is a caninized murine anti-canine PD-1 antibody. or an antigen-binding fragment of a caninized mouse anti-canine PD-1 antibody.

[0059] Accordingly, the present invention provides a pharmaceutical composition comprising one, two, three or more of the following: Provided: anti-canine PD-L1 antibody, anti-canine PD-1 antibody, anti-canine CTLA-4 antibody, anti-canine antigen-binding fragment of PD-L1 antibody, antigen-binding fragment of anti-canine PD-1 antibody, or an antigen-binding fragment of an anti-canine CTLA-4 antibody. anti-canine protein (i.e., anti-canine PD-L1, PD-1, or CTLA-4) antibodies or In another embodiment, the antigen-binding fragment is a murine anti-canine protein antibody. Such anti-canine protein antibodies or antigen-binding fragments thereof are caninized anti-canine protein antibodies. In a more particular embodiment, the anti-canine protein antibody or its antigen-binding fragment is The antibody is a caninized mouse anti-dog protein antibody.

[0060] Furthermore, the present invention provides a method for increasing the activity of immune cells, wherein the method comprises: The method comprises administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of the present invention. In an embodiment, the method is used in the treatment of cancer. In another embodiment, the method comprises: In yet another embodiment, the compounds of the present invention are used in the treatment of an infection or infectious disease. Nucleated antibodies or antigen-binding fragments thereof are used as vaccine adjuvants. In certain embodiments, a caninized murine anti-canine CTLA-4 antibody or antigen-binding fragment thereof The pharmaceutical composition comprises a caninized murine anti-canine PD-1 antibody or an antigen-binding fragment thereof. before the caninized and / or caninized murine anti-canine PD-L1 antibody or antigen-binding fragment thereof The compound may be administered to the patient at a later time or simultaneously.

[0061] These and other aspects of the present invention are described in the "Brief Description of the Drawings" and "Detailed Description" below. This will be better understood by reference to [Brief explanation of the drawings]

[0062] [Figure 1] Figure 1 shows the binding activity of six antibodies to canine CTLA-4 (cCTLA-4). Accordingly, Figure 1 represents a plot of the amount of individual canine CTLA-4 antibodies in ng / mL (Ab Log) added to canine CTLA-4 in an ELISA demonstrating the binding activity of the antibodies to cCTLA-4. The individual antibodies to canine CTLA-4 are designated 27G12, 110E3, 12B3, 45A9, 39A11, and 22A11. [Figure 2]Figure 2 shows antibodies that block the interaction between canine CD86 and CTLA-4. The figure shows a plot of the amount of individual canine CTLA-4 antibodies in ng / mL (Ab Log) added to cCTLA-4 to block the binding of canine CTLA-4 to CD86. The individual antibodies against canine CTLA-4 are identified as 39A11, 27G12, 45A9, 12B3, 110E3, and 22A11. As shown in the figure, the antibodies are able to block the interaction between canine CD86 and CTLA-4. [Figure 3] Figure 3 shows antibodies that block the interaction of canine CD80 with CTLA-4. The figure shows a plot of the amount of individual canine CTLA-4 antibodies in ng / mL (Ab Log) added to cCTLA-4 to block the binding of canine CTLA-4 to CD80. The individual antibodies against canine CTLA-4 are designated 39A11, 27G12, 45A9, 12B3, 110E3, and 22A11. As shown in the figure, the antibodies can also block the interaction of canine CD80 with CTLA-4. [Figure 4] Figures 4A-4G show antibodies binding to CHO cells expressing canine CTLA-4. Figure 4A is the Iso control, Figure 4B is 39A11, Figure 4C is 27G12, Figure 4D is 12B3, Figure 4E is 45A9, Figure 4F is 110E3, and Figure 4G is 22A11. As shown in the figures, the antibodies are capable of binding to CHO cells expressing cCTLA-4. [Figure 5] Figure 5 shows a bar graph quantifying the reduction in three concentrations of individual canine CTLA-4 antibodies added at 25 μg / mL, 50 μg / mL, or 100 μg / mL (Ab) to activate canine PBMC cells to produce IFNγ in the presence of concanavalin A (CoA). The antibodies tested are on the horizontal axis and are labeled CTLA-4 monoclonal antibodies (xCTLA-4 mAb). As shown in the figure, the antibodies are capable of activating canine PBMC cells to produce IFNγ. [Figure 6]Figure 6 shows a plot of the amount of CTLA-4 monoclonal antibody (xCTLA-4; Ab Log ng / mL) that has the same reactivity with canine CTLA-4 as the parent antibody. ELISA results demonstrate that both 12B3 and 39A11 were successfully caninized. Caninized c12B3L3H2 and L3H3 have similar reactivity with cCTLA-4 as the parent 12B3, and caninized c39A11L3H3 has similar reactivity with cCTLA-4 as the parent 39A11. [Figure 7] Figures 7A-7B show the binding epitopes on cCTLA-4 for c12B3 (Figure 7A) and c39A11 (Figure 7B). Two regions of the canine CTLA-4 protein are represented, and the two regions 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 of SEQ ID NO: 136 (which contains the MYPPPY motif (SEQ ID NO: 137)) and 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

[0063] Abbreviation The following abbreviations are used throughout the detailed description and examples of the present invention: [Table 1] TIFF2025159730000002.tif70165

[0064] definition In order that the present invention may be more readily understood, certain technical and scientific terms will now be used: Unless expressly defined elsewhere herein, All other technical and scientific terms used are understood to be within the skill of those skilled in the art to which this invention belongs. Thus, it has its generally understood meaning.

[0065] As used herein, including the appended claims, "a" means The singular forms of words such as "," "an" and "the" are used unless the context clearly indicates otherwise. Unless otherwise specified, the terms "a," "b," "c," "d," "e," "f," "g," "h," "i," "j ..., "including their corresponding plural references.

[0066] CTLA-4 functions as an immune checkpoint, downregulating immune responses. The protein receptor, also known as CD152 (cluster of differentiation 152), It is an abbreviation for "cytotoxic T lymphocyte-associated protein 4," which is used to identify canine CTLA-4. The amino acid sequence is SEQ ID NO: 126. The present invention further provides a canine CTLA-4 antibody against canine CTLA-4. The present invention provides a purified mouse antibody.

[0067] "Activation" as applied to a cell or receptor is used unless the context or explicit indication indicates otherwise. Unless otherwise specified, "activation" refers to the activation or treatment of a cell or receptor by a ligand. Cell activation regulated by internal mechanisms as well as by external or environmental factors It can refer to cell activation.

[0068] "Ligand" refers to natural and synthetic ligands, e.g., cytokines, Ligands include variants, analogs, muteins and antibody-derived binding compounds. "Do" also includes small molecules, e.g., peptide mimetics of cytokines and peptide mimetics of antibodies. Also included are chidomimetics.

[0069] The "activity" of a molecule can be expressed or indicated in terms of: ligand or These include binding of the molecule to a receptor, catalytic activity; gene expression or cell signaling, differentiation, or the ability to stimulate maturation; antigenic activity; modulation of the activity of other molecules, etc. The "activity" of a molecule can also be Additionally, they may be shown to regulate cell-cell interactions (e.g., adhesion). activity in forming or maintaining cell structures (e.g., cell membranes or cell bones) Activity in maintaining the integrity of the product. "Activity" can also mean: Specific activity, e.g., [catalytic activity] / [mg protein] or [immunological activity] / [mg g protein], concentration in biological compartments, etc. "Activity" refers to the activity of the innate or adaptive immune system The regulation of the components of the

[0070] "Administration" and "treatment" refer to the administration of an animal (e.g., a canine subject), a cell, When applied to tissues, organs, or biological fluids, animals (e.g., canine subjects), cells, tissues, organs or contacting a biological fluid with an exogenous pharmaceutical, therapeutic, diagnostic agent or composition. This involves contact of reagents with cells and contact of reagents with body fluids (where the body fluids come into contact with the cells). Includes.

[0071] "Administration" and "treatment" also include administering to a subject therapies, such as by a reagent, diagnostic, binding compound, or by administering to another cell. It also refers to in vitro and ex vivo treatments, for example of cells, by

[0072] The term "subject" includes any living organism, preferably an animal, More preferably, it includes a mammal (e.g., a dog, cat, or human), and most preferably, an iguanosine monophosphate (II) or iodide (II). Includes Nu.

[0073] "Treat" or "treating" refers to the use of a therapeutic agent ( For example, a composition comprising any of the antibodies or antigen-binding fragments of the invention may be administered to a subject in need thereof for treatment. Patients suspected of having or suffering from one or more disease indications for which a drug has therapeutic activity For example, administration to a canine subject or patient is meant to be internally or externally administered.

[0074] Typically, a therapeutic agent induces or reduces the clinically measurable regression of the symptom(s). By preventing the progression of the disease(s), such a disease is prevented in the subject or population being treated. The compound is administered in an amount effective to reduce and / or ameliorate the symptoms of the disease. An amount of a therapeutic agent effective to alleviate symptoms (also referred to as a "therapeutically effective amount") is determined by the amount of the therapeutic agent administered to a patient (e.g., The symptoms of the disease, age and weight of the dog, and the medication that will elicit the desired response in the subject. The efficacy of the pharmaceutical composition may vary depending on factors such as whether the symptoms of the disease are reduced or ameliorated. consult a veterinarian or another trained healthcare provider to assess the severity or progression of the signs. The present invention (e.g., For example, embodiments of the method of treatment or article of manufacture may alleviate the target disease symptom(s) in all subjects. Although any method known in the art may not be effective in reducing Statistical tests (e.g., Student's t-test, chi-square test, Mann-Whitney U test, Kruskal-Wallis test (H test), Joncke-Tapstra test and Wilcox test Target disease symptom(s) in a statistically significant number of subjects, as determined by a statistic test (Son test) It should be alleviated.

[0075] "Treatment" refers to a treatment administered to a human subject, a veterinary subject (e.g., a dog), or a research subject. "Treatment" refers to human subjects, veterinary medicine, therapeutic treatment, and research and diagnostic uses. applied to a subject (e.g., a dog) or research subject, or to a cell, tissue, or organ In such cases, the antibodies or antigen-binding fragments of the invention may be administered to, for example, dogs or other animal subjects, cells, or the like. This includes contacting a cell, tissue, physiological compartment, or physiological fluid.

[0076] As used herein, the term "dog" refers to all dogs, unless otherwise indicated. Domestic dog, domestic dog (Canis lupus familiaris) or canine Includes the genus (Canis familiaris).

[0077] As used herein, the term "cat" refers to any member of the family Felidae. This shows members of the family, including wild, zoo and domestic members. - e.g., domestic cats, purebred and / or mixed breed companion cats, show cats, experimental This includes domestic cats, cloned cats, and wild or feral cats.

[0078] As used herein, the term "canine frame" refers to a CDR residue The amino acid sequences of the heavy and light chains of a canine antibody, excluding the hypervariable region residues defined as In the context of caninized antibodies, in most embodiments, the amino acids of the native canine CDRs are The amino acid sequences are aligned with the corresponding heterologous CDRs (e.g., CDRs from a murine antibody) in both chains. Optionally, the heavy and / or light chains of the canine antibody are, for example, replaced by and / or to preserve the conformation of heterologous CDRs in a canine antibody, as exemplified in Some heterologous non-CDR residues may be included to alter Fc function.

[0079] Canine CTLA-4 has the amino acid sequence of SEQ ID NO: 126 (which includes the signal sequence). In a specific embodiment, canine CTLA-4 is found to contain the sequence set forth in SEQ ID NO: 1. The canine CTLA-4 sequence is encoded by a nucleic acid comprising a 25 nucleotide sequence: Canine CT may differ, for example, by having conserved mutations in non-conserved regions. CTLA-4 is substantially the same as canine CTLA-4, which is composed of the amino acid sequence of SEQ ID NO: 126. They have essentially the same biological function.

[0080] As used herein, a substitution of another amino acid within the amino acid sequence of, for example, an antibody, "Substitution of an amino acid residue" by an acid residue means "replacing an amino acid residue" with another amino acid residue. and differs in specific amino acid residues at specific positions within the amino acid sequence. It means that it is replaced (or substituted) by an amino acid residue. Such substitutions can be specifically designed, i.e., inserted by, for example, recombinant DNA techniques. This allows the deliberate replacement of alanine with serine at specific positions in the amino acid sequence. Alternatively, specific amino acid residues or sequences of amino acid residues of the antibody can be selected by more natural selection processes. Through this process, for example, antibodies produced by a cell can be directed to a given region of its antigen (e.g., based on their ability to bind to the target antigen or a region containing a portion thereof, and / or The nucleotide sequence is designed to contain a specific CDR that retains the same canonical structure as the CDR it replaces. , can be replaced by one or more amino acid residues. Such substitutions / replacements are This can result in "mutated" CDRs and / or mutated antibodies.

