Patent Application
20220251208
The present invention relates to antibodies to proteins involved in co-stimulatory or co-inhibitory signaling pathways, including CTLA-4. More particularly, the present invention further relates to caninized antibodies to canine CTLA-4 that have specific sequences and a high binding affinity for canine CTLA-4. The present invention also relates to use of the antibodies of the present invention in the treatment of cancer in canines.
The initiation or termination of immune responses is mediated via signaling pathways that are activated by complex interactions between a set 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 immune responses and have been shown to be mediated most importantly through the interaction of CD28 on the surface of T cells and B7.1 (also known as CD80) and B7.2 (also known as CD86) family members on the surface of APCs. B7.1 and B7.2 are thought to perform similar functions.
In contrast, co-inhibitory pathways lead to the inhibition or termination of the immune responses and have been shown to be mediated via the interaction between Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4) on T cells and CD80/CD86 proteins on APCs. Additional co-inhibitory signaling pathways have been shown to be mediated via the interaction between programmed cell death receptor 1 (PD-1) on T cells and programmed cell death receptor ligands 1 or 2 (PD-L1/PD-L2) proteins on APCs. Furthermore, it has also been shown that the interaction between PD-L1 and CD80 can also result in inhibitory signals in T cells.
CD80 and CD86 are members of the immunoglobulin (Ig) superfamily [Sharpe and Freeman, Nature Reviews, 2:116-126 (2002)]. CD80 is expressed on activated B cells, activated T cells, as well as macrophages, and dendritic cells [Swanson and Hall, Eur J. Immunol., 23:295-298 (1993); Razi-Wolfe et al., PNAS, 89:4210-4214 (1992)]. CD86 is constitutively expressed on dendritic cells, Langerhans cells, and B cells. In addition, CD86 is expressed on monocytes and is up-regulated following IFN-gamma stimulation [Larsen et al., Immunol, 152:5208-5219 (1994); Inaba, J. Exp. Med. 180:1849-1860 (1994)].
CD80 and CD86 bind 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)]. The binding of CD80 and CD86 to CTLA-4 has a much higher affinity than the binding of CD80/CD86 to CD28 [van der Merwe, J. Exp. Med. 185:393-402 (1997)].
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 containing a hexa-peptide 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 the majority of 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, B7.1/B7.2 binding to CD28 provides a critical co-stimulatory signal to the T cell allowing for T cell activation and subsequent development of the immune response [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 signal, the T cells undergo apoptosis or enter a state of unresponsiveness [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 alter the threshold level of TCR ligation (e.g., the amount of antigen-MHC complex) required for activation, reduce the time needed to stimulate naïve cells and enhance the magnitude of the T cell response [Soskic et al., Advances in Immunology, 124:96-123 (2014)].
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 naïve T cells, although it is rapidly up-regulated soon after CD28 ligation and T cell activation with a peak expression level of CTLA-4 at about 48-96 hours after the initial T cell activation [Alegre et al., J. Immunol., 157:4762-4770 (1996); Freeman et al., J. Immunol., 149:3795-3801 (1992)]. CTLA-4 binds to both B7.1 and B7.2 with a much higher affinity than CD28 [van der Merwe et al., J. Exp. Med., 185:393-402 (1997)]. However, in contrast to the stimulatory effects of CD28 binding B7.1 or B7.2, CTLA-4 acts as an inhibitory receptor that is vital for down-modulation of the immune response [Walnus et al., Immunity, 1:405-413 (1994); Walnus, J. Exp. Med., 183:2541-2550 (1996); Krummel and Allison, J. Exp. Med., 183:2533-2540 (1996)]. The mechanism by which CTLA-4 mediates its immune inhibitory functions are related to its capacity to act as a competitive inhibitor of the interaction between CD28 and CD80/CD86 [reviewed in Swanson, Immunology, 1010:169-177 (2000)]. The critical role of CTLA-4 in immune down-regulation is demonstrated in CTLA-4 deficient mice, which die by 3-5 weeks of age because of 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 was also demonstrated that the consequences of CTLA-4 knockout is dependent on the interaction of CD28 with its ligands CD80 and CD86 as shown by the lack of disease in the CTLA-4/CD80/CD86 triple knockout mice [Mandelbrot et al., J. Exp. Med., 189:435-440 (1999)]. This is also confirmed by the protection against lymphoproliferation afforded by repeated administration of CTLA-4 Ig in CTLA-4 knockout mice [Tivol et al., J Immunol., 158:5091-5094 (1997)].
In addition, blocking the effect of CTLA-4 with antibodies has been shown to enhance in vitro and in vivo T cell responses and to increase anti-tumor immune responses [Leach et al., Science, 271:1734-1736 (1996)]. Based on these findings, the development of CTLA-4 blockers such as monoclonal antibodies were undertaken to provide therapeutic modalities for treatment of cancer [Hodi et al., PNAS, 100(8):4712-4717 (2003); Phan G Q 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; U.S. Pat. No. 8,017,114 B2; WO2010097597A1; WO2012120125 A1; and Boutros et al., Nat Rev Clin Oncol., 13(8):473-486 (2016)].
PD-1 is a member of the CD28/CTLA-4 family of immune modulatory receptors. PD-1 is also a member of the Ig superfamily and contains an extracellular variable domain that binds its ligands and a cytoplasmic tail that binds signaling molecules [reviewed in Zak et al., Cell Structure, 25:1163-1174 (2017)]. The cytoplasmic tail of PD-1 contains two tyrosine-based signaling motifs [Zhang et al., Immunity 20:337-347 (2004)]. PD-1 expression is not found on unstimulated T cells, B cells, or myeloid cells. However, PD-1 expression is up-regulated on these cells following activation [Chemnitz et al., J. Immunol., 173:945-954 (2004); Petrvas et al., J. Exp. Med., 203:2281-2292 (2006)]. PD-1 is most closely related to CTLA-4, sharing approximately 24% amino acid identity [Jin et al., Current Topics in Microbiology and Immunology, 350:17-37 (2010)]. PD-1 attenuates T cell activation when bound to PD-L1 and PD-L2, which are expressed on the surface of APCs. The binding of either 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 function as ligands for PD-1. As with CTLA-4, PD-1 ligation appears to transmit a negative immunomodulatory signal. Ligation of PD-1 by PD-L1 or PD-L2 results in the 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 display signs of autoimmunity although the severity of the observed effects is not as profound as those exhibited 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 pathways are currently under intense investigation, research to date suggests that the PD-L1/PD-L2/PD-1 interactions are involved in the negative regulation of some immune responses because of diminishing the signals downstream of TCR stimulation leading to decreased cytokine secretion and impairment of T cell proliferation and decrease in the production of cytotoxic molecules by T cells [Freeman et al., J. Exp. Med., 192 (7):1027-1034 (2000)].
