Caninized antibodies to human and canine CTLA-4

Abstract

To provide caninized anti-human CTLA-4 antibodies that have specific sequences and a high binding affinity for canine CTLA-4, and to provide use of these antibodies in the treatment of cancer in canines and other companion animals.SOLUTION: Provided is an isolated caninized antibody or antigen binding fragment thereof comprising a canine IgG heavy chain and a canine kappa light chain, which specifically binds canine CTLA-4, and comprises complementary determining regions comprising a specific amino acid sequence.SELECTED DRAWING: Figure 5

Description

[Technical field]

[0001] Cross-reference to related applications This application was filed under 35 U.S.C. § 119(e) on July 15, 2019. This application claims priority to U.S. Provisional Patent Application No. 62 / 874,287, filed on This application is incorporated herein by reference in its entirety.

[0002] The present invention relates to a protein involved in a costimulatory or co-inhibitory signaling pathway (which is a CTL More specifically, the present invention further relates to antibodies against certain The human CTLA-4 has the sequence and has high binding affinity to canine CTLA-4. The present invention further relates to a caninized antibody against 4. It also concerns the use of the body. [Background technology]

[0003] The initiation or termination of an immune response is determined by the activity of many types of immune cells, particularly T lymphocytes and antigen-presenting cells. Activation occurs through complex interactions among a series of proteins expressed on the surface of the adenocarcinoma (APC) The co-stimulatory signaling pathway regulates the onset of immune responses. These two factors contribute to the survival of the immune system, and 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 the interaction between the B7.1 and B7.2 proteins and the B7.3 protein is mediated through interactions with the B7.1 and B7.2 proteins. 7.2 is believed to perform a similar function.

[0004] In contrast, the coinhibitory pathway leads to the inhibition or termination of the immune response and involves the inhibition of C on T cells. Mediated through the interaction between TLA-4 and B7.1 / B7.2 proteins on APCs has been shown. Additional co-inhibitory signaling pathways are mediated through the interaction between programmed cell death receptor 1 (PD-1) on T cells and programmed cell death receptor ligand 1 or 2 (PD- L1 / PD-L2) proteins on APCs, as has been shown. Furthermore, the interaction between PD-L1 and B7.1 has also been shown to potentially bring about inhibitory signals within T cells.

[0005] B7.1 and B7.2 are members of the immunoglobulin (Ig) superfamily [Sharpe and Freeman, Nature Reviews, 2: 116-126 (2002)]. B7.1 is expressed on activated B cells, activated T cells, and macrophages and dendritic cells [Swanson and Hall, Eur J. Immunol., 23:295-298 (1993); Razi-Wo lfe et al., PNAS, 89:4210-4214 (1992)]. B 7.2 is constitutively expressed on dendritic cells, Langerhans cells, and B cells. Furthermore, B7 .2 is expressed on monocytes and is upregulated after IFN-γ stimulation [L arsen et al., Immunol.,152:5208-5219 (19 94); Inaba, J. Exp. Med. 180:1849-1860 ( 1994)].

[0006] B7.1 and B7.2 bind to CD28 and CTLA-4, resulting in different functional outcomes [Linsley et al., PNAS, 87:5031-5035 ( 1990); Linsley et al., J. Exp. Med., 173 :721-730(1991); Azuma et al., Nature 366 :76-79 (1993); Freeman et al., Science 2 62:909-912 (1993)]. Binding of B7.1 and B7.2 to CTLA-4 shows a much higher affinity than the binding of B7.1 and B7.2 to CD28 [van der Merwe, J. Exp. Med. 185:393-402 (199 7)].

[0007] CD28 is a homodimeric glycoprotein that is a member of the Ig superfamily [Aruffo and Seed, PNAS, 84:8573-8577 (19 87)]. The mature protein has a single extracellular variable domain consisting of 134 amino acid residues that contains the hexapeptide motif MYPPPY essential for counter-receptor binding and is [Riley and June, Blood, 105:13-21 (20 05)]. The 41-amino acid cytoplasmic domain of CD28 contains 4 tyrosine residues that can be phosphorylated upon activation [Sharpe and Freeman, Nat. Rev. Immunol., 2:116-126 (2002)]. CD28 is expressed on most CD4 + T cells and approximately 50% of CD8 + T cells [Gross et al ., J. Immunol., 149:380-388 (1992); Rile y and June, Blood, 105:13-21 (2005)]. T cells After receptor (TCR) ligation, B7.1 / B7.2 bound to CD28 provides an important costimulatory signal to T cells, enabling T cell activation and subsequent generation of an immune response [Reiser et al., PNAS, 89:271-275 (1 992); Jenkins et al., J. Immunol., 147:2 461-2466 (1991)]. In the absence of the CD28 signal, T cells have been shown to undergo apoptosis or become unresponsive [Jenkins et a l., J. Exp. Med. 165:302-319 (1987); Jen kins et al., PNAS, 84:5409-5413 (1987); Schwartz, Science, 248:1349-1356 (1990)] . CD28-B7.1 / B7.2 binding changes the threshold level (e.g., the amount of antigen-MHC complex) of TCR ligation required for activation, shortens the time required to stimulate naive cells, and can enhance the magnitude of the T cell response [Soskic et al ., Advances in Immunology, 124:96-123 (2

[0008] CTLA-4 (CD152) is also a member of the Ig superfamily and is composed of a single extracellular domain, a transmembrane domain, and a short cytoplasmic tail [Swan son, Immunology; 1010:169-177 (2000)]. Further more, CTLA-4 shares approximately 30% amino acid identity with CD28. CTLA- 4 is not constitutively expressed in naive T cells but is induced upon CD28 ligation and T cell activationIt is rapidly upregulated immediately after activation, approximately 48–96 h after initial T cell activation. CTLA-4 expression levels peak at 14 days after tumor initiation [Alegre et al., J. I mmunol., 157:4762-4770 (1996); Freeman e et al., J. Immunol., 149:3795-3801 (1992) CTLA-4 binds to both B7.1 and B7.2 with much higher affinity than CD28. [van der Merwe et al., J. Exp. Med., 185:393-402 (1997)]. However, CD28-binding B7.1 or In contrast to the stimulatory effects of B7.2, CTLA-4 down-modulates immune responses. It functions as an essential inhibitory receptor for the immune system [Walnus et al., Immuni ty, 1:405-413 (1994); Walnus, J. Exp. Me d., 183:2541-2550 (1996); Krummel and Al lison, J. Exp. Med., 183:2533-2540 (1996 )]. The mechanism by which CTLA-4 mediates its immunosuppressive function is through its interaction with CD28 and B7.1 / B7. This is related to their ability to act as competitive inhibitors of the interaction between the two ["Swanson , Immunology, 1010:169-177 (2000) The important role of CTLA-4 in immune downregulation is This has been demonstrated in CTLA-4-deficient mice, which show reduced expression of CTLA-4 in multiple organs. Death occurs at 3-5 weeks of age due to the development of a lymphoproliferative disorder characterized by T cell infiltration [Tivo l et al., Immunity, 3:541-5417 (1995); Waterhouse et al., Science, 270:985-988 ( 1995)]. The results of CTLA-4 knockout, as shown by the absence of disease in CTLA-4 / B7.1 / B7.2 triple knockout mice, also demonstrated dependence on the interaction of CD28 with its ligands B7.1 and B7.2 [Mandelbrot et al., J. Exp. Med., 189:435-4 40 (1999)]. This was also confirmed by the protection against lymphoproliferation brought about by repeated administration of CTLA-4Ig to CTLA-4 knockout mice [Tivol et al., J Immunol., 158:5091 -5094 (1997)].

[0009] Furthermore, blocking the effect of CTLA-4 with antibodies has been shown to enhance T cell responses in vitro and in vivo and increase the anti-tumor immune response [Leach et al., Science, 271:1734-1736 (1996)]. Based on these findings, the development of CTLA-4 blockers such as monoclonal antibodies has been carried out to provide treatment modalities for treating cancer [Hodi et al., P NAS, 100(8):4712-4717 (2003); Phan GQ et al., PNAS,100(14):8372-8377 (2003); Att ia, Journal of Clinical Oncology, 23(25) :6043-6053 (2005); Comin-Anduix et al., Journal of Translational Medicine, 6:22- 22 (2008); WO2000037504A2; US8,017,114 B2; WO2010097597A1; WO2012120125A1; and Bo Utros 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 receptor and a cytoplasmic tail that binds to a signaling molecule. [Zak et al., Cell Structure, 25:1163-1 174 (2017)]. The cytoplasmic tail of PD-1 is divided into two It contains a lysine-based signaling motif [Zhang et al., Im munity 20:337-347 (2004)]. PD-1 expression is stimulated PD-1 expression is not seen on T cells, B cells, or myeloid cells that do not express PD-1. It is upregulated in these cells after activation [Chemnitz et al. l., J. Immunol., 173:945-954 (2004); Pet rvas et al., J. Exp. Med., 203:2281-2292 (2006)]. PD-1 is most closely related to CTLA-4, accounting for approximately 24% of all cell deaths. They share amino acid identity [Jin et al., Current Topics in Microbiology and Immunology, 350:17- 37 (2010)]. PD-1 reduces T cell activation when it binds to PD-L1 and PD -L2, which are expressed on the surface of APCs. When any of these ligands binds to PD -1, antigen signaling via the T cell receptor (TCR) is negatively regulated . To date, only PD-L1 and PD-L2 have been known to function as ligands for PD-1 . Similar to the case of CTLA-4, PD-1 ligation appears to transmit negative immune regulatory signals. Ligation of PD-1 by PD-L1 or PD-L2 results in inhibition of proliferation and cytokine production via the TCR [Jin et al., Current Topics in Microbiology and Immunology, 350:17-37 (2010)]. In contrast to CTLA-4-deficient animals, PD-1-deficient mice die much later in life and show signs of auto immunity, but the severity of the observed effects is not as severe as that shown by CTLA-4-deficient animals [Nishimura et al., Immunity, 11(2):1 41-151 (1999); Nishimura et al., Science , 291(5502):319-322 (2001)]. The PD-1 signaling pathway is currently being intensively studied, and previous studies have shown that the PD-L1 / PD-L2 / PD- 1 interaction reduces signals downstream of TCR stimulation, which results in reduced cytokine secretion , impaired T cell proliferation, and reduced production of cytotoxic molecules by T cells , suggesting that it is involved in the negative regulation of some immune responses [Freeman et al., J. Exp. Med., 192 (7):1027-1034 (2000)]. (2000)].