[0081] Costimulatory signaling pathways lead to the development of an immune response, and CD2 on the surface of T cells 8 and CD80 (also known as B7.1) and CD86 (also known as B7.2) CTLA-4 has been shown to be mediated by the interaction of CD28 binds to both CD80 and CD86 with significantly higher affinity than In fact, CTLA-4 functions as an inhibitory receptor essential for down-modulation of The mechanism by which CD28 mediates its immunosuppressive function is through interactions between CD28 and CD80 and CD86. The present invention relates to the ability of canine CD8 to act as a competitive inhibitor of the activity of canine CD8. 0 and canine CD86 from binding to CTLA-4, thereby inhibiting the binding of canine CD28 Monoclonal antibody that binds to canine CD80 and CD86 to enable costimulatory signaling. The production and characterization of cloned antibodies are described herein. Useful in treating cancer as well as other diseases in companion animals as disclosed It has a sexual nature.

[0082] A particular canine CTLA-4 amino acid sequence generally corresponds to SEQ ID NO:1, excluding the signal sequence. It is at least 90% identical to canine CTLA-4, which contains a 26 amino acid sequence. In the present specification, canine CTLA-4 has the amino acid sequence of SEQ ID NO: 126, excluding the signal sequence. Canine CTLA-4 containing at least 95%, or even at least 96%, 97% , 98% or 99% identical. 4. The amino acid sequence of canine C α-glucan nucleotides is SEQ ID NO: 126, excluding the signal sequence. In a specific embodiment, the canine CTLA-4 has no more than 10 amino acid differences from canine CTLA-4. The amino acid sequence is that of canine CT comprising the amino acid sequence of SEQ ID NO: 126, excluding the signal sequence. LA-4 and 5 or less amino acids, or further 4 or less, 3 or less, 2 or less, or 1 or less The percent identity may represent the amino acid difference between the This can be verified.

[0083] The term "immune response" refers to, for example, the response to cancer cells, cells or tissues infected by a pathogen, or an invading selective damage to pathogens, their destruction or their mammalian body (e.g., canine body) lymphocytes, antigen-presenting cells, phagocytes, granulocytes and the like, resulting in removal from the liver The effects of soluble macromolecules (e.g., antibodies, cytokines, and complement) produced by the are.

[0084] Anti-canine CTLA-4 antibody The present invention provides isolated antibodies that bind to canine CTLA-4 (particularly murine anti-canine CTLA -4 antibody and its caninized antibody) or antigen-binding fragment thereof and such antibody or In certain embodiments, the present invention provides the use of a murine anti-canine CTLA-4 antibody fragment of provided are murine anti-canine CTLA-4 CDRs derived from canine CTLA-4. CTLA-4 and its ligand (canine CD86 or CD8 0) and have been shown to block binding to one or both of these CDRs. inserted into the modified canine frame of the canine antibody to produce a caninized mouse anti-canine CTLA-4 antibody. It can generate a body.

[0085] As used herein, an "anti-canine CTLA-4 antibody" refers to an antibody that specifically binds to canine CTLA-4. (e.g., in a mammal such as a mouse or rabbit) against canine CTLs The antibody specifically binds to canine CTLA-4. Antibodies, and in particular antibodies that specifically bind to canine CTLA-4, or "canine CTLA- The antibody that specifically binds to a polypeptide containing the amino acid sequence of 4 is Although this antibody shows preferential binding to canine CTLA-4 compared to human CTLA-4, this binding is not absolute. Binding specificity is not required. Anti-canine CTLA-4 antibodies are not limited to canine proteins. When determining the presence of canine CTLA-4 in a sample to be determined, or when determining that it is a canine sample without unduly interfering with the activity of other molecules in the pool (e.g., to prevent false positives or of canine CTLA-4 (without causing undesirable consequences such as side effects in therapeutic situations) If the antibody is capable of altering the activity of canine CTLA-4, it is considered "specific" for canine CTLA-4. The degree of specificity required for a CTLA-4 antibody may depend on the intended use of the antibody. In any case, it is defined by its suitability for use for the intended purpose. The antibody or the binding compound derived from the antigen-binding site of the antibody in the method is capable of binding to the antigen or its variants. The affinity of the antibody or mutant protein to the canine antigen is at least as high as that of any other canine antigen. is twice as large, preferably at least 10 times as large, and more preferably at least 20 times as large It binds with greater affinity, most preferably at least 100-fold greater.

[0086] As used herein, an antibody refers to an antibody that contains a portion of the amino acid sequence of canine CTLA-4. and a polypeptide containing the canine CTLA-4 sequence, but lacking that portion of the canine CTLA-4 sequence. If it does not bind to the canine protein, it will bind to the given antigen sequence (in this case, the canine CTLA-4 antigen). It is said to specifically bind to polypeptides containing a portion of the amino acid sequence. The antibody that specifically binds to a polypeptide containing CTLA-4 is a canine CTLA-4 FLAG. FLAG®-tagged form, but not another FLAG®-tagged canine Binding compounds derived from antibodies or the antigen-binding site of antibodies do not bind to proteins. Others that have been tested against the canine antigen or its variants or muteins and more preferably at least 10 times greater than its affinity for the canine antigen of At least 20 times greater, and even more preferably at least 100 times greater, affinity If the antigen is "specifically" bound to the canine antigen or its variant or mutein, Combine.

[0087] As used herein, the term "antibody" refers to any antibody that exhibits a desired biological activity. It is therefore used in the broadest sense and is not intended to be limiting. monoclonal antibodies (which includes full-length monoclonal antibodies), Polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), caninized antibodies (can monoclonalized antibody), whole dog antibody, chimeric antibody and camelized single domain antibody "Parent antibody" includes modifications of the antibody for the intended use (e.g., canine antibodies). by exposure of the immune system to an antigen prior to caninization (caninization of the antibody for use as a therapeutic antibody). The antibody obtained by this method is

[0088] 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 that binds to an antigen of a full-length antibody. Antibody fragments (e.g., one or more Examples of antigen-binding fragments include fragments that retain the CDR regions of the target gene. These 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 antibody fragments.

[0089] A "Fab fragment" is a fragment consisting of one light chain and one heavy chain. H 1 region and variable region The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab fragment" may be the product of papain cleavage of an antibody.

[0090] The "fragment crystallizable" ("Fc") region of an antibody H 3 and C H Contains 2 domains It contains two heavy chain fragments, which are separated by two or more disulfide bonds. By and C H The three domains are held together by hydrophobic interactions.

[0091] A "Fab' fragment" is a fragment that contains one light chain and one V H Domain and C H 1 domain Contains C H 1 Domain and C H A portion of one heavy chain, including the region between the two domains or fragment, thereby forming a fusion protein between the two heavy chains of two Fab' fragments. Interchain disulfide bonds can form to form F(ab')2 molecules.

[0092] "F(ab')2 fragment" refers to a fragment that contains two light chains and a C H 1 Domain and C H 2D The heavy chains each contain a portion of the constant region between the heavy chains, thereby providing a Interchain disulfide bonds are formed at the F(ab')2 fragment. Two Fab' fragments linked by a disulfide bond between the heavy chains A "F(ab')2 fragment" may be the product of pepsin cleavage of an antibody.

[0093] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions. There are.

[0094] The term "single chain Fv antibody" or "scFv antibody" refers to the V of an antibody. H Domain and V L Domain The present invention shows an antibody fragment containing two or more domains, where these domains are separated into a single polypeptide. Generally, Fv polypeptides are characterized by the fact that scFvs are the desired target for antigen binding. V, which allows the formation of the structure H Domains and V L Polypeptide link between domains [See: Pluckthun, THE P HARMACOLOGY OF MONOCLONALANTIBODIES, vol. 113 Rosenburg and Moore eds., Springer -Verlag, New York, pp. 269-315 (1994); W O88 / 01649; and US 4,946,778 and US 5,260 ,203].

[0095] As used herein, the term "canine CTLA-4" refers to its binding partner (ligand), e.g., For example, canine CD80 or canine CD86) Anti-canine CTLA-4 antibodies or their antigen-binding flags that "block" or "block" binding Assays can be performed using standard binding assays (e.g., BIACore®, ELISA, or canine CTLA-4, canine CD86, and / or canine CD86 as determined by flow cytometry Anti-canine CTLA-4 antibodies that block (partially or completely) binding to canine CD80 or Such "blocking" antibodies are antigen-binding fragments thereof. This is exemplified in Example 4 below using a blocking assay based on

[0096] As used herein, the term "canonical structure" refers to the structure of the heavy and light chains of an antibody. Each of the hypervariable regions has a local conformation that it can adopt within the framework in which it resides. For each hypervariable region, a small number of canonical structures (generally one or two) are identified. These are expressed as simple integers such as (In particular, the amino acids of the framework for the corresponding anti-canine CTLA-4 variable domains) These canonical structures can be predicted with high accuracy from the amino acid sequence. Modifications in the amino acid sequence of a given CDR result in retention of the ability to bind to its antigen-binding partner may be decisive as to whether the othia and Lesk, Canonical Structures for the hypervariable regions of immunoglob ulins, J. Mol. Biol. 196:901-917(1987); Chothia et al., Conformation of immunoglobulin obulin hypervaribale regions, Nature, 34 :877-883(1989); and Al-Lazikani et al., St. andard Conformations for the canonical s Structures of immunoglobulins, J. Mol. Bi ol. 273:927-948 (1997)].

[0097] "Domain antibodies" are immunologically functional antibodies that contain only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more V H The region is a peptide covalently linked with a linker to generate a bivalent domain antibody. Two Vs H The regions can target the same antigen or different antigens.

[0098] A "bivalent antibody" contains two antigen-binding sites. In some instances, the two binding sites However, bivalent antibodies can be bispecific. (see below).

[0099] In certain embodiments, the monoclonal antibodies herein are camelized single domain antibodies. [See, e.g., Muyldermans et al. l.,Trends Biochem.Sci.26:230(2001);Reich mann et al., J. Immunol. Methods 231:25(199 9);WO94 / 04678;WO94 / 25591;US6,005,079]. In one embodiment, the present invention provides a method for the production of a single domain antibody comprising administering to a subject a therapeutic agent ... H Single domain antibodies containing domains are provided.

[0100] As used herein, the term "diabody" refers to a diabody having two antigen-binding sites. The fragments are small antibody fragments that are located within the same polypeptide chain. Chain variable domain (V L ) linked to a heavy chain variable domain (V H ) including (V H -V L ,also is V L -V H ) The linker is too short to allow pairing between the two domains on the same chain. By using a carrier, the domain is forced to pair with a complementary domain on another strand. to generate two antigen-binding sites. [See: EP04040 97B1; WO93 / 11161; and Holliger et al., Proc. Natl.Acad.Sci.USA 90:6444-6448(1993)]. Genetics For a review of engineered antibody variants, see generally: Holt et al. liger and Hudson Nat.Biotechnol.23:1126- 1136(2005)].

[0101] Typically, the antibody or antigen-binding fragment of the invention has a canine CTLA-4 binding activity. When expressed on a molar basis, the activity is at least 10% of that of the parent antibody. Preferably, the antibody or antigen-binding fragment of the present invention has the same structure as the parent antibody. at least 20%, 50%, 70%, 80%, 90% of the canine CTLA-4 binding affinity as %, 95%, or 100% or more of the antibody or antibody of the present invention. A native binding fragment may contain conservative amino acid substitutions or modifications that do not substantially alter its biological activity. Non-conservative amino acid substitutions (also called "conservative variants" or "function-conservative variants" of antibodies) It is also contemplated that the compound may contain hydroxybenzoates, such as hydroxybenzoates.

[0102] "Isolated antibody" refers to a purified state, and in that context, the antibody The molecule may be a molecule that is a biological molecule (e.g., a nucleic acid, a protein, a lipid, a carbohydrate) or a substance (e.g., This means that the product is substantially free of any substances (e.g., cell debris and growth medium). The term "isolated" means that such material or water, buffers or salts are not present in the solution described herein. As long as it is not present in an amount that substantially interferes with the experimental or therapeutic use of the binding compound being used. and the complete absence of such substances or the absence of water, buffers or salts. It is not intended to imply that

[0103] As used herein, a "chimeric antibody" refers to an antibody that contains variable domains derived from a first antibody. an antibody having a constant domain derived from a first antibody and a second antibody, The two antibodies are derived from different species [US 4,816,567; and Morrison n et al.,Proc.Natl.Acad.Sci.USA 81:6851- 6855 (1984)]. Typically, the variable domains are derived from laboratory animals such as rodents. The resulting chimeric antibody is derived from a corresponding antibody (the "parent antibody"), and the constant domain sequences are more deleterious immune responses than the parent (e.g., rodent) antibody in human and canine subjects, respectively The antibodies are derived from animal subjects so that they are less likely to cause side effects.

