Canine-derived antibodies against human and canine CTLA-4

Abstract

The present invention provides canylated anti-human CTLA-4 antibodies having specific sequences and having a high binding affinity for canine CTLA-4. The invention also relates to the use of these antibodies in the treatment of cancer in canine and other companion animals.

Description

[0001] This application is a divisional application of an invention patent application with an application date of July 15, 2020, application number 202080050832.8, and invention name “Caninized antibodies against human and canine CTLA-4”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. §119(e) to provisional application U.S. Serial No. 62 / 874,287, filed on July 15, 2019, the entire contents of which are incorporated herein by reference. Field of the Invention

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

[0006] The initiation or termination of an immune response is mediated via signaling pathways activated by a complex interaction between a group of proteins expressed on the surface of many immune cells, most notably T lymphocytes and antigen presenting cells (APCs). Co-stimulatory signaling pathways lead to the development of an immune response and have been shown to be most importantly mediated by the interaction of CD28 on the surface of T cells with members of the B7.1 (also known as CD80) and B7.2 (also known as CD86) families on the surface of APCs. B7.1 and B7.2 are thought to perform similar functions.

[0007] In contrast, the co-inhibitory pathway results in the inhibition or termination of the immune response and has been shown to be mediated by the interaction between CTLA-4 on T cells and B7.1 / B7.2 proteins on APCs. An additional co-inhibitory signaling pathway has been shown to be mediated by the interaction between the programmed cell death receptor 1 (PD-1) on T cells and the programmed cell death receptor ligand 1 or 2 (PD-L1 / PD-L2) proteins on APCs. In addition, it has also been shown that the interaction between PD-L1 and B7.1 can also generate inhibitory signals in T cells.

[0008] 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, as well as macrophages and dendritic cells [Swanson and Hall, Eur J. Immunol., 23: 295-298 (1993); Razi-Wolfe et al., PNAS, 89: 4210-4214 (1992)]. B7.2 is constitutively expressed on dendritic cells, Langerhans cells and B cells. In addition, B7.2 is expressed on monocytes and is upregulated following IFN-γ stimulation [Larsen et al., Immunol., 152:5208-5219 (1994); Inaba, J. Exp. Med. 180:1849-1860 (1994)].

[0009] B7.1 and B7.2 bind to CD28 and CTLA-4 with different functional consequences [Linsley et al., PNAS, 87:5031-5035 (1990); Linsley et al., J. Exp. Med., 173:721-730 (1991); Azuma et al., Nature 366:76-79 (1993); Freeman et al., Science 262:909-912 (1993)]. B7.1 and B7.2 bind to CTLA-4 with much higher affinity than B7.1 / B7.2 bind to CD28 [van der Merwe, J. Exp. Med. 185:393-402 (1997)].

[0010] CD28 is a homodimeric glycoprotein that is a member of the Ig superfamily [Aruffo and Seed, PNAS, 84: 8573-8577 (1987)]. The mature protein has a single extracellular variable domain of 134 amino acid residues, which contains the hexapeptide motif MYPPPY that is essential for counter receptor binding [Riley and June, Blood, 105: 13-21 (2005)]. The 41 amino acid cytoplasmic domain of CD28 contains four tyrosine residues that can be phosphorylated upon activation [Sharpe and Freeman, Nat. Rev. Immunol., 2: 116-126 (2002)]. CD28 is expressed on most CD4+ T cells and about 50% of CD8+ T cells [Gross et al., J. Immunol., 149: 380-388 (1992); Riley and June, Blood, 105: 13-21 (2005)]. After T cell receptor (TCR) ligation, the binding of B7.1 / B7.2 to CD28 provides a critical co-stimulatory signal for T cells, allowing T cell activation and subsequent immune response development [Reiser et al., PNAS, 89: 271-275 (1992); Jenkins et al., J. Immunol., 147: 2461-2466 (1991)]. It has been shown that in the absence of CD28 signals, T cells undergo apoptosis or enter an unresponsive state [Jenkins et al., J. Exp. Med. 165: 302-319 (1987); Jenkins et al., PNAS, 84: 5409-5413 (1987); Schwartz, Science, 248: 1349-1356 (1990)]. CD28-B7.1 / B7.2 binding can change the threshold level of TCR ligation required for activation (e.g., the amount of antigen-MHC complex), reduce the time required to stimulate naive cells and enhance the amplitude of T cell responses [Soskic et al., Advances in Immunology, 124: 96-123 (2014)].

[0011] CTLA-4 (CD152) is also a member of the Ig superfamily and consists of a single extracellular domain, a transmembrane domain, and a short cytoplasmic tail [Swanson, Immunology; 1010: 169-177 (2000)]. In addition, CTLA-4 shares about 30% amino acid identity with CD28. CTLA-4 is not constitutively expressed on naive T cells, although it is rapidly upregulated shortly after CD28 ligation and T cell activation, with the peak expression level of CTLA-4 being about 48-96 hours after naive T cell activation [Alegret al., J. Immunol., 157: 4762-4770 (1996); Freeman et al., J. Immunol., 149: 3795-3801 (1992)]. CTLA-4 binds to both B7.1 and B7.2 with much higher affinity than CD28 [van der Merwe et al., J. Exp. Med., 185:393-402 (1997)]. However, in contrast to the stimulatory effects of CD28 binding to B7.1 or B7.2, CTLA-4 acts as an inhibitory receptor that is critical for downregulation of immune responses [Walnus et al., Immunity, 1:405-413 (1994); Walnus, J. Exp. Med., 183:2541-2550 (1996); Krummel and Allison, J. Exp. Med., 183:2533-2540 (1996)]. The mechanism by which CTLA-4 mediates its immunosuppressive function is related to its ability to act as a competitive inhibitor of the interaction between CD28 and B7.1 / B7.2 [reviewed in Swanson, Immunology, 1010: 169-177 (2000)]. The key role of CTLA-4 in immune downregulation was demonstrated in CTLA-4-deficient mice, which died at 3-5 weeks of age due to the development of a lymphoproliferative disease characterized by T cell infiltration of multiple organs [Tivol et al., Immunity, 3: 541-5417 (1995); Waterhouse et al., Science, 270: 985-988 (1995)]. It has also been demonstrated that the consequences of CTLA-4 knockout depend on the interaction of CD28 with its ligands B7.1 and B7.2, as shown by the lack of disease in CTLA-4 / B7-1 / B7-2 triple knockout mice [Mandelbrot et al., J. Exp. Med., 189:435-440 (1999)].This was also confirmed by the protection against lymphoproliferation obtained by repeated administration of CTLA-4Ig in CTLA-4 knockout mice [Tivol et al., J Immunol., 158:5091-5094 (1997)].

[0012] In addition, blocking the action of CTLA-4 with antibodies has been shown to enhance T cell responses in vitro and in vivo and increase anti-tumor immune responses [Leach et al., Science, 271: 1734-1736 (1996)]. Based on these findings, development of CTLA-4 blockers such as monoclonal antibodies is underway to provide a therapeutic modality for treating cancer [Hodi et al., PNAS, 100(8):4712-4717 (2003); Phan GQ et al., PNAS, 100(14):8372-8377 (2003); Attia, Journal of Clinical Oncology, 23(25):6043-6053 (2005); Comin-Anduix et al., Journal of Translational Medicine, 6:22-22 (2008); WO2000037504 A2; US8,017,114 B2; WO2010097597A1; WO2012120125A1; and Boutros et al., Nat Rev Clin Oncol.,13(8):473-486(2016)].

[0013] PD-1 is a member of the CD28 / CTLA-4 immunomodulatory receptor family. PD-1 is also a member of the Ig superfamily and contains an extracellular variable domain that binds its ligand and a cytoplasmic tail that binds a signaling molecule [reviewed in Zak et al., Cell Structure, 25: 1163-1174 (2017)]. The cytoplasmic tail of PD-1 contains two tyrosine-based signaling motifs [Zhang et al., Immunity 20: 337–347 (2004)]. PD-1 expression is not found on unstimulated T cells, B cells, or bone marrow cells. However, after activation, PD-1 expression is upregulated on these cells [Chemnitz et al., J. Immunol., 173: 945–954 (2004); Petrvas et al., J. Exp. Med., 203: 2281-2292 (2006)]. PD-1 is most closely related to CTLA-4, sharing approximately 24% amino acid identity [Jin et al., Current Topics in Microbiology and Immunology, 350: 17-37 (2010)]. When bound to PD-L1 and PD-L2 expressed on the surface of APCs, PD-1 attenuates T cell activation. Binding of any of these ligands to PD-1 negatively regulates antigen signaling via the T cell receptor (TCR). To date, only PD-L1 and PD-L2 have been found to act as ligands for PD-1. Like CTLA-4, PD-1 binding appears to transmit negative immunoregulatory signals. Ligation of PD-1 by PD-L1 or PD-L2 results in inhibition of TCR-mediated proliferation and cytokine production [Jin et al., Current Topics in Microbiology and Immunology, 350: 17-37 (2010)]. In contrast to CTLA-4-deficient animals, PD-1-deficient mice die much later in life and show signs of autoimmunity, although the severity of the effects observed is less severe than those displayed by CTLA-4-deficient animals [Nishimura et al., Immunity, 11(2):141–151 (1999); Nishimura et al., Science, 291(5502):319–322 (2001)].Although the PD-1 signaling pathway is currently under intensive investigation, studies to date indicate that the PD-L1 / PD-L2 / PD-1 interaction is involved in the negative regulation of some immune responses, as reduced signaling downstream of TCR stimulation leads to reduced cytokine secretion and impaired T cell proliferation as well as reduced production of cytotoxic molecules by T cells [Freeman et al., J. Exp. Med., 192(7):1027–1034 (2000)].

[0014] PD-L1 (CD274) is a type 1 membrane protein consisting of IgV-like and IgC-like extracellular domains, a hydrophobic transmembrane domain, and a short cytoplasmic tail (consisting of 30 amino acids) with unknown signal transduction properties. PD-L1 is considered to be a member of the B7 family and shares approximately 20% amino acid identity with B7 family members. PDL1 binds to its receptor PD-1 found on activated T cells, B cells, and myeloid cells. PD-L1 also binds to the co-stimulatory molecule B7.1, but not CD86 [Butte et al., Immunology, 45(13):3567–3572(2008)]. The affinity of B7.1 for PD-L1 is between its affinity for CD28 and CTLA-4. The related molecule PD-L2 has no affinity for CD80 or CD86, but shares PD-1 as a receptor. Engagement of PD-L1 with its receptor PD-1 on T cells delivers signals that inhibit TCR-mediated IL-2 production and T cell proliferation. Binding of PD-L1 to PD-1 also contributes to ligand-induced TCR downregulation during antigen presentation to naive T cells. In addition, binding of PD-L1 to B7.1 on T cells leads to T cell apoptosis. The role of PD1 and PD-L1 as inhibitors of T cell activation has been demonstrated in many studies. Based on these findings, the development of PD-1 and PD-L1 blockers such as monoclonal antibodies is being conducted to provide a therapeutic modality for the treatment of cancer and infectious diseases.

[0015] Humanized monoclonal antibodies that block the binding and activity of canine PD-1, PD-L1, and CTLA-4 have been developed and are currently available for treating human subjects diagnosed with one of several different types of cancer. Similarly, caninized monoclonal antibodies that block the binding and activity of canine PD-1 and PD-L1 have also been reported [US9,944,704B2, US10,106,607B2, and US2018 / 0237535A1, the entire contents of which are incorporated herein by reference]. However, there have been no reports to date of caninized monoclonal antibodies that block the binding and activity of canine CTLA-4.

[0016] Citation of any reference herein should not be construed as an admission that such reference is available as "prior art" to the present application. Brief description of the invention

[0018] The present invention relates to caninized anti-human CTLA-4 antibodies, which have specific binding affinity for canine CTLA-4 and have the ability to block the binding of canine CTLA-4 to canine CD80 and / or CD86. The present invention also relates to the use of such antibodies in treating diseases such as cancer and / or diseases caused by infection.