[0011] PD-L1 (CD274) is a type 1 membrane protein and has an IgV-like extracellular domain. IgC- and IgC-like extracellular domains, hydrophobic transmembrane domains, and signaling properties unknown PD-L1 is a member of the B7 family of PD-L1 receptors. It is recognized as a member of the B7 family 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 B7.1, but not to CD86. does not bind [Butte et al., Immunology, 45 (13) :3567-3572 (2008)]. The affinity of B7.1 for PD-L1 was The affinity of PD-L2 is intermediate between that of CD80 and CTLA-4. Although they have no affinity for CD86, they share the receptor PD-1. When D-L1 binds to its receptor PD-1 on T cells, it triggers TCR-mediated IL-2 production. PD-L1 binds to PD-1 and delivers a signal that inhibits T cell proliferation. It also contributes to ligand-induced TCR downmodulation during antigen presentation to target T cells. Furthermore, binding of PD-L1 to B7.1 on T cells induces apoptosis of T cells. The role of PD-1 and PD-L1 as inhibitors of T cell activation has been demonstrated in many studies. Based on these findings, therapeutic modalities for treating cancer and infectious diseases have been developed. Development of PD-1 and PD-L1 blockers, such as monoclonal antibodies, to provide was held.

[0012] Humanized monoclonal antibodies that block the binding and activity of canine PD-1, PD-L1, and CTLA-4 have been developed and are now available for use in the treatment of human subjects diagnosed with one of several different types of cancer. Similarly, canine monoclonal antibodies that block the binding and activity of canine PD-1 and PDL 1 have also been reported [U.S.9 ,944,704B2, U.S.10,106,607B2 and U.S.2018 / 02 37535A1; these are incorporated herein by reference in their entirety]. However, to date, no canine monoclonal antibodies that block the binding and activity of canine CTLA-4 have been reported. The citation of references in this specification should not be construed as an admission that such references are available as "prior art" to this application.

[0013] References cited herein are not to be construed as an admission that such references are available as "prior art" to this application.

Prior Art Documents

Patent Documents

[0014]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Non-Patent Documents

[0015] [Non-Patent Document 1] Sharpe and Freeman, Nature Reviews, 2:116-126 (2002) [Non-Patent Document 2] Swanson and Hall, Eur J. Immunol., 23:295-298 (1993) [Non-Patent Document 3] Razi-Wolfe et al., PNAS, 89:4210-4214 (1992) [Non-Patent Document 4] Larsen et al., Immunol.,152:5208-5219 (1994) [Non-Patent Document 5] Inaba, J. Exp. Med. 180:1849-1860 (1994) [Non-Patent Document 6] Linsley et al., PNAS, 87:5031-5035 (1990) [Non-Patent Document 7] Linsley et al., J. Exp. Med., 173:721-730(1991) [Non-Patent Document 8] Azuma et al., Nature 366:76-79 (1993) [Non-Patent Document 9] Freeman et al., Science 262:909-912 (1993) [Non-Patent Document 10] van der Merwe, J. Exp. Med. 185:393-402 (1997) [Non-Patent Document 11] Aruffo and Seed, PNAS, 84:8573-8577 (1987) [Non-Patent Document 12] Riley and June, Blood, 105:13-21 (2005) [Non-Patent Document 13] Sharpe and Freeman, Nat. Rev. Immunol., 2:116-126 (2002)

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Non-Patent Document 19

Non-Patent Document 20

Non-Patent Document 21

Non-Patent Document 22

Non-Patent Document 23

Non-Patent Document 24

Non-Patent Document 25

Non-Patent Document 38

Non-Patent Document 39

Non-Patent Document 40

Non-Patent Document 41

Non-Patent Document 42

Non-Patent Document 43

Non-Patent Document 44

Non-Patent Document 45

Non-Patent Document 46

Non-Patent Document 47

Non-Patent Document 48

Non-Patent Document 49

Summary of the Invention

Problems to be Solved by the Invention

[0016] The present invention relates to a caninized anti-human CTLA-4 antibody having specific binding affinity for canine CTLA-4 and the ability to block the binding of canine CTLA-4 to canine CD80 and / or CD86. The present invention further relates to the use of such antibodies in the treatment of diseases such as cancer and / or diseases caused by infections. Accordingly, the present invention provides an isolated caninized antibody or an antigen-binding fragment of the caninized antibody that specifically binds to CTLA-4 and comprises a canine IgG heavy chain and a canine kappa or lambda light chain. In certain embodiments of this type, the canine kappa or lambda light chain comprises three light chain complementarity determining regions (CDRs) [i.e., CDR light 1 (CDRL1), CDR light 2 (CDRL2) and CDR light 3 (CDRL3)], and the canine IgG heavy chain comprises three heavy chain CDRs [i.e., CDR heavy 1 (CDRH1), CDR heavy 2 (CDRH2) and CDR heavy 3 (CDRH3)], wherein all six CDRs are obtained from mammalian CTLA-4 antibodies. Certain embodiments of the caninized antibodies and fragments thereof of the present invention bind to canine CTLA-4 and / or block the binding of canine CTLA-4 to canine CD80 and / or CD86.

[0017] ​​​​​​​​​​​​

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

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

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

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

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

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

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

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

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

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

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

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

[0030] In certain embodiments, the kappa light chain of the canine antibody contains the amino acid sequence of SEQ ID NO: 44 . In more specific embodiments, the kappa light chain is encoded by the nucleotide sequence of SEQ ID NO: 43 . In related embodiments, the kappa light chain contains the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within a conservative variant of the amino acid sequence of SEQ ID NO: 44 . In yet other embodiments, the kappa light chain contains the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within a functionally conserved variant of the amino acid sequence of SEQ ID NO: 44 . In yet other embodiments, the kappa light chain of the canine antibody contains the amino acid sequence of SEQ ID NO: 46 . In more specific embodiments, the kappa light chain is encoded by the nucleotide sequence of SEQ ID NO: 45

[0031] . In related embodiments, the kappa light chain contains the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within a conservative variant of the amino acid sequence of SEQ ID NO: 46 . In yet other embodiments, the kappa light chain contains the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within a functionally conserved variant of the amino acid sequence of SEQ ID NO: 46 . In related embodiments, the kappa light chain contains the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within a conservative variant of the amino acid sequence of SEQ ID NO: 46 . In yet other embodiments, the kappa light chain contains the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within a functionally conserved variant of the amino acid sequence of SEQ ID NO: 46 and contains the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58. Within the functionally conserved mutants of the amino acid sequence, there are the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 which are included.

[0032] The present invention further provides a canine antibody comprising any light chain of the present invention and any heavy chain of the present invention. In certain embodiments, the isolated canine antibody has a heavy chain comprising the amino acid sequences of SEQ ID NO: 36, SEQ ID NO: 62 or a conservative variant of the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 62, and the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within the conservative variant, and a kappa light chain comprising the amino acid sequence of SEQ ID NO: 46 or a conservative variant of the amino acid sequence of SEQ ID NO: 46 and the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within the conservative variant. In more specific embodiments, the isolated canine antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 6 and a kappa light chain comprising the amino acid sequence of SEQ ID NO: 46. In other specific embodiments, the isolated canine antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 62 and a kappa light chain comprising the amino acid sequence of SEQ ID NO: 46. In other specific embodiments, the isolated canine antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 40, SEQ ID NO: 66

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

[0034] The present invention further provides an isolated Nucleic acid encoding any one of the light chains of the canine antibody of the present invention. Similarly, the present invention further provides an isolated nucleic acid encoding any one of the heavy chains of the canine antibody of the present invention. The present invention further provides an expression vector containing one or more of the isolated nucleic acids of the present invention. The present invention further Provides a host cell containing one or more expression vectors of the present invention. In certain embodiments, the antibody is a recombinant antibody or an antigen-binding fragment thereof. In related embodiments, the variable heavy chain domain and the variable light chain domain are linked by a flexible linker To form a single-chain antibody.