PD-L1 (CD274) is a type 1 membrane protein and consists of IgV-like and IgC-like extracellular domains, a hydrophobic transmembrane domain, and a short cytoplasmic tail made from 30 amino acids, with unknown signal transduction properties. PD-L1 is recognized as a member of the B7 family and shares approximately 20% amino acid identity with B7 family members. PD-L1 binds to its receptor, PD-1, found on activated T cells, B cells, and myeloid cells. PD-L1 also binds to the costimulatory molecule CD80, but not to CD86 [Butte et al., Immunology, 45 (13):3567-3572 (2008)]. The affinity of CD80 for PD-L1 is intermediate between its affinities for CD28 and CTLA-4. The related molecule PD-L2 has no affinity for either CD80 or CD86, but shares PD-1 as a receptor. Engagement of PD-L1 with its receptor PD-1 on T cells delivers a signal that inhibits TCR-mediated IL-2 production and T cell proliferation. PD-L1 binding to PD-1 also contributes to ligand-induced TCR down-modulation during antigen presentation to naive T cells. Additionally, PD-L1 binding to CD80 on T cells leads to T cell apoptosis. The role of PD-1 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, were undertaken to provide therapeutic modalities for treatment of cancer and infectious diseases.
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 use in the treatment of 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 [U.S. Pat. No. 9,944,704 B2, U.S. Pat. No. 10,106,607 B2, and U.S.2018/0237535 A1, the contents of which are hereby incorporated by reference in their entireties]. However, heretofore there have been no reports of a caninized monoclonal antibody that blocks the binding and activity of canine CTLA-4.
The citation of any reference herein should not be construed as an admission that such reference is available as “prior art” to the instant application.
The present invention relates to anti-canine Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4) antibodies that bind canine CTLA-4. In particular embodiments, the antibodies to canine CTLA-4 bind canine CTLA-4 with specificity. In more particular embodiments, the antibodies to canine CTLA-4 also have the ability to block the binding of canine CTLA-4 with canine CD80. In other particular embodiments, the antibodies to canine CTLA-4 also have the ability to block the binding of canine CTLA-4 with canine CD86. In still other particular embodiments, the antibodies to canine CTLA-4 have the ability to both block the binding of canine CTLA-4 with canine CD80 and to block the binding of canine CTLA-4 with canine CD86.
Moreover, the present invention relates to the complementary determining regions (CDRs) comprised by these antibodies and the combination of these CDRs (e.g., obtained from murine anti-canine CTLA-4 antibodies) into canine frames to form caninized anti-canine CTLA-4 antibodies. The present invention also relates to use of such antibodies in the treatment of conditions such as cancer.
Accordingly, the present invention provides unique sets of CDRs from six (6) exemplified murine anti-canine CTLA-4 antibodies. The six exemplified murine anti-canine CTLA-4 antibodies have unique sets of CDRs, i.e., three light chain CDRs: CDR light 1 (CDRL1), CDR light 2 (CDRL2), and CDR light 3 (CDRL3) and three heavy chain CDRs: CDR heavy 1 (CDRH1), CDR heavy 2 (CDRH2) and CDR heavy 3 (CDRH3). As detailed below, there is substantial sequence homology within each group of CDRs, and even some redundancy (e.g., see, the set of VL CDR-3's below in Table 1). Therefore, the present invention not only provides the amino acid sequences of the six CDRs from the six exemplified murine anti-canine CTLA-4 antibodies, but further provides conservatively modified variants of these CDRs, as well as variants that comprise (e.g., share) the same canonical structure and/or bind to one or more (e.g., 1, 2, 3, 4, or more) amino acid residues of canine CTLA-4 that are comprised by an epitope of canine CTLA-4.
One aspect of the present invention provides mammalian antibodies that bind canine Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4). In particular embodiments, a mammalian antibody or antigen binding fragment thereof of the present invention is a murine antibody. In preferred embodiments, the mammalian antibodies of the present invention, including murine antibodies of the present invention, or antigen binding fragments thereof are caninized antibodies or a caninized antigen binding fragment thereof.
In particular embodiments, the mammalian antibodies bind canine CTLA-4 with specificity. In more particular embodiments, the mammalian antibodies to canine CTLA-4 also have the ability to block the binding of canine CTLA-4 with canine CD80. In other particular embodiments, the mammalian antibodies to canine CTLA-4 also have the ability to block the binding of canine CTLA-4 with canine CD86. In still other particular embodiments, the mammalian antibodies to canine CTLA-4 have the ability to both block the binding of canine CTLA-4 with canine CD80 and to block the binding of canine CTLA-4 with canine CD86.
In certain embodiments the mammalian antibodies that bind canine CTLA-4 are isolated antibodies. The present invention further provides antigenic binding fragments of any of these mammalian antibodies that bind canine CTLA-4. In particular embodiments the antibodies comprises three light chain complementary determining regions (CDRs): CDR light 1 (CDRL1), CDR light 2 (CDRL2), and CDR light 3 (CDRL3); and three heavy chain CDRs: CDR heavy 1 (CDRH1), CDR heavy 2 (CDRH2) and CDR heavy 3 (CDRH3).