[0104] As used herein, the term "caninized antibody" refers to both canine antibodies and non-canine (e.g., This refers to a form of antibody that contains sequences derived from both caninized and mouse antibodies. The antibody has all or substantially all of its hypervariable loops corresponding to those of a non-canine immunoglobulin. (e.g., containing the six mouse anti-canine CTLA-4 CDRs exemplified below), contain substantially all of at least one, and typically two, variable domains, and All or substantially all of the framework (FR) area (and typically the rest of the frame All or substantially all of the sequences are canine immunoglobulin sequences. As described above, caninized antibodies can be produced by combining a canine frame or a modified canine frame with a mammalian Both the three heavy chain CDRs and the three light chain CDRs derived from the mouse anti-canine CTLA-4 antibody were used. The modified canine frame, for example, increases its binding to canine CTLA-4. and / or canine CTLA-4 to canine CD86 and / or canine CD80 The efficacy of the caninized antibody was further optimized to increase its ability to block binding of The amino acid sequence may include one or more of the amino acid changes exemplified herein.

[0105] The term "full canine antibody" refers to an antibody that contains only canine immunoglobulin protein sequences. Whole canine antibodies are produced in mice, in mouse cells, or derived from mouse cells. If produced in a hybridoma, it may contain mouse sugar chains. "Antibody" refers to an antibody containing only mouse immunoglobulin sequences. Alternatively, a complete canine antibody The cells were grown in rats, in rat cells, or in hybridomas derived from rat cells. Similarly, a "rat antibody" may contain rat glycosylation if produced in a rat immunoglobulin. Antibodies containing only immunoglobulin sequences are shown.

[0106] There are four known IgG heavy chain subtypes in dog IgG: IgG-A, IgG-I The two known light chain subtypes are called IgG-B, IgG-C, and IgG-D. They are called da and kappa.

[0107] The variable regions of each light / heavy chain pair form the antibody binding site. Thus, generally, an intact antibody Except in bifunctional or bispecific antibodies, Therefore, the two binding sites are generally identical.

[0108] Typically, the variable domains of both the heavy and light chains contain relatively conserved framework regions. Three hypervariable regions, also called complementarity-determining regions (CDRs), located within the FR The CDRs are usually aligned by the framework regions, thereby Generally, from the N-terminus to the C-terminus, the light chain variable domain and Each heavy chain variable domain has FR1, CDR1, FR2, CDR2, FR3, CDR The amino acid assignments for each domain generally follow the definitions below: :Sequences of Proteins of Immunological Interest,Kabat,et al.;National Institute s of Health, Bethesda, Md.;5 thed.;NIH Pub l.No.91-3242(1991);Kabat,Adv.Prot.Chem.3 2:1-75(1978);Kabat,et al.,J.Biol.Chem.25 2:6609-6616(1977);Chothia,et al.,J.Mol.B iol. 196:901-917 (1987); or Chothia, et al., Nature 342:878-883(1989)].

[0109] As used herein, the term "hypervariable region" refers to the region of an antibody that is responsible for antigen binding. The amino acid residues are shown. The hypervariable regions are referred to as "complementarity determining regions" or "CDRs" (i.e., CDRL1, CDRL2 and CDRL3 in the light chain variable domain and CDRL4 in the heavy chain variable domain The sequence contains amino acid residues derived from CDRH1, CDRH2, and CDRH3. The CDR regions of antibodies are defined by Kabat et al. es of Proteins of Immunological Interest , 5th Ed. Public Health Service, Nationa l Institutes of Health, Bethesda, Md. (1 991); and further, structures defining antibody CDR regions. 'Chothia and Lesk, J. Mol. Biol. 196: 9 01-917 (1987)]. The term "framework" or "FR" residues are defined herein as CDR residues. Variable domain residues other than the hypervariable region residues are shown.

[0110] In certain embodiments of the present invention, in addition to binding and activating canine immune cells, CTLA-4 A canine or caninized antibody to optimally has the two following attributes: 1. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) lack of effector functions such as 2. Large-scale purification using industry-standard techniques such as those based on Protein A chromatography It is easily purified on a large scale.

[0111] No naturally occurring canine IgG isotype meets both criteria. For example, IgG-B can be purified using Protein A, but high levels of AD are observed. On the other hand, IgG-A binds weakly to Protein A, but also Furthermore, both IgG-C and IgG-D can be purified using a Protein A column. However, IgG-D does not exhibit ADCC activity. (IgG-C exhibits considerable ADCC activity.) One way in which the present invention addresses these issues is by using effectors such as ADCC. - lacking functionality and easily purified using industry-standard Protein A chromatography The present invention provides an engineered canine IgG-B antibody specific for CTLA-4 that can be produced in vivo. By providing.

[0112] In an alternative embodiment of the invention, canine IgG-B or IgG-C antibodies eliminate / substantially reduce effector functions such as ADCC They have not been intentionally modified to inhibit effector functions such as ADCC. It holds.

[0113] "Homology" refers to the degree of homology between two polynucleotide sequences or between two polynucleotides when optimally aligned. It shows the sequence similarity between peptide sequences. When the positions are occupied by the same base or amino acid monomer subunit, e.g., If a position in each of two DNA molecules is occupied by an adenine, this These molecules are homologous at that position. The percentage of homology is the number of positions shared by the two sequences. The number of homologous positions found is divided by the total number of positions compared and multiplied by 100. For example, when two sequences are optimally aligned, 6 out of 10 positions in the two sequences are If there is a match or homology, the two sequences are 60% homologous. A proper comparison is made when two sequences are aligned to obtain the maximum percent identity. can be.

[0114] An "isolated nucleic acid molecule" is a polynucleotide that is found in nature. Polynucleotides that are not linked to all or part of a nucleotide or that are not linked in nature DNA of genomic, mRNA, cDNA or synthetic origin linked to nucleotides or RNA or any combination thereof. For purposes of this disclosure, specific nucleotides It should be understood that a "nucleic acid molecule comprising" a chromosome sequence does not encompass intact chromosomes. An isolated nucleic acid molecule "comprising" a designated nucleic acid sequence comprises, in addition to the designated sequence, 10 up to or even up to 20 or more other proteins or parts thereof or flags The coding sequence for the nucleic acid sequence described may be included. It may contain operably linked regulatory sequences that control expression of the region and / or The vector sequence may include a vector sequence.

[0115] The term "control sequences" refers to sequences that regulate the expression of an operably linked coding sequence in a particular host organism. The control sequences shown are the DNA sequences necessary to activate the promoter. A promoter is included, and optionally an operator sequence and a ribosome binding site are included. Eukaryotic cells use promoters, polyadenylation signals, and enhancers. is known.

[0116] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader may be used if it contributes to the secretion of a polypeptide. When expressed as a preprotein, the polypeptide is operable to encode DNA. a promoter or enhancer is a gene that affects the transcription of a coding sequence; or a ribosome binding site; is operably linked to a coding sequence when positioned so as to facilitate translation Generally, "operably linked" means that the DNA sequences being linked are contiguous. and, in the case of a secretory leader, adjacent and in reading phase; However, enhancers do not have to be contiguous. Linkage can be convenient. This is accomplished by ligation at a restriction site. If no such site exists, Synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0117] As used herein, the terms "cells," "cell lines," and "cell cultures" are used interchangeably. Present and future are used interchangeably, and all such designations include progeny. The terms "transformant" and "transformed cell" refer to the primary subject cell and the transformed cell without regard to the number of passages. Furthermore, it also includes cultures derived from deliberate or accidental mutations. It is also understood that not all progeny will have the exact same DNA content. have the same function or biological activity as screened for in the transformed cells Mutant progeny are included. Where a different designation is intended, it shall be clear from the context. It becomes clear.

[0118] As used herein, "germline sequence" refers to an unrearranged immunoglobulin sequence. The sequence of the unrearranged immunoglobulin DNA sequence is shown. Any suitable source of human germline sequences can be used. Human germline sequences can be found, for example, at the National Institute of National Institute of Arthritis and Musculoskeletal / Skin Diseases of the National Institutes of Health itute of Arthritis and Musculoskeletal a nd Skin Diseases of the United States Na JOI on the National Institutes of Health website Germline sequences are available from the NSOLVER® germline database. Germline sequences are described, for example, in Giudicelli et al. Acids Res. 33:D256-D261 (2005)] You can get it like this.

[0119] Characterization of murine anti-canine CTLA-4 and caninized murine anti-canine CTLA-4 antibodies The present invention relates to an isolated murine anti-canine CTLA-4 antibody and its caninized form, and to the treatment of diseases. Use of antibodies or antigen-binding fragments thereof (e.g., in treating cancer in dogs) In dogs, there are four IgG heavy chains, designated A, B, C, and D. These heavy chains are called IgGA, IgGB, IgGC, and IgGD. Each of the two heavy chains contains one variable domain (V H) and three constant domains called CH-1, CH-2 and CH-3. The -1 domain is connected to the ribosome via an amino acid sequence called the "hinge" or alternatively the "hinge region." It is linked to the CH-2 domain via a

[0120] The DNA and amino acid sequences of these four heavy chains are described by Tang et al. [Vet Immunol. Immunopathol. 80: 259-270 (20 The amino acid and DNA sequences for these heavy chains were first identified by the Sequences are also available from the GenBank database. For example, the amino acid sequence of the IgGA heavy chain is The amino acid sequence has the accession number AAL35301.1, and the IgGB has the accession number AAL353 02.1, IgGC has the accession number AAL35303.1, and IgGD The canine antibody has the accession number (AAL35304.1). The DNA and amino acid sequences of these light chains are also included in GenB The amino acid sequence of the kappa light chain is available from the .ank database. The lambda light chain has accession number ABY57289.1, and the lambda light chain has accession number ABY5556 It has 9.1.

[0121] In the present invention, the amino acid sequences for each of the four canine IgG Fc fragments are: The identification of the CH1 and CH2 domains determined by Tang et al. (supra) Caninized mouse anti-canine CTLA-4 binds to canine CTLA-4. Antibodies include, but are not limited to: Mouse anti-dog CTL Canine IgG-A, IgG-B, IgG-C, and IgG-D heavy chains and Thus, the present invention provides an antibody that binds to canine CTLA-4 and inhibits canine CTLA-4. An isolated antibody that blocks the binding of canine CTLA-4 to canine CD86 and / or canine CD80. mouse anti-canine CTLA-4 and / or caninized mouse anti-canine CTLA-4 antibody or its Antigen-binding fragments are provided.

[0122] The present invention further provides a complete antibody that can be combined with a corresponding light chain to produce a caninized antibody. The present invention further provides a caninized mouse anti-canine antigen antibody ( This includes isolated caninized murine anti-canine CTLA-4 antibodies) and methods for treating diseases (e.g., Methods for using antibodies or antigen-binding fragments thereof in the treatment of cancer in dogs are provided. do.

[0123] The present invention further provides a method for producing a canine crystalline fragment region (cFc region) comprising administering to one or more effectors - a cFc region that has been genetically engineered to enhance, reduce or eliminate function In one aspect of the present invention, a caninized mouse anti-canine CTLA-4 antibody is provided, comprising: The engineered cFc reduces or eliminates one or more effector functions. In another embodiment, the engineered cFc has enhanced one or more effector functions. In certain embodiments, the engineered cFc region is an engineered canine Ig In another such embodiment, the engineered cFc region is a GB Fc region. In certain embodiments, the effector mechanism is a genetically engineered canine IgGC Fc region. The ability is antibody-dependent cellular cytotoxicity (ADCC), which is increased, reduced or eliminated. In another embodiment, the effector function is complement dependent cytotoxicity (CDC), In yet another embodiment, the cFc region is enhanced, reduced or eliminated. They have been genetically engineered to increase, decrease, or eliminate both CDC and CDC.

[0124] To generate canine IgG mutants lacking effector function, multiple mutations were performed. Variant canine IgGB heavy chains were generated. These variants contained the following amino acid sequences within the Fc portion of the heavy chain: It may contain one or more of the following single or compound substitutions: P4A, D31A, N63A, G64P, T65A, A93G, and P95A. The heavy chain containing the amino acid substitutions was cloned into an expression plasmid containing the gene encoding the light chain. HEK293 cells were transfected with the corresponding plasmids. Expressed and purified intact antibodies are analyzed for their potential to mediate immune effector functions. To investigate Fc γ Binding to RI and C1q was assessed. See US Pat. No. 06,607B2, the contents of which are incorporated herein by reference in their entirety. [Will be included]

[0125] The present invention further relates to a method for producing an IgG1A antibody comprising, in place of its native IgGD hinge region, the following hinge region: , a modified canine IgGD is provided. [Table 2]

[0126] Alternatively, the IgGD hinge region may be modified by replacing serine residues with proline residues. The gene can be modified by the following sequence: PKESTCKCI of SEQ ID NO: 131 P PCPVP ES (proline residues (P) substituting naturally occurring serine residues are bold and underlined). Such modifications result in canine IgGD lacking Fab arm exchange. The modified canine IgGD can be produced using standard methods of recombinant DNA technology. can be constructed [see, e.g., Maniatis et al., Molecular Cloning, A Laboratory Manual (1982)]. To construct these variants, a nucleic acid encoding the amino acid sequence of canine IgGD is modified The modified nucleic acid can then be modified to encode the modified IgGD. The nucleic acid sequence is cloned into an expression plasmid for protein expression.