[0019] Thus, the present invention provides an isolated caninized antibody that specifically binds to CTLA-4, comprising a canine IgG heavy chain and a canine κ or λ light chain, or an antigen-binding fragment of the caninized antibody. In a specific embodiment of this type, the canine κ or λ light chain comprises three light chain complementary determining regions (CDRs), namely CDR light chain 1 (CDRL1), CDR light chain 2 (CDRL2), and CDR light chain 3 (CDRL3), and the canine IgG heavy chain comprises three heavy chain CDRs, namely CDR heavy chain 1 (CDRH1), CDR heavy chain 2 (CDRH2), and CDR heavy chain 3 (CDRH3), all six CDRs being derived from mammalian CTLA-4 antibodies. Specific 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.

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

[0021] In a specific embodiment, the 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 a related embodiment, the 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.

[0022] In a more specific embodiment, the CDRL1 of the caninized antibody comprises the amino acid sequence of SEQ ID NO:54, CDRL2 comprises the amino acid sequence of SEQ ID NO:56, and CDRL3 comprises the amino acid sequence of SEQ ID NO:58, and in addition, the CDRH1 of the caninized antibody comprises the amino acid sequence of SEQ ID NO:48, CDRH2 comprises the amino acid sequence of SEQ ID NO:50, and CDRH3 comprises the amino acid sequence of SEQ ID NO:52.

[0023] For certain embodiments of the invention, the heavy chain of the caninized 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. In 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 functional conservative variant of the amino acid sequence of SEQ ID NO: 34.

[0024] In still other embodiments, the heavy chain of the caninized 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. 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 functional conservative variant of the amino acid sequence of SEQ ID NO: 36.

[0025] In still other embodiments, the heavy chain of the caninized antibody comprises the amino acid sequence of SEQ ID NO: 38. In more specific embodiments, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 39. In related embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a conservative variant of the amino acid sequence of SEQ ID NO: 38. 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 functional conservative variant of the amino acid sequence of SEQ ID NO: 38.

[0026] In still other embodiments, the heavy chain of the caninized antibody comprises the amino acid sequence of SEQ ID NO: 40. In more specific embodiments, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 39. In related embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a conservative variant of the amino acid sequence of SEQ ID NO: 40. In 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 functional conservative variant of the amino acid sequence of SEQ ID NO: 40.

[0027] For certain embodiments of the invention, the heavy chain of the caninized antibody comprises the amino acid sequence of SEQ ID NO: 60, and in more specific embodiments, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 59. In related embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a conservative variant of the amino acid sequence of SEQ ID NO: 60. In 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 functional conservative variant of the amino acid sequence of SEQ ID NO: 60.

[0028] In still other embodiments, the heavy chain of the caninized antibody comprises the amino acid sequence of SEQ ID NO: 62. In more specific embodiments, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 61. In related embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a conservative variant of the amino acid sequence of SEQ ID NO: 62. In 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 functional conservative variant of the amino acid sequence of SEQ ID NO: 62.

[0029] In still other embodiments, the heavy chain of the caninized antibody comprises the amino acid sequence of SEQ ID NO: 64. In more specific embodiments, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 63. In related embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a conservative variant of the amino acid sequence of SEQ ID NO: 64. In 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 functional conservative variant of the amino acid sequence of SEQ ID NO: 64.

[0030] In still other embodiments, the heavy chain of the caninized antibody comprises the amino acid sequence of SEQ ID NO: 66. In more specific embodiments, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 65. In related embodiments, the heavy chain comprises the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52 within a conservative variant of the amino acid sequence of SEQ ID NO: 66. In 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 functional conservative variant of the amino acid sequence of SEQ ID NO: 66.

[0031] In certain embodiments, the canine light chain of the caninized antibody is a kappa chain. In alternative embodiments, the canine light chain is a lambda chain. In a specific embodiment, the kappa light chain comprises the amino acid sequence of SEQ ID NO: 42, and in a more specific embodiment, the kappa light chain is encoded by the nucleotide sequence of SEQ ID NO: 41. In a related embodiment, the kappa light chain comprises the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within a conservative variant of the amino acid sequence of SEQ ID NO: 42. In still other embodiments, the kappa light chain comprises the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within a functional conservative variant of the amino acid sequence of SEQ ID NO: 42.

[0032] In a specific embodiment, the κ light chain of the caninized antibody comprises the amino acid sequence of SEQ ID NO: 44. In a more specific embodiment, the κ light chain is encoded by the nucleotide sequence of SEQ ID NO: 43. In a related embodiment, the κ light chain comprises the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within a conservative variant of the amino acid sequence of SEQ ID NO: 44. In still other embodiments, the κ light chain comprises the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within a functional conservative variant of the amino acid sequence of SEQ ID NO: 44.

[0033] In still other embodiments, the kappa light chain of the caninized antibody comprises 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. In related embodiments, the kappa light chain comprises the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within a conservative variant of the amino acid sequence of SEQ ID NO: 46. In still other embodiments, the kappa light chain comprises the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 within a functional conservative variant of the amino acid sequence of SEQ ID NO: 46.

[0034] The present invention further provides a caninized antibody comprising any light chain of the present invention and any heavy chain of the present invention. In a specific embodiment, the isolated caninized antibody comprises a heavy chain and a kappa light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:36, SEQ ID NO:62 or the amino acid sequence 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 or SEQ ID NO:62, and the kappa light chain comprises the amino acid sequence of SEQ ID NO:46 or the amino acid sequence of SEQ ID NO:54, SEQ ID NO:56 and SEQ ID NO:58 within a conservative variant of the amino acid sequence of SEQ ID NO:46. In a more specific embodiment, the isolated caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:36 and a kappa light chain comprising the amino acid sequence of SEQ ID NO:46. In other specific embodiments, the isolated caninized 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.

[0035] In other specific embodiments, the isolated caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:40, SEQ ID NO:66, or an amino acid sequence 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 or SEQ ID NO:66, and a kappa light chain comprising the amino acid sequence of SEQ ID NO:42 or an amino acid sequence 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 more specific embodiments, the isolated caninized 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 still other specific embodiments, the isolated caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:66 and a kappa light chain comprising the amino acid sequence of SEQ ID NO:42.

[0036] The present invention further provides an isolated nucleic acid encoding any light chain of the caninized antibody of the present invention. Similarly, the present invention further provides an isolated nucleic acid encoding any heavy chain of the caninized antibody of the present invention. The present invention further provides an expression vector comprising one or more isolated nucleic acids of the present invention. The present invention also provides a host cell comprising one or more expression vectors of the present invention.

[0037] In a specific embodiment, the antibody is a recombinant antibody or an antigen-binding fragment thereof. In a related embodiment, the variable heavy chain domain and the variable light chain domain are connected by a flexible linker to form a single chain antibody.

[0038] In a specific embodiment, the antibody or antigen-binding fragment is a Fab fragment.

[0039] In other embodiments, the antibody or antigen binding fragment is a Fab' fragment. In other embodiments, the antibody or antigen binding fragment is a (Fab')2 fragment. In still other embodiments, the antibody or antigen binding fragment is a diabody. In specific embodiments, the antibody or antigen binding fragment is a domain antibody. In more specific embodiments, the antibody or antigen binding fragment is a camelized single domain antibody.

[0040] In specific embodiments, the caninized anti-human CTLA-4 antibody or antigen-binding fragment increases the immune response of a treated canine subject.

[0041] Thus, the present invention further provides an isolated nucleic acid encoding a caninized anti-human CTLA-4 antibody or antigen-binding fragment as disclosed herein. In a related embodiment, such an antibody or antigen-binding fragment can be used to prepare a medicament for treating cancer in a canine subject. Alternatively or in combination, the present invention provides for use of any antibody or antibody fragment of the present invention for diagnostic purposes. In still further embodiments, a kit comprising any caninized antibody or antigen-binding fragment disclosed herein is provided.

[0042] In still other embodiments, an expression vector is provided, comprising an isolated nucleic acid encoding any caninized anti-human CTLA-4 antibody or antigen-binding fragment of the invention. The invention also relates to a host cell comprising any expression vector described herein. In a specific embodiment, these nucleic acids, expression vectors or polypeptides of the invention can be used in a method for preparing an antibody.

[0043] The present invention further includes a pharmaceutical composition comprising a caninized antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier or diluent. In addition, the present invention provides a method for increasing the activity of immune cells, comprising administering a therapeutically effective amount of such a pharmaceutical composition to a subject in need thereof. In a specific embodiment, the subject is a canine. In an alternative embodiment, the subject is a feline. In still other embodiments, the subject is an equine. In certain embodiments, the method is used to treat cancer. In other embodiments, the method is used to treat an infection or infectious disease. In still other embodiments, the caninized antibody or antigen-binding fragment thereof of the present invention is used as a vaccine adjuvant.

[0044] These and other aspects of the invention will be better understood by reference to the following brief description and detailed description of the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 ELISA results against canine CTLA-4 for a series of selected chimeric antibodies (as indicated) are shown, confirming their binding activity: ·3B10, Δ8H5, ▼611, ▲10D1, ▽3B3, ▽418, 411, 1E2 and +Iso-control. The individual chimeric antibodies are described in Table 1 below. The Iso-control is a caninized murine antibody directed against an antigen unrelated to CTLA-4.

[0047] Figure 2 ELISA results are shown for a series of caninized 3B-10 antibodies (as indicated), demonstrating that caninized 3B-10 antibodies strongly bind to canine CTLA-4·3B10 MC, c3B10L2-H3,▲c3B10L3-H3,▼c3B10L2-H2,Oc3B10L3-H2, c3B10L3-H4 and Δc3B10L1-H1. L1, L2 and L3 correspond to VL1, VL2 and VL3 in Table 5 below, respectively, and H1, H2, H3 and H4 correspond to VH1, VH2, VH3 and VH4 in Table 4 below, respectively. 3B10 MC is Figure 1 The same chimeric antibody described in .

[0048] Figure 3 Provides ELISA results using canine CD86 and canine CTLA-4 in the presence of chimeric or caninized 3B10 antibodies. ·3B10 MC, c3B10L1-H1, ▲c3B10L3-H2, ▼c3B10L3-H4 and Iso-control. The results demonstrate the antibody blocking activity against the binding interaction of canine CD86 with canine CTLA-4 for both chimeric and caninized 3B10 antibodies. The Iso-control is a caninized murine antibody against an antigen not related to CTLA-4.

[0049] Figure 4 IFNγ production by canine PBMCs following activation with selected CTLA-4 chimeric antibodies is shown, as shown in [1E2, 3B10, 10D1, 8H5, 611, 411, and 418] and as above Figure 1 described.

[0050] Figure 5 An epitope of canine CTLA-4 was identified, comprising the amino acid sequence of prior art SEQ ID NO: 68, which interacts with the caninized 3B10L3H2 (3B10 VL3VH2) antibody. DETAILED DESCRIPTION OF THE INVENTION

[0052] abbreviation:

[0053] Throughout the detailed description and examples of the present invention, the following abbreviations will be used:

[0054] ADCC antibody-dependent cellular cytotoxicity

[0055] CDC complement-dependent cytotoxicity

[0056] CDRs are complementarity determining regions in immunoglobulin variable regions, defined using the Kabat numbering system.

[0057] CHO Chinese Hamster Ovary

[0058] EC50 is the concentration that results in 50% efficacy or binding

[0059] ELISA enzyme-linked immunosorbent assay

[0060] FR antibody framework region: the immunoglobulin variable region excluding the CDR region.

[0061] HRP Horseradish Peroxidase

[0062] IFN

[0063] IC50 is the concentration that causes 50% inhibition

[0064] IgG

[0065] Kabat Immunoglobulin alignment and numbering system pioneered by Elvin A. Kabat [Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)]

[0066] mAb Monoclonal Antibody (also called Mab or MAb)

[0067] MES2-(N-morpholino)ethanesulfonic acid

[0068] MOA mechanism of action

[0069] PCR polymerase chain reaction

[0070] PK Pharmacokinetics

[0071] SEB Staphylococcal enterotoxin B

[0072] TT Tetanus Toxoid

[0073] The V region is a segment of the IgG chain that is variable in sequence between different antibodies. It extends to Kabat residue 109 in the light chain and Kabat residue 113 in the heavy chain.