In particular embodiments, the mammalian antibody or an antigen binding fragment thereof comprises a CDRH3 that comprises the amino acid sequence of SEQ ID NO: 90, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 90, or a variant of SEQ ID NO: 90 that comprises the canonical structure class of 7. In more particular embodiments, the mammalian antibody or an antigen binding fragment thereof further comprises a CDRH2 that comprises the amino acid sequence of SEQ ID NO: 88, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 88, or a variant of SEQ ID NO: 88 that comprises the canonical structure class of 2A. In even more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRH1 that comprises the amino acid sequence of SEQ ID NO: 86, a CDRH1 that comprises a conservatively modified variant of the amino acid sequence of SEQ ID NO: 86, or a variant of SEQ ID NO: 86 that comprises the canonical structure class of 1. In still more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRL3 that comprises the amino acid sequence of SEQ ID NO: 96, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 96, or a variant of SEQ ID NO: 96 that comprises the canonical structure class of 1. In yet more particular embodiments, the mammalian antibody or an antigen binding fragment thereof further comprises a CDRL2 that comprises the amino acid sequence of SEQ ID NO: 94, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 94, or a variant of SEQ ID NO: 94 that comprises the canonical structure class of 1. In even more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRL1 that comprises the amino acid sequence of SEQ ID NO: 92, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 92, or a variant of SEQ ID NO: 92 that comprises the canonical structure class of 4.
In alternative embodiments, the mammalian antibody or an antigen binding fragment thereof comprises a CDRH3 that comprises the amino acid sequence of SEQ ID NO: 102, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 102, or a variant of SEQ ID NO: 102 that comprises the canonical structure class of 9. In more particular embodiments, the mammalian antibody or an antigen binding fragment thereof further comprises a CDRH2 that comprises the amino acid sequence of SEQ ID NO: 100, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 100, or a variant of SEQ ID NO: 100 that comprises the canonical structure class of 4. In even more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRH1 that comprises the amino acid sequence of SEQ ID NO: 98, a CDRH1 that comprises a conservatively modified variant of the amino acid sequence of SEQ ID NO: 98, or a variant of SEQ ID NO: 98 that comprises the canonical structure class of 1. In still more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRL3 that comprises the amino acid sequence of SEQ ID NO: 108, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 108, or a variant of SEQ ID NO: 108 that comprises the canonical structure class of 1. In yet more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRL2 that comprises the amino acid sequence of SEQ ID NO: 106, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 106, or a variant of SEQ ID NO: 106 that comprises the canonical structure class of 1. In even more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRL1 that comprises the amino acid sequence of SEQ ID NO: 104, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 104, or a variant of SEQ ID NO: 104 that comprises the canonical structure class of 1.
In other alternative embodiments, the mammalian antibody or an antigen binding fragment thereof comprises a CDRH3 that comprises the amino acid sequence of SEQ ID NO: 113, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 113, or a variant of SEQ ID NO: 113 that comprises the canonical structure class of 7. In more particular embodiments, the mammalian antibody or an antigen binding fragment thereof further comprises a CDRH2 that comprises the amino acid sequence of SEQ ID NO: 88, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 88, or a variant of SEQ ID NO: 88 that comprises the canonical structure class of 2A. In even more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRH1 that comprises the amino acid sequence of SEQ ID NO: 86, a CDRH1 that comprises a conservatively modified variant of the amino acid sequence of SEQ ID NO: 86, or a variant of SEQ ID NO: 86 that comprises the canonical structure class of 1. In still more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRL3 that comprises the amino acid sequence of SEQ ID NO: 96, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 96, or a variant of SEQ ID NO: 96 that comprises the canonical structure class of 1. In yet more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRL2 that comprises the amino acid sequence of SEQ ID NO: 94, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 94, or a variant of SEQ ID NO: 94 that comprises the canonical structure class of 1. In even more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRL1 that comprises the amino acid sequence of SEQ ID NO: 117, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 117, or a variant of SEQ ID NO: 117 that comprises the canonical structure class of 4.
In yet other alternative embodiments, the mammalian antibody or an antigen binding fragment thereof comprises a CDRH3 that comprises the amino acid sequence of SEQ ID NO: 115, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 115, or a variant of SEQ ID NO: 115 that comprises the canonical structure class of 7. In more particular embodiments, the mammalian antibody or an antigen binding fragment thereof further comprises a CDRH2 that comprises the amino acid sequence of SEQ ID NO: 88, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 88, or a variant of SEQ ID NO: 88 that comprises the canonical structure class of 2A. In even more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRH1 that comprises the amino acid sequence of SEQ ID NO: 86, a CDRH1 that comprises a conservatively modified variant of the amino acid sequence of SEQ ID NO: 86, or a variant of SEQ ID NO: 86 that comprises the canonical structure class of 1. In still more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRL3 that comprises the amino acid sequence of SEQ ID NO: 96, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 96, or a variant of SEQ ID NO: 96 that comprises the canonical structure class of 1. In yet more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRL2 that comprises the amino acid sequence of SEQ ID NO: 122, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 122, or a variant of SEQ ID NO: 122 that comprises the canonical structure class of 1. In even more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRL1 that comprises the amino acid sequence of SEQ ID NO: 119, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 119, or a variant of SEQ ID NO: 119 that comprises the canonical structure class of 4.
In still other alternative embodiments, the mammalian antibody or an antigen binding fragment thereof comprises a CDRH3 that comprises the amino acid sequence of SEQ ID NO: 114, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 114, or a variant of SEQ ID NO: 114 that comprises the canonical structure class of 7. In more particular embodiments, the mammalian antibody or an antigen binding fragment thereof further comprises a CDRH2 that comprises the amino acid sequence of SEQ ID NO: 111, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 111, or a variant of SEQ ID NO: 111 that comprises the canonical structure class of 2A. In even more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRH1 that comprises the amino acid sequence of SEQ ID NO: 109, a CDRH1 that comprises a conservatively modified variant of the amino acid sequence of SEQ ID NO: 109, or a variant of SEQ ID NO: 109 that comprises the canonical structure class of 1. In still more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRL3 that comprises the amino acid sequence of SEQ ID NO: 96, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 96, or a variant of SEQ ID NO: 96 that comprises the canonical structure class of 1. In yet more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRL2 that comprises the amino acid sequence of SEQ ID NO: 121, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 121, or a variant of SEQ ID NO: 121 that comprises the canonical structure class of 1. In even more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRL1 that comprises the amino acid sequence of SEQ ID NO: 118, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 118, or a variant of SEQ ID NO: 118 that comprises the canonical structure class of 4.