[0127] The antibodies or antigen-binding fragments thereof that bind to canine CTLA-4 are described herein. The antibody contains 3, 4, 5, or 6 of the complementarity determining regions (CDRs) of a known mouse anti-dog antibody. The three, four, five or six CDRs may be selected from the CDR sequences provided below. In a further embodiment, an isolated antibody that binds to canine CTLA-4 can be independently selected from the following: The antibody or antigen-binding fragment thereof may comprise a mouse light chain CDR-1, CDR-2 and / or a canine antibody kappa or lambda light chain containing CDR-3 and a mouse heavy chain CDR-1; The dog antibody heavy chain IgG includes CDR-2 and / or CDR-3.

[0128] In another embodiment, the present invention provides an antibody that specifically binds to canine CTLA-4 and SEQ ID NO: 92 for VLCDR-1, VLCDR-2 and VLCDR-3, respectively , 94 and 96 amino acid sequences and at least 80%, 85%, 90%, 95%, 98% or is a canine antibody kappa or lambda containing a given set of three CDRs that contain 99% sequence identity. For the VHCDR-1, VHCDR-2, and VHCDR-3 light chains, respectively, and at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140 Canine antibodies comprising a given set of three CDRs that contain 5%, 98%, or 99% sequence identity. heavy chain IgG; or SEQ ID NO: 10 for VLCDR-1, VLCDR-2 and VLCDR-3, respectively 4, 106 and 108 amino acid sequences and at least 80%, 85%, 90%, 95%, 9 Canine antibody kappa or ribozymes containing a given set of three CDRs containing 8% or 99% sequence identity. for lambda light chain and VHCDR-1, VHCDR-2 and VHCDR-3, respectively. 98, 100 and 102 and at least 80%, 85%, A given set of different CDRs that contain 90%, 95%, 98% or 99% sequence identity. Canine antibody heavy chain IgG; or SEQ ID NO: 11 for VLCDR-1, VLCDR-2 and VLCDR-3, respectively 7, 94 and 96 amino acid sequences and at least 80%, 85%, 90%, 95%, 98% or a canine antibody kappa or lambda containing a given set of three CDRs containing 99% sequence identity. The untranslated light chains and VHCDR-1, VHCDR-2, and VHCDR-3, respectively, All of the amino acid sequences of SEQ ID NOs: 86, 88 and 113 have at least 80%, 85%, 90% , 95%, 98% or 99% sequence identity. antibody heavy chain IgG; or SEQ ID NO: 11 for VLCDR-1, VLCDR-2 and VLCDR-3, respectively 9, 122 and 96 amino acid sequences and at least 80%, 85%, 90%, 95%, 98 100% or 99% sequence identity of a given set of three CDRs of a canine antibody kappa or Lambda light chain and VHCDR-1, VHCDR-2 and VHCDR-3, respectively. The amino acid sequences of SEQ ID NOs: 86, 88 and 115 are at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity. antibody heavy chain IgG; while at the same time still maintaining favorable binding and functional properties In another embodiment, the antibody of the present invention is or an antigen-binding fragment having 0, 1, 2, 3, 4, or 5 conservative or non-conservative amino acids. and one or more of said sets of three light chain CDRs and three heavy chain CDRs having an acid substitution. While containing a combination of kappa or lambda light chain and IgG heavy chain sequences, which are preferred It includes a canine frame that still exhibits binding and functional properties.

[0129] Sequence identity is the degree to which the amino acids of two polypeptides match when the two sequences are optimally aligned. As used herein, 1 indicates the degree of identity at equal positions. One amino acid sequence is a sequence of amino acids that has the same amino acid residues as the second amino acid sequence. If they are identical, they are 100% "identical." Thus, a given amino acid sequence is For amino acid sequences, 50% of the amino acid residues in the two amino acid sequences are identical. If the sequence is 50% identical, the comparison is 50% identical. A contiguous block of amino acid residues contained in a polypeptide or protein In certain embodiments, the correspondence between the two amino acid sequences is varied. Take into consideration the selected deletion or insertion that can be altered.

[0130] Sequence similarity encompasses identical residues and biochemically related non-identical amino acids. Biochemically related amino acids that share similar properties and may be interchangeable are considered.

[0131] "Conservatively modified variants" or "conservative substitutions" refer to amino acids in a protein that are similarly modified. properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.) The resulting change is often a substitution of another amino acid with that This can be done without altering the biological activity of the protein. It is recognized that single amino acid substitutions within non-essential regions of the peptide do not substantially alter biological activity. [See, e.g., Watson et al., Molecular Biology of the Gene,The Benjamin / Cummings Pub. C o., p.224 (4th Ed.; 1987).” In addition, structurally or functionally similar amino acid substitutions are less likely to destroy biological activity. Exemplary conservative substitutions are shown in Table A immediately below. [Table 3]

[0132] Function-conservative variants of the antibodies of the invention are also contemplated by the present invention. " as used herein refers to a compound having desired characteristics such as antigen affinity and / or specificity. The term "antibody" refers to an antibody or fragment in which one or more amino acid residues have been altered without altering the antibody's properties. Such variants include, but are not limited to, variants in which the particular amino acid is a conserved amino acid in Table A above. This includes substitutions of amino acids with amino acids having similar properties, such as conservative amino acid substitutions. do.

[0133] nucleic acid The present invention further provides the murine anti-canine CTLA-4 and / or Immunoglobulin chains of caninized murine anti-canine CTLA-4 antibodies and their antigen-binding fragments (See, for example, the Examples below.)

[0134] Furthermore, when the comparison is performed by the BLAST algorithm, the The amino acid sequence of the caninized antibody is at least about 70% identical, preferably at least more 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% Nucleic acids encoding immunoglobulin polypeptides comprising an amino acid sequence that is The algorithm parameters are: The sequences are selected to give the greatest match between the respective sequences. When the comparison is performed using the ST algorithm, it has at least about 7 amino acid residues with any of the reference amino acid sequences. 0% similar, preferably at least about 80% similar, more preferably at least about 90% similar. % similar, and most preferably at least about 95% similar (e.g., 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, Immunoglobulin polypeptides containing amino acid sequences that are identical (7%, 98%, 99%, 100%) Nucleic acids encoding the sequences are also provided, wherein the parameters of the algorithm are The sequences are chosen to give the greatest match between the respective sequences over the entire length of the reference sequence. are encompassed by the present invention.

[0135] As used herein, percent identity of nucleotide and amino acid sequences are the default parameters for alignment and identity Using C,MacVector (MacVector, Inc. Cary, NY C 27519), Vector NTI (Informax, Inc. MD), O Xford Molecular Group PLC (1996) and Clustal This can be verified using the W algorithm. further uses the same or similar default parameters to check sequence similarity. Alternatively, for example, GCG(G enetics Computer Group, Program Manual f or the GCG Package, Version 7, Madison, Wisconsin) pileup program under default filter conditions. It is also possible to use an Advanced Blast search.

[0136] The following references concern the BLAST algorithm, which is often used for sequence analysis: BLAST ALGORITHMS: Altschul,SF,e t al.,J.Mol.Biol.215:403-410 (1990);Gish , W., et al.,Nature Genet.3:266-272 (199 3);Madden,T.L., et al.,Meth.Enzymol.266: 131-141(1996); Altschul,S.F., et al., Nu cleic Acids Res. 25:3389-3402 (1997); Zh ang, J., et al.,Genome Res. 7:649-656 (1 997);Wootton, J.C., et al., Comput. Chem . 17:149-163 (1993); Hancock, J.M. et al ., Comput.Appl. Biosci. 10:67-70 (1994); ALIGNMENT SCORING SYSTEMS: Dayhoff, M.O ., et al., “A model of evolutionary chan ge in proteins.” in Atlas of Protein Seq uence and Structure, vol. 5, suppl. 3. M .O. Dayhoff (ed.), pp. 345-352, (1978); Natl. Biomed. Res. Found., Washington, D C; Schwartz, R.M., et al., “Matrices for detecting distant relationships.” in At las of Protein Sequence and Structure, v ol. 5, suppl. 3.“ (1978), M.O. Dayhoff (ed.), pp. 353-358 (1978), Natl. Biomed. Res. Found., Washington, DC; Altschul, S.F., J. Mol. Biol. 219:555-565 (1991); States, D.J., et al., Methods 3:66-70(19 91); Henikoff, S., et al., Proc. Natl. A cad. Sci. USA 89:10915-10919 (1992); Alt schul, S.F., et al., J. Mol. Evol. 36:29 0-300 (1993); ALIGNMENT STATISTICS: Karl in, S., et al., Proc. Natl. Acad. Sci. U SA 87:2264-2268 (1990); Karlin, S., et a l., Proc. Natl. Acad. Sci. USA 90:5873- 5877 (1993); Dembo, A., et al., Ann. Pro b. 22:2022-2039 (1994); and, Altschul, S.F . “Evaluating the statistical significan ce of multiple distinct local alignments .” in Theoretical and Computational Meth ods in Genome Research (S. Suhai, ed.), pp. 1-14, Plenum, New York (1997).

[0137] The present invention further provides an expression vector comprising a nucleic acid of the present invention, wherein the nucleic acid is capable of expressing a gene in a host It is engineered into a regulatory sequence that is recognized by the host cell when the cell is transfected with the vector. Furthermore, a host cell comprising the expression vector of the present invention and a Methods for producing the antibodies or antigen-binding fragments thereof disclosed herein are also provided. wherein the method comprises carrying an expression vector encoding the antibody or antigen-binding fragment. and culturing a host cell in a medium that binds to the antigen or an antigen-binding fragment thereof. This includes isolating it from cells or culture medium.

[0138] Caninized murine anti-canine CTLA-4 antibodies can be produced recombinantly by methods known in the art. For expression of the antibodies or fragments disclosed herein, Mammalian cell lines available as hosts are well known in the art and are available from the American American Type Culture Collect These include many immortalized cell lines available from the American College of Cardiology (ATCC). Specifically, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, and HeLa cells , baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g. For example, Hep G2, A549 cells, 3T3 cells, HEK-293 cells, and many other cells Mammalian host cells include human, mouse, rat, dog, monkey, and Particularly preferred cell lines include mouse, goat, bovine, horse and hamster cells. Other cells that can be used are selected by confirming whether they have high expression levels. Cell lines include insect cell lines, e.g., Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. The heavy chain or its antigen-binding portion or fragment, the light chain and / or its antigen-binding portion or fragment When a recombinant expression vector encoding the ligation fragment is introduced into a mammalian host cell, for a period of time sufficient to allow expression of the antibody in the host cell, or more preferably for a period of time sufficient to allow secretion of the antibody into the medium in which the host cells are grown, The antibody is produced by culturing the host cells.

[0139] Antibodies can be recovered from the culture medium using standard protein purification methods. Furthermore, expression of the antibodies of the invention (or other components derived therefrom) from production cell lines can be achieved in many ways. Many known techniques can be used to enhance the expression of glutamine synthetase. The current system (GS system) is a general approach to enhance expression under specific conditions. The S series is disclosed in European Patent No. 0216846, European Patent No. 0256055 and European Patent No. 03 23997 and in whole or in part in connection with European Patent Application No. 89303964.4 It is considered in this context.

[0140] Generally, glycoproteins produced in a particular cell line or transgenic animal are glycosylation characteristic of glycoproteins produced in the cell line or transgenic animal Therefore, the particular glycosylation pattern of an antibody determines the specific glycosylation pattern of the antibody produced. However, this will depend on the particular cell line or transgenic animal used to generate the and the nucleic acid molecules provided herein or All antibodies containing the amino acid sequences provided herein are intended to be free of any glycosylation that the antibody may have. Regardless of the fucosylation pattern, the present invention is contemplated. Similarly, in certain embodiments, non-fucosylated N Antibodies with a glycosylation pattern containing only -glycans are characterized as these antibodies typically exhibit greater potency than their fucosylated counterparts both in vitro and in vivo This can be advantageous since it has been shown that nkawa et al.,J.Biol.Chem.278:3466-3473(2 003); U.S. Patent No. 6,946,292 and U.S. Patent No. 7,214,775].

[0141] The present invention further provides antibodies to the murine anti-canine CTLA-4 antibodies disclosed herein. Antibody fragments include fragments obtained by enzymatically cleaving IgG with, for example, pepsin. Fab fragments include F(ab)2 fragments, which can be generated by cleavage. The method involves reducing F(ab)2 with, for example, dithiothreitol or mercaptoethylamine. Fab fragments can be generated by disulfide bridges. TeV H -C H1 V added to the chain L -C L The F(ab)2 fragment is a two-chain F(ab)2 is two Fab fragments joined by a disulfide bridge. The Fab portion of the molecule is composed of F c Contains a portion of the area. F V The fragment is V L Area or V H It is an area.