[0035] In certain embodiments, the antibody is a Fab fragment. In another embodiment, the antibody or antigen-binding fragment is a Fab' fragment. In another embodiment, the antibody or antigen-binding fragment is a (Fab') Fragment. In yet another embodiment, the antibody or antigen-binding fragment is a diabody. In certain embodiments, the antibody or antigen-binding fragment is a domain antibody. In a more

[0036] Specific embodiment, the antibody or antigen-binding fragment is a canine single-domain antibody. In another embodiment, the antibody or antigen-binding fragment is a Fab' fragment. In another embodiment, the antibody or antigen-binding fragment is a (Fab') Fragment. In yet another embodiment, the antibody or antigen-binding fragment is a diabody. In certain embodiments, the antibody or antigen-binding fragment is a domain antibody. In a more 2 Specific embodiment, the antibody or antigen-binding fragment is a canine single-domain antibody. There is. In yet another embodiment, the antibody or antigen-binding fragment is a diabody. In certain embodiments, the antibody or antigen-binding fragment is a domain antibody. In a more Specific embodiment, the antibody or antigen-binding fragment is a canine single-domain antibody. In a more specific embodiment, the antibody or antigen-binding fragment is a canine single-domain antibody. There is.

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

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

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

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

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

[0042]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

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

Table 1

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

[0045] ​As used herein, including in the appended claims, the singular forms of the words "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise. As used herein, including in the appended claims, the singular forms of the words "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise. As used herein, including in the appended claims, the singular forms of the words "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0046] As used herein, including in the appended claims, the singular forms of the words "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise. As used herein, including in the appended claims, the singular forms of the words "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise. As used herein, including in the appended claims, the singular forms of the words "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise. As used herein, including in the appended claims, the singular forms of the words "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise. As used herein, including in the appended claims, the singular forms of the words "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise. As used herein, including in the appended claims, the singular forms of the words "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise. As used herein, including in the appended claims, the singular forms of the words "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0047] As used herein, including in the appended claims, the singular forms of the words "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise. As used herein, including in the appended claims, the singular forms of the words "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise. As used herein, including in the appended claims, the singular forms of the words "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise. As used herein, including in the appended claims, the singular forms of the words "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise. As used herein, including in the appended claims, the singular forms of the words "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise. As used herein, including in the appended claims, the singular forms of the words "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise. As used herein, including in the appended claims, the singular forms of the words "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise. As used herein, including in the appended claims, the singular forms of the words "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise. As used herein, including in the appended claims, the singular forms of the words "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise.

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

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

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

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

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

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

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

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

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

[0057] Typically, the canineized antibody or antigen-binding fragment thereof of the present invention, when its canine CTLA -4 binding activity is expressed on a molar basis, retains at least 10% of its activity (when compared to its corresponding parent antibody). Preferably, the antibody or antigen-binding fragment of the present invention retains at least 20%, 50%, 7 0%, 80%, 90%, 95% or 100% or more of the canine CTLA-4 binding affinity as the parent antibody.

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

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

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

[0061] Typically, the variable domains of both the heavy and light chains contain three hypervariable regions, also referred to as complementary determining regions (CDRs), which are located within relatively conserved framework regions (FRs). The CDRs are usually adjacent to the framework regions, thereby enabling binding to a specific epitope. Generally, from the N-terminus to the C-terminus, both the light chain variable domain and the heavy chain variable domain contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignment to each domain is generally as follows ​​in accordance with the definition: Sequences of Proteins of Immunol ogical Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md., 5 t h ed., NIH Publ. No. 91-3242 (1991); Kab at, Adv. Prot. Chem. 32:1-75 (1978); Kab at, et al., J. Biol. Chem. 252:6609-6616 (1977); Chothia, et al., J. Mol. Biol. 196:901-917 (1987) or Chothia, et al., N ature 342:878-883 (1989).

[0062] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody involved in antigen binding. The hypervariable regions include amino acid residues derived from "complementary determining regions" or "CDRs" (i.e., CDRL1, CDRL2, and CDRL3 within the light chain variable domain, and CDRH1, CDRH2, and CDRH3 within the heavy chain variable domain). [See "Kabat et al. Sequences of Proteins of Immunological Inter est, 5th Ed. Public Health Service, Nati onal Institutes of Health, Bethesda, Md. (1991)", which defines the CDR regions of antibodies by sequence; see also "Chothia et al., J. Mol. Biol. 196:901-917 (1987) or Chothia et al., Nature 342:878-883 (1989)", which defines the CDR regions of antibodies by structure; ​“Chothia and Lesk, J. Mol. Biol. 196 : 901-917 (1987)” should also be referred to].

[0063] As used herein, the term "framework" or "FR" residue refers to variable domain residues other than the hypervariable region residues defined herein as CD R residues. The framework of a chimeric antibody represents part of the canine framework. As used herein, the term "canine" refers to all domestic dogs, Canis lupus familiaris or Canidae dogs

[0064] unless otherwise indicated. including the genus Canis familiaris.

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

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

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

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

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

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

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

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

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

[0074] ​​​​​​​​​It shows the DNA sequence necessary to cause. Appropriate control sequences for prokaryotes include, for example, a promoter, optionally including an operator sequence, and a ribosome binding site site. Eukaryotic cells are known to use promoters, polyadenylation signals and enhancers.

[0075] Nucleic acids are "operably linked" when placed in a functional relationship with another nucleic acid sequence. For example, DNA regarding a presequence or a secretion leader is operably linked to the DNA for a polypeptide when it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to its coding sequence when it affects the transcription of the coding sequence; or a ribosome binding site is operably linked to the coding sequence when it is positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are adjacent, and in the case of a secretion leader, adjacent and in the reading frame. However, an enhancer does not have to be adjacent. Ligation is achieved by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional practice. When a nucleic acid sequence is provided herein, it can be readily understood that it may include a stop codon. However, since the stop codon is interchangeable, including a specific stop codon in the sequence should not be regarded as a necessary part of the sequence. synthetic oligonucleotide adapters or linkers are used according to conventional practice. When a nucleic acid sequence is provided herein, it can be readily understood that it may include a stop codon. However, since the stop codon is interchangeable, including a specific stop codon in the sequence should not be regarded as a necessary part of the sequence. However, since the stop codon is interchangeable, including a specific stop codon in the sequence should not be regarded as a necessary part of the sequence.

[0076] ​​​​As used herein, the terms "cell", "cell line", and "cell culture" are used interchangeably and all such designations include progeny. Thus, the terms "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom without regard to the number of passages. Further, it is understood that due to intentional or accidental mutations, not all progeny may have exactly the same DNA content. Mutant progeny that have the same function or biological activity as screened in the originally transformed cell are included. Where distinct designations are intended, they will be apparent from the context.

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

[0078] ​​​​​​​​​​​​​​Characteristics of representative canine anti-human CTLA-4 antibodies The present invention provides an isolated canine anti-human CTLA-4 antibody and methods of using the antibody or antigen-binding fragment thereof in the treatment of diseases (e.g., treatment of cancer in dogs). Examples of canine anti-human CTLA-4 antibodies that bind to canine CTLA-4 include, but are not limited to, antibodies that contain canine IgG-A, IgG-B, IgG-C, and IgG-D heavy chains and / or or canine kappa light chains together with anti-human CTLA-4 CDRs. Accordingly, the present invention provides an isolated canine anti-human CTLA- 4 antibody or antigen-binding fragment thereof that binds to canine CTLA-4 and blocks the binding of canine CTLA-4 to canine CD8 0 and / or CD86. A "conservatively modified variant" or "conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation, and rigidity), such that the change can often be made without altering the biological activity of the protein. One of ordinary skill in the art will generally recognize that one amino acid substitution within a non-essential region of a polypeptide does not substantially alter biological activity [see, e.g., "Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Pub lishing Co., p.224 (4th Ed.; 1987)"]. Additionally, substitutions of amino acids that are structurally or functionally similar are less likely to disrupt biological activity. Various embodiments of the antibodies or antigen-binding fragments of the present invention are described in this specification.

[0079] lishing Co., p.224 (4th Ed.; 1987)"]. Additionally, substitutions of amino acids that are structurally or functionally similar are less likely to disrupt biological activity. Various embodiments of the antibodies or antigen-binding fragments of the present invention are described in this specification. In addition, substitutions of amino acids that are structurally or functionally similar are less likely to disrupt biological activity. Various embodiments of the antibodies or antigen-binding fragments of the present invention are described in this specification. A polypeptide chain having the sequences disclosed in the specification (e.g., SEQ ID NO: 34, 36, 38, 40, 42, 44, 60, 62, 64 or 66) or a region other than the CDR region with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more conservative amino acid substitutions, is included. Exemplary conservative substitutions are listed in Table A.

Table 2

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

[0081] Nucleic acid The invention further includes nucleic acids encoding the immunoglobulin chains of the canine anti-human CTLA-4 antibodies and their antigen-binding fragments disclosed herein. For example, the invention includes all of the novel nucleic acids described in the following table.