In yet other alternative embodiments, the mammalian antibody or an antigen binding fragment thereof comprises a CDRH3 that comprises the amino acid sequence of SEQ ID NO: 116, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 116, or a variant of SEQ ID NO: 116 that comprises the canonical structure class of 12. In more particular embodiments, the mammalian antibody or an antigen binding fragment thereof further comprises a CDRH2 that comprises the amino acid sequence of SEQ ID NO: 112, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 112, or a variant of SEQ ID NO: 112 that comprises the canonical structure class of 2A. In even more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRH1 that comprises the amino acid sequence of SEQ ID NO: 110, a CDRH1 that comprises a conservatively modified variant of the amino acid sequence of SEQ ID NO: 110, or a variant of SEQ ID NO: 110 that comprises the canonical structure class of 1. In still more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRL3 that comprises the amino acid sequence of SEQ ID NO: 124, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 124, or a variant of SEQ ID NO: 124 that comprises the canonical structure class of 1. In yet more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRL2 that comprises the amino acid sequence of SEQ ID NO: 123, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 123, or a variant of SEQ ID NO: 123 that comprises the canonical structure class of 1. In even more particular embodiments, the mammalian antibody or an antigen binding fragment thereof also further comprises a CDRL1 that comprises the amino acid sequence of SEQ ID NO: 120, a conservatively modified variant of the amino acid sequence of SEQ ID NO: 120, or a variant of SEQ ID NO: 120 that comprises the canonical structure class of 2.
As indicated above, caninized antibodies to canine CTLA-4 or caninized antigen binding fragments thereof are an important aspect of the present invention and the present invention provides caninized mammalian antibodies, including caninized murine antibodies, of all of such mammalian antibodies. Accordingly, the present invention also provides an isolated caninized antibody or antigen binding fragment thereof that specifically binds CTLA-4 comprising a canine IgG heavy chain and a canine kappa or lambda light chain. In particular embodiments of this type, the canine kappa or lambda light chain comprises three light chain complementary determining regions (CDRs): CDR light 1 (CDRL1), CDR light 2 (CDRL2), and CDR light 3 (CDRL3); and the canine IgG heavy chain comprises three heavy chain CDRs: CDR heavy 1 (CDRH1), CDR heavy 2 (CDRH2) and CDR heavy 3 (CDRH3) that are obtained from murine anti-canine CTLA-4 antibodies. Particular embodiments of the caninized antibodies and antigen binding fragments thereof of the present invention bind canine CTLA-4 and/or block the binding of canine CTLA-4 to canine CD80 and/or to canine CD86.
A caninized antibody of the present invention or caninized antigen binding fragment thereof, can comprise a IgGD that comprises a hinge region that comprises the amino acid sequence of SEQ ID NO: 128. In a related embodiment, the hinge region comprises the amino acid sequence of SEQ ID NO: 129. In yet another related embodiment, the hinge region comprises the amino acid sequence of SEQ ID NO: 130. In still another related embodiment, the hinge region comprises the amino acid sequence of SEQ ID NO: 131.
In alternative embodiments, a caninized antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO: 62. In specific embodiments of this type, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 61. In other embodiments, a caninized antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO: 64. In specific embodiments of this type, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 63. In still other embodiments, a caninized antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO: 66. In specific embodiments of this type, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 65. In more particular embodiments, the caninized antibody further comprises a light chain that comprises the amino acid sequence of SEQ ID NO: 50. In specific embodiments of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 49. In other particular embodiments, the caninized antibody further comprises a light chain that comprises the amino acid sequence of SEQ ID NO: 52. In specific embodiments of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 51. In still other particular embodiments, the caninized antibody further comprises a light chain that comprises the amino acid sequence of SEQ ID NO: 54. In specific embodiments of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 53.
In alternative embodiments, a caninized antibody comprises a modified heavy chain that comprises the amino acid sequence of SEQ ID NO: 74. In specific embodiment of this type, the modified heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 73. In other embodiments, a caninized antibody comprises a modified heavy chain that comprises the amino acid sequence of SEQ ID NO: 76. In specific embodiment of this type, the modified heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 75. In yet other embodiments, a caninized antibody comprises a modified heavy chain that comprises the amino acid sequence of SEQ ID NO: 78. In specific embodiments of this type, the modified heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 77. In more particular embodiments, the caninized antibody further comprises a light chain that comprises the amino acid sequence of SEQ ID NO: 50. In specific embodiment of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 49. In other particular embodiments, the caninized antibody further comprises a light chain that comprises the amino acid sequence of SEQ ID NO: 52. In specific embodiment of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 51. In still other particular embodiments, the caninized antibody further comprises a light chain that comprises the amino acid sequence of SEQ ID NO: 54. In specific embodiment of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 53.
In particular embodiments, the caninized antibodies comprise a heavy chain that comprises the amino acid sequence of SEQ ID NO: 66 and a light chain that comprises the amino acid sequence of SEQ ID NO: 52. In other embodiments, the caninized antibodies comprise a heavy chain that comprises the amino acid sequence of SEQ ID NO: 66 and a light chain that comprises the amino acid sequence of SEQ ID NO: 54.
In alternative embodiments, the caninized antibodies comprise a modified heavy chain that comprises the amino acid sequence of SEQ ID NO: 78 and a light chain that comprises the amino acid sequence of SEQ ID NO: 52. In other embodiments, the caninized antibodies comprise a modified heavy chain that comprises the amino acid sequence of SEQ ID NO: 78 and a light chain that comprises the amino acid sequence of SEQ ID NO: 54.
In other embodiments, a caninized antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO: 68. In specific embodiments of this type, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 67. In other embodiments, a caninized antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO: 70. In specific embodiments of this type, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 69. In still other embodiments, a caninized antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO: 72. In specific embodiments of this type, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 71. In more particular embodiments, the caninized antibody further comprises a light chain that comprises the amino acid sequence of SEQ ID NO: 56. In specific embodiments of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 55. In other particular embodiments, the caninized antibody further comprises a light chain that comprises the amino acid sequence of SEQ ID NO: 58. In specific embodiments of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 57. In still other particular embodiments, the caninized antibody further comprises a light chain that comprises the amino acid sequence of SEQ ID NO: 60. In specific embodiments of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 59.