[0074] VH immunoglobulin heavy chain variable region

[0075] VK immunoglobulin kappa light chain variable region

[0076] definition:

[0077] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by ordinary technicians in the field to which the present invention belongs.

[0078] As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0079] Unless the context indicates otherwise or explicitly states, "activation" as it applies to cells or receptors refers to the activation or treatment of cells or receptors with a ligand. "Ligand" encompasses natural and synthetic ligands, such as cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies. "Ligand" also encompasses small molecules, such as peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" can refer to cell activation regulated by internal mechanisms as well as external or environmental factors.

[0080] The "activity" of a molecule can describe or refer to the binding of a molecule to a ligand or receptor, catalytic activity; the ability to stimulate gene expression or cell signaling, differentiation or maturation; antigenic activity, regulation of other molecular activities, etc. The "activity" of a molecule can also refer to the activity of regulating or maintaining cell-to-cell interactions (e.g., adhesion), or the activity of maintaining cell structure (e.g., cell membrane or cytoskeleton). "Activity" can also mean specific activity, such as [catalytic activity] / [mg protein] or [immune activity] / [mg protein], concentration in a biological compartment, etc. "Activity" can refer to the regulation of components of the innate or adaptive immune system.

[0081] "Administering" and "treating" refer to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal (e.g., a canine subject), cell, tissue, organ, or biological fluid as they apply to an animal (e.g., a canine subject), cell, tissue, organ, or biological fluid. Treatment of cells encompasses contact of an agent with a cell, as well as contact of an agent with a fluid, wherein the fluid is in contact with the cell.

[0082] "Administering" and "treating" also refer to in vitro and ex vivo manipulation of cells, for example, by an agent, diagnostic agent, binding compound or by another cell. The term "subject" includes any organism, preferably an animal, more preferably a mammal (e.g., a canine, a feline, or a human), most preferably a canine.

[0083] "Treatment" or "treatment" refers to the internal or external administration of a therapeutic agent, such as a composition containing any of the antibodies or antigen-binding fragments of the invention, to a canine subject or patient, for example, having one or more symptoms of a disease or suspected of having a disease, wherein the agent has therapeutic activity for the canine subject or patient. Typically, the agent is administered in an amount effective to alleviate and / or improve one or more symptoms of a disease in the treated subject or population, whether to induce regression of such symptoms or inhibit the progression of such symptoms to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to factors such as the disease state, age, and weight of the patient (e.g., canine, feline, or equine) and the ability of the pharmaceutical composition to elicit a desired response in the subject. Whether the disease symptom has been alleviated or improved can be evaluated by any clinical measurement commonly used by a veterinarian or other skilled health care provider to assess the severity or progression of the symptom. Although an embodiment of the invention (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the target disease symptom in every subject, it should alleviate the target disease symptom 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, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra-test, and Wilcoxon-test).

[0084] "Treatment" refers to therapeutic treatment as well as research and diagnostic applications when applied to humans, veterinary (e.g., canines), or research subjects. "Treatment" covers contact of an antibody or antigen-binding fragment of the invention with, for example, a canine or other animal subject (e.g., feline), cell, tissue, physiological compartment, or physiological fluid when applied to humans, veterinary (e.g., canines), or research subjects, or cells, tissues, or organs.

[0085] The term "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement), which results in selective damage, destruction, or elimination of cells or tissues of cancer cells, infectious pathogens, or invasive pathogens in the mammalian body (e.g., dog).

[0086] Caninized anti-human CTLA-4 antibody

[0087] The present invention provides isolated caninized anti-human CTLA-4 antibodies or antigen-binding fragments thereof that bind to canine CTLA-4, and uses of such antibodies or fragments.

[0088] As used herein, a caninized anti-human CTLA-4 antibody refers to a caninized antibody that specifically binds to mammalian CTLA-4. An antibody that specifically binds to mammalian CTLA-4 (particularly canine CTLA-4) is an antibody that exhibits preferential binding to mammalian CTLA-4 compared to other antigens, but such specificity does not require absolute binding specificity. A caninized anti-human CTLA-4 antibody is considered to be "specific" for canine CTLA-4 if its binding determines the presence of canine CTLA-4 in a biological sample obtained from a canine, or if it is able to alter the activity of canine CTLA-4 without excessively interfering with the activity of other unrelated canine proteins in a canine sample, such as without producing undesirable results, such as false positives in a diagnostic setting or side effects in a therapeutic setting. The degree of specificity required of a caninized anti-human CTLA-4 antibody may depend on the intended use of the antibody and is at least limited by its suitability for the intended purpose. The antibodies of the contemplated methods, or binding compounds derived from the antigen binding sites of the antibodies, bind to their antigens, or variants or muteins thereof, with an affinity that is at least two times greater, preferably at least 10 times greater, more preferably at least 20 times greater, and most preferably at least 100 times greater than to any other canine antigen. However, an isolated antibody that specifically binds canine CTLA-4 may cross-react with other antigens, particularly closely related antigens, such as feline CTLA-4, equine CTLA-4, and / or human CTLA-4.

[0089] As used herein, an antibody is said to specifically bind to a polypeptide comprising a given sequence (in this case, canine CTLA-4) if the antibody binds to a polypeptide comprising the sequence of canine CTLA-4 but does not bind to other canine proteins that lack the canine CTLA-4 amino acid sequence at nearly any location. For example, an antibody that specifically binds to a polypeptide comprising canine CTLA-4 may bind Tagged form of canine CTLA-4, but does not specifically bind to other Tagged canine proteins.

[0090] As used herein, unless otherwise indicated, "antibody fragment" or "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen (e.g., canine CTLA-4) bound by a full-length antibody, such as a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; nanobodies and multispecific antibodies formed from multiple antibody fragments.

[0091] Typically, when the activity is expressed on a molar basis, the caninized antibodies or antigen-binding fragments thereof of the invention retain at least 10% of their canine CTLA-4 binding activity (when compared to the corresponding parent antibody). Preferably, the antibodies or antigen-binding fragments of the invention retain at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the canine CTLA-4 binding affinity of the parent antibody.

[0092] The present invention includes antibodies referred to as "conservative variants" of antibodies with a defined amino acid sequence. "Conservative antibodies" as used herein have one, two, three or more conservative amino acid substitutions in their amino acid sequence relative to the caninized antibodies of the present invention with a defined amino acid sequence. The present invention further includes antibodies referred to as "functional conservative variants" of caninized antibodies with a defined amino acid sequence. "Functional conservative variants" as used herein have one, two, three or more non-conservative amino acid substitutions relative to the amino acid sequence of caninized antibodies. The terms "conservative variants" and "functional conservative variants" are used only for amino acid residue changes in the canine framework of the corresponding caninized antibodies of the present invention, and are not used for the specific CDRs of caninized antibodies. Importantly, "conservative variants" and / or "functional conservative variants" do not substantially change the biological activity of the corresponding caninized antibodies of the present invention comprising the defined amino acid sequence.

[0093] An "isolated antibody" refers to a purified state, and in this case refers to a molecule that is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium. In general, the term "isolated" is not intended to refer to the complete absence of such materials or the absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with the experimental or therapeutic use of the binding compounds as described herein.

[0094] The variable regions of each light chain / heavy chain pair form the antigen binding site of the antibody. Thus, typically, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are typically identical.

[0095] Typically, the variable domains of both the heavy and light chains contain three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FRs). The CDRs are typically flanked by framework regions to enable binding to specific epitopes. Typically, from N-terminus to C-terminus, both the light and heavy chain variable domains (regions) contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain is typically based on the definitions in: Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md., 5 th ed., NIH Publ. No. 91-3242 (1991); Kabat, Adv. Prot. Chem. 32: 1-75 (1978); Kabat, et al., J. Biol. Chem. 252: 6609-6616 (1977); Chothia, et al. al., J. Mol. Biol. 196:901-917 (1987) or Chothia, et al., Nature 342:878-883 (1989).

[0096] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from the "complementarity determining region" or "CDR" (i.e., CDRL1, CDRL2, and CDRL3 in the light chain variable domain and CDRH1, CDRH2, and CDRH3 in the heavy chain variable domain). [See Kabat et al. Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), which define the CDR regions of antibodies by sequence; see also Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), which define the CDR regions of antibodies by structure].

[0097] The term "framework" or "FR" residues as used herein refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues. The framework of a caninized antibody refers to a portion of a canine framework.

[0098] Unless otherwise indicated, the term "canine" as used herein includes all domestic dogs, Canis lupus familiaris or Canis familiaris.

[0099] As used herein, the term "feline" refers to any member of the family Felidae. Domestic cats, purebred and / or hybrid companion cats, and wild or feral cats are all felines.

[0100] As used herein, the term "canine framework" refers to the amino acid sequence of the heavy and light chains of a canine antibody excluding the hypervariable region residues defined herein as CDR residues. For caninized antibodies, in most embodiments, the amino acid sequence of the native canine CDR is replaced in both chains by the corresponding exogenous CDR (e.g., CDR from a mouse antibody). Optionally, the heavy and / or light chains of the canine antibody may include some exogenous non-CDR residues, for example, in order to maintain the conformation of the exogenous CDR in the canine antibody, and / or to modify Fc function, as discussed below.

[0101] There are four known IgG heavy chain subtypes of dog IgG, referred to as IgG-A, IgG-B, IgG-, and IgG-D. The two known light chain subtypes are referred to as λ and κ.

[0102] In addition to binding and activating canine immune cells, canine or caninized antibodies against CTLA-4 can also be designed to have two additional properties:

[0103] 1. Lack of effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and

[0104] 2. Ease of large-scale purification using industry standard techniques (e.g., Protein A chromatography-based techniques).

[0105] None of the naturally occurring canine IgG isotypes meet these two criteria. For example, IgG-B can be purified using protein A, but has a high level of ADCC activity. On the other hand, IgGA weakly binds to protein A, but also shows undesirable ADCC activity. In addition, neither IgG-C nor IgG-D can be purified on a protein A column, although IgG-D does not show ADCC activity. (IgG-C has considerable ADCC activity). The present invention overcomes this difficulty by providing mutant canine IgG-B antibodies specific for CTLA-4 [see, US10,106,607B2, the entire contents of which are incorporated herein by reference]. These antibodies all lack effector functions such as ADCC and can be easily purified using industry standard protein A chromatography.

[0106] As used herein, the term "caninized antibody" refers to an antibody comprising three heavy chain CDRs and three light chain CDRs from a non-canine source, such as an anti-human CTLA-4 antibody and a canine framework or a modified canine framework. The modified canine framework comprises one or more amino acid changes. In certain embodiments, the modified canine framework further optimizes the potency of the caninized antibody, for example, increasing its binding to canine CTLA-4 and / or its ability to block the binding of canine CTLA-4 to canine CD80 and / or CD86.

[0107] "Homology" refers to the sequence similarity between two polynucleotide sequences or two polypeptide sequences when they are optimally aligned. When one position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if one position in each of the two DNA molecules is occupied by adenine, the molecules are homologous at that position. The homology percentage is the number of homologous positions shared by the two sequences divided by the total number of positions compared × 100. For example, when the sequences are optimally aligned, if 6 of the 10 positions of the two sequences are matched or homologous, the two sequences are 60% homologous. Typically, the comparison is made when the two sequences are aligned to give the maximum homology percentage.

[0108] As used herein, an amino acid sequence is 100% "identical" or has 100% "identity" to a second amino acid sequence when the amino acid residues of the two sequences are identical. 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 alignment is performed over a stretch of contiguous amino acid residues comprised in a given protein, e.g., to compare portions of a protein or polypeptide. In a specific embodiment, selected deletions or insertions are contemplated that would otherwise alter the correspondence between the two amino acid sequences.