[0082] Furthermore, when compared by the BLAST algorithm, at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical, and most preferably at least about 95% identical to the in-frame amino acid sequence of the antibodies provided herein. ​​or an immunoglobulin polypeptide comprising an in-frame amino acid sequence that is at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) Nucleic acids encoding are also encompassed by the present invention, wherein the parameters of the algorithm are selected to give the maximum match between the respective sequences over the entire length of each reference sequence. The present invention further provides a nucleic acid encoding an in-frame of an immunoglobulin polypeptide comprising an amino acid sequence that is at least about 70% similar, preferably at least about 80% similar, more preferably at least about 90% similar, and most preferably at least about 95% similar (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to any of the reference amino acid sequences when compared using the BLAST algorithm, wherein the parameters of the algorithm are selected to give the maximum match between the respective sequences over the entire length of each reference sequence and are similarly encompassed by the present invention. Sequence identity indicates the degree to which the amino acids of two polypeptides are identical at positions where the two sequences are optimally aligned. Sequence similarity encompasses identical residues and non-identical amino acids that are biochemically related. Biochemically related amino acids that share similar properties and may be exchangeable are discussed above. The following references are related to the BLAST algorithms often used for sequence analysis: BLAST ALGORITHMS: Altschul, S.F ., et al., J. Mol. Biol. 215:403-410 (19

[0083]

[0084] ., et al., J. Mol. Biol. 215:403-410 (19 ., et al., J. Mol. Biol. 215:403-410 (19 ​​​​​​​​​90); Gish, W., et al., Nature Genet. 3:2 66 - 272 (1993); Madden, T.L., et al., Met h. Enzymol. 266:131 - 141(1996); Altschul, S.F., et al., Nucleic Acids Res. 25:338 9 - 3402 (1997); Zhang, J., et al., Genome Res. 7:649 - 656 (1997); Wootton, J.C., e t al., Comput. Chem. 17:149 - 163 (1993); Hancock, J.M. et al., Comput. Appl. Bios ci. 10:67 - 70 (1994); ALIGNMENT SCORING S YSTEMS: Dayhoff, M.O., et al., “A model of evolutionary change in proteins.” in Atlas of Protein Sequence and Structure, vol. 5, suppl. 3. M.O. Dayhoff (ed.), p p. 345 - 352, (1978); Natl. Biomed. Res. F ound., Washington, DC; Schwartz, R.M., e t al., “Matrices for detecting distant r elationships.” in Atlas of Protein Seque nce and Structure, vol. 5, suppl. 3.“ (1 978), M.O. Dayhoff (ed.), pp. 353 - 358 ( 1978), Natl. Biomed. Res. Found., Washin gton, DC; Altschul, S.F., J. Mol. Biol. 219:555-565 (1991); States, D.J., et al. , Methods 3:66-70(1991); Henikoff, S., e t al., Proc. Natl. Acad. Sci. USA 89:109 15-10919 (1992); Altschul, S.F., et al., J. Mol. Evol. 36:290-300 (1993); ALIGNM ENT STATISTICS: Karlin, S., et al., Proc . Natl. Acad. Sci. USA 87:2264-2268 (199 0); Karlin, S., et al., Proc. Natl. Acad . Sci. USA 90:5873-5877 (1993); Dembo, A., et al., Ann. Prob. 22:2022-2039 (199 4); and, Altschul, S.F. “Evaluating the st atistical significance of multiple disti nct local alignments.” in Theoretical an d Computational Methods in Genome Resear ch (S. Suhai, ed.), pp. 1-14, Plenum, Ne w York (1997).

[0085] The present invention further provides an expression vector containing the isolated nucleic acid of the present invention, wherein The nucleic acid is recognized by a host cell when the host cell is transfected with the vector. In addition, the expression vector of the present invention is operably linked to a control sequence that recognizes the gene. and a host cell containing the antibody or antigen-binding fragment thereof disclosed herein. Also provided is a method of making an antibody or antigen-binding fragment thereof, the method comprising: and culturing a host cell carrying an expression vector encoding the antigen or its antigen in a medium. and isolating the antigen-binding fragment from the host cell or medium.

[0086] Epitope binding and binding affinity The present invention further comprises the amino acid sequence of SEQ ID NO: 36 and / or SEQ ID NO: 46. Caninized anti-human CTLA-4 antibody and an antibody that binds to the same epitope on canine CTLA-4 The caninized anti-human CTLA-4 antibody or its antigen-binding fragment is provided. The binding fragment inhibits the binding of canine CTLA-4 to canine CD80 and / or CD86. can harm.

[0087] The caninized anti-human CTLA-4 antibody can be produced recombinantly as described in the Examples below. The expression of the antibodies or fragments disclosed herein can be produced in a Mammalian cell lines available as hosts for the production of ribozymes are well known in the art and are available from American Type Culture Collection These include many immortalized cell lines available from the American College of Cancer Cell Lines (ATCC). , among others, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeL a cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and many other cell lines are included. Mammalian host cells include cells of humans, mice, rats, dogs, rabbits, pigs, goats, cows, horses and hamsters. Particularly preferred cell lines are selected by checking which cell line has a high expression level. Other cell lines that can be used are insect cell lines, e.g., Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells. When introducing a recombinant expression vector encoding the heavy chain or its antigen-binding portion or fragment, the light chain and / or its antigen-binding fragment into a mammalian host cell, the host cell is cultured for a period sufficient to enable the expression of the antibody in the host cell, or, more preferably, for a period sufficient to enable the secretion of the antibody into the medium in which the host cell grows, and the antibody is produced by culturing the host cell. The antibody can be recovered from the medium using standard protein purification methods. Furthermore, the expression of the antibody of the present invention (or other components derived therefrom) from the production cell line can be enhanced using many known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common approach for enhancing expression under specific conditions. The GS system is considered in whole or in part in relation to European Patent No. 0216846, European Patent No. 0256055, European Patent No. 0 323997 and European Patent Application No. 89303964.4.

[0088] In general, glycoproteins produced in a specific cell line or transgenic animal

[0089] , glycosylation patterns characteristic of the glycoproteins produced by the cell line or transgenic animal. Thus, the specific glycosylation pattern of an antibody depends on the specific cell line or transgenic animal used to produce the antibody. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein constitute the invention, regardless of the glycosylation pattern the antibody may have. Similarly, in certain embodiments, antibodies having glycosylation patterns comprising only non-fucosylated N-glycans may be advantageous because these antibodies have been shown to typically exhibit greater potency than their fucosylated counterparts both in vitro and in vivo [see, for example: Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Patent No. 6,946,292 and U.S. Patent No. 7,214,775]. It has. The invention further encompasses antibody fragments of the canine anti-human CTLA-4 antibody disclosed herein. Such antibody fragments include, for example, F(ab) fragments that can be generated by enzymatic cleavage of IgG with pepsin. Fab fragments can be generated, for example, by reducing F(ab) with dithiothreitol or mercaptoethylamine. A Fab fragment is a V -C chain linked to a V -C chain by a disulfide bridge. F(ab) fragments. 3466-3473 (2003); U.S. Patent No. 6,946,292 and U.S. Patent No. 7,214,775].

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

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

[0092] Antibody engineering The humanized anti-human CTLA-4 antibody of the present invention is engineered, for example, to include modifications to the framework residues within the variable domain of the parental (i.e., canine) monoclonal antibody in order to improve the properties of the antibody. In order to improve the properties of the antibody, for example, modifications to the framework residues within the variable domain of the parental (i.e., canine) monoclonal antibody are included. In order to improve the properties of the antibody, for example, modifications to the framework residues within the variable domain of the parental (i.e., canine) monoclonal antibody are included.

[0093] Experimental and diagnostic uses The humanized anti-human CTLA-4 antibody or antigen-binding fragment thereof of the present invention further comprises In a diagnostic assay for the CTLA-4 protein, for example, it may be useful in detecting its expression in certain tumor cells, tissues or sera. Such diagnostic methods may be useful in the diagnosis of various diseases (particularly certain cancers in dogs). For example, such a method may include the following steps: (a) Coating a substrate (e.g., the surface of a microtiter plate well, e.g., the surface of a plastic plate) with a canine anti-human CTLA-4 antibody or an antigen-binding fragment thereof;

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

[0095] In a further embodiment, the labeled antibody is labeled with a peroxidase that reacts with ABTS [e.g., 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] or 3,3',5,5'-tetramethylbenzidine to produce a detectable color change. Alternatively, the labeled antibody is labeled with a detectable radioisotope (e.g., (3-ethylbenzothiazoline-6-sulfonic acid)] or 3,3',5,5'-tetramethylbenzidine to produce a detectable color change. Alternatively, the labeled antibody is labeled with a detectable radioisotope (e.g., (3-ethylbenzothiazoline-6-sulfonic acid)] or 3,3',5,5'-tetramethylbenzidine to produce a detectable color change. Alternatively, the labeled antibody is labeled with a detectable radioisotope (e.g., H) that can be detected by a scintillation counter in the presence of a scintillant. H) that can be detected by a scintillation counter in the presence of a scintillant. 3 H) that can be detected by a scintillation counter in the presence of a scintillant. It can be used in Western blotting or immunoblotting methods. Such methods form part of the present invention and include, for example:

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

[0097]

[0098] (iii) Detecting the bound canine anti-human CTLA-4 antibody or an antigen-binding fragment thereof. (iii) Detecting the bound canine anti-human CTLA-4 antibody or an antigen-binding fragment thereof. The detection of the bound antibody or antigen-binding fragment can be carried out by binding it to a secondary antibody (anti-immunoglobulin antibody) labeled detectably and then detecting the presence of the secondary antibody.