In alternative embodiments, a caninized antibody comprises a modified heavy chain that comprises the amino acid sequence of SEQ ID NO: 80. In specific embodiment of this type, the modified heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 79. In other embodiments, a caninized antibody comprises a modified heavy chain that comprises the amino acid sequence of SEQ ID NO: 82. In specific embodiment of this type, the modified heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 81. In yet other embodiments, a caninized antibody comprises a modified heavy chain that comprises the amino acid sequence of SEQ ID NO: 84. In specific embodiments of this type, the modified heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 83. In more particular embodiments, the caninized antibody further comprises a light chain that comprises the amino acid sequence of SEQ ID NO: 56. In specific embodiments of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 55. In other particular embodiments, the caninized antibody further comprises a light chain that comprises the amino acid sequence of SEQ ID NO: 58. In specific embodiments of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 57. In still other particular embodiments, the caninized antibody further comprises a light chain that comprises the amino acid sequence of SEQ ID NO: 60. In specific embodiments of this type, the light chain is encoded by the nucleotide sequence of SEQ ID NO: 59.
In particular embodiments, the caninized antibodies comprise a modified heavy chain that comprises the amino acid sequence of SEQ ID NO: 72 and a light chain that comprises the amino acid sequence of SEQ ID NO: 58. In other embodiments, the caninized antibodies comprise a heavy chain that comprises the amino acid sequence of SEQ ID NO: 72 and a light chain that comprises the amino acid sequence of SEQ ID NO: 60.
In alternative embodiments, the caninized antibodies comprise a modified heavy chain that comprises the amino acid sequence of SEQ ID NO: 84 and a light chain that comprises the amino acid sequence of SEQ ID NO: 58. In other embodiments, the caninized antibodies comprise a modified heavy chain that comprises the amino acid sequence of SEQ ID NO: 84 and a light chain that comprises the amino acid sequence of SEQ ID NO: 60.
The present invention further provides mammalian antibodies or antigen binding fragments thereof that bind to canine CTLA-4 with a dissociation constant (Kd) that is lower than 1×10−12 M (e.g., 5×10−13 M, or lower). In other embodiments the mammalian antibodies or antigen binding fragments thereof bind to canine CTLA-4 with a dissociation constant of 1×10−5 M to 1×10−12 M. In more particular embodiments the mammalian antibodies or antigen binding fragments thereof bind to canine CTLA-4 with a dissociation constant of 1×10−7 M to 1×10−11 M. In still more particular embodiments the mammalian antibodies or antigen binding fragments thereof bind to canine CTLA-4 with a dissociation constant of 1×10−8 M to 1×10−11 M. In yet more particular embodiments the mammalian antibodies or antigen binding fragments thereof bind to canine CTLA-4 with a dissociation constant of 1×10−8 M to 1×10−10 M.
The present invention also provides mammalian antibodies or antigen binding fragments thereof that bind to canine CTLA-4 with an on rate (k0) that is greater than 1×107 M−1s−1. In other embodiments the mammalian antibodies or antigen binding fragments thereof bind to canine CTLA-4 with an on rate of 1×102 M−1s−1 to 1×107 M−1s−1. In more particular embodiments the mammalian antibodies or antigen binding fragments thereof bind to canine CTLA-4 with an on rate of 1×103 M−1s−1 to 1×106 M−1s−1. In still more particular embodiments the mammalian antibodies or antigen binding fragments thereof bind to canine CTLA-4 with an on rate of 1×103 M−1s−1 to 1×105 M−1s−1. In yet more particular embodiments the mammalian antibodies or antigen binding fragments thereof bind to canine CTLA-4 on rate of 1×104 M−1s−1 to 1×105 M−1s−1.
The present invention further provides mammalian antibodies or antigen binding fragments thereof that bind to canine CTLA-4 with an off rate (kw) slower than 1×10−7 s−1. In other embodiments, the mammalian antibodies or antigen binding fragments thereof bind to canine CTLA-4 with an off rate of 1×10−3 s−1 to 1×10's−1. In more particular embodiments the mammalian antibodies or antigen binding fragments thereof bind to canine CTLA-4 with an off rate of 1×104 s−1 to 1×10−7 s−1. In still more particular embodiments the mammalian antibodies or antigen binding fragments thereof bind to canine CTLA-4 with an off rate of 1×10−5s−1 to 1×10−7s−1.
In particular embodiments, a mammalian antibody of the present invention (including chimeric antibodies) blocks the binding of canine CD80 and/or CD86 with canine CTLA-4. In more particular embodiments the antibody blocks the binding of canine CD80 and/or CD86 with canine CTLA-4 with a minimum EC50 of 1×10−8 M to 1×10−9 M or an even lower concentration. In still more particular embodiments the EC50 is 5×10−9 M to 5×10−13 M. In still more particular embodiments the EC50 is between 5×10−9 M and 5×10−11 M. Accordingly, in particular embodiments, the antibodies of the present invention can exhibit one, two, three, four, or all these properties, i.e., the aforesaid dissociation constants with canine CTLA-4, the aforesaid on rates for binding with canine CTLA-4, the aforesaid off rates for dissociating from the antibody-canine CTLA-4 binding complex, or effective treating cancer in an animal subject.
The present invention further provides caninized mammalian antibodies and antigen-binding fragments that cross-compete with the mammalian antibodies disclosed herein. In particular embodiments, the caninized mammalian antibodies cross-compete with an antibody comprising the 6 CDRs of 45A9 [see, Table 1 below]. In related embodiments, the caninized mammalian antibodies cross-compete with an antibody comprising the 6 CDRs of 27G12 [see, Table 1 below]. In still other related embodiments, the caninized mammalian antibodies cross-compete with an antibody comprising the 6 CDRs of 22A11 [see, Table 1 below]. In yet other related embodiments, the caninized mammalian antibodies cross-compete with an antibody comprising the 6 CDRs of 110E3 [see, Table 1 below]. In specific embodiments, the caninized mammalian antibodies cross-compete with an antibody comprising the 6 CDRs of 12B3 [see, Tables 1 and 3 below]. In other specific embodiments, the caninized mammalian antibodies cross-compete with an antibody comprising the 6 CDRs of 39A11 [see, Tables 1 and 3 below]. In particular embodiments, the assay is a standard binding assay. In one such embodiment, the standard binding assay is performed with BIACore®. In another such embodiment, the standard binding assay is performed with an ELISA. In yet another such embodiment, the standard binding assay is performed by flow cytometry.