[0109] An "isolated nucleic acid molecule" refers to a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, that is unrelated to all or part of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature. For the purposes of this disclosure, it is understood that a "nucleic acid molecule comprising a specific nucleotide sequence" does not encompass a complete chromosome. An isolated nucleic acid molecule "comprising" a specified nucleic acid sequence may include, in addition to the specified sequence, coding sequences for up to 10 or even up to 20 or more other proteins, or portions or fragments thereof, or may include operably linked regulatory sequences that control the expression of the coding region of the enumerated nucleic acid sequence, and / or may include vector sequences.

[0110] The phrase "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes include, for example, a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

[0111] When a nucleic acid is functionally associated with another nucleic acid sequence, the nucleic acid is "operably linked". For example, if the DNA of a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, it is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a sequence, it is operably linked to a coding sequence; or if a ribosome binding site is positioned to facilitate translation, it is operably linked to a coding sequence. Generally, "operably linked" refers to that the DNA sequence being linked is continuous, and in the case of a secretory leader, it is continuous and in the reading phase. However, enhancers do not have to be continuous. The connection is accomplished by connecting at a convenient restriction site. If such a site does not exist, a synthetic oligonucleotide adapter or linker is used according to conventional practice. It should also be readily understood that when a nucleic acid sequence is provided herein, it may include a stop codon. However, since stop codons are interchangeable, the inclusion of a specific stop codon in a sequence should not be considered an essential part of the sequence.

[0112] As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all of these names include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It should also be understood that not all progeny will have exactly the same DNA content, due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. Where different names are contemplated, this will be clear from the context.

[0113] As used herein, "germline sequence" refers to the sequence of an unrearranged immunoglobulin DNA sequence. Any suitable source of unrearranged immunoglobulin sequences may be used. Human germline sequences can be obtained, for example, from the website of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the United States National Institutes of Health. Mouse germline sequences can be obtained, for example, as described in Giudicelli et al. [Nucleic Acids Res. 33: D256-D261 (2005)].

[0114] Properties of Exemplary Caninized Anti-Human CTLA-4 Antibodies

[0115] The present invention provides isolated caninized anti-human CTLA-4 antibodies and methods of using the antibodies or antigen-binding fragments thereof in treating diseases (e.g., treating cancer in canines). Examples of caninized anti-human CTLA-4 antibodies that bind to canine CTLA-4 include, but are not limited to, antibodies comprising canine IgG-A, IgG-B, IgG-C, and IgG-D heavy chains and / or canine kappa light chains together with anti-human CTLA-4 CDRs. Thus, the present invention provides isolated caninized anti-human CTLA-4 and / or antigen-binding fragments thereof that bind to canine CTLA-4 and block the binding of canine CTLA-4 to canine CD86 and / or canine CD80.

[0116] "Conservatively modified variants" or "conservative substitutions" refer to amino acids in a protein that are substituted with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.), such that changes can often be made without altering the biological activity of the protein. Those skilled in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity [see, e.g., Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.; 1987)]. In addition, substitutions of amino acids with similar structure or function are unlikely to destroy biological activity. Various embodiments of the antibodies or antigen-binding fragments of the invention comprise polypeptide chains having sequences disclosed herein, e.g., SEQ ID NO: 34, 36, 38, 40, 42, 44, 60, 62, 64 or 66, or polypeptide chains comprising up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more conservative amino acid substitutions in non-CDR regions. Exemplary conservative substitutions are listed in Table A.

[0117] Table A

[0118] Exemplary conservative amino acid substitutions

[0119]

[0120]

[0121] The present invention also contemplates functional conservative variants of the antibodies of the present invention. As used herein, "functional conservative variants" refer to antibodies or fragments in which one or more amino acid residues have been altered without changing desired properties (such as antigen affinity and / or specificity). Such variants include, but are not limited to, replacing amino acids with amino acids having similar properties, such as conservative amino acid substitutions of Table A above.

[0122] Nucleic Acids

[0123] The present invention further includes nucleic acids encoding the immunoglobulin chains of the caninized anti-human CTLA-4 antibodies disclosed herein and antigen-binding fragments thereof. For example, the present invention includes all novel nucleic acids listed in the following table.

[0124] The present invention also includes nucleic acids encoding immunoglobulin polypeptides comprising an amino acid sequence of a canine framework that is at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical, and most preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the amino acid sequence of the canine framework of the antibodies provided herein, when an alignment is performed by the BLAST algorithm (wherein the parameters of the algorithm are selected to give the largest match between the corresponding sequences over the entire length of the corresponding reference sequences). The present invention further provides nucleic acids encoding immunoglobulin polypeptides, which, when compared with the BLAST algorithm (wherein the parameters of the algorithm are selected to give the maximum match between the corresponding sequences over the entire length of the corresponding reference sequences), comprise an amino acid sequence that is at least about 70% similar, preferably at least about 80% similar, more preferably at least about 90% similar, and most preferably at least about 95% similar (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to any reference amino acid sequence, and the nucleic acids are also included in the present invention.

[0125] Sequence identity refers to the degree to which the amino acids in equivalent positions of two polypeptides are identical when the two sequences are optimally aligned. Sequence similarity includes identical residues and biochemically related amino acids that are not identical. Biochemically related amino acids that have similar properties and are interchangeable are discussed above.

[0126] The following references relate to the BLAST algorithm which is frequently used for sequence analysis: BLAST ALGORITHMS: Altschul, SF, et al., J. Mol. Biol. 215:403-410 (1990); Gish, W., et al., Nature Genet. 3:266-272 (1993); Madden, TL, et al., Meth. Enzymol. 266:131-141 (1996); Altschul, SF, et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang, J., et al., Genome Res. 7:649-656 (1997); Wootton, JC, et al., Comput. Chem. 17:149-163 (1993); Hancock, J. Met. al., Comput.Appl.Biosci.10:67-70(1994); ALIGNMENT SCORING SYSTEMS: Dayhoff, MO, etal., "A model of evolutionary change in proteins." in Atlas of Protein SequenceandStructure, Volume 5, Supplement 3, MODayhoff (ed.), pp.345-352, (1978); Natl.Biomed.Res.Found., Washington, DC; Schwartz, RM, et al., "Matrices for detecting distant relationships." in Atlas of ProteinSequence and Structure, vol.5, suppl.3." (1978), MODayhoff (ed.), pp.353-358 (1978), Natl.Biomed.Res.Found., Washington, DC; Altschul, SF, J.Mol.Biol.219:555-565 (1991); States, DJ, et al., Methods 3:66-70(1991); Henikoff, S., et al., Proc. Natl. Acad. Sci. USA89: 10915-10919 (1992); Altschul, SF, et al., J. Mol. Evol.36:290-300(1993);ALIGNMENT STATISTICS:Karlin,S.,et al.,Proc.Natl.Acad.Sci.USA 87:2264-2268(1990);Karlin,S.,et al.,Proc.Natl.Acad.Sci.USA 90:5873-5877(1993); Dembo, A., et al., Ann. Prob. 22: 2022-2039 (1994); and Altschul, SF "Evaluating the statistical significance of multipledistinct local alignments." in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), pp. 1-14, Plenum, New York (1997). .

[0127] The present invention also provides an expression vector comprising a nucleic acid of the present invention, wherein the nucleic acid is operably linked to a control sequence that is recognized by a host cell when the host cell is transfected with the vector. Also provided are a host cell comprising the expression vector of the present invention and a method for producing an antibody or antigen-binding fragment thereof disclosed herein, the method comprising: culturing a host cell carrying an expression vector encoding an antibody or antigen-binding fragment in a culture medium, and isolating the antigen or antigen-binding fragment thereof from the host cell or culture medium.

[0128] Epitope Binding and Binding Affinity

[0129] The present invention further provides an antibody or antigen-binding fragment thereof that binds to the same epitope on canine CTLA-4 as a caninized anti-human CTLA-4 antibody, wherein the caninized anti-human CTLA-4 comprises the amino acid sequence of SEQ ID NO: 36 and / or SEQ ID NO: 46. The caninized anti-human CTLA4 antibody or antigen-binding fragment thereof is capable of inhibiting the binding of canine CTLA4 to canine CD80 and / or CD86.

[0130] Caninized anti-human CTLA-4 antibodies can be recombinantly produced by the methods described in the following examples. Mammalian cell lines that can be used as hosts for expressing antibodies or fragments disclosed herein are well known in this embodiment and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and many other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other cell lines that can be used are insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. When recombinant expression vectors encoding heavy chains or antigen-binding portions or fragments thereof, light chains and / or antigen-binding fragments thereof are introduced into mammalian host cells, antibodies are produced by culturing the host cells for a period of time sufficient to allow expression of the antibodies in the host cells or, more preferably, secretion of the antibodies into the culture medium in which the host cells are grown.

[0131] The antibody can be recovered from the culture medium using standard protein purification methods. In addition, many known techniques can be used to enhance the expression of the antibodies of the invention (or other parts thereof) from production cell lines. For example, the glutamine synthetase gene expression system (GS system) is a common method for enhancing expression under certain conditions. The GS system is discussed in whole or in part in conjunction with European Patent Nos. 0 216 846, 0 256 055 and 0 323 997 and European Patent Application No. 89303964.4.

[0132] In general, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern specific to the glycoprotein produced in the cell line or transgenic animal. Therefore, the specific glycosylation pattern of an antibody will depend on the specific cell line or transgenic animal used to produce the antibody. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein constitute the present invention, regardless of the glycosylation pattern that the antibody may have. Similarly, in certain embodiments, antibodies with a glycosylation pattern comprising only non-fucosylated N-glycans may be advantageous because these antibodies have been shown to generally show more effective efficacy than their fucosylated counterparts both in vitro and in vivo [see, e.g., Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Pat. Nos. 6,946,292 and 7,214,775].

[0133] The present invention also includes antibody fragments of the caninized anti-human CTLA-4 antibodies disclosed herein. The antibody fragments include F(ab)2 fragments, which can be produced by enzymatic cleavage of IgG by, for example, pepsin. Fab fragments can be produced by, for example, reducing F(ab)2 with dithiothreitol or mercaptoethylamine. Fab fragments are VL-CL chains attached to VH-CH1 chains by disulfide bonds. F(ab)2 fragments are two Fab fragments attached by two disulfide bonds. The Fab portion of the F(ab)2 molecule includes a portion of the Fc region between which a disulfide bridge is located. Fv fragments are V L or V H area.

[0134] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region, e.g., a canine constant region, such as an IgG-A, IgG-B, IgG-C, and IgG-D canine heavy chain constant region or a variant thereof. In another embodiment, the antibody or antigen-binding fragment comprises a light chain constant region, e.g., a canine light chain constant region, such as a lambda or kappa canine light chain region or a variant thereof. By way of example, and not limitation, the canine heavy chain constant region may be from IgG-B [e.g., modified IgG-B, US10,106,607B2, the entire contents of which are incorporated herein by reference], and the canine light chain constant region may be from kappa.

[0135] Antibody Engineering

[0136] The caninized anti-human CTLA-4 antibodies of the invention have been engineered to include modifications to the canine framework (including canine framework residues) within the variable domains of the parent (ie, canine) monoclonal antibody, e.g., to improve the properties of the antibody.

[0137] Experimental and diagnostic uses

[0138] The caninized anti-human CTLA-4 or antigen-binding fragments thereof of the present invention can also be used in diagnostic assays for canine CTLA-4 protein, for example, to detect its expression in specific tumor cells, tissues or serum. Such diagnostic methods can be used for various disease diagnosis, especially certain cancers of canines.

[0139] For example, this method includes the following steps:

[0140] (a) coating a substrate (e.g., the surface of a microtiter plate well, e.g., a plastic plate) with a caninized anti-human CTLA-4 antibody or an antigen-binding fragment thereof;

[0141] (b) applying to the matrix a sample to be tested for the presence of canine CTLA-4;

[0142] (c) washing the plate to remove unbound substances in the sample;

[0143] (d) applying a detectably labeled antibody (e.g., an enzyme-linked antibody) that is also specific for the CTLA-4 antigen;

[0144] (e) washing the matrix to remove unbound, labeled antibodies;

[0145] (f) if the labeled antibody is enzyme-linked, administering a chemical substance that is converted into a fluorescent signal by the enzyme; and

[0146] (g) detecting the presence of the labeled antibody.