[0142] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region, e.g., a canine constant regions, such as IgGA, IgGB, IgGC, and IgGD canine heavy chain constant regions or variants thereof. In another embodiment, the antibody or antigen-binding fragment comprises a light chain constant region, e.g., For example, 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 is derived from IgG-B and the canine light chain constant region can be derived from kappa.

[0143] antibody engineering The caninized murine anti-canine CTLA-4 antibodies of the present invention can be used to, for example, improve the properties of the antibody. To achieve this, the canine framework and and / or can be engineered to contain modifications to canine frame residues.

[0144] Epitope binding and binding affinity The present invention further provides a method for producing the same murine anti-canine CTLA-4 antibodies as disclosed herein. Antibodies that bind to amino acid residues of epitopes of canine CTLA-4 or their antigen-binding fragments In certain embodiments, a murine anti-canine CTLA-4 antibody or antigen-binding The fragment may further comprise a fragment that binds canine CTLA-4 to canine CD86 and / or CD80. In a related embodiment, caninized murine anti-dog CTLs can be inhibited / blocked. The A-4 antibody or antigen-binding fragment thereof also binds to canine CD86 and / or canine CTLA-4. can inhibit / block binding to CD80.

[0145] Experimental and diagnostic uses The murine anti-canine CTLA-4 and / or caninized murine anti-canine CTLA-4 antibody of the present invention The antigen-binding fragment is further used as a diagnostic assay for canine CTLA-4 protein. In particular, they are useful in detecting their expression in conjunction with and / or associated with cancer. could be.

[0146] For example, such a method may include the following steps: (a) a substrate (e.g., the surface of a microtiter plate well, e.g., a plastic The surface of the plate was coated with mouse anti-canine CTLA-4 antibody or its antigen-binding fragment. Coating stage; (b) applying a sample to be tested for the presence of canine CTLA-4 to the substrate; (c) washing the plate to remove unbound material in the sample; (d) a detectably labeled antibody (e.g., an enzyme-linked antibody) that is also specific for the CTLA-4 antigen. applying a conjugated antibody; (e) washing the substrate to remove unbound labeled antibody; (f) If the labeled antibody is enzyme-linked, the compound is converted by the enzyme into a fluorescent signal. applying a chemical substance; and (g) detecting the presence of the labeled antibody.

[0147] In a further embodiment, the labeled antibody is ABTS [e.g., 2,2'-azino-bis( 3-ethylbenzthiazoline-6-sulfonic acid)] or 3,3',5,5'-tetramethyl Labeled with peroxidase that reacts with rubenzidine (TMB) to produce a detectable color change Alternatively, the labeled antibody can be visualized in a scintillation counter in the presence of a scintillant. A detectable radioisotope (e.g., 3 H) The murine anti-canine CTLA-4 antibodies of the present invention can be used in Western blot procedures or immunoassays. It can be used in protein blot procedures.

[0148] Such procedures form part of the present invention and include, for example: (i) Membrane or tissue samples to be tested for the presence of bound canine CTLA-4 or fragments thereof. Alternatively, another solid substrate may be coated with the caninized murine anti-canine CTLA-4 antibody of the present invention or its antigen-binding fragment. Such membranes are suitable for non-denaturing PAGE (polyacrylamide gel electrophoresis). electrophoresis) gel or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) The proteins to be tested for the presence of canine CTLA-4 in the gel (e.g., After electrophoretic separation) transferred to nitrocellulose or vinyl [e.g., polyfluoride The membrane can be in the form of a polyvinylidene fluoride (PVDF) membrane. Before contacting the membrane with the A-4 antibody or antigen-binding fragment thereof, the membrane may be optionally Block nonspecific protein binding sites on the antibody with, for example, nonfat dry milk. ; (ii) an unconjugated caninized mouse anti-canine CTLA-4 antibody or an antigen-binding fragment thereof; and washing the membrane one or more times to remove any unbound material; and (iii) a conjugated caninized mouse anti-canine CTLA-4 antibody or antigen-binding fragment thereof To detect the

[0149] Detection of the bound antibody or antigen-binding fragment is performed by detecting the antibody or antigen-binding fragment. The antibody is then allowed to bind to a detectably labeled second antibody (anti-immunoglobulin antibody), and the second antibody is then This can be done by detecting the presence of a secondary antibody.

[0150] The murine anti-canine CTLA-4 antibodies disclosed herein, caninized murine anti-canine CTLA-4 antibodies, The LA-4 antibody and / or antigen-binding fragment thereof may further be used for immunohistochemistry. Such methods form part of the present invention and include, for example: (1) Cells to be tested for the presence of canine CTLA-4 are cultured using, for example, a mouse antibody of the present invention. (2) contacting the canine CTLA-4 antibody or antigen-binding fragment thereof with the canine CTLA-4 antibody or antigen-binding fragment thereof; and Detecting antibodies or fragments on the surface or inside cells of a subject. If the fragment itself is detectably labeled, it can be detected directly. Alternatively, the antibody or antigen-binding fragment may be conjugated to a detectably labeled secondary antibody. It can be detected.

[0151] Imaging techniques include SPECT imaging (single photon emission computed tomography) Imaging (positron emission tomography) or PET imaging (positron emission tomography). Examples include: iodine-1, e.g., in combination with SPECT imaging; twenty three( 123 I) and technetium-99m ( 99m Tc), or e.g., PET imaging Combined with Jing, 11 C. 13 N, 15 O or 18 F or Indium-1 11 [See, e.g., Gordon et al., International Rev . Neurobiol. 67:385-440 (2005)].

[0152] Cross-blocking antibodies Furthermore, the anti-canine CTLA-4 antibody or antigen-binding fragment thereof of the present invention may have the following characteristics: Also included are: canine C to which the antibodies and fragments discussed herein bind. Any antibody or antigen-binding fragment thereof that binds to the same epitope as TLA-4, and In addition, the antibodies or fragments discussed herein for canine CTLA-4 binding The present invention relates to cross-blocking (partially or completely) canine CTLA-4 binding. Cross-blocking (partially or fully) by the antibodies or fragments discussed therein Any antibody or antigen-binding fragment that is locked; and any variant thereof. .

[0153] The cross-blocking antibodies and antigen-binding fragments thereof discussed herein are Standard binding assays (e.g., BIACore®, exemplified below) In LISA or flow cytometry, the antibodies disclosed herein ( Based on the CDRs provided in Example 5 below) (i.e., 45A9, 27G12 , 22A11, 110E3; and more particularly 12B3 and / or 39A11) These can be identified based on their ability to cross-compete, e.g., by standard ELISA. A can be used, in which the assay uses recombinant canine CTLA- 4 proteins are immobilized on a plate, one of the antibodies is fluorescently labeled, and the target Additionally or alternatively, the ability of unlabeled antibodies to compete with the binding of the labeled antibody is assessed. Acore® analysis can be used to assess the ability of antibodies to cross-compete. For example, 27G12, 45A9, 110E3 and / or 22A11; and and more specifically inhibits the binding of 12B3 and / or 39A11 to canine CTLA-4. The ability of the test antibody to bind to canine CTLA-4 is determined by comparing the test antibody to 27G12, 4 5A9, 110E3 and / or 22A11 and / or 12B3 and / or 39 A11, so in some cases, 27G12, 45A9, 110E3 and / or 22A11 and / or 12B3 and / or 39A11 It has been shown that it can bind to the same epitope on canine CTLA-4. As noted, the same enzyme as any of the anti-canine CTLA-4 antibodies or fragments of the present invention may be used. Antibodies and fragments that bind to the pitope also form part of the present invention.

[0154] Pharmaceutical Compositions and Administration A pharmaceutical composition of a caninized murine anti-canine CTLA-4 antibody or an antigen-binding fragment thereof To prepare a sterile composition, it is mixed with a pharmaceutically acceptable carrier or excipient. [See, for example, Remington's Pharmaceuticals Maceutical Sciences and US Pharmacopei a: National Formulary, Mack Publishing C company, Easton, PA (1984)].

[0155] Therapeutic and diagnostic formulations may be in the form of, for example, a lyophilized powder, a slurry, an aqueous solution, or a suspension. It can be prepared by mixing with an acceptable carrier, excipient or stabilizer as listed in [See, e.g., Hardman, et al. (2001 ) Goodman and Gilman's The Pharmacologic al Basis of Therapeutics,McGraw-Hill,New York, NY; Gennaro(2000) Remington: The Sc. ience and Practice of Pharmacy,Lippincott t, Williams, and Wilkins, New York, NY; Avis ,et al. (eds.) (1993) Pharmaceutical Dos age forms: Parenteral Medications, Marce l Dekker, NY; Lieberman, et al. (eds.) (199 0) Pharmaceutical Dosage Forms:Tablets,M arcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disp. erse Systems,Marcel Dekker,NY;Weiner and Kotkoskie(2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY]. one In an embodiment, the anti-CTLA-4 antibody of the present invention is dissolved in sodium acetate solution (pH 5-6). Dilute to appropriate concentration and add NaCl or sucrose for tonicity. To enhance the can be added.

[0156] The toxicity and therapeutic efficacy of the antibody composition administered alone or in combination with another agent may be determined by: For example, LD 50 (a dose lethal to 50% of the population) and ED50 (50% of the population Standard pharmacological studies in cell cultures or experimental animals to determine the therapeutically effective dose The dose ratio between toxic and therapeutic effects is called the therapeutic index (LD 50 / ED 50 In certain embodiments, antibodies that exhibit high therapeutic indices are desirable. Data from these cell culture assays and animal studies are not sufficient for use in dogs. The dosage of such compounds may be used to determine the dosage range of the compound. or ED with little or no toxicity 50 The circulating concentration range includes: The amount may vary within this range depending upon the dosage form and route of administration used.

[0157] The method of administration can vary. Suitable routes of administration include: oral , rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous , intraperitoneally, intranasally, intraocularly, by inhalation, by insufflation, topically, cutaneously, transdermally, or intra-arterially. The caninized mouse anti-canine CTLA-4 antibody or antigen-binding fragment thereof can be administered by injection. In a further embodiment of the present invention, the caninized mouse The anti-canine CTLA-4 antibody or antigen-binding fragment thereof, or a pharmaceutical composition thereof, It may be administered intravenously, subcutaneously, intramuscularly, intraarterially, or by inhalation or aerosol delivery. Administration by oral routes (e.g., orally; e.g., in pills, capsules, or tablets) is also contemplated. It is within the scope of the invention.

[0158] The compositions can be administered using medical devices known in the art, for example The pharmaceutical composition of the present invention can be administered using a hypodermic needle (which can be, for example, a prefilled syringe or an autologous syringe). The present invention can be administered by injection using a syringe. The pharmaceutical composition may further be administered by a needleless hypodermic injection device (e.g., U.S. Pat. No. 6,620,133). No. 5; No. 6,096,002; No. 5,399,163; No. 5,383,851; No. No. 5,312,335; No. 5,064,413; No. 4,941,880; No. 4,79 Administering the drug using a device such as those disclosed in US Pat. Nos. 0,824 or 4,596,556 It is also possible.

[0159] The pharmaceutical compositions disclosed herein may also be administered by injection. Examples of well-known implants and modules for administering compositions include: No. 4,487,603, which provides a controlled rate of delivery of a drug. U.S. Pat. No. 4,447,227 discloses an implantable microinfusion pump for administering No. 33, which discloses a drug infusion pump for delivering drugs at precise infusion rates. U.S. Pat. No. 4,447,224, which describes a variable flow rate implant for sustained drug delivery; U.S. Pat. No. 4,439,196, which discloses a multi-channel injection device; Many other such systems have been described. Plants, delivery systems and modules are well known to those skilled in the art.

[0160] Alternatively, for example, immunopathology, often in depot or sustained release formulations. By injecting antibodies directly into the interior of a joint with a characterized pathogen-induced lesion or arthritis. Therefore, mouse anti-canine CTLA-4 antibody or caninized mouse anti-canine CTLA-4 antibody was administered systemically. Furthermore, the administration of the antibody can be localized rather than localized. Targeted drug delivery systems that target pathogen-induced lesions or arthritic joints, e.g. For example, the antibody can be administered in liposomes coated with the tissue-specific antibody. The liposomes are targeted to and taken up selectively by the affected tissue.

[0161] The dosing regimen may be adjusted based on serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, and the therapeutic antibody Several factors, including the immunogenicity of the protein and the accessibility of target cells in the biological matrix, have been implicated. Preferably, the dosing regimen should be tailored to minimize unwanted side effects. while delivering sufficient therapeutic antibody to bring about improvement at the targeted disease site. The amount of biologic delivered will depend in part on the specific therapeutic antibody and the severity of the condition being treated. Guidance is available on selecting an appropriate dose of a therapeutic antibody. [See, e.g., Wawrzynczak Antibody T herapy,Bios Scientific Pub.Ltd,Oxfordshi re,UK(1996);Kresina (ed.) Monoclonal Ant ibodies, Cytokines and Arthritis,Marcel Dekker, New York, NY (1991); Bach (ed.) Mono clonal Antibodies and Peptide Therapy in Autoimmune Diseases,Marcel Dekker,New Y ork,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);Beniami novitz et al. New Engl. J. Med. 342:613-619( 2000);Ghosh et al.New Engl.J.Med.348:24- 32(2003);Lipsky et al.New Engl.J.Med.343 :1594-1602(2000)].