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

[0098] The anti-human CTLA-4 canine antibody and its antigen-binding fragment disclosed in this specification can further be used in immunohistochemistry. Such a method forms part of the present invention and, for example, includes the following: (1) contacting cells to be tested for the presence of canine CTLA-4 with the anti-human CTLA-4 canine antibody or its antigen-binding fragment of the present invention; and (2) detecting the antibody or fragment on or inside the surface of the cells. If the antibody or antigen-binding fragment itself is detectably labeled, it can be directly detected. Alternatively, a secondary antibody detectably labeled can be bound to the antibody or antigen-binding fragment and detected. The specific anti-human CTLA-4 canine antibody and its antigen-binding fragment disclosed in this specification can further be used in in vivo tumor imaging. Such a method can include injecting a radio-labeled anti-human CTLA-4 canine antibody or its antigen-binding fragment into a dog whose body is to be tested for the presence of a tumor associated with the expression of canine CTLA-4, and subsequently subjecting the body of the patient to nuclear imaging to detect the presence of the labeled antibody or antigen-binding fragment, for example, at locations containing a high concentration of the antibody or antigen-binding fragment bound to the tumor. Imaging techniques include SPECT imaging (single photon emission computed tomography) or PET imaging (positron emission tomography), etc. Labels include, for example, the following: for example, iodine in combination with SPECT imaging. In the case of immunohistochemistry, the method may include: (1) contacting cells to be tested for the presence of canine CTLA-4 with the anti-human CTLA-4 canine antibody or its antigen-binding fragment of the present invention; and (2) detecting the antibody or fragment on or inside the surface of the cells. If the antibody or antigen-binding fragment itself is detectably labeled, it can be directly detected. Alternatively, a secondary antibody detectably labeled can be bound to the antibody or antigen-binding fragment and detected. For in vivo tumor imaging, the method may include injecting a radio-labeled anti-human CTLA-4 canine antibody or its antigen-binding fragment into a dog whose body is to be tested for the presence of a tumor associated with the expression of canine CTLA-4, and subsequently subjecting the body of the patient to nuclear imaging to detect the presence of the labeled antibody or antigen-binding fragment, for example, at locations containing a high concentration of the antibody or antigen-binding fragment bound to the tumor. Examples of imaging techniques include SPECT imaging (single photon emission computed tomography) or PET imaging (positron emission tomography), etc. Examples of labels include, for example, the following: for example, iodine in combination with SPECT imaging. In immunohistochemistry, cells to be tested for the presence of canine CTLA-4 are contacted with the anti-human CTLA-4 canine antibody or its antigen-binding fragment of the present invention, and then the antibody or fragment on or inside the cell surface is detected. If the antibody or antigen-binding fragment itself is detectably labeled, it can be directly detected. Otherwise, a detectably labeled secondary antibody can be bound to the antibody or antigen-binding fragment for detection. For in vivo tumor imaging, a radio-labeled anti-human CTLA-4 canine antibody or its antigen-binding fragment is injected into a dog to be tested for the presence of a tumor associated with canine CTLA-4 expression. Then, the patient's body is subjected to nuclear imaging to detect the presence of the labeled antibody or antigen-binding fragment, such as at locations with a high concentration of the antibody or antigen-binding fragment bound to the tumor.

[0099] In immunohistochemistry, the method typically involves: (1) bringing cells to be tested for the presence of canine CTLA-4 into contact with the anti-human CTLA-4 canine antibody or its antigen-binding fragment of the present invention; and (2) detecting the antibody or fragment on or within the cell surface. If the antibody or antigen-binding fragment is itself detectably labeled, it can be directly detected. Alternatively, a detectably labeled secondary antibody can be attached to the antibody or antigen-binding fragment for detection. Regarding in vivo tumor imaging, the procedure may include injecting a radio-labeled anti-human CTLA-4 canine antibody or its antigen-binding fragment into a dog whose body is being examined for the presence of a tumor related to the expression of canine CTLA-4, followed by subjecting the patient's body to nuclear imaging to detect the presence of the labeled antibody or antigen-binding fragment, such as at sites where a high concentration of the antibody or antigen-binding fragment is bound to the tumor. Imaging techniques include SPECT imaging (single photon emission computed tomography) or PET imaging (positron emission tomography), etc. Labels include, for example, the following: for instance, iodine in combination with SPECT imaging. In the context of immunohistochemistry, the method may consist of: (1) contacting cells to be tested for the presence of canine CTLA-4 with the anti-human CTLA-4 canine antibody or its antigen-binding fragment of the present invention; and (2) detecting the antibody or fragment on or inside the cell surface. If the antibody or antigen-binding fragment itself is detectably labeled, it can be directly detected. Otherwise, a detectably labeled secondary antibody can be bound to the antibody or antigen-binding fragment for detection. For in vivo tumor imaging, the method may involve injecting a radio-labeled anti-human CTLA-4 canine antibody or its antigen-binding fragment into a dog whose body is being tested for the presence of a tumor associated with the expression of canine CTLA-4, and then subjecting the patient's body to nuclear imaging to detect the presence of the labeled antibody or antigen-binding fragment, such as at locations where a high concentration of the antibody or antigen-binding fragment is bound to the tumor. Examples of imaging techniques include SPECT imaging (single photon emission computed tomography) or PET imaging (positron emission tomography), etc. Examples of labels include, for example, the following: for example, iodine in combination with SPECT imaging. In immunohistochemistry, the method usually includes: (1) making cells to be tested for the presence of canine CTLA-4 contact with the anti-human CTLA-4 canine antibody or its antigen-binding fragment of the present invention; and (2) detecting the antibody or fragment on or within the cell surface. If the antibody or antigen-binding fragment is itself detectably labeled, it can be directly detected. Alternatively, a detectably labeled secondary antibody can be attached to the antibody or antigen-binding fragment for detection.

[0100] In the case of in vivo tumor imaging, the method may include injecting a radio-labeled anti-human CTLA-4 canine antibody or its antigen-binding fragment into a dog whose body is being tested for the presence of a tumor associated with the expression of canine CTLA-4, and then subjecting the patient's body to nuclear imaging to detect the presence of the labeled antibody or antigen-binding fragment, such as at locations where a high concentration of the antibody or antigen-binding fragment is bound to the tumor. Examples of imaging techniques include SPECT imaging (single photon emission computed tomography) or PET imaging (positron emission tomography), etc. Examples of labels include, for example, the following: for example, iodine in combination with SPECT imaging. Labels include, for example, the following: for example, iodine in combination with SPECT imaging. Iodine-123( 123 I) and Technetium-99m( 99m Tc), or, for example, in combination with PE T imaging, 11 C, 13 N, 15 O or 18 F, or Indium -111 [see, for example, "Gordon et al., International Rev. Neurobiol. 67:385-440 (2005)"].

[0101] Pharmaceutical compositions and administration To prepare a pharmaceutical composition or a sterile composition of a canine anti-human CTLA-4 antibody or an antigen-binding fragment thereof, the canine anti-human CTLA-4 antibody or an antigen-binding fragment thereof is mixed with a pharmaceutically acceptable carrier or excipient. [See, for example: Remington’s Pharmaceutical Sciences a nd U.S. Pharmacopeia: National Formulary , Mack Publishing Company, Easton, PA (1 984)]. , Mack Publishing Company, Easton, PA (1 984).

[0102] Formulations of therapeutic and diagnostic agents can be prepared by mixing them with acceptable carriers, excipients or stabilizers, for example, in the form of lyophilized powders, slurries, aqueous solutions or suspensions [see, for example: Hardman, et al. (2 001) Goodman and Gilman’s The Pharmacolo gical Basis of Therapeutics, McGraw-Hill gical Basis of Therapeutics, McGraw-Hill , New York, NY; Gennaro (2000) Remington : The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New Y ork, NY; Avis, et al. (eds.) (1993) Phar maceutical Dosage Forms: Parenteral Medi cations, Marcel Dekker, NY; Lieberman, e t al. (eds.) (1990) Pharmaceutical Dosag e Forms: Tablets, Marcel Dekker, NY; Lie berman, et al. (eds.) (1990) Pharmaceuti cal Dosage Forms: Disperse Systems, Marc el Dekker, NY; Weiner and Kotkoskie (200 0) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY]. In one embodiment, the anti-CTLA-4 antibody of the present invention is diluted to an appropriate concentration in a sodium acetate solution (pH 5-6), and then NaCl or sucrose is added for tonicity. To enhance stability, additional agents such as polysorbate 20 or polysorbate 80 can be added. .

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

[0104] The methods of administration can vary. Suitable routes of administration include, among others, the following: Oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, directly into the cerebral ventricle, intravenous , intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intraarterial.

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

[0106] The composition can be administered using medical devices known in the art. For example , the pharmaceutical composition of the present invention can be administered by injection with a subcutaneous injection needle (which includes, for example, a prefilled syringe or an auto injector). The pharmaceutical compositions disclosed herein can also be administered using a needleless subcutaneous injection device (for example, the devices disclosed in U.S. Patents: No. 6,620,13 5; No. 6,096,002; No. 5,399,163; No. 5,383,851; 5,312,335; No. 5,064,413; No. 4,941,880; No. 4,79 0,824 or No. 4,596,556).