[0147] In a further embodiment, the labeled antibody is labeled with a peroxidase that reacts with ABTS [e.g., 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] or 3,3',5,5'-tetramethylbenzidine (TMB) to produce a detectable color change. Alternatively, the labeled antibody is labeled with a detectable radioisotope (e.g., 3H) that can be detected with a scintillation counter in the presence of a scintillator. The caninized anti-human CTLA-4 antibodies of the invention can be used in a Western blot or immunowestern blot procedure.

[0148] Such procedures form part of the present invention and include, for example:

[0149] (i) contacting a membrane or other solid matrix to which the canine CTLA-4 or fragment thereof to be tested for binding is present with the caninized anti-human CTLA-4 antibody or antigen-binding fragment thereof of the invention. Such a membrane can take the form of a nitrocellulose or vinyl [e.g., polyvinylidene fluoride (PVDF)] based membrane to which the protein to be tested for the presence of canine CTLA-4 has been transferred in a non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel (e.g., after electrophoretic separation in the gel). Prior to contacting the membrane with the caninized mouse anti-human CTLA-4 antibody or antigen-binding fragment thereof, the membrane is optionally blocked with, for example, skim milk powder, to bind nonspecific protein binding sites on the membrane.

[0150] (ii) washing the membrane one or more times to remove unbound caninized anti-human CTLA-4 antibody or antigen-binding fragment thereof and other unbound substances; and

[0151] (iii) detecting bound caninized anti-human CTLA-4 antibody or antigen-binding fragment thereof.

[0152] The bound antibody or antigen-binding fragment can be detected by binding the antibody or antigen-binding fragment to a detectably labeled second antibody (anti-immunoglobulin antibody) and then detecting the presence of the second antibody.

[0153] The caninized anti-human CTLA-4 antibodies and antigen-binding fragments thereof disclosed herein can also be used for immunohistochemistry. Such methods form part of the invention and include, for example, (1) contacting cells to be tested for the presence of canine CTLA-4 with, for example, a caninized anti-human CTLA-4 antibody or antigen-binding fragment thereof of the invention; and (2) detecting the antibody or fragment on or in the cell.

[0154] If the antibody or antigen-binding fragment itself is detectably labeled, it can be detected directly. Alternatively, the antibody or antigen-binding fragment can be bound by a detectably labeled secondary antibody.

[0155] Certain caninized anti-human CTLA4 antibodies and antigen-binding fragments thereof disclosed herein can also be used for in vivo tumor imaging. Such methods may include injecting a radiolabeled caninized anti-human CTLA4 antibody or antigen-binding fragment thereof into the body of a canine to be tested for the presence of a tumor associated with canine CTLA-4 expression, followed by nuclear imaging of the patient's body to detect the presence of the labeled antibody or antigen-binding fragment, for example at a locus containing a high concentration of the antibody or antigen-binding fragment bound to the tumor.

[0156] Imaging techniques include SPECT imaging (single photon emission computed tomography) or PET imaging (positron emission tomography). Labels include, for example, iodine-123 (123I) and technetium-99m (99mTc), for example in combination with SPECT imaging, or 11 C. 13 N. 15 O or 18 F, for example in combination with PET imaging, or indium- 111 [See, e.g., Gordon et al., International Rev. Neurobiol. 67:385-440 (2005)].

[0157] Pharmaceutical compositions and administration

[0158] To prepare a pharmaceutical or sterile composition of a caninized murine anti-human CTLA-4 antibody or antigen-binding fragment thereof, it can be mixed with a pharmaceutically acceptable carrier or excipient [see, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984)].

[0159] The preparation of therapeutic and diagnostic agents can be prepared by mixing with acceptable carriers, excipients or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions [see, for example, Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY]. In one embodiment, the anti-CTLA-4 antibody of the invention is diluted to an appropriate concentration in a sodium acetate solution at pH 5-6, and NaCl or sucrose is added to improve tonicity. Additional agents such as polysorbate 20 or polysorbate 80 may be added to enhance stability.

[0160] Toxicity and therapeutic efficacy of an antibody composition administered alone or in combination with another agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as for determining LD 50 (a dose lethal to 50% of the population) and ED 50(the dose that is therapeutically effective in 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index (LD50 / ED50). In certain aspects, antibodies that exhibit a high therapeutic index are desirable. The data obtained from these cell culture assays and animal studies can be used to formulate a dosage range for canines. The dosage of such a compound is preferably within a circulating concentration range that includes the ED50 with little or no toxicity. The dosage can vary within this range, depending on the dosage form used and the route of administration.

[0161] The mode of administration can vary. Suitable routes of administration include oral, rectal, transmucosal, enteral, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, dermal, transdermal or intraarterial.

[0162] In a specific embodiment, the caninized murine anti-human CTLA-4 antibody or antigen-binding fragment thereof can be administered by an invasive route, such as by injection. In a further embodiment of the invention, the caninized murine anti-human CTLA-4 antibody or antigen-binding fragment thereof or a pharmaceutical composition thereof is administered intravenously, subcutaneously, intramuscularly, intraarterially, or by inhalation, aerosol delivery. Administration by a non-invasive route (e.g., oral, such as in pills, capsules, or tablets) is also within the scope of the invention.

[0163] The composition can be administered with medical devices known in the art. For example, the pharmaceutical composition of the present invention can be administered by injection with a hypodermic needle (including, for example, a prefilled syringe or an automatic injector). The pharmaceutical compositions disclosed herein can also be administered with a needle-free hypodermic injection device; such as the devices disclosed in U.S. Patent Nos.: 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.

[0164] The pharmaceutical composition disclosed herein can also be administered by infusion. Examples of well-known implants and module forms for administering pharmaceutical compositions include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses an implantable infusion device with a variable flow rate for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with a multi-chamber compartment. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0165] Alternatively, caninized anti-human CTLA-4 antibodies can be administered in a local rather than systemic manner, for example, by direct injection of the antibody into an arthritic joint or pathogen-induced lesion characterized by immunopathology, often in a depot or sustained release formulation. Moreover, the antibody can be administered in a targeted drug delivery system, for example, in liposomes coated with tissue-specific antibodies that target, for example, arthritic joints or pathogen-induced lesions characterized by immunopathology. The liposomes will selectively target and be taken up by the diseased tissue.

[0166] The administration regimen depends on several factors, including serum or tissue turnover of the therapeutic antibody, symptom levels, immunogenicity of the therapeutic antibody, and accessibility of target cells in the biomatrix. Preferably, the administration regimen delivers enough therapeutic antibodies to achieve improvement in the target disease state while minimizing undesirable side effects. Therefore, the amount of the biologic delivered depends in part on the specific therapeutic antibody and the severity of the condition being treated. Guidance for selecting appropriate doses of therapeutic antibodies is available [see, e.g., Wawrzynczak Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK (1996); Kresina (ed.) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY (1991); Bach (ed.) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY (1993); Baert, et al. New Engl. J. Med. 348:601-608 (2003); Milgrom et al. New Engl. J. Med. 341:1966-1973 (1999); Slamon et al. New Engl. J. Med. 344:783-792 (2001); Beniaminovitz et al. New Engl. J. Med. 345:783-792 (2002); et al. New Engl. J. Med. 342:613-619 (2000); Ghosh et al. New Engl. J. Med. 348: 24-32 (2003); Lipsky et al. New Engl. J. Med. 343: 1594-1602 (2000)].

[0167] Determination of the appropriate dosage is made by a veterinarian, for example using parameters or factors known in the art or suspected to affect treatment. Typically, the dosage is started with an amount slightly less than the optimal dosage and increased in small increments thereafter until the desired or optimal effect is obtained relative to any negative side effects. Important diagnostic measures include those such as symptoms of inflammation or levels of inflammatory cytokines produced.

[0168] The antibodies or antigen-binding fragments thereof disclosed herein can be provided by continuous infusion or by dosages administered, e.g., daily, 1-7 times per week, weekly, biweekly, monthly, bimonthly, quarterly, semi-annually, yearly, etc. The dosage can be provided, e.g., intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. The total weekly dose is usually at least 0.05 μg / kg body weight, more usually at least 0.2 μg / kg, 0.5 μg / kg, 1 μg / kg, 10 μg / kg, 100 μg / kg, 0.25 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 5.0 mg / ml, 10 mg / kg, 25 mg / kg, 50 mg / kg or more [see, e.g., Yang, et al. New Engl. J. Med. 349:427-434 (2003); Herold, et al. New Engl. J. Med. 346:1692-1698 (2002); Liu, et al. J. Neurol. Neurosurg. Psych. 67:451-456 (1999); Portielji, et al. Cancer Immunol. Immunother. 52: 133-144 (2003)]. Dosages can also be provided to achieve a predetermined target concentration of caninized anti-human CTLA-4 antibody in the subject's serum, e.g., 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg / ml or more. In other embodiments, the caninized 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 weekly, biweekly, "every 4 weeks", monthly, bimonthly or quarterly.

[0169] As used herein, "inhibit" or "treat" or "treatment" includes a delay in the development of symptoms associated with a disorder and / or a reduction in the severity of symptoms of such a disorder. The term also includes amelioration of existing uncontrolled or unwanted symptoms, prevention of additional symptoms, and amelioration or prevention of the underlying causes of these symptoms. Thus, the term means that a beneficial result has been conferred on a vertebrate subject having a disorder, disease or symptom or having the potential to develop such a disorder, disease or symptom.

[0170] As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of a caninized anti-human CTLA-4 antibody or antigen-binding fragment thereof of the invention that, when administered to a cell, tissue, or subject alone or in combination with another therapeutic agent, is effective to cause a measurable improvement in one or more symptoms of a disease or condition, or the progression of such a disease or condition. A therapeutically effective dose further refers to an amount of a binding compound sufficient to result in at least a partial improvement in symptoms, such as treatment, healing, prevention, or amelioration of a related medical condition, or an increase in the rate of treatment, healing, prevention, or amelioration of such a condition. When applied to a single active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to the combined amount of the active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent will result in an improvement in a diagnostic measure or parameter of at least 10%; typically at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably at least 50%. In cases where subjective measurements are used to assess disease severity, an effective amount may also result in an improvement in a subjective measurement.

[0171] Other combination therapies

[0172] As previously described, caninized anti-human CTLA4 antibodies or antigen-binding fragments thereof can be co-administered with one or more therapeutic agents (e.g., chemotherapeutic agents). The antibody can be linked to the agent (as an immune complex) or can be administered separately from the agent. In the latter case (administered alone), the antibody can be administered before, after, or simultaneously with the agent, or can be co-administered with other known therapies.

[0173] Reagent test kit

[0174] Kits are further provided that include one or more components, including but not limited to an antibody or antigen-binding fragment as discussed herein that specifically binds to CTLA-4 (e.g., a caninized anti-human CTLA-4 antibody of the invention or an antigen-binding fragment thereof) in combination with one or more additional components (including, but not limited to, a pharmaceutically acceptable carrier and / or a chemotherapeutic agent, as discussed herein). The binding composition and / or chemotherapeutic agent can be formulated as a neat composition or combined in a pharmaceutical composition with a pharmaceutically acceptable carrier.

[0175] In one embodiment, the kit includes a binding composition of the invention (e.g., a caninized murine anti-human CTLA-4 antibody (comprising the amino acid sequences of SEQ ID NO:36 and SEQ ID NO:46) in one container (e.g., in a sterile glass or plastic vial) and a pharmaceutical composition thereof and / or a chemotherapeutic agent in another container (e.g., in a sterile glass or plastic vial).

[0176] In another embodiment, a kit comprises a combination of the invention, which includes a combination composition component (e.g., a caninized anti-human CTLA-4 antibody comprising the amino acid sequences of SEQ ID NO: 36 and SEQ ID NO: 46) and a pharmaceutically acceptable carrier, optionally in combination with one or more therapeutic agent components, together, optionally formulated into a pharmaceutical composition in a single common container.