As indicated above, the antibodies (and antigen binding fragments thereof) of the present invention, including the aforesaid antibodies (and antigen binding fragments thereof), can be monoclonal antibodies (and antigen binding fragments thereof), mammalian antibodies (and antigen binding fragments thereof), e.g., murine (mouse) antibodies (and antigen binding fragments thereof), caninized antibodies (and antigen binding fragments thereof) including caninized murine antibodies (and antigen binding fragments thereof). In certain embodiments, the antibodies (and antigen binding fragments thereof) are isolated.
In preferred embodiments, a caninized antibody of the present invention or antigenic fragment thereof, binds to an epitope of the amino acid sequence of canine CTLA-4. In a particular embodiment, the caninized antibody interacts with one or more of the amino acid residue at positions T35, R38, T51, T53, Y90, K93, Y98 and Y102 of the amino acid sequence of SEQ ID NO: 138. In another embodiment the caninized antibody interacts with one or more of the amino acid residue at positions 35T, R38, S42, K93 and Y102 of the amino acid sequence of SEQ ID NO: 138.
The present invention further provides caninized antibodies that bind to one or more epitopes or portions thereof of the amino acid sequences of SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, and SEQ ID NO: 137. In particular embodiments, a caninized antibody of the present invention or antigenic fragment thereof, binds to an epitope or a portion thereof comprised by the amino acid sequence of SEQ ID NO: 132. In a more particular embodiment of this type, the epitope or portion thereof is comprised by the amino acid sequence of SEQ ID NO: 134. In another embodiment of this type, the epitope or a portion thereof is comprised by the amino acid sequence of SEQ ID NO: 135. In certain embodiments, the epitope or a portion thereof is comprised by the amino acid sequence of SEQ ID NO: 133. In a more particular embodiment of this type, the epitope or portion thereof is comprised by the amino acid sequence of SEQ ID NO: 136. In related embodiments, the caninized antibodies bind to one or more epitopes or portions thereof that are comprised by the amino acid sequences of SEQ ID NO: 134 and/or SEQ ID NO: 136 and/or SEQ ID NO: 135.
The present invention further provides nucleic acids (including isolated and/or recombinant nucleic acids) that encode any one of the light chains of the caninized antibody of the present invention. Similarly, the present invention provides isolated nucleic acids (including isolated and/or recombinant nucleic acids) that encode any one of the heavy chains of the caninized antibody of the present invention.
The present invention further provides expression vectors that comprise one or more of the nucleic acids (including isolated nucleic acids) of the present invention. The present invention also provides host cells that comprise one or more expression vectors of the present invention.
In particular embodiments, the antibody is a recombinant antibody or an antigen binding fragment thereof. In related embodiments, the variable heavy chain domain and variable light chain domain are connected by a flexible linker to form a single-chain antibody. In particular embodiments, the antibody or antigen binding fragment is a Fab fragment. In other embodiments, the antibody or antigen binding fragment is a Fab′ fragment. In yet 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 particular embodiments, the antibody or antigen binding fragment is a domain antibody. In particular embodiments, the antibody or antigen binding fragment is a single domain antibody.
In particular embodiments, a caninized murine anti-canine CTLA-4 antibody or antigen binding fragment binds to CTLA-4 in an animal subject (e.g., canine) being treated for cancer. In more particular embodiments, administration of a caninized murine anti-canine CTLA-4 antibody or antigen binding fragment of the present invention serves to ameliorate one or more symptom of cancer in the animal subject (e.g., canine) being treated.
The present invention further provides isolated nucleic acids that encode caninized murine anti-canine CTLA-4 antibodies or portions thereof. In related embodiments such antibodies or antigen binding fragments can be used for the preparation of a medicament to treat cancer in a canine subject. Alternatively, or in conjunction, the present invention provides for the use of any of the antibodies or antibody fragments of the present invention for diagnostic use. In yet additional embodiments, a kit is provided comprising any of the caninized antibodies or antigen binding fragments disclosed herein.
The present invention further provides isolated peptides that bind to a caninized antibody of the present invention, that comprise 5 to 25 amino acid residues, and are 90% identical or more to the amino acid sequence of SEQ ID NO: 132. In particular embodiments, the isolated peptides are identical to the amino acid sequence of SEQ ID NO: 132. In more particular embodiments, the isolated peptides comprise 10 to 20 amino acid residues. In related embodiments, the isolated peptides bind to a caninized antibody of the present invention, comprise 5 to 25 amino acid residues, and are 90% identical or more to the amino acid sequence of SEQ ID NO: 133. In particular embodiments, the isolated peptides are identical to the amino acid sequence of SEQ ID NO: 133. In more particular embodiments of this type, the isolated peptides comprise 10 to 20 amino acid residues.
In still other embodiments, the isolated peptides that bind to a caninized antibody of the present invention comprise amino acid sequences that are 90% identical or more to the amino acid sequence of SEQ ID NO: 134. In yet other embodiments, the isolated peptides comprise amino acid sequences that are identical to the amino acid sequence of SEQ ID NO: 134. In other embodiments, the isolated peptides that bind to a caninized antibody of the present invention comprise amino acid sequences that are 90% identical or more to the amino acid sequence of SEQ ID NO: 135. In still other embodiments, the isolated peptides comprise amino acid sequences that are identical to the amino acid sequence of SEQ ID NO: 135. In other embodiments, the isolated peptides that bind to a caninized antibody of the present invention comprise amino acid sequences that are 90% identical or more to the amino acid sequence of SEQ ID NO: 136. In yet other embodiments, the isolated peptides comprise amino acid sequences that are identical to the amino acid sequence of SEQ ID NO: 136.