[0162] Determination of appropriate dosages is known in the art, e.g., by a veterinarian to affect treatment. Generally, administration is performed using known or suspected parameters or factors. It is recommended to start with a somewhat lower dose and then wait until the desired or optimal effect is achieved relative to the negative side effects. The dose should be increased gradually until this is achieved. Important diagnostic criteria include the diagnosis of symptoms such as tumor size. Includes rejection criteria.

[0163] The antibodies or antigen-binding fragments thereof disclosed herein can be administered by continuous infusion. or may be provided, for example, once daily, 1-7 times per week, once weekly, once every other week, or once monthly. , delivered in doses administered every other month, every three months, every six months, or every year. Dosages can be administered, for example, intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, or spinally. The total weekly dose is generally at least 0.05 mg / kg. 5 μg / kg body weight, more commonly at least 0.2 μg / kg, 0.5 μg / kg, 1μg / kg, 10μg / kg, 100μg / kg, 0.25mg / kg, 1.0 mg / kg, 2.0mg / kg, 5.0mg / mL, 10mg / kg, 25mg / kg, 50 mg / kg or more [see, for example, Yang, e t al.New Engl.J.Med.349:427-434 (2003);H erold,et al.New Engl. J.Med.346:1692-169 8(2002);Liu,et al. J. Neurol.Neurosurg.P sych.67:451-456 (1999);Portielji,et al.C ancer Immunol.Immunother.52:133-144(2003 )]. The dose also determines the level of caninized mouse anti-canine CTLA-4 antibodies in the subject's serum. Predefined target concentrations (e.g., 0.1, 0.3, 1, 3, 10, 30, 10 In another embodiment, the saturation concentration may be provided to achieve a concentration of 0, 300 μg / mL or more. The caninized mouse anti-canine CTLA-4 antibody of the present invention is administered once a week, once every two weeks, or once every four weeks. 10, 20, 50, 80, 10 times, monthly, bimonthly or quarterly 0, 200, 500, 1000 or 2500 mg / subject, administered subcutaneously or intravenously. do.

[0164] Antigenic peptides recognized by anti-canine CTLA-4 mAbs (e.g., CTLA- 4) also inhibits canine CTLA-4 and canine CD80 and / or use as a vaccine to induce antibodies that block binding to CD86 Such vaccines can be used as therapeutic vaccines against diseases such as cancer. In order to use these antigenic peptides as vaccines, it may be useful to In order to increase the immunogenicity of peptides and induce peptide-specific antibodies, these peptides are One or more of the peptides is conjugated to another carrier protein, either chemically or via recombinant DNA technology techniques. Techniques for coupling peptides to carrier proteins are known to those skilled in the art. Peptide vaccines are administered IM, S / C, orally, by spray, or in ovo. Peptide vaccines can be used to vaccinate animals via the immunization route. Subunits expressed from fungal, viral, yeast or baculovirus systems Alternatively, such peptide vaccines can be used as Various peptide vaccines that can be produced by methods known to those skilled in the art. Peptide vaccines can be delivered after administration of viral or bacterial vectors. It can be administered at a dose of 1-1000 μg, and optionally with an adjuvant and and an acceptable pharmaceutical carrier.

[0165] As used herein, "inhibit" or "treat" or "treatment" means to inhibit This includes postponing the onset of symptoms related to harm and / or reducing the severity of symptoms of such disorders. These terms also refer to the improvement of existing uncontrolled or undesirable symptoms. to prevent additional symptoms and to ameliorate or prevent the underlying causes of such symptoms. Thus, these terms include the term "preventing" or "preventing" a disorder, disease, or symptom. or in a vertebrate subject having the potential to develop such a disorder, disease or symptom. It means that beneficial results are being given.

[0166] As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," and "effective "Amount" refers to the amount of a compound administered to a cell, tissue, or subject, either alone or in combination with additional therapeutic agents. In some cases, one or more symptoms of a disease or condition or the progression of such a disease or condition caninized murine anti-canine CTLA antibodies of the present invention effective to produce a measurable improvement in A therapeutically effective dose is defined as the amount of a 100-mg / kg body weight or 100-mg / kg body weight of a 100-mg / kg body weight or an antigen-binding fragment thereof. At least partial amelioration of symptoms, e.g., treatment, cure, prevention or treatment of an associated medical condition. sufficient to bring about an improvement or to increase the rate of treatment, cure, prevention or improvement of such symptoms. The amount of combined compound that is sufficient for the individual active ingredients administered alone is shown. When applied to a combination, the therapeutically effective dose refers to that component alone. A therapeutically effective dose is a therapeutically effective dose whether administered in combination, sequentially or simultaneously. The effective amount of a therapeutic agent is the total amount of active ingredients that produces a therapeutic effect. At least 10%, usually at least 20%, preferably at least about 30% of the meters; More preferably, it provides an improvement of at least 40%, and most preferably at least 50%. The effective amount may also be determined by subjective criteria, if used to assess the severity of the disease. It can also result in improved standards.

[0167] Other combination therapies As described above, the caninized mouse anti-canine CTLA-4 antibody of the present invention or an antigen thereof The binding fragments and / or antigenic peptides may be administered in combination with one or more additional therapeutic agents, such as inhibitors discussed in paragraph (1)) and / or caninized murine anti-canine PD-1 antibodies [e.g., U See US Pat. No. 9,944,704 B2 and US Pat. No. 10,106,107 B2. the contents of both of which are incorporated herein by reference in their entireties] and / or caninization Mouse anti-canine PD-L1 antibodies [see, e.g., US20180237535A1 and the contents of which are incorporated herein by reference in their entirety. The antibody(ies) can be linked to a drug (as an immunoconjugate) and / or The antibody may be administered separately from the drug or another antibody. In the latter case (separate administration), the antibody The agent may be administered before, after, or simultaneously with another known therapy, or may be administered in the same manner as another known therapy. It can be administered at the same time.

[0168] kit Additionally, one or more components, including but not limited to, those specific for CTLA-4 The antibodies or antigen-binding fragments (e.g., caninized antibodies) discussed herein that bind to a mouse anti-canine CTLA-4 antibody or an antigen-binding fragment thereof) The above additional components (which may be caninized mouse anti-canine PD-1 antibodies and / or caninized mouse and a kit comprising the above-described antibody (including an anti-canine PD-L1 antibody). The conjugate composition may be used in pharmaceutical preparations as a pure composition or in combination with a pharmaceutically acceptable carrier. It can be formulated into a pharmaceutical composition.

[0169] In one embodiment, the kit comprises a binding composition of the invention (e.g., caninized murine anti-dog CTL A-4 or a pharmaceutical composition thereof) in one container (e.g., a sterile glass or plastic container). vial) containing caninized mouse anti-canine PD-1 antibody and / or caninized mouse anti The canine PD-L1 antibody or pharmaceutical composition thereof is placed in a separate container (e.g., a glass or plastic container). Contained in a sterile vial (made by Pharma).

[0170] Where the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit may be used in conjunction with a For example, the kit may include one or more of the components discussed above. A hypodermic needle or other injection device may be included. The kit may further include The product may also include a package insert containing information regarding the pharmaceutical composition and dosage form. Such information is provided to pet owners and veterinarians to assist them in understanding the enclosed pharmaceutical compositions and dosage forms. For example, the following information about the combination of the present invention may be found in the package insert: The following information may be provided in the literature: pharmacokinetics, pharmacodynamics, clinical trials, efficacy parameters, indications and dosage , contraindications, warnings, precautions, adverse reactions, overdose, proper dosage and administration, method of supply, proper storage conditions Subject matter, references, manufacturer / distributor information, and patent information.

[0171] For convenience, the antibodies or specific binding agents disclosed herein may be provided in kits, i.e. A packaged combination of reagents in predetermined amounts together with instructions for performing a diagnostic or detection assay. If the antibody(ies) are labeled with an enzyme, the kit may include a substrate and the enzyme. containing the required cofactor (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives, such as stabilizers, buffers (e.g., block buffers or The relative amounts of the various reagents can substantially affect the sensitivity of the assay. The concentration of the reagents in solution can be varied widely to provide a specifically optimized The reagents typically contain excipients that, when dissolved, provide a reagent solution having the appropriate concentration. may be provided as a lyophilized, dry powder. [Example]

[0172] Example Example 1 Mouse monoclonal antibodies against canine CTLA-4 and corresponding mouse-canine chimeras Antibody generation The mouse monoclonal antibody was used in combination with recombinant canine CTLA-4 (cCTLA-4) as the immunogen. The positive hybrids were generated using mouse hybridoma technology using the IgG1 protein. The clones were tested for antibody reactivity with cCTLA-4 and for antibody responses in ELISA and FACS assays. Blocking the interaction of canine CD86 or CD80 with cCTLA-4 (blocking activity) The selected hybridoma clones were selected based on their V H and V L Sequence anti The entire fragment was sequenced by rapid amplification of cDNA ends (RACE). The six selected monoclonal antibodies were 12B3, 27G12, 39A11, and The amino acid sequences of the six antibodies are shown as 45A9, 110E3, and 22A11. The columns are SEQ ID NOs: 2, 4, 6, 8, 10 and 12 for the heavy chain variable region, respectively. and for the light chain variable region, SEQ ID NOs: 14, 16, 18, 20, and 21, respectively. 2 and 24. The CDRs are underlined in the sequences provided below. See also Table 1 below.] The corresponding nucleic acids encoding the amino acid sequences identified above are The peptide sequences for the heavy chain variable region are SEQ ID NOs: 1, 3, 5, 7, 9 and 10, respectively. 11, and for the light chain variable region, SEQ ID NO: 13, respectively. , 15, 17, 19, 21 and 23. Nucleotides of the heavy chain variable region The sequence is the nucleotide sequence of the modified canine constant heavy chain (CH1-hinge-CH2-CH3). The sequences were fused to the nucleotide sequences designated as SEQ ID NOs: 25, 27, 29, 31, 33 and 35, respectively. The Mera mouse-dog heavy chain nucleotide sequences were generated, with variable regions shown in bold. The nucleotide sequence of the light chain variable region was aligned with the nucleotide sequence of the canine constant kappa light chain domain. The resulting chimeras are designated SEQ ID NOs: 37, 39, 41, 43, 45 and 47, respectively. The mouse-dog light chain nucleotide sequences were generated. The variable regions are shown in bold. The amino acid sequence encoded by the mouse-dog heavy chain nucleotide sequence is set forth in SEQ ID NO: The chimeric mouse-dog light chain nucleotides were designated 26, 28, 30, 32, 34, and 36. The amino acid sequences encoded by the sequences are shown in SEQ ID NOs: 38, 40, 42, 44, and 46. and 48. Variable regions are in bold and CDRs are underlined. - The canine heavy and light chains were cloned into separate expression plasmids using standard molecular biology techniques. Plasmids containing the heavy and light chain genes were transfected into HEK293 cells. The expressed antibody was purified from HEK293 cell supernatant using protein A.

[0173] Example 2 Mouse CDR amino acid sequence The CDRs of the mouse anti-canine CTLA-4 monoclonal antibodies are listed in Table 1 below. . [Table 4]

[0174] The individual canonical structure assignments for the six CDRs of each of the six antibodies are shown in the table below. 2. [Table 5]

[0175] Example 3 Reactivity of chimeric antibodies with canine CTLA-4 Chimeric antibodies usually have the same reactivity as their parent mouse antibodies. To confirm the reactivity with TLA-4, a mouse-dog chimeric antibody was prepared and analyzed as follows. were tested for reactivity with cCTLA-4 by ELISA.

[0176] 1. Coat the immunoplate with 200 ng / well of cCTLA-4. Plates were incubated overnight at 4°C; 2. Wash the plate three times with PBS containing 0.05% Tween 20 (PBST). Ta; 3. Block the plate with 0.5% BSA in PBS for 45-60 minutes at room temperature. did; 4. The plate was washed three times with PBST; 5. Antibody was diluted 3-fold in each row or column of the dilution plate; 6. Transfer the diluted antibody to each column or row of the plate and incubate the plate at room temperature for 45-60 minutes. Incubated for minutes; 7. The plate was washed three times with PBST; 8. Add a 1:2000 dilution of horseradish peroxidase-conjugated antibody to each well of the plate. Canine IgG Fc was added and the plate was incubated at room temperature for 45–60 min; 9. The plate was washed three times with PBST; 10. Add TMB substrate to each well of the plate and let the plate sit at room temperature for 10-15 minutes. Incubate for minutes to allow color development; 11. The reaction was stopped by adding 100 μL of 1.5 M phosphoric acid to each well; 12. The plate was read at 450 nm with a reference wavelength of 540 nm.

[0177] ELISA results showed that the chimeric antibody was able to bind to cCTLA-4. [See Figure 1].