[0177] If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit may also include a device for performing such administration. For example, the kit may include one or more hypodermic needles or other injection devices as described above. The kit may also include a package insert including information about the pharmaceutical composition and dosage form in the kit. Typically, such information helps pet owners and veterinarians to effectively and safely use the encapsulated pharmaceutical composition and dosage form. For example, the following information about the combination of the present invention may be provided in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdose, appropriate dosage and administration, supply specifications, appropriate storage conditions, references, manufacturer / distributor information, and patent information.

[0178] For convenience, the antibody or specific binding agent disclosed herein can be provided in a kit, i.e., a predetermined amount of reagent and a packaged combination of instructions for diagnosis or detection. When the antibody is labeled with an enzyme, the kit will include substrates and cofactors required for the enzyme (e.g., a substrate precursor that can detect a chromophore or a fluorophore). In addition, other additives may be included, such as stabilizers, buffers (e.g., blocking buffer or lysis buffer), etc. The relative amounts of various reagents can vary widely to provide a concentration of the reagent in solution that substantially optimizes the sensitivity of the assay. In particular, the reagent can be provided as a dry powder, typically lyophilized, including an excipient, which will provide a reagent solution with an appropriate concentration when dissolved. Example

[0179] Example 1

[0180] Construction of anti-CTLA-4 chimeric antibody

[0181] It is believed that it is possible that the prior art monoclonal antibodies raised against human CTLA-4, which can bind to human CTLA-4 and block the binding of human CTLA-4 to human CD86, can also bind to canine CTLA-4 and can be expected to block the binding of canine CTLA-4 to canine CD86. To test this possibility, the known nucleotide sequence 3B10 (as disclosed in WO2012120125) corresponding to the heavy chain variable region of the anti-human CTLA-4 monoclonal antibody was fused to the nucleotide sequence of the modified canine constant heavy chain (CH1-hinge-CH2-3) to generate a chimeric mouse-canine heavy chain nucleotide sequence, designated SEQ ID NO: 1. A second known nucleotide sequence encoding the amino acid sequence of the light chain variable region corresponding to the anti-human CTLA-4 monoclonal antibody was fused to the nucleotide sequence of the canine kappa light chain constant region to generate a chimeric mouse-canine light chain nucleotide sequence, designated SEQ ID NO: 3. The protein encoded by the chimeric mouse-canine heavy chain nucleotide sequence comprises the amino acid sequence of SEQ ID NO: 2, and the protein encoded by the chimeric mouse-canine light chain nucleotide sequence comprises the amino acid sequence of SEQ ID NO: 4. Using standard molecular biology techniques, the chimeric human-canine heavy and light chains were cloned into separate expression plasmids. The plasmids containing the heavy and light chain genes were transfected into HEK293 cells, and the expressed antibodies were purified from the HEK293 cell supernatant using protein A.

[0182] Similarly, chimeric antibody 8H5 containing the variable domain previously disclosed in WO2012120125 was also constructed as exemplified by antibodies formed by combinations of heavy and light chains with DNA and amino acid sequences listed in SEQ ID NOs: 5 to 8, respectively. In addition, chimeric antibodies 411, 418 and 611 containing the variable domain disclosed in WO2000037504 were constructed as exemplified by antibodies formed by combinations of heavy and light chains with nucleic acid and amino acid sequences listed in SEQ ID NOs: 9-20, respectively. Chimeric antibodies 10D1 and 1E2 containing the variable domain disclosed in US8,017,114B2 were also constructed as exemplified by antibodies formed by combinations of heavy and light chains with nucleotide and amino acid sequences listed in SEQ ID NOs: 21-28, respectively. In addition, chimeric antibody 3B3 containing the variable regions disclosed in WO2010097597 was constructed, as exemplified by antibodies formed by combining heavy and light chains having nucleotide and amino acid sequences as set forth in SEQ ID NOs: 29-32, respectively. The amino acid sequence identification numbers of the chimeric antibodies are provided in Table 1 below.

[0183] Table 1 Amino acid sequences of chimeric antibodies

[0184]

[0185]

[0186] Example 2

[0187] Reactivity of anti-CTLA-4 chimeric antibodies

[0188] The chimeric antibody of Example 1 was cultured in Expi293 cells [obtained from THERMO FISHER The chimeric antibody was expressed in the Expi293 expression system of [Epi293] and purified using a protein A column. The chimeric antibody was then tested for reactivity with canine CTLA-4 by ELISA as follows:

[0189] 1. Coat 200 ng / well of CTLA-4 in the immunoplate and incubate the plate overnight at 4°C.

[0190] 2. Wash the plate three times with PBS containing 0.05% Tween 20 (PBST).

[0191] 3. Block the plates with 0.5% BSA in PBS for 45-60 minutes at room temperature.

[0192] 4. Wash the plate 3 times with PBST.

[0193] 5. Dilute the antibody three-fold in each column or row of the dilution plate.

[0194] 6. Transfer the diluted antibodies to each column or row of the immunoplate and incubate the plate for 45-60 minutes at room temperature.

[0195] 7. Wash the plate 3 times with PBST.

[0196] 8. Add 1:2000 dilution of horseradish peroxidase-labeled anti-canine IgG Fc to each well of the plate and incubate the plate for 45-60 minutes at room temperature.

[0197] 9. Wash the plate 3 times with PBST.

[0198] 10. Add TMB substrate to each well of the plate and incubate the plate for 10 to 15 minutes at room temperature to develop color.

[0199] 11. Add 100 μL of 1.5 M phosphoric acid to each well to stop the reaction.

[0200] 12. Read the plate at 450 nm using a reference wavelength of 540 nm.

[0201] ELISA results are described in Figure 1In the results, it was shown that the selected antibodies can bind to canine CTLA-4. In addition, the 3B10 chimeric antibody was found to be the best binder among the chimeric antibodies in this group.

[0202] Example 3

[0203] Construction of canine anti-human CTLA-4 monoclonal antibody 3B10

[0204] The 3B10 chimeric antibody was determined to have the strongest binding affinity for canine CTLA-4 (see Figure 1 ), a group of 6 CDRs of the chimeric antibody was selected to be added to the canine framework. In order to perform the caninization process, the DNA sequences encoding the canine IgG heavy and light chains were determined [see US10,106,607B2, the entire contents of which are incorporated herein by reference]. The DNA and protein sequences of canine heavy and light chains are known in the art and can be obtained by searching the NCBI gene and protein database. There are four known IgG subtypes of dog IgG, which are referred to as IgG-A, IgG-B, IgG-C and IgG-D. There are two types of light chains in canine antibodies, referred to as κ and λ.

[0205] Table 2 DNA sequences of prior art CDRS in caninized antibodies

[0206] CDR Nucleotide sequence SEQ ID NO: H-1 gattataacatggat 47 H-2 aacattaacccgaacagcgaaagcaccagctataaccagaaatttaaaggc 49 H-3 gatggcaaccgctatgatgcgtggtttgcgtat 51 L-1 agcgcgagcagcagcgtgacctatatgcat 53 L-2 agcaccagcattctggcgagc 55 L-3 cagcagcgcaccagctatccgctgacc 57

[0207] Table 3 1 Amino acid sequences of prior art CDRS in caninized antibodies

[0208]

[0209]

[0210]

[0211] Without being bound by any particular method, the overall process of preparing caninized heavy and light chains, which can be mixed in different combinations to produce caninized anti-human CTLA-4 mAbs, can include the following scheme:

[0212] i) Identify the CDRs of the H and L chains of the desired anti-CTLA-4 mAb. Translate the amino acid sequences of the CDRs back into the appropriate DNA sequences.

[0213] ii) Identification of suitable DNA sequences for the H and L chains of canine IgG (eg, modified heavy and light kappa chains of IgG-B as described in US 10,106,607 B2).

[0214] iii) Identify DNA sequences encoding the endogenous CDRs of canine IgG H and L chain DNAs of the above sequences.

[0215] iv) Replace the DNA sequences encoding the endogenous canine H and L chain CDRs with DNA sequences encoding the desired anti-human CTLA-4 CDRs. When a modified canine IgG B is used, modifications of D31A and N63A as numbered in SEQ ID NO: 69 are made in canine IgG B as previously described in US 10,106,607 B2 (incorporated herein in its entirety). In addition, the DNA encoding some canine framework amino acid residues is optionally replaced with DNA encoding selected amino acid residues from the desired anti-human CTLA-4 mAb canine region. This process is called back mutation. Two sets of four variants for each heavy chain and one set of three for the light chain variable region listed in Tables 4 and 5 were developed.

[0216] variants, wherein each variant in the group contains a different back mutation site.

[0217] v) Synthesizing the DNA from step (iv) and cloning it into a suitable expression plasmid.

[0218] vi) Plasmids containing the desired caninized H and L chains were transfected into HEK293 cells.

[0219] vii) Purification of the expressed caninized antibody from HEK293 supernatant.

[0220] viii) Purified caninized antibodies were tested for binding to canine CTLA-4.

[0221] A panel of caninized H and L chain sequences was developed. SEQ ID NO: are listed in Tables 4 and 5 below.

[0222] Table 4 SEQ ID NO of caninized 3B10 heavy chain.

[0223]

[0224]

[0225]

[0226] Table 5 SEQ ID NO. of caninized 3B10 light chain.

[0227] Caninized light chain SEQ ID NO: Nucleic acid SEQ ID NO: Amino Acid 3B10 VL1 41 42 3B10VL2 43 44 3B10VL3 45 46

[0228] The present invention provides caninized antibodies formed by any combination of caninized heavy chains and light chains listed in Tables 4 and 5 above. Next, the antibodies were expressed in Expi293 cells and then purified. Figure 2The ELISA results shown in confirm that 3B10 has been successfully caninized, and 3B10L3H2, 3B10L3H3, and 3B10L3H4 all have tight binding activity similar to that of the parental mouse-canine chimeric antibody of 3B10.

[0229] Example 4

[0230] Blocking activity of chimeric and caninized antibody 3B10 on the interaction between canine CD86 and CTLA-4

[0231] To ensure that the caninized 3B10 antibody retains the neutralizing activity against canine CTLA-4 as the parent antibody, the caninized antibody was tested for its blocking activity against the interaction between canine CD-80 and canine CTLA-4 by ELISA as follows:

[0232] 1. Coat 200 ng / well of CTLA-4 in the immunoplate and incubate the plate overnight at 4°C.

[0233] 2. Wash the plate three times with PBS containing 0.05% Tween 20 (PBST).

[0234] 3. Block the plates with 0.5% BSA in PBS for 45-60 minutes at room temperature.

[0235] 4. Wash the plate 3 times with PBST.

[0236] 5. Dilute the antibody in each column or row of the dilution plate three-fold, then add 100 ng / well of biotinylated CD86 and mix with the antibody.

[0237] 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.

[0238] 7. Wash the plate 3 times with PBST.

[0239] 8. Add 1:2000 dilution of horseradish peroxidase-conjugated streptavidin to each well of the plate and incubate the plate for 45-60 minutes at room temperature.

[0240] 9. Wash the plate 3 times with PBST.

[0241] 10. Add TMB substrate to each well of the plate and incubate the plate for 10 to 15 minutes at room temperature to allow color development.

[0242] 11. Add 100 μL of 1.5 M phosphoric acid to each well to stop the reaction.

[0243] 12. Read the plate at 450 nm using a reference wavelength of 540 nm.

[0244] Depicted in Figure 3 The results of the ELISA plate in the figure show that the caninized 3B10 variant has similar blocking activity as the parental 3B10.

[0245] Example 5

[0246] IFNγ production by canine PBMC activated by chimeric antibody introduction:

[0247] Interferon-γ (IFN-γ; also called type II interferon) is produced primarily by activated T lymphocytes and possibly natural killer cells. This property is widely used as an indicator of T cell activation by quantitatively detecting IFN-γ production on an ELISA-based assay. In order to identify functional antibodies to CTLA-4 (anti-CTLA-4 antibodies), selected antibodies were tested for their activity in stimulating IFN-γ production on canine peripheral blood mononuclear cells using the following protocol.