[0107] The pharmaceutical compositions disclosed herein can also be administered by infusion. Examples of well-known implants and modules for administering pharmaceutical compositions include, among others, the following: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for administering a drug at a controlled rate; U.S. Patent No. 4,4 47,233, which discloses a drug infusion pump for delivering a drug at an accurate infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow rate implantable infusion device for sustained drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments. Many other such implants, delivery systems and modules are well known to those skilled in the art. Alternatively, for example, in many cases, in a depot formulation or a sustained release formulation, into the interior of a pathogen-induced lesion or an arthritic joint characterized by immunopathology, the canine anti-human CTLA-4

[0108] By directly injecting the antibody, the canine anti-human CTLA-4 antibody can be administered locally rather than systemically. Furthermore, for example, in a targeted drug delivery system targeting pathogen-induced lesions or joints of arthritis characterized by immunopathology, the canine anti-human CTLA-4 antibody can be administered, for example, in liposomes coated with tissue-specific antibodies. The liposomes target the diseased tissue and are selectively taken up by the diseased tissue.

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

[0110] The determination of the appropriate dosage is performed by a veterinarian using, for example, parameters or factors that are known or can be inferred in the art to affect the treatment. Generally, the administration starts at a somewhat lower amount than the optimal dosage and then is gradually increased until the desired or optimal effect is achieved as compared to negative side effects. Important diagnostic criteria include diagnostic criteria for symptoms, for example, diagnostic criteria for symptoms such as inflammation or the level of inflammatory cytokines produced. The antibodies or antigen-binding fragments thereof disclosed herein can be provided by continuous infusion or, for example, by dosages administered once a day, 1 to 7 times a week, once a week, once every other week, once a month, once every other month, once every three months, once every six months, once a year, etc. The dosages can be, for example, intravenous, subcutaneous, topical, oral, nasal, rectal, intramuscular, intracerebral, intraspinal

[0111] ​​​​​​​It can be provided orally, or by inhalation. The total weekly dose is generally at least 0. 05 μg / kg body weight, and more generally at least 0.2 μg / kg, 0.5 μg / kg, 1 μg / kg, 10 μg / kg, 100 μg / kg, 0.25 mg / kg, 1. 0 mg / kg, 2.0 mg / kg, 5.0 mg / mL, 10 mg / kg, 25 mg / kg , 50 mg / kg or more [see, for example: Yang , et al. New Engl. J. Med. 349:427-434 ( 2003); Herold, et al. New Engl. J. Med. 346:1692-1698 (2002); Liu, et al. J. Neu rol. Neurosurg. Psych. 67:451-456 (1999) ; Portielji, et al. Cancer Immunol. Immu nother. 52:133-144 (2003)]. The dose may also be further adjusted to achieve a predetermined target concentration (e.g., 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg / mL or more) of the canine anti-human CTLA-4 antibody in the serum of the subject . In another embodiment, the canine anti-human CTLA-4 antibody of the invention is administered subcutaneously or intravenously at 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg / subject, once a week, once every two weeks, "once every four weeks", once a month, once every two months or once every three months . As used herein, "inhibiting" or "treating" or "treatment" includes delaying the onset of symptoms associated with a disorder and / or reducing the severity of the symptoms of such a disorder

[0112] and . Do. These terms further improve existing uncontrolled or undesirable symptoms To do, prevent additional symptoms, and improve or prevent the underlying causes of such symptoms Including. Thus, these terms refer to vertebrate subjects having a disorder, disease or condition Or having the potential to develop such a disorder, disease or condition It means that a beneficial result is given.

[0113] As used herein, the terms "therapeutically effective amount", "therapeutically effective dose" and "effective Amount" alone or in combination with additional therapeutic agents, when administered to cells, tissues or subjects In the case, in one or more symptoms of a disease or condition or in the progression of such a disease or condition The amount of the canine anti-human CTLA-4 antibody of the present invention or an antigen-binding fragment thereof effective to produce a measurable improvement Indicates. The therapeutically effective dose further reduces at least some of the symptoms At least partial improvement, e.g., treatment, cure, prevention or improvement of a related medical condition or Or an amount of a combination compound sufficient to increase the rate of treatment, cure, prevention or improvement of such a condition When applied to an individual active ingredient administered alone The therapeutically effective dose indicates that component alone. When applied to a combination, the therapeutically effective Amount indicates the total amount of active ingredients that, whether administered together, sequentially or simultaneously, produce a therapeutic effect The effective amount of a therapeutic agent results in at least a 10% improvement in diagnostic criteria or parameters, usually at least a 20% improvement, preferably at least about a 30% improvement, more preferably At least a 40% improvement, most preferably at least a 50% improvement. Effective amount ​Furthermore, when subjective criteria are used to evaluate the severity of a disease, improvement of the subjective criteria can also be achieved.

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

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

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

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

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

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

Example

[0120] Example 1 Construction of anti-CTLA-4 chimeric antibody It is known that a prior art monoclonal antibody produced against human CTLA-4, which both binds to human CTLA-4 and blocks the binding of human CTLA-4 to human CD86, may also bind to canine CTLA-4 and possibly block the binding of canine CD86 to canine CTLA-4 . To test this possibility, a known nucleotide sequence corresponding to the heavy chain variable region of the anti-human CTLA-4 monoclonal antibody 3B10 (disclosed in WO2012120125 ) was fused to the nucleotide sequence of a modified canine constant heavy chain (CH1-hinge-CH2-3) to generate a chimeric mouse-canine heavy chain nucleotide sequence shown as SEQ ID NO: 1 . A second known nucleotide sequence encoding the amino acid sequence corresponding to the light chain variable region of anti-human CTLA-4 was used ​​​​​​Fused to the nucleotide sequence of the canine constant kappa light chain domain to generate the chimeric mouse-canine light chain nucleotide sequence shown as SEQ ID NO: 3. The protein encoded by the chimeric mouse-canine heavy chain nucleotide sequence contains the amino acid sequence of SEQ ID NO: 2; and the protein encoded by the chimeric mouse-canine light chain nucleotide sequence contains the amino acid sequence of SEQ ID NO: 4. The chimeric human-canine heavy and light chains were cloned into separate expression plasmids using standard molecular biology techniques. Plasmids containing the heavy and light chain genes were transfected into HEK293 cells, and the expressed antibody was purified from the HEK293 cell supernatant using Protein A. Similarly, chimeric antibody 8H5 containing the variable domains previously disclosed in WO2012120125 was also constructed as exemplified by an antibody formed by a combination of a heavy chain and a light chain containing the DNA sequences and amino acid sequences described as SEQ ID NOs: 5 to SEQ ID NO: 8, respectively. Furthermore, chimeric antibodies 411, 418, and 611 containing the variable domains disclosed in WO2000037504 were also constructed as exemplified by an antibody formed by a combination of a heavy chain and a light chain containing the nucleic acid sequences and amino acid sequences described as SEQ ID NOs: 9 to SEQ ID NO: 20, respectively. Chimeric antibodies 10D1 and 1E2 containing the variable domains disclosed in U.S. 8,017,114B2 were also exemplified by an antibody formed by a combination of a heavy chain and a light chain containing the nucleotide sequences and amino acid sequences described as SEQ ID NOs: 21 to SEQ ID NO: 28, respectively. The protein encoded by the chimeric mouse-canine heavy chain nucleotide sequence contains the amino acid sequence of SEQ ID NO: 2; and the protein encoded by the chimeric mouse-canine light chain nucleotide sequence contains the amino acid sequence of SEQ ID NO: 4. The chimeric human-canine heavy and light chains were cloned into separate expression plasmids using standard molecular biology techniques. Plasmids containing the heavy and light chain genes were transfected into HEK293 cells, and the expressed antibody was purified from the HEK293 cell supernatant using Protein A. The protein encoded by the chimeric mouse-canine heavy chain nucleotide sequence contains the amino acid sequence of SEQ ID NO: 2; and the protein encoded by the chimeric mouse-canine light chain nucleotide sequence contains the amino acid sequence of SEQ ID NO: 4. The chimeric human-canine heavy and light chains were cloned into separate expression plasmids using standard molecular biology techniques. Plasmids containing the heavy and light chain genes were transfected into HEK293 cells, and the expressed antibody was purified from the HEK293 cell supernatant using Protein A. The protein encoded by the chimeric mouse-canine heavy chain nucleotide sequence contains the amino acid sequence of SEQ ID NO: 2; and the protein encoded by the chimeric mouse-canine light chain nucleotide sequence contains the amino acid sequence of SEQ ID NO: 4. The chimeric human-canine heavy and light chains were cloned into separate expression plasmids using standard molecular biology techniques. Plasmids containing the heavy and light chain genes were transfected into HEK293 cells, and the expressed antibody was purified from the HEK293 cell supernatant using Protein A. The protein encoded by the chimeric mouse-canine heavy chain nucleotide sequence contains the amino acid sequence of SEQ ID NO: 2; and the protein encoded by the chimeric mouse-canine light chain nucleotide sequence contains the amino acid sequence of SEQ ID NO: 4. The chimeric human-canine heavy and light chains were cloned into separate expression plasmids using standard molecular biology techniques. Plasmids containing the heavy and light chain genes were transfected into HEK293 cells, and the expressed antibody was purified from the HEK293 cell supernatant using Protein A. The protein encoded by the chimeric mouse-canine heavy chain nucleotide sequence contains the amino acid sequence of SEQ ID NO: 2; and the protein encoded by the chimeric mouse-canine light chain nucleotide sequence contains the amino acid sequence of SEQ ID NO: 4. The chimeric human-canine heavy and light chains were cloned into separate expression plasmids using standard molecular biology techniques. Plasmids containing the heavy and light chain genes were transfected into HEK293 cells, and the expressed antibody was purified from the HEK293 cell supernatant using Protein A. The protein encoded by the chimeric mouse-canine heavy chain nucleotide sequence contains the amino acid sequence of SEQ ID NO: 2; and the protein encoded by the chimeric mouse-canine light chain nucleotide sequence contains the amino acid sequence of SEQ ID NO: 4. The chimeric human-canine heavy and light chains were cloned into separate expression plasmids using standard molecular biology techniques. Plasmids containing the heavy and light chain genes were transfected into HEK293 cells, and the expressed antibody was purified from the HEK293 cell supernatant using Protein A.