The present invention further provides fusion proteins that comprise such isolated peptides that bind to a caninized antibody of the present invention. The present invention further provides fusion proteins that comprise any of the aforesaid peptides. In a particular embodiment, the fusion protein comprises such an antigenic peptide and an Fc region of a non-canine mammalian IgG antibody. In a more particular embodiment the fusion protein comprises an Fc region of a non-canine mammalian IgG antibody. In certain embodiments the non-canine mammalian IgG antibody is a murine IgG. In alternative embodiments the non-canine mammalian IgG antibody is a human IgG. In other embodiments the non-canine mammalian IgG antibody is an equine IgG. In still other embodiments the non-canine mammalian IgG antibody is a porcine IgG. In yet other embodiments the non-canine mammalian IgG antibody is a bovine IgG.
In particular embodiments the non-canine mammalian IgG antibody is an IgG1. In other embodiments the non-canine mammalian IgG antibody is an IgG2a. In still other embodiments the non-canine mammalian IgG antibody is an IgG3. In yet other embodiments the non-canine mammalian IgG antibody is an IgG4. In other embodiments the fusion protein comprises any of the aforesaid antigenic peptides and maltose-binding protein. In yet other embodiments, the fusion protein comprises any of the aforesaid antigenic peptides and beta-galactosidase. In still other embodiments the fusion protein comprises any of the aforesaid antigenic peptides and glutathione S-transferase. In yet other embodiments, the fusion protein comprises any of the aforesaid antigenic peptides and thioredoxin. In still other embodiments the fusion protein comprises any of the aforesaid antigenic peptides and Gro EL. In yet other embodiments the fusion protein comprises any of the aforesaid antigenic peptides and NusA.
The present invention also provides nucleic acids (including isolated and/or recombinant nucleic acids) that encode one or more isolated immunogenic and/or antigenic peptide and/or the fusion proteins of the present invention. The present invention further provides expression vectors comprising such isolated nucleic acids, as well as host cells that comprise one or more expression vectors of the present invention.
Pharmaceutical compositions can also comprise antigenic peptides (including isolated antigenic peptides) from canine CTLA-4, fusion proteins comprising the antigenic peptides from canine CTLA-4 of the present invention, nucleic acids (including isolated nucleic acids) encoding the antigenic fragments and/or fusion proteins of the present invention, the expression vectors comprising such nucleic acids, or any combination thereof, and a pharmaceutically acceptable carrier or diluent. In addition, the present invention includes pharmaceutical compositions comprising anti-canine CTLA-4 antibodies (including caninized murine anti-canine CTLA-4 antibodies) or antigen binding fragments thereof of the present invention. Such pharmaceutical compositions can be used to treat cancer, an infection or infective disease, be used as a vaccine adjuvant, and/or, in a method of increasing the activity of an immune cell, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition.
In particular embodiments, such pharmaceutical compositions further comprise an anti-canine PD-1 antibody (including a caninized murine anti-canine PD-1 antibody) or antigen binding fragment thereof. In more particular embodiments, the anti-canine PD-1 antibody is a caninized murine anti-canine PD-1 antibody or a antigen binding fragment of the caninized murine anti-canine PD-1 antibody.
In related embodiments, such pharmaceutical compositions further comprise an anti-canine PD-L1 antibody (including a caninized murine anti-canine PD-L1 antibody) or an antigen binding fragment thereof. In particular embodiments the anti-canine PD-L1 antibody is a caninized murine anti-canine PD-1 antibody or an antigen binding fragment of a caninized murine anti-canine PD-1 antibody.
Accordingly, the present invention provides pharmaceutical compositions that comprise one, two, three, or more of the following: an anti-canine PD-L1 antibody, an anti-canine PD-1 antibody, an anti-canine CTLA-4 antibody, an antigen binding fragment of an anti-canine PD-L1 antibody, an antigen binding fragment of an anti-canine PD-1 antibody, or an antigen binding fragment of an anti-canine CTLA-4 antibody. In particular embodiments, such anti-canine protein (i.e., anti-canine PD-L1, PD-1, or CTLA-4) antibodies or the antigen binding fragments thereof are murine anti-canine protein antibodies. In other embodiments, such anti-canine protein antibodies or the antigen binding fragments thereof are caninized anti-canine protein antibodies. In more particular embodiments, the anti-canine protein antibodies or the antigen binding fragments thereof are caninized murine anti-canine protein antibodies.
In addition, the present invention provides methods of increasing the activity of an immune cell, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the present invention. In certain embodiments the method is used in the treatment of cancer. In other embodiments, the method is used in the treatment of an infection or infectious disease. In still other embodiments, a caninized antibody of the present invention or antigen binding fragment thereof is used as a vaccine adjuvant. In particular embodiments a pharmaceutical composition comprising a caninized murine anti-canine CTLA-4 antibody or antigen binding fragment thereof can be administered before, after or concurrently with a caninized murine anti-canine PD-1 antibody or antigen binding fragment thereof and/or a caninized murine anti-canine PD-L1 antibody or antigen binding fragment thereof.
These and other aspects of the present invention will be better appreciated by reference to the following Brief Description of the Drawings and the Detailed Description.
Throughout the detailed description and examples of the invention the following abbreviations will be used:
So that the invention may be more readily understood, certain 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 meaning commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, including the appended claims, the singular forms of words such as “a,” “an,” and “the,” include their corresponding plural references unless the context clearly dictates otherwise.
“CTLA-4” is an abbreviation for “cytotoxic T-lymphocyte-associated protein 4”, also known as CD152 (cluster of differentiation 152), which is a protein receptor that functions as an immune checkpoint and downregulates immune responses. The amino acid sequence of canine CTLA-4 is SEQ ID NO: 126. The present invention further provides caninized murine antibodies to canine CTLA-4.
“Activation” as it applies to cells or to receptors refers to the activation or treatment of a cell or receptor with a ligand, unless indicated otherwise by the context or explicitly. Activation” can refer to cell activation as regulated by internal mechanisms as well as by external or environmental factors.