[0248] Experiments and Results

[0249] Isolation of canine peripheral blood mononuclear cells:

[0250] 1. Collect ~20 mL of whole blood in EDTA or sodium heparin tubes.

[0251] 2. Transfer the blood to a 50 mL polystyrene tube and dilute 50:50 with Hank's Balanced Salt Solution (HBSS).

[0252] 3. Add 15 mL of Ficoll-Plaque to four x 50 mL SepMate TM Then, slowly add ~10 mL of 50:50 diluted blood to each SepMate tube containing Ficoll. TM Side of the tube.

[0253] 4. Centrifuge the tubes at 1200 x g for 20 minutes.

[0254] 5. Harvest cells from the gradient interface and transfer cells to a 50 mL polypropylene tube. Add HBSS to the 40-45 mL mark and centrifuge the cells at 800 x g for 10 minutes.

[0255] 6. Discard the supernatant, resuspend the cells in 40-45 mL of HBSS, and centrifuge the tube again at 800 x g for 10 minutes.

[0256] 7. Discard the supernatant and resuspend the cells from each tube with 2 mL of canine lymphocyte medium (pooled cells from the same animal).

[0257] 8. Take a small aliquot of the cell suspension, mix it with 0.04% trypan blue, and count the number of cells.

[0258] 9. Store the cell suspension at 2-7°C until use [but no longer than 24 hours before use.]

[0259] Cell proliferation assay of canine peripheral blood mononuclear cells:

[0260] 1. Dilute the antibody in canine lymphocyte medium [RPMI medium, purchased from LONZA, catalog number 12-167Q, or equivalent] to a final concentration of 40 μg / mL (make 160 μg / mL) and sterilize it using a 0.2 μm syringe filter. Place the two-fold diluted antibodies under a sterile dilution plate and set them aside.

[0261] 2. Dilute the cells to 2.5 × 10 in canine lymphocyte culture medium. 6 cells / mL and dispensed into an entire 96-well tissue culture plate at 100 μL / well.

[0262] 3. Dilute Con A in Canine Lymphocyte Medium to a final concentration of 250 ng / mL (prepared 1000 ng / mL), sterilize using a 0.2 μm syringe filter, and add 50 μL to all wells. (Note - Con A was not added to one column of eight wells for the cells only control, and is not expected to be added to one column of eight wells for the cells + mAb only control).

[0263] 4. Add 100 μL / well canine lymphocyte culture medium to the wells containing only cells and

[0264] 50 μL of medium was added to the column in control wells containing Con A (Con A+ cells, no mAb treatment).

[0265] 5. Add 50 μL of diluted mAb to duplicate wells.

[0266] 6. Incubate the plates at 36±2°C, 4.0-6.0% CO2 in a humidified incubator for 68 to 124 hours.

[0267] IFN-γ ELISA:

[0268] 1. After 68-124 hours of incubation, centrifuge the plates at 800 x g for 10 minutes.

[0269] 2. Collect supernatant from each well and pool replicates. These samples can be frozen at ≤ -50°C for later use or tested immediately.

[0270] 3. If necessary, dilute the supernatant sample appropriately and analyze according to the canine IFN-γQuantikin TM

[0271] Instructions for the ELISA kit [R&D Systems catalog number CAIF00] ELISA.

[0272] Figure 4 The results presented in demonstrate that the chimeric antibodies tested, especially 3B10, 411 and 611, significantly activated canine T cells to produce IFN-γ.

[0273] Example 6

[0274] Epitope mapping of caninized 3B10 antibody

[0275] The binding epitope of the caninized antibody 3B10L3H2 on canine CTLA-4 (NCBI reference sequence: NP_001003106; SEQ ID NO: 68) was mapped by chemical cross-linking and mass spectrometry. The results showed that the antibody binds to two separate linear regions on the antigen CTLA-4, including amino acids R33, R38, S42, T45, R83, T87, Y90, K93 and Y98 (see Figure 5 ).exist Figure 5 In FIG. 5 , the amino acid residues of the discontinuous epitope of CTLA-4 bound by the caninized antibody 3B10L3H2 are shown to consist of two linear portions, amino acid residues 30 to 50 and amino acid residues 80 to 100, respectively.

[0276] sequence

[0277] SEQ ID NO: 1: Chimeric mouse-canine heavy chain DNA (3B10):

[0278]

[0279]

[0280] SEQ ID NO:2: Chimeric mouse-canine heavy chain amino acid sequence (3B10):

[0281]

[0282] SEQ ID NO:3: Chimeric mouse-canine light chain DNA (3B10):

[0283]

[0284] SEQ ID NO:4: Chimeric mouse-canine light chain amino acid sequence (3B10):

[0285]

[0286]

[0287] SEQ ID NO:5: Chimeric mouse-canine heavy chain DNA (8H5):

[0288]

[0289]

[0290] SEQ ID NO: 6. Chimeric mouse-canine heavy chain amino acid sequence (8H5):

[0291]

[0292] SEQ ID NO:7: Chimeric mouse-canine light chain DNA (8H5):

[0293]

[0294]

[0295] SEQ ID NO:8: Chimeric mouse-canine light chain amino acid sequence (8H5):

[0296]

[0297] SEQ ID NO:9: Chimeric human-canine heavy chain DNA (411)

[0298]

[0299]

[0300] SEQ ID NO: 10: Chimeric human-canine heavy chain amino acid sequence (411):

[0301]

[0302]

[0303] SEQ ID NO: 11. Chimeric human-canine light chain DNA (411):

[0304]

[0305] SEQ ID NO: 12: Chimeric human-canine light chain amino acid sequence (411):

[0306]

[0307] SEQ ID NO: 13. Chimeric human-canine heavy chain DNA (418):

[0308]

[0309]

[0310] SEQ ID NO: 14. Chimeric human-canine heavy chain amino acid sequence (418):

[0311]

[0312] SEQ ID NO: 15 Chimeric human-canine light chain DNA (418):

[0313]

[0314]

[0315] SEQ ID NO: 16. Chimeric human-canine light chain amino acid sequence (418):

[0316]

[0317] SEQ ID NO: 17. Chimeric human-canine heavy chain DNA (611):

[0318]

[0319]

[0320] SEQ ID NO: 18: Chimeric human-canine heavy chain amino acid sequence (611):

[0321]

[0322] SEQ ID NO: 19: Chimeric human-canine light chain DNA (611)

[0323]

[0324]

[0325] SEQ ID NO: 20. Chimeric human-canine light chain amino acid sequence (611):

[0326]

[0327] SEQ ID NO: 21. Chimeric human-canine heavy chain DNA (10D1):

[0328]

[0329]

[0330] SEQ ID NO: 22. Chimeric human-canine heavy chain amino acid sequence (10D1):

[0331]

[0332]

[0333] SEQ ID NO: 23. Chimeric human-canine light chain DNA (10D1):

[0334]

[0335] SEQ ID NO: 24. Chimeric human-canine light chain amino acid sequence (10D1):

[0336]

[0337]

[0338] SEQ ID NO: 25. Chimeric human-canine heavy chain DNA (1E2):

[0339]

[0340]

[0341] SEQ ID NO: 26. Chimeric human-canine heavy chain amino acid sequence (1E2)

[0342]

[0343] SEQ ID NO:27: Chimeric human-canine light chain DNA (1E2):

[0344]

[0345]

[0346] SEQ ID NO:28: Chimeric human-canine light chain amino acid sequence (1E2):

[0347]

[0348] SEQ ID NO:29: Chimeric mouse-canine heavy chain DNA (3B3):

[0349]

[0350]

[0351] SEQ ID NO: 30. Chimeric mouse-canine heavy chain amino acid sequence (3B3):

[0352]

[0353]

[0354] SEQ ID NO:31: Chimeric mouse-canine light chain DNA (3B3):

[0355]

[0356] SEQ ID NO:32: Chimeric mouse-canine light chain amino acid sequence (3B3):

[0357]

[0358] SEQ ID NO:33: Caninized 3B10 heavy chain DNA (VH1)

[0359]

[0360]

[0361] SEQ ID NO: 34. Caninized 3B10 heavy chain amino acid sequence (VH1)

[0362]

[0363] SEQ ID NO:35: Caninized 3B10 heavy chain DNA (VH2)

[0364]

[0365]

[0366] SEQ ID NO:36: Caninized 3B10 heavy chain amino acid sequence (VH2)

[0367]

[0368]

[0369] SEQ ID NO:37: Caninized 3B10 heavy chain DNA (VH3)

[0370]

[0371]

[0372] SEQ ID NO: 38. Caninized 3B10 heavy chain amino acid sequence (VH3)

[0373]

[0374] SEQ ID NO:39: Caninized 3B10 heavy chain DNA (VH4)

[0375]

[0376]

[0377] SEQ ID NO:40: Caninized 3B10 heavy chain amino acid sequence (VH4)

[0378]

[0379]

[0380] SEQ ID NO:41: Caninized 3B10 light chain DNA (VL1)

[0381]

[0382] SEQ ID NO:42: Caninized 3B10 light chain amino acid sequence (VL1)

[0383]

[0384]

[0385] SEQ ID NO:43: Caninized 3B10 light chain DNA (VL2)

[0386]

[0387] SEQ ID NO:44: Caninized 3B10 light chain amino acid sequence (VL2)

[0388]

[0389] SEQ ID NO:45: Caninized 3B10 light chain DNA (VL3)

[0390]

[0391] SEQ ID NO:46: Caninized 3B10 light chain amino acid sequence (VL3)

[0392]

[0393] SEQ ID NO:59: Caninized 3B10 heavy chain IgGBm DNA (VH1)

[0394]

[0395]

[0396] SEQ ID NO:60: Caninized 3B10 heavy chain IgGBm amino acid sequence (VH1)

[0397]

[0398] SEQ ID NO:61: Caninized 3B10 heavy chain IgGBm DNA (VH2)

[0399]

[0400]

[0401] SEQ ID NO:62: Caninized 3B10 heavy chain IgGBm amino acid sequence (VH2)

[0402]

[0403] SEQ ID NO:63: Caninized 3B10 heavy chain IgGBm DNA (VH3)

[0404]

[0405]

[0406] SEQ ID NO:64: Caninized 3B10 heavy chain IgGBm amino acid sequence (VH3)

[0407]

[0408] SEQ ID NO:65: Caninized 3B10 heavy chain IgGBm DNA (VH4)

[0409]

[0410]

[0411] SEQ ID NO:66: Caninized 3B10 heavy chain amino acid IgGBm sequence (VH4)

[0412]

[0413]

[0414] SEQ ID NO:67: Canine CTLA-4 NA sequence (NCBI reference sequence: NP_001003106)

[0415] ATGGCGGGCTTTGGCTTTCGCCGCCATGGCGCGCAGCCGGATCTGGCGAGCCGCACCTGGCCGTGCACCGCGCTGTTTAGCCTGCTGTTTATTCCGGTGTTTAGCAAAGGCATGCATGTGGCGCAGCCGGCGGTGGTGCTGGCGAGCAGCCGCGGCGTGGCGAGCTTTGTGTGCGAATATGGCAGCAGCGGCAACGCGGCGGAAGTGCGCGTGACCGTGCTGCGCCAGGCGGGCAGCCAGATGACCGAAGTGTGCGCGGCGACCTATACCGTGGAAGATGAACTGGCGTTTCTGGATGATAGCACCTGCACCGGCACCAGCAGCGGCAACAAAGTGAACCTGACCATTCAGGGCCTGCGCGCGATGGATACCGGCCTGTATATTTGCAAAGTGGAACTGATGTATCCGCCGCCGTATTATGTGGGCATGGGCAACGGCACCCAGATTTATGTGATTGATCCGGAACCGTGCCCGGATAGCGATTTTCTGCTGTGGATTCTGGCGGCGGTGAGCAGCGGCCTGTTTTTTTATAGCTTTCTGATTACCGCGGTGAGCCTGAGCAAAATGCTGAAAAAACGCAGCCCGCTGACCACCGGCGTGTATGTGAAAATGCCGCCGACCGAACCGGAATGCGAAAAACAGTTTCAGCCGTATTTTATTCCGATTAAC

[0416] SEQ ID NO:68: Canine CTLA-4 AA sequence (NCBI reference sequence: NP_001003106).