[0121] Similarly, chimeric antibody 8H5 containing the variable domains previously disclosed in WO2012120125 was also constructed as exemplified by an antibody formed by a combination of a heavy chain and a light chain containing the DNA sequences and amino acid sequences described as SEQ ID NOs: 5 to SEQ ID NO: 8, respectively. Similarly, chimeric antibody 8H5 containing the variable domains previously disclosed in WO2012120125 was also constructed as exemplified by an antibody formed by a combination of a heavy chain and a light chain containing the DNA sequences and amino acid sequences described as SEQ ID NOs: 5 to SEQ ID NO: 8, respectively. Similarly, chimeric antibody 8H5 containing the variable domains previously disclosed in WO2012120125 was also constructed as exemplified by an antibody formed by a combination of a heavy chain and a light chain containing the DNA sequences and amino acid sequences described as SEQ ID NOs: 5 to SEQ ID NO: 8, respectively. Furthermore, chimeric antibodies 411, 418, and 611 containing the variable domains disclosed in WO2000037504 were also constructed as exemplified by an antibody formed by a combination of a heavy chain and a light chain containing the nucleic acid sequences and amino acid sequences described as SEQ ID NOs: 9 to SEQ ID NO: 20, respectively. Furthermore, chimeric antibodies 411, 418, and 611 containing the variable domains disclosed in WO2000037504 were also constructed as exemplified by an antibody formed by a combination of a heavy chain and a light chain containing the nucleic acid sequences and amino acid sequences described as SEQ ID NOs: 9 to SEQ ID NO: 20, respectively. Furthermore, chimeric antibodies 411, 418, and 611 containing the variable domains disclosed in WO2000037504 were also constructed as exemplified by an antibody formed by a combination of a heavy chain and a light chain containing the nucleic acid sequences and amino acid sequences described as SEQ ID NOs: 9 to SEQ ID NO: 20, respectively. Chimeric antibodies 10D1 and 1E2 containing the variable domains disclosed in U.S. 8,017,114B2 were also exemplified by an antibody formed by a combination of a heavy chain and a light chain containing the nucleotide sequences and amino acid sequences described as SEQ ID NOs: 21 to SEQ ID NO: 28, respectively. Chimeric antibodies 10D1 and 1E2 containing the variable domains disclosed in U.S. 8,017,114B2 were also exemplified by an antibody formed by a combination of a heavy chain and a light chain containing the nucleotide sequences and amino acid sequences described as SEQ ID NOs: 21 to SEQ ID NO: 28, respectively. Chimeric antibodies 10D1 and 1E2 containing the variable domains disclosed in U.S. 8,017,114B2 were also exemplified by an antibody formed by a combination of a heavy chain and a light chain containing the nucleotide sequences and amino acid sequences described as SEQ ID NOs: 21 to SEQ ID NO: 28, respectively. Chimeric antibodies 10D1 and 1E2 containing the variable domains disclosed in U.S. 8,017,114B2 were also exemplified by an antibody formed by a combination of a heavy chain and a light chain containing the nucleotide sequences and amino acid sequences described as SEQ ID NOs: 21 to SEQ ID NO: 28, respectively. Furthermore, the present invention was constructed so as to include the variable domains disclosed in WO2010097597. The chimeric antibody 3B3 is also shown as SEQ ID NO: 29 to SEQ ID NO: 32, respectively. The antibody is formed by combining heavy and light chains that contain the nucleotide and amino acid sequences The chimeric antibody was constructed as exemplified by the antibody shown in Table 1, having the amino acid sequence ID No. , as shown in Table 1 below. [Table 3]

[0122] Example 2 Reactivity of anti-CTLA-4 chimeric antibody The chimeric antibody of Example 1 was cultured in Expi293 cells [THERMO FISHER SCI The protein was expressed in the Expi293 expression system obtained from NETIFIC®. The chimeric antibody was then purified using a phenylalanine A column. The chimeric antibody was then detected by ELISA as follows: Reactivity with canine CTLA-4 was tested: 1. Coat immunoplates with 200 ng / well of CTLA-4 and plate the The rates were incubated overnight at 4°C; 2. Wash the plate three times with PBS containing 0.05% Tween 20 (PBST). Washed; 3. Block the plate with 0.5% BSA in PBS for 45-60 minutes at room temperature. did; 4. The plate was washed 3 times with PBST; 5. Antibody was diluted 3-fold in each row or column of the dilution plate; 6. Transfer the diluted antibodies to each row or column of the immunoplate and incubate the plate at room temperature for 45 Incubated for ~60 min; 7. The plate was washed 3 times with PBST; 8. Add 1:2000 dilution of horseradish peroxidase-conjugated anti-IgE to each well of the plate. NuIgG Fc was added and the plate was incubated at room temperature for 45-60 min; 9. The plate was washed 3 times with PBST; 10. Add TMB substrate to each well of the plate and incubate the plate at room temperature for 10-15 minutes. 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. Plates were read at 450 nm with a reference wavelength of 540 nm.

[0123] The ELISA results are shown in Figure 1 and show that the selected antibodies inhibited canine CTLA- 4. Furthermore, the 3B10 chimeric antibody was able to bind to the chimeric It was found to be the best binder among the Ra antibodies.

[0124] Example 3 Construction of canine anti-human CTLA-4 monoclonal antibody 3B10 It was confirmed that the 3B10 chimeric antibody has the strongest binding affinity to canine CTLA-4. Since we confirmed that the chimeric antibody has the same structure as the IgG1 antibody (see FIG. 1), we selected a set of six CDRs from this chimeric antibody. To carry out the caninization process, the heavy chain of canine IgG was selected and added to the canine frame. The DNA sequences encoding the IL-1 and IL-2 chains were confirmed [see US 10,106,607 B2].

[0033] , the contents of which are incorporated herein by reference in their entirety. The DNA and protein sequences of the NC and light chains are known in the art and are available from can be obtained by searching the BI gene and protein database. There are four known IgG subtypes: IgG-A, IgG-B, and IgG There are two types of dog antibodies called kappa and lambda. Light chains are present. [Table 4] [Table 5]

[0125] While not bound to a specific approach, various combinations of mixes are possible. To produce caninized heavy and light chains capable of producing caninized anti-human CTLA-4 mAbs, The overall process may include the following scheme: (i) Identify the CDRs of the heavy and light chains of the desired anti-CTLA-4 mAb. Back-translate the amino acid sequence into the appropriate DNA sequence; (ii) Canine IgG heavy and light chains (see, e.g., US 10,106,607 B2) Identify the appropriate DNA sequences for the modified heavy and light kappa chains of IgG-B described do; (iii) encoding the endogenous CDRs of the canine IgG heavy and light chain DNA of the above sequence; Identifying DNA sequences; (iv) Binding DNA sequences encoding endogenous canine heavy and light chain CDRs to the desired anti-human The modified canine IgG-B is replaced with a DNA sequence encoding the CTLA-4 CDR. When used, the modifications at D31A and N63A numbered in SEQ ID NO:69 are U. No. 10,106,607B2, which is incorporated herein in its entirety. The antibody was then tested in dog IgG-B as previously described. , DNA encoding several canine in-frame amino acid residues was added to the desired anti-human CTLA- It was replaced with DNA encoding selected amino acid residues from the 4mAb canine region. This process is referred to as a reverse mutation. Two sets of four mutants each for each heavy chain and one set of three mutants for the light chain variable region, described in Tables 4 and 5, were developed using each mutant of the set containing different reverse mutation sites; (v) Synthesize the DNA from step (iv) and clone it into an appropriate expression plasmid. (vi) Transfect HEK293 cells with the plasmid containing the desired canineized H chain and L chain. (vii) Purify the expressed canineized antibody from the HEK293 supernatant. (viii) Test the purified canineized antibody for binding to canine CTLA-4.

[0126] A set of canineized H chain and L chain sequences was developed. The sequence numbers are listed in Tables 4 and 5 below.