[0178] Example 4 Blockade of chimeric antibodies against the interaction of canine CD86 or CD80 with canine CTLA-4 Binding activity To examine the blocking activity of the chimeric antibodies, an ELISA-based blocking assay was performed. The test was carried out as follows: 1. Coat the immunoplate with 200 ng / well of cCTLA-4. Incubate the plate overnight at 4°C; 2. Wash the plate three times with PBS containing 0.05% Tween 20 (PBST). do; 3. Block the plate with 0.5% BSA in PBS for 45-60 minutes at room temperature. do; 4. Wash the plate three times with PBST; 5. In each row or column of the dilution plate, dilute the antibody 3-fold, then add 100 ng / well. Biotinylated CD86 or CD80 was added to the cells and then mixed with the antibody. 6. Transfer the mixture to each row or column of the immunoplate and incubate the plate at room temperature for 45-60 minutes. Incubated for minutes; 7. The plate was washed three times with PBST; 8. Add a 1:2000 dilution of horseradish peroxidase to each well of the plate. Add conjugated streptavidin and incubate the plate at room temperature for 45-60 minutes. cubated; 9. The plate was washed three times with PBST; 10. Add TMB substrate to each well of the plate and let the plate sit at room temperature for 10-15 minutes. Incubate for minutes to allow color development; 11. The reaction was stopped by adding 100 μL of 1.5 M phosphoric acid to each well; 12. The plate was read at 450 nm with a reference wavelength of 540 nm.

[0179] The chimeric antibody inhibited the interaction between cCTLA-4 and CD86 [Figure 2] and the interaction with CD80. It was found to block the effect [Figure 3].

[0180] Example 5 FACS to test the binding activity of chimeric antibodies to CHO-cCTLA-4 Assay We generated a CHO-K1 cell line that stably expresses cCTLA-4. The antibodies were tested for binding and blocking activity in a FACS flow assay. To test the cCTLA-4 binding activity of the chimeric antibodies, a FACS assay was performed as follows. It was implemented as follows: 1. CHO-K1-cCTLA-4 cells were grown in culture medium in a T-75 flask. The cells were passaged when they reached 90% confluency; Culture medium: F12K (Gibco, Cat. No. 21127-022), 10% FBS (Gi bco, Catalog No. 10099-141), and 4 μg / mL puromycin (G ibco, catalog number A1113803); 2. Detach the cells with trypsin-EDTA solution, resuspend the cells in medium, and Viable cells were counted with a viability of ≥ %; 3. Spin down the cells, aspirate the supernatant, and then resuspend the cells in FACS buffer (T hermo Fisher Scientific, catalog number BDB554656) Resuspend in 1 x 10 7 expressed as cells / mL; 4. Add antibody to 100 μL of cells and incubate at room temperature with gentle shaking for 30 minutes. Baited; 5. Wash the cells 3 times with 250 μL of FACS buffer and then resuspend the cells in 100 μL of resuspended in L of FACS buffer; 6. Cells were stained with FITC-conjugated anti-dog IgG and gently shaken. Incubated at room temperature for 30 minutes; 7. Wash the cells 3x with 250 μL of FACS buffer and then resuspend the cells in 500 μL of FACS buffer. resuspended in CS buffer; 8. 10,000 cells were read by flow cytometry.

[0181] FACS results showed that the chimeric antibody could bind to CHO-cCTLA-4 cells. This shows that [see Figures 4A-4G].

[0182] Example 6 Interferon gamma (IFNγ) in canine PBMCs activated by chimeric antibodies Generation of Isolation of canine peripheral blood mononuclear cells 1. Approximately 20 mL of whole blood was collected in an EDTA or sodium heparin tube; 2. Transfer the blood to a 50 mL polystyrene tube and add HBSS (Thermo Fisher Scientific) r Scientific catalogue number 21022CM) diluted 50:50; 3. Mix 15 mL of Ficoll-PlaquePlus with 4 x 50 mL SepMat e TMtube (STEMCELL Technologies, Cat. No. 15460) Approximately 10 mL of the 50:50 diluted blood was then slowly added to the Ficoll Each SepMate containing TM added to the side of the tube; 4. The tubes were centrifuged at 1200 × g for 20 minutes; 5. The cells were collected from the gradient interface and transferred to a 50 mL polypropylene tube. Add HBSS up to the 40-45 mL mark and centrifuge the cells at 800 x g for 10 minutes. Heart separated; 6. Discard the supernatant, resuspend the cells in 40-45 mL HBSS, and resuspend the tube. Centrifuged at 800 × g for 10 min; 7. Discard the supernatant and resuspend the cells from each tube in 2 mL of canine lymphocyte medium (RPMI 1.0). The cells were resuspended in 100% ethanol (100% ethanol medium, Lonza, Cat. No. 12-167Q). Pooled from; 8. Take a small aliquot of the cell suspension, mix with 0.04% trypan blue, and The number of cells was counted; 9. The cell suspension was stored at 2-7°C until use, but should be kept at 24 hours before use. Not stored at 2-7°C.

[0183] Cell proliferation assay for canine peripheral blood mononuclear cells 1. Dilute the antibody in canine lymphocyte medium to a final concentration of 40 μg / mL (prepared 160 μg / mL) and sterilized using a 0.2 μm syringe filter. was diluted two-fold in a sterile dilution plate and set aside; 2. Cells were cultured in canine lymphocyte medium at 2.5 × 10 6 Dilute to 100 cells / mL and place in a 96-well plate. 100 μL was dispensed per well across the woven culture plate; 3. Dilute Con A in canine lymphocyte medium to a final concentration of 250 ng / mL. (Preparation is 1000 ng / mL), sterilized using a 0.2 μm syringe filter, and 50 μL was added to all wells (one row of eight wells for the cell-only control and one column for the thin film). Cell + mAb only control wells received no Con A); 4. Add 100 μL of canine lymphocyte medium per well to the cell-only wells. Then, 50 μL of medium was added to the Con A control wells (Con A+ cells without mAb treatment). Added to the containing column; 5. 50 μL of diluted mAb was added to replicate wells; 6. Place the plate in a humidified incubator at 36±2°C and 4.0-6.0% CO2. and incubated for 68 to 124 hours.

[0184] IFNγ ELISA 1. After 68-124 hours of incubation, centrifuge the plate at 800 x g for 10 minutes. Heart separated; 2. Supernatant was collected from each well and pooled. These samples were used later. can be frozen at -50°C or below for further testing or can be tested immediately; 3. Dilute the supernatant sample appropriately as needed and use it for Canine IFN-γ Quan tikine ELISA kit [R&D Systems Catalog No. CAIF00] IFN-γ ELISA was performed according to the instructions.

[0185] The results showed that selected antibodies, including 12B3, activated canine T cells to produce IFNγ. This shows that it is possible to generate

[0186] Example 7 Construction of caninized anti-cCTLA-4 monoclonal antibodies 12B3 and 39A11 Their strong binding affinity to cCTLA-4 and cCTLA-4 and its ligands Due to their blocking activity against CD86 and CD80, the murine antibodies 12B3 and and 39A11 were selected for the generation of the first caninized antibodies. To this end, the DNA sequences encoding the heavy and light chains of canine IgG were determined. The DNA and protein sequences of the strands are known in the art and are available from NCBI Gene and The four amino acid sequences of canine IgG can be obtained by searching protein databases. There are known IgG subtypes, which are called IgGA, IgGB, IgGC, and IgGD. Similar to human IgG1, canine IgGB has potent effector functions. To knock out the effector function of IgGB, we constructed a modified IgGB (Ig GBm), which has been depleted of its native ADCC and CDC functions [US 10,106,107B 2, which is incorporated herein by reference in its entirety]. Canine Antibodies There are two types of light chains in IgG, called kappa and lambda. Although not all antibodies are specifically designed, various combinations of caninized anti-canine CTLA-4mA antibodies were mixed together. The overall process for producing caninized heavy and light chains capable of producing caninized IgG is as follows: It may contain: (i) The CDRs of the H and L chains of the selected antibody were identified. The amino acid sequences of the CDRs were then analyzed appropriately. reverse-translated to the appropriate DNA sequence; (ii) Antibodies against the heavy and light chains of canine IgG (e.g., heavy and light kappa chains of IgGB) identified the appropriate DNA sequence; (iii) encoding the endogenous CDRs of the canine IgG heavy and light chain DNA of the above sequence; DNA sequences were identified; (iv) DNA sequences encoding endogenous canine heavy and light chain CDRs are cloned into selected The CDRs of the antibody were replaced with DNA sequences encoding the CDRs of the antibody. DNA encoding the canine framework amino acid residues is inserted into the selected antibody framework. Selected amino acid residues from the work region were substituted into the coding DNA; (v) synthesizing the DNA from step (iv) and substituting it into a suitable expression plasmid; cloned; (vi) The synthesized plasmids were transfected into HEK293 cells; (vii) Expressed caninized antibodies were purified from HEK293 supernatants; (viii) Purified caninized antibodies were tested for binding to canine CTLA-4.

[0187] The nucleotide and amino acid sequences of the CDRs of 12B3 and 39A11 are shown in Table 3 below. is described in. [Table 6]

[0188] A set of caninized light and heavy chain sequences was constructed, the sequence identification numbers of which are listed in Table 4- 6. [Table 7] [Table 8] [Table 9] [Table 10]

[0189] The present invention is formed by the combination of caninized heavy and light chains of each antibody listed in the table above. The present invention provides caninized antibodies of 12B3 and 39A11; such antibodies are As shown in Figure 6, the ELISA results show particularly tight binding to TLA-4. , showing that both 12B3 and 39A11 were successfully caninized. B3L3H2 and L3H3 have similar reactivity with cCTLA-4 as the parent 12B3. Caninized c39A11L3H3 exhibits similar reactivity with cCTLA-4 as the parent 39A11. The chimeras of 12B3 and 39A11 represent their parent antibodies. [Table 11] TIFF2025159730000013.tif210169TIFF2025159730000014.tif205166

[0190] TIFF2025159730000015.tif211169TIFF2025159730000016.tif208170

[0191] TIFF2025159730000017.tif207166TIFF2025159730000018.tif209166TIFF2025159730000019.tif202166TIFF2025159730000020.tif201166

[0192] TIFF2025159730000021.tif217169TIFF2025159730000022.tif207166TIFF2025159730000023.tif205166TIFF2025159730000024.tif213169 TIFF2025159730000025.tif205166TIFF2025159730000026.tif216170TIFF2025159730000027.tif217170TIFF2025159730000028.tif207166

[0193] TIFF2025159730000029.tif203166TIFF2025159730000030.tif208166TIFF2025159730000031.tif237170

[0194] Example 8 Epitope maps of caninized anti-cCTLA-4 monoclonal antibodies 12B3 and 39A11 Ping The interaction of antibodies with their cognate protein antigens is mediated by specific amino acids (paratopes) of the antibody. ) to specific amino acids (epitopes) on the target antigen. An epitope is an antigenic determinant that generates a specific response by immunoglobulins. The protein of interest consists of a group of amino acids on the surface of a protein that are recognized by different antibodies. The epitope recognized by the antibody may be a linear epitope. Linear epitopes are classified as conformational or linear epitopes. Conformational epitopes are formed by a continuous sequence of amino acids, whereas conformational epitopes are formed by a continuous sequence of amino acids. The three-dimensional protein structure is composed of non-contiguous (e.g., widely separated) amino acids. They will be together after the wedding.

[0195] Epitope mapping is the process of identifying the amino acid sequence ( This refers to the process of identifying a specific antigen (i.e., an epitope) on a target antigen. Identification of epitopes recognized by mAbs (mAbs) has important applications. For example, Epitope mapping can be useful for the development of novel therapeutic agents, diagnostic agents, and vaccines. It may also aid in the selection of optimized therapeutic mAbs and help elucidate their mechanisms of action. Further epitope information on canine CTLA-4 is available, allowing identification of unique epitopes. The epitope identification can also be used to define the protective or pathogenic effect of the vaccine. The method further involves conjugating the identified peptide epitopes to a carrier protein or another immunostimulatory agent. This could lead to the development of subunit vaccines based on biological or genetic conjugation.

[0196] Epitope mapping can be performed using polyclonal or monoclonal antibodies. and, depending on the predicted nature of the epitope (i.e., linear vs. conformational), Several methods are used to identify epitopes: Mapping linear epitopes This is more straightforward and relatively easy to implement. For this purpose, linear epitope maps Commercial services for ping often use peptide scanning. In this method, a set of overlapping short peptide sequences of the target protein is chemically synthesized and related These antibodies are then tested for their ability to bind to the antibody of interest. This strategy allows for rapid, high-throughput On the other hand, mapping discontinuous epitopes is technically difficult. More difficult and specialized techniques, such as using monoclonal antibodies to target X-ray co-crystallography with proteins, hydrogen-deuterium (H / D) exchange, enzyme digestion Mass spectrometry in combination with cleavage, as well as several other methods known to those skilled in the art. Need.