[0417] MAGFGFRRHGAQPDLASRTWPCTALFSLLFIPVFSKGMHVAQPAVVLASSRGVASFVCEYGSSGNAAEVRVTVLRQAGSQMTEVCAATYTVEDELAFLDDSTCTGTSSGNK VNLTIQGLRAMDTGLYICKVELMYPPPYYVGMGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLITAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN

[0418] SEQ ID NO:69: Genetically modified cFc region of canine IgG B (from US10,106,107B2)

[0419] LGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARG QAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK

[0420] In all of the aforementioned nucleotide sequences, the variable regions are indicated in bold, while in the corresponding amino acid sequences, the sequences of the variable regions are indicated in bold and the CDRs are indicated in underline and bold.

[0421] Preferred embodiment:

[0422] 1. An isolated caninized antibody or antigen-binding fragment thereof that specifically binds to canine CTLA-4, comprising a canine IgG heavy chain and a canine κ light chain; wherein the canine κ light chain comprises three light chain complementary determining regions (CDRs): CDR light chain 1 (CDRL1), CDR light chain 2 (CDRL2), and CDR light chain 3 (CDRL3); and the canine IgG heavy chain comprises three heavy chain CDRs: CDR heavy chain 1 (CDRH1), CDR heavy chain 2 (CDRH2), and CDR heavy chain 3 (CDRH3):

[0423] (a) wherein CDRL1 comprises the amino acid sequence of SEQ ID NO: 54;

[0424] (b) wherein CDRL2 comprises an amino acid sequence comprising SEQ ID NO: 56;

[0425] (c) wherein CDRL3 comprises the amino acid sequence of SEQ ID NO: 58;

[0426] (d) wherein CDRH1 comprises the amino acid sequence of SEQ ID NO: 48;

[0427] (e) wherein CDRH2 comprises the amino acid sequence of SEQ ID NO: 50; and

[0428] (f) wherein the CDRH3 comprises the amino acid sequence of SEQ ID NO:52.

[0429] 2. The isolated caninized antibody of claim 1, wherein the canine IgG heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, and SEQ ID NO:66.

[0430] 3. The isolated caninized antibody of item 1 or 2, wherein the κ light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 46.

[0431] 4. The isolated caninized antibody of item 1, wherein the canine IgG heavy chain comprises the amino acid sequence of SEQ ID NO: 36 and the canine κ light chain comprises the amino acid sequence of SEQ ID NO: 46.

[0432] 5. The isolated caninized antibody of item 1, wherein the canine IgG heavy chain comprises the amino acid sequence of SEQ ID NO: 62 and the canine κ light chain comprises the amino acid sequence of SEQ ID NO: 46.

[0433] 6. An isolated nucleic acid encoding the canine kappa light chain of the caninized antibody of item 1, 2, 3, 4 or 5.

[0434] 7. An isolated nucleic acid encoding the canine IgG heavy chain of the caninized antibody of item 1, 2, 3, 4 or 5.

[0435] 8. An expression vector comprising the isolated nucleic acid of item 6 or 7.

[0436] 9. A host cell comprising one or more expression vectors according to item 8.

[0437] 10. A pharmaceutical composition comprising the antibody of item 1, 2, 3, 4 or 5 and a pharmaceutically acceptable carrier or diluent.

[0438] 11. A method for increasing the activity of immune cells, comprising administering a therapeutically effective amount of the pharmaceutical composition of item 10 to a subject in need thereof.

[0439] 12. The method of item 11, wherein the method is used for:

[0440] a) Treatment of cancer;

[0441] b) treatment of infection or infectious disease; or

[0442] c) As a vaccine adjuvant.

[0443] 13. A method for producing a caninized antibody or an antigen-binding fragment thereof that specifically binds to CTLA-4, comprising:

[0444] a. culturing the host cell of item 9 in a culture medium under conditions wherein the nucleic acid is expressed, thereby producing a polypeptide comprising light chain and heavy chain variable regions; and

[0445] b. recovering the polypeptide from the host cell or culture medium.

Claims

1. A nucleic acid comprising a first coding sequence selected from the group consisting of SEQ ID NO: 1, 33, 35, 37, 39, 59, 61, 63 and 65.

2. A nucleic acid comprising a second coding sequence selected from the group consisting of SEQ ID NO: 3, 41, 43 and 45.

3. A nucleic acid comprising the first coding sequence of claim 1 and the second coding sequence of claim 2.

4. A nucleic acid comprising: (a) a first coding sequence of SEQ ID NO: 1 and a second coding sequence of SEQ ID NO: 3; (b) a first coding sequence of SEQ ID NO:33 and a second coding sequence of SEQ ID NO:41; (c) a first coding sequence of SEQ ID NO:33 and a second coding sequence of SEQ ID NO:43; (d) a first coding sequence of SEQ ID NO:33 and a second coding sequence of SEQ ID NO:45; (e) a first coding sequence of SEQ ID NO:35 and a second coding sequence of SEQ ID NO:41; (f) a first coding sequence of SEQ ID NO:35 and a second coding sequence of SEQ ID NO:43; (g) a first coding sequence of SEQ ID NO:35 and a second coding sequence of SEQ ID NO:45; (h) a first coding sequence of SEQ ID NO:37 and a second coding sequence of SEQ ID NO:41; (i) a first coding sequence of SEQ ID NO:37 and a second coding sequence of SEQ ID NO:43; (j) a first coding sequence of SEQ ID NO:37 and a second coding sequence of SEQ ID NO:45; (k) a first coding sequence of SEQ ID NO:39 and a second coding sequence of SEQ ID NO:41; (1) a first coding sequence of SEQ ID NO:39 and a second coding sequence of SEQ ID NO:43; (m) a first coding sequence of SEQ ID NO:39 and a second coding sequence of SEQ ID NO:45; (n) a first coding sequence of SEQ ID NO:59 and a second coding sequence of SEQ ID NO:41; (o) a first coding sequence of SEQ ID NO:59 and a second coding sequence of SEQ ID NO:43; (p) a first coding sequence of SEQ ID NO:59 and a second coding sequence of SEQ ID NO:45; (q) a first coding sequence of SEQ ID NO:61 and a second coding sequence of SEQ ID NO:41; (r) a first coding sequence of SEQ ID NO:61 and a second coding sequence of SEQ ID NO:43; (s) a first coding sequence of SEQ ID NO:61 and a second coding sequence of SEQ ID NO:45; (t) a first coding sequence of SEQ ID NO:63 and a second coding sequence of SEQ ID NO:41; (u) a first coding sequence of SEQ ID NO:63 and a second coding sequence of SEQ ID NO:43; (v) a first coding sequence of SEQ ID NO:63 and a second coding sequence of SEQ ID NO:45; (w) a first coding sequence of SEQ ID NO:65 and a second coding sequence of SEQ ID NO:41; (x) a first coding sequence of SEQ ID NO:65 and a second coding sequence of SEQ ID NO:43; or (y) a first coding sequence of SEQ ID NO:65 and a second coding sequence of SEQ ID NO:

45.

5. A caninized antibody or antigen-binding fragment thereof that specifically binds to canine CTLA-4, comprising a heavy chain and a light chain; wherein: (a) the heavy chain comprises the amino acid sequence of SEQ ID NO: 34 and the light chain comprises the amino acid sequence of SEQ ID NO: 42; (b) the heavy chain comprises the amino acid sequence of SEQ ID NO:34 and the light chain comprises the amino acid sequence of SEQ ID NO:44; (c) the heavy chain comprises the amino acid sequence of SEQ ID NO:34 and the light chain comprises the amino acid sequence of SEQ ID NO:46; (d) the heavy chain comprises the amino acid sequence of SEQ ID NO:36 and the light chain comprises the amino acid sequence of SEQ ID NO:42; (e) the heavy chain comprises the amino acid sequence of SEQ ID NO:36 and the light chain comprises the amino acid sequence of SEQ ID NO:44; (f) the heavy chain comprises the amino acid sequence of SEQ ID NO:38 and the light chain comprises the amino acid sequence of SEQ ID NO:42; (g) the heavy chain comprises the amino acid sequence of SEQ ID NO:38 and the light chain comprises the amino acid sequence of SEQ ID NO:44; (h) the heavy chain comprises the amino acid sequence of SEQ ID NO:38 and the light chain comprises the amino acid sequence of SEQ ID NO:46; (i) the heavy chain comprises the amino acid sequence of SEQ ID NO: 40 and the light chain comprises the amino acid sequence of SEQ ID NO: 42; (j) the heavy chain comprises the amino acid sequence of SEQ ID NO:40 and the light chain comprises the amino acid sequence of SEQ ID NO:44; (k) the heavy chain comprises the amino acid sequence of SEQ ID NO:40 and the light chain comprises the amino acid sequence of SEQ ID NO:46; (1) the heavy chain comprises the amino acid sequence of SEQ ID NO: 60 and the light chain comprises the amino acid sequence of SEQ ID NO: 42; (m) the heavy chain comprises the amino acid sequence of SEQ ID NO: 60 and the light chain comprises the amino acid sequence of SEQ ID NO: 44; (n) the heavy chain comprises the amino acid sequence of SEQ ID NO:60 and the light chain comprises the amino acid sequence of SEQ ID NO:46; (o) the heavy chain comprises the amino acid sequence of SEQ ID NO: 62 and the light chain comprises the amino acid sequence of SEQ ID NO: 42; (p) the heavy chain comprises the amino acid sequence of SEQ ID NO:62 and the light chain comprises the amino acid sequence of SEQ ID NO:44; (q) the heavy chain comprises the amino acid sequence of SEQ ID NO: 64 and the light chain comprises the amino acid sequence of SEQ ID NO: 42; (r) the heavy chain comprises the amino acid sequence of SEQ ID NO:64 and the light chain comprises the amino acid sequence of SEQ ID NO:44; (s) the heavy chain comprises the amino acid sequence of SEQ ID NO:64 and the light chain comprises the amino acid sequence of SEQ ID NO:46; (t) the heavy chain comprises the amino acid sequence of SEQ ID NO:66 and the light chain comprises the amino acid sequence of SEQ ID NO:42; (u) the heavy chain comprises the amino acid sequence of SEQ ID NO: 66 and the light chain comprises the amino acid sequence of SEQ ID NO: 44; or (v) the heavy chain comprises the amino acid sequence of SEQ ID NO:66 and the light chain comprises the amino acid sequence of SEQ ID NO:

46.

6. A chimeric caninized antibody or antigen-binding fragment thereof that specifically binds to canine CTLA-4, comprising a heavy chain and a light chain; wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 2 and the light chain comprises the amino acid sequence of SEQ ID NO:

4.

7. A pharmaceutical composition comprising the nucleic acid of any one of claims 1 to 4 and a pharmaceutically acceptable carrier or diluent.

8. A pharmaceutical composition comprising the antibody of claim 5 or 6 and a pharmaceutically acceptable carrier or diluent.

9. An expression vector comprising the nucleic acid of any one of claims 1 to 4.

10. A host cell comprising one or more nucleic acids according to any one of claims 1 to 4 or one or more expression vectors according to claim 9.

11. A method for producing an antibody or antigen-binding fragment thereof that specifically binds to CTLA-4, comprising: (a) culturing the host cell of claim 10 in a culture medium under conditions wherein the nucleic acid is expressed, thereby producing one or more polypeptides; as well as (b) recovering the polypeptide from the host cell or culture medium.

12. The method of claim 11, wherein the antibody or antigen-binding fragment thereof that specifically binds to CTLA-4 is a caninized antibody or a chimeric antibody.

13. The method of claim 11 or 12, wherein the antibody or antigen-binding fragment thereof specifically binds canine CTLA-4.

14. Use of the pharmaceutical composition of claim 7 or 8 in the preparation of a medicament for increasing the activity of immune cells in a subject in need thereof.

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