Table 6

Table 7

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

[0128] ​​​​​​​​​ Example 4 Blocking activity of chimeric and canineized antibody 3B10 against the interaction between canine CD86 and CTLA-4 Example 5 The canineized 3B10 antibody maintains the same neutralizing activity as the parental antibody against canine CTLA-4. To confirm this, the canineized antibody was tested by ELISA for its blocking activity against the interaction of canine-CD-80 and canine CTLA-4 as follows: 1. Coat the immunoplate with 200 ng / well of CTLA-4 and incubate the plate at 4°C overnight; 2. Wash the plate three times with PBS containing 0.05% Tween 20 (PBST); 3. Block the plate with 0.5% BSA in PBS for 45 - 60 minutes at room temperature; 4. Wash the plate three times with PBST; 5. In each column or row of the dilution plate, dilute the antibody three-fold and then add 100 ng / well of biotinylated CD-86 and mix with the antibody; 6. Transfer the diluted antibody and CD-86 mixture to each column or row of the immunoplate and incubate the plate at room temperature for 45 - 60 minutes; 7. Wash the plate three times with PBST; 8. Add streptavidin conjugated with horseradish peroxidase diluted 1:2000 to each well of the plate and incubate the plate at room temperature for 45 - 60 minutes; 9. Wash the plate three times with PBST; 10. Add TMB substrate to each well of the plate and incubate the plate at room temperature for 10 - 15 minutes to develop color; 11. Add 100 μL of 1.5 M phosphoric acid to each well to stop the reaction. ​ 12. The plate was read at 450 nm using a reference wavelength of 540 nm.

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

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

[0131] Isolation of canine peripheral blood mononuclear cells Cell proliferation assay for canine peripheral blood mononuclear cells 1. Approximately 20 mL of whole blood was collected into EDTA or sodium heparin tubes; 2. The blood was transferred to 50 mL polystyrene tubes and diluted 50:50 with Hank's balanced salt solution (HBSS); 3. 15 mL of Ficoll-PlaquePlus was added to four 50 mL SepMat e TM tubes. Then, approximately 10 mL of the 50:50 diluted blood was slowly added to the side of each SepMate tube containing Ficoll; TM 5. The blood was centrifuged at 400 x g for 30 minutes at room temperature; 4. The tube was centrifuged at 1200×g for 20 minutes; 5. Cells were collected from the gradient interface and transferred to 50 mL polypropylene tubes. HBSS was added up to the 40 - 45 mL mark, and the cells were centrifuged at 800×g for 10 minutes ; 6. The supernatant was discarded, the cells were resuspended in 40 - 45 mL of HBSS, and the tube was centrifuged again at 800 ×g for 10 minutes; 7. The supernatant was discarded, and the cells from each tube were resuspended in 2 mL of canine lymphocyte medium (the cells were pooled from the same animal); 8. A small aliquot of the cell suspension was taken, mixed with 0.04% trypan blue, and the cell count was determined; 9. The cell suspension was stored at 2 - 7°C until use [however, it was not stored at 2 - 7°C starting 24 hours before use].

[0132] IFNγ ELISA 1. The antibody was diluted in canine lymphocyte medium [RPMI medium (catalog number 12 - 167Q) purchased from LONZA, or an equivalent] to a final concentration of 40 μg / mL ( prepared at 160 μg / mL), and sterilized using a 0.2 μm syringe filter. The antibody was doubly diluted in a sterile dilution plate and left; 2. The cells were diluted in canine lymphocyte medium to 2.5×10 6 cells / mL, and 100 μL per well was dispensed across all wells of a 96 - well tissue culture plate; 3. Con A was diluted in canine lymphocyte medium to a final concentration of 250 ng / mL (prepared at 1000 ng / mL), sterilized using a 0.2 μm syringe filter, and 50 μL was added to all wells. (Note: Cells only in one row of 8 wells for control and Wells for the cell + mAb only control received no Con A); 4. 100 μL of canine lymphocyte medium was added to the wells with cells only, and 50 μL of medium was added to the columns containing the Con A control wells (Con A + cells without mAb treatment). ; 5. 50 μL of the diluted mAb was added to the replicate wells; 6. The plate was incubated in a humidified incubator at 36 ± 2 °C, 4.0 - 6.0% CO 2 for 68 - 124 hours.

[0133] Example 6 1. After incubation for 68 - 124 hours, the plate was centrifuged at 800 × g for 10 minutes; ; 2. Supernatants were collected from each well and pool replicate. These samples can be frozen at -50 °C or below for later use, or tested immediately; ; 3. If necessary, the supernatant samples were appropriately diluted and IFN-γ ELISA was performed according to the instructions of the Canine IFN-γ Quantikine ELISA kit [R&D Systems catalog number CAIF TM 00]. ;

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

[0135] Epitope mapping of canineized 3B10 antibody ​ The binding epitope of the canineized antibody 3B10L3H2 on canine CTLA-4 (NCBI reference sequence: NP_001003106; accession number 68) was mapped by chemical cross-linking and mass spectrometry. ; Ping was performed. The results show that the antibody binds to two separate linear regions on the antigen CTLA-4 containing amino acids R33, R38, S42, T45, R83, T 87, Y90, K93 and Y98 (see Figure 5). In Figure 5, the amino acid residues related to the non-adjacent epitopes of CTLA-4 to which the canineized antibody 3B1 0L3H2 binds are shown to be composed of two linear parts (amino acid residues 30-50 and amino acid residues 80-100, respectively).

Table 8

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

Claims

1. 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to canine cytotoxic T-lymphocyte antigen 4 (CTLA-4), comprising a chimeric heavy chain and a chimeric light chain, wherein the isolated antibody or antigen-binding fragment thereof comprises: a. a chimeric light chain comprising the amino acid sequence of SEQ ID NO: 12 and a chimeric heavy chain comprising the amino acid sequence of SEQ ID NO: 10; b. a chimeric light chain comprising the amino acid sequence of SEQ ID NO: 16 and a chimeric heavy chain comprising the amino acid sequence of SEQ ID NO: 14; c. a chimeric light chain comprising the amino acid sequence of SEQ ID NO: 20 and a chimeric heavy chain comprising the amino acid sequence of SEQ ID NO: 18; d. a chimeric light chain comprising the amino acid sequence of SEQ ID NO: 24 and a chimeric heavy chain comprising the amino acid sequence of SEQ ID NO: 22; e. a chimeric light chain comprising the amino acid sequence of SEQ ID NO: 28 and a chimeric heavy chain comprising the amino acid sequence of SEQ ID NO: 26; f. a chimeric light chain comprising the amino acid sequence of SEQ ID NO: 32 and a chimeric heavy chain comprising the amino acid sequence of SEQ ID NO: 30; or g. a chimeric light chain comprising the amino acid sequence of SEQ ID NO: 8 and a chimeric heavy chain comprising the amino acid sequence of SEQ ID NO: 6; The isolated antibody or antigen-binding fragment thereof blocks the binding of canine CTLA-4 to canine CD80, blocks the binding of canine CTLA-4 to canine CD86, or blocks the binding of canine CTLA-4 to both canine CD80 and canine CD86.

2. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the constant region of the chimeric heavy chain comprises the amino acid sequence of SEQ ID NO:

69.

3. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the constant region of the chimeric light chain comprises the constant region of a canine kappa light chain.

4. An isolated nucleic acid encoding the light chain of the antibody or antigen-binding fragment thereof of claim 1 , 2 or 3.

5. 5. The isolated nucleic acid of claim 4, comprising a nucleic acid sequence selected from SEQ ID NOs: 7, 11, 15, 19, 23, 27 and 31.

6. 4. An isolated nucleic acid encoding the IgG heavy chain of the antibody or antigen-binding fragment thereof of claim 1 , 2 or 3.

7. 7. The isolated nucleic acid of claim 6, comprising a nucleic acid sequence selected from SEQ ID NOs: 5, 9, 13, 17, 21, 25 and 29.

8. 8. An expression vector comprising the isolated nucleic acid of any one of claims 4, 5, 6 or 7.

9. A host cell comprising one or more expression vectors according to claim 8.

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of claim 1, 2 or 3 and a pharmaceutically acceptable carrier or diluent.

11. A method for enhancing immune cell activity, comprising administering to a non-human subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 10.

12. The method comprises: a. To treat cancer: b. to treat an infection or infectious disease, or c) As a vaccine adjuvant The method according to claim 11, wherein

13. 13. The method of claim 11 or 12, wherein the non-human subject is a dog.

14. 11. Use of the pharmaceutical composition of claim 10 in the manufacture of a medicament for enhancing immune cell activity in a non-human subject in need thereof.

15. The pharmaceutical a. To treat cancer: b. to treat an infection or infectious disease, or c) As a vaccine adjuvant 15. Use of the pharmaceutical composition in the manufacture of a medicament according to claim 14.

16. 1. A method for producing an isolated antibody or antigen-binding fragment thereof that specifically binds to CTLA-4, comprising: a. culturing the host cell of claim 9 in a medium under conditions in which the nucleic acid is expressed, thereby producing a polypeptide comprising the light chain and heavy chain variable regions; and b. recovering the polypeptide from the host cell or culture medium. A method comprising:

17. A method for producing an antibody that specifically binds to canine CTLA-4, comprising: (i) an expression vector comprising a first nucleic acid encoding an IgG heavy chain comprising an amino acid sequence selected from the group of SEQ ID NOs: 2, 6, 10, 14, 34, 18, 22, 26, 30, 36, 38, 40, 60, 62, 64, and 66; and (ii) an expression vector comprising a second nucleic acid encoding a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 42, 44, and 46. in a culture medium under conditions in which the first nucleic acid and the second nucleic acid are expressed, thereby producing a polypeptide comprising an IgG heavy chain and a polypeptide comprising an IgG light chain; and b. Recovering antibodies that specifically bind to canine CTLA-4 from the host cells, the medium, or both the host cells and the medium. A method comprising:

Images