[0197] Mapping the canine CTLA-4 receptor alpha epitope using mass spectrometry: Caninized 12B3 (exemplified by 12B3L2H3) and To identify the epitopes of 39A11 and 39A11 (exemplified by 39A11L3H3), cCTLA-4 / c12B3L2H3 and cCTLA-4 / c39A11L2H3 Each of the complexes was incubated with a deuterated crosslinker and subjected to multienzymatic cleavage. After enrichment of cross-linked peptides, the samples were subjected to high-resolution mass spectrometry (nLC-LTQ-Orbi The data was analyzed using XQuest and Stavrox software. The analysis was carried out using software.

[0198] Analysis revealed that c12B3L2H3 is a cCTLA-4 clone containing the amino acid sequence of SEQ ID NO: 138. Interacting with amino acid residues at positions 35, 38, 51, 53, 90, 93, 98, and 102 c39A11L2H3 has the amino acid sequence of SEQ ID NO: 138 (FIG. 7A). Interacts with amino acid residues at positions 35, 38, 42, 93, and 102 of cCTLA-4, including Two specific regions of the canine CTLA-4 protein were shown to be involved in the regulation of CTLA-4 expression (Fig. 7B). 7A and 7B: the sequences of SEQ ID NO: 132 and SEQ ID NO: 133, respectively. amino acid sequence (see Table 8 below). In particular, both antibodies bind to the M Binds to SEQ ID NO: 134 and SEQ ID NO: 136, which contain the YPPPY motif (SEQ ID NO: 137) The MYPPPY motif forms a loop bond with CD80 and CD86, which , a conserved motif for CTLA-4 across species. c12B3 further comprises SEQ ID NO: It also binds to one additional region on canine CTLA-4, which contains the amino acid sequence of amino acid sequence 135. Combined with the results of Example 4, the epitope mapping results indicate that c12 Both B3 and c39A11 bind to canine CTLA-4 and its ligands CD80 and CD86. This further supports the idea that the antibody is a functional antibody capable of blocking interaction with Additionally, caninized antibodies that bind to the epitopes of SEQ ID NO: 134 and SEQ ID NO: 136 are also included in the present invention. It's a part of it. [Table 12]

Claims

1. An isolated nucleic acid comprising a nucleotide sequence encoding the light chain of an antibody or antigen-binding fragment thereof 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 canine CTLA-4 to canine CD86, wherein the light chain comprises three light chain CDRs: VLCDR-1, VLCDR-2, and VLCDR-3, (a) VLCDR-1 is encoded by the nucleotide sequence of SEQ ID NO: 103; (b) VLCDR-2 is encoded by the nucleotide sequence of SEQ ID NO: 105; and (c) VLCDR-3 is encoded by the nucleotide sequence of SEQ ID NO: 107; Isolated nucleic acid.

2. 2. The isolated nucleic acid of claim 1, comprising a nucleotide sequence having at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:55, SEQ ID NO:57, and SEQ ID NO:

59.

3. An expression vector comprising the isolated nucleic acid of claim 1.

4. A host cell comprising the expression vector of claim 3.

5. An isolated nucleic acid comprising a nucleotide sequence encoding the heavy chain of an antibody or antigen-binding fragment thereof 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 canine CTLA-4 to canine CD86, wherein the heavy chain comprises three heavy chain CDRs: VHCDR-1, VHCDR-2, and VHCDR-3, (a) VHCDR-1 is encoded by the nucleotide sequence of SEQ ID NO:97; (b) VHCDR-2 is encoded by the nucleotide sequence of SEQ ID NO:99; and (c) VHCDR-3 is encoded by the nucleotide sequence of SEQ ID NO: 101; Isolated nucleic acid.

6. 6. The isolated nucleic acid of claim 5, comprising a nucleotide sequence having at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:79, SEQ ID NO:81, and SEQ ID NO:

83.

7. An expression vector comprising the isolated nucleic acid of claim 5.

8. A host cell comprising the expression vector of claim 7.

9. A nucleic acid composition comprising: i) a first nucleic acid encoding an antibody light chain comprising the nucleotide sequence of SEQ ID NO: 57 and a second nucleic acid encoding an antibody heavy chain comprising the nucleotide sequence of SEQ ID NO: 71 or SEQ ID NO: 83; ii) a first nucleic acid encoding an antibody light chain comprising the nucleotide sequence of SEQ ID NO:59 and a second nucleic acid encoding an antibody heavy chain comprising the nucleotide sequence of SEQ ID NO:71 or SEQ ID NO:83; iii) a first nucleic acid encoding an antibody light chain comprising the nucleotide sequence of SEQ ID NO: 55 and a second nucleic acid encoding an antibody heavy chain comprising the nucleotide sequence of SEQ ID NO: 67 or SEQ ID NO: 79; iv) a first nucleic acid encoding an antibody light chain comprising the nucleotide sequence of SEQ ID NO: 55 and a second nucleic acid encoding an antibody heavy chain comprising the nucleotide sequence of SEQ ID NO: 69 or SEQ ID NO: 81; v) a first nucleic acid encoding an antibody light chain comprising the nucleotide sequence of SEQ ID NO: 55 and a second nucleic acid encoding an antibody heavy chain comprising the nucleotide sequence of SEQ ID NO: 71 or SEQ ID NO: 83; vi) a first nucleic acid encoding an antibody light chain comprising the nucleotide sequence of SEQ ID NO:57 and a second nucleic acid encoding an antibody heavy chain comprising the nucleotide sequence of SEQ ID NO:67 or SEQ ID NO:79; Vii) a first nucleic acid encoding an antibody light chain comprising the nucleotide sequence of SEQ ID NO: 57 and a second nucleic acid encoding an antibody heavy chain comprising the nucleotide sequence of SEQ ID NO: 69 or SEQ ID NO: 81; viii) a first nucleic acid encoding an antibody light chain comprising the nucleotide sequence of SEQ ID NO:59 and a second nucleic acid encoding an antibody heavy chain comprising the nucleotide sequence of SEQ ID NO:67 or SEQ ID NO:79; ix) a first nucleic acid encoding an antibody light chain comprising the nucleotide sequence of SEQ ID NO:59 and a second nucleic acid encoding an antibody heavy chain comprising the nucleotide sequence of SEQ ID NO:69 or SEQ ID NO:81; x) a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 57 and the nucleotide sequence set forth in SEQ ID NO: 71 or SEQ ID NO: 83; xi) a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 59 and the nucleotide sequence set forth in SEQ ID NO: 71 or SEQ ID NO: 83; xii) a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 55 and the nucleotide sequence set forth in SEQ ID NO: 67 or SEQ ID NO: 79; xiii) a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 55 and the nucleotide sequence set forth in SEQ ID NO: 69 or SEQ ID NO: 81; xiv) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 55, SEQ ID NO: 71 or SEQ ID NO: 83; xv) a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 57 and the nucleotide sequence set forth in SEQ ID NO: 67 or SEQ ID NO: 79; xvi) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 57 and the nucleotide sequence of SEQ ID NO: 69 or SEQ ID NO: 81; xvii) a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 59 and the nucleotide sequence set forth in SEQ ID NO: 67 or SEQ ID NO: 79; or xviii) A nucleic acid comprising the nucleotide sequence of SEQ ID NO: 59 and the nucleotide sequence of SEQ ID NO: 69 or SEQ ID NO:

81.

10. An expression vector system comprising the nucleic acid composition of claim 9.

11. A host cell comprising the expression vector system of claim 10.

12. 12. A method for producing an antibody or antigen-binding fragment thereof, comprising culturing a host cell of any one of claims 4, 8, or 11 in a culture medium and isolating the antibody heavy chain and the antibody light chain from the host cell or the culture medium.

13. An isolated nucleic acid comprising a nucleotide sequence encoding the light chain of an antibody or antigen-binding fragment thereof 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 canine CTLA-4 to canine CD86, wherein the light chain comprises three light chain CDRs: VLCDR-1, VLCDR-2, and VLCDR-3, (a) VLCDR-1 is encoded by the nucleotide sequence of SEQ ID NO:91; (b) VLCDR-2 is encoded by the nucleotide sequence of SEQ ID NO:93; and (c) VLCDR-3 is encoded by the nucleotide sequence of SEQ ID NO: 95; Isolated nucleic acid.

14. 14. The isolated nucleic acid of claim 13, comprising a nucleotide sequence having at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NO:51, and SEQ ID NO:

53.

15. 14. An expression vector comprising the isolated nucleic acid of claim 13.

16. A host cell comprising the expression vector of claim 15.

17. An isolated nucleic acid comprising a nucleotide sequence encoding the heavy chain of an antibody or antigen-binding fragment thereof 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 canine CTLA-4 to canine CD86, wherein the heavy chain comprises three heavy chain CDRs: VHCDR-1, VHCDR-2, and VHCDR-3, (a) VHCDR-1 is encoded by the nucleotide sequence of SEQ ID NO:85; (b) VHCDR-2 is encoded by the nucleotide sequence of SEQ ID NO:87; and (c) VHCDR-3 is encoded by the nucleotide sequence of SEQ ID NO: 89; Isolated nucleic acid.

18. 18. The isolated nucleic acid of claim 17, comprising a nucleotide sequence having at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, and SEQ ID NO:

77.

19. 18. An expression vector comprising the isolated nucleic acid of claim 17.

20. A host cell comprising the expression vector of claim 19.

21. A nucleic acid composition comprising: (i) a first nucleic acid encoding an antibody light chain comprising the nucleotide sequence of SEQ ID NO:53 and a second nucleic acid encoding an antibody heavy chain comprising the nucleotide sequence of SEQ ID NO:63 or SEQ ID NO:75; (ii) a first nucleic acid encoding an antibody light chain comprising the nucleotide sequence of SEQ ID NO:53 and a second nucleic acid encoding an antibody heavy chain comprising the nucleotide sequence of SEQ ID NO:65 or SEQ ID NO:77; (iii) a first nucleic acid encoding an antibody light chain comprising the nucleotide sequence of SEQ ID NO:49 and a second nucleic acid encoding an antibody heavy chain comprising the nucleotide sequence of SEQ ID NO:61 or SEQ ID NO:73; (iv) a first nucleic acid encoding an antibody light chain comprising the nucleotide sequence of SEQ ID NO:49 and a second nucleic acid encoding an antibody heavy chain comprising the nucleotide sequence of SEQ ID NO:63 or SEQ ID NO:75; (v) a first nucleic acid encoding an antibody light chain comprising the nucleotide sequence of SEQ ID NO:49 and a second nucleic acid encoding an antibody heavy chain comprising the nucleotide sequence of SEQ ID NO:65 or SEQ ID NO:77; (vi) a first nucleic acid encoding an antibody light chain comprising the nucleotide sequence of SEQ ID NO:51 and a second nucleic acid encoding an antibody heavy chain comprising the nucleotide sequence of SEQ ID NO:61 or SEQ ID NO:73; (vii) a first nucleic acid encoding an antibody light chain comprising the nucleotide sequence of SEQ ID NO: 51 and a second nucleic acid encoding an antibody heavy chain comprising the nucleotide sequence of SEQ ID NO: 63 or SEQ ID NO: 57; (viii) a first nucleic acid encoding an antibody light chain comprising the nucleotide sequence of SEQ ID NO:51 and a second nucleic acid encoding an antibody heavy chain comprising the nucleotide sequence of SEQ ID NO:65 or SEQ ID NO:77; (ix) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:53 and a second nucleic acid encoding an antibody heavy chain comprising the nucleotide sequence of SEQ ID NO:61 or SEQ ID NO:7; (x) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 53, SEQ ID NO: 63, or SEQ ID NO: 75; (xi) a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 53 and the nucleotide sequence set forth in SEQ ID NO: 65 or SEQ ID NO: 77; (xii) a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 49 and the nucleotide sequence set forth in SEQ ID NO: 61 or SEQ ID NO: 73; (xiii) a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 49 and the nucleotide sequence set forth in SEQ ID NO: 63 or SEQ ID NO: 75; (xiv) a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 49 and the nucleotide sequence set forth in SEQ ID NO: 65 or SEQ ID NO: 77; (xv) a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 51 and the nucleotide sequence set forth in SEQ ID NO: 61 or SEQ ID NO: 73; (xvi) a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 51 and the nucleotide sequence set forth in SEQ ID NO: 63 or SEQ ID NO: 57; (xvii) a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 51 and the nucleotide sequence set forth in SEQ ID NO: 65 or SEQ ID NO: 77; or (xviii) A nucleic acid comprising the nucleotide sequence of SEQ ID NO: 53 and the nucleotide sequence of SEQ ID NO: 61 or SEQ ID NO:

7.

22. 22. An expression vector system comprising the nucleic acid composition of claim 21.

23. A host cell comprising the expression vector system of claim 22.

24. 26. A method for producing an antibody or antigen-binding fragment thereof, comprising culturing a host cell of any one of claims 16, 20, or 23 in a culture medium and isolating the antibody heavy chain and the antibody light chain from the host cell or the culture medium.

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