“Ligand” encompasses natural and synthetic ligands, e.g., cytokines, cytokine variants, analogues, muteins, and binding compounds derived from antibodies. “Ligand” also encompasses small molecules, e.g., peptide mimetics of cytokines and peptide mimetics of antibodies.”
“Activity” of a molecule may describe or refer to the binding of the molecule to a ligand or to a receptor, to catalytic activity; to the ability to stimulate gene expression or cell signaling, differentiation, or maturation; to antigenic activity, to the modulation of activities of other molecules, and the like. “Activity” of a molecule may also refer to activity in modulating or maintaining cell-to-cell interactions, e.g., adhesion, or activity in maintaining a structure of a cell, e.g., cell membranes or cytoskeleton. “Activity” can also mean specific activity, e.g., [catalytic activity]/[mg protein], or [immunological activity]/[mg protein], concentration in a biological compartment, or the like. “Activity” may refer to modulation of components of the innate or the adaptive immune systems.
“Administration” and “treatment,” as it applies to an animal, e.g., a canine subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal e.g., a canine subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell.
“Administration” and “treatment” also means in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell.
The term “subject” includes any organism, preferably an animal, more preferably a mammal (e.g., canine, feline, or human) and most preferably a canine.
“Treat” or “treating” means to administer a therapeutic agent, such as a composition containing any of the antibodies or antigen binding fragments of the present invention, internally or externally to e.g., a canine subject or patient having one or more disease symptoms, or being suspected of having a disease, for which the agent has therapeutic activity.
Typically, the agent is administered in an amount effective to alleviate and/or ameliorate one or more disease symptoms in the treated subject or population, whether by inducing the regression of or inhibiting the progression of such symptom(s) by any clinically measurable degree. The amount of a therapeutic agent that is effective to alleviate any particular disease symptom (also referred to as the “therapeutically effective amount”) may vary according to factors such as the disease state, age, and weight of the patient (e.g., canine), and the ability of the pharmaceutical composition to elicit a desired response in the subject. Whether a disease symptom has been alleviated or ameliorated can be assessed by any clinical measurement typically used by veterinarians or other skilled healthcare providers to assess the severity or progression status of that symptom. While an embodiment of the present invention (e.g., a treatment method or article of manufacture) may not be effective in alleviating the target disease symptom(s) in every subject, it should alleviate the target disease symptom(s) in a statistically significant number of subjects as determined by any statistical test known in the art such as the Student's t-test, the chi2-test, the U-test according to Mann and Whitney, the Kruskal-Wallis test (H-test), Jonckheere-Terpstra-test and the Wilcoxon-test.
“Treatment,” as it applies to a human, veterinary (e.g., canine), or research subject, refers to therapeutic treatment, as well as research and diagnostic applications. “Treatment” as it applies to a human, veterinary (e.g., canine), or research subject, or cell, tissue, or organ, encompasses contact of the antibodies or antigen binding fragments of the present invention to e.g., a canine or other animal subject, a cell, tissue, physiological compartment, or physiological fluid.
As used herein, the term “canine” includes all domestic dogs, Canis lupus familiaris or Canis familiaris, unless otherwise indicated.
As used herein, the term “feline” refers to any member of the Felidae family. Members of this family include wild, zoo, and domestic members, including domestic cats, pure-bred and/or mongrel companion cats, show cats, laboratory cats, cloned cats, and wild or feral cats.
As used herein the term “canine frame” refers to the amino acid sequence of the heavy chain and light chain of a canine antibody other than the hypervariable region residues defined herein as CDR residues. With regard to a caninized antibody, in the majority of embodiments the amino acid sequences of the native canine CDRs are replaced with the corresponding foreign CDRs (e.g., those from a mouse antibody) in both chains. Optionally the heavy and/or light chains of the canine antibody may contain some foreign non-CDR residues, e.g., so as to preserve the conformation of the foreign CDRs within the canine antibody, and/or to modify the Fc function, as exemplified below.
Canine CTLA-4 has been found to comprise the amino acid sequence of SEQ ID NO: 126 (including the signal sequence]. In a specific embodiment canine CTLA-4 is encoded by a nucleic acid that comprises the nucleotide sequence of SEQ ID NO: 125. Canine CTLA-4 sequences may differ by having, for example, conserved variations in non-conserved regions, but the canine CTLA-4 will have substantially the same biological function as the canine CTLA-4 comprised by the amino acid sequence of SEQ ID NO: 126.
As used herein, a “substitution of an amino acid residue” with another amino acid residue in an amino acid sequence of an antibody for example, is equivalent to “replacing an amino acid residue” with another amino acid residue and denotes that a particular amino acid residue at a specific position in the amino acid sequence has been replaced by (or substituted for) by a different amino acid residue. Such substitutions can be particularly designed i.e., purposefully replacing an alanine with a serine at a specific position in the amino acid sequence by e.g., recombinant DNA technology. Alternatively, a particular amino acid residue or string of amino acid residues of an antibody can be replaced by one or more amino acid residues through more natural selection processes e.g., based on the ability of the antibody produced by a cell to bind to a given region on that antigen, e.g., one containing an epitope or a portion thereof, and/or for the antibody to comprise a particular CDR that retains the same canonical structure as the CDR it is replacing. Such substitutions/replacements can lead to “variant” CDRs and/or variant antibodies.
Co-stimulatory signaling pathways lead to the development of immune responses and have been shown to be mediated through the interaction of CD28 on the surface of T cells and CD80 (also known as B7.1) and CD86 (also known as B7.2). CTLA-4 binds to both CD80 and CD86 with a much higher affinity than CD28 and thereby acts as an inhibitory receptor that is vital for down-modulation of the immune response. Indeed, the mechanism by which CTLA-4 mediates its immune inhibitory functions is related to its capacity to act as a competitive inhibitor of the interaction of CD28 with CD80 and CD86. Accordingly, the present invention describes the generation and characterization of monoclonal antibodies that block the binding of canine CD80 and canine CD86 to CTLA-4 and thereby, permits the co-stimulatory signaling due to the binding of canine CD28 to canine CD80 and CD86. These antibodies therefore have utility in treatment of cancer, as well as other diseases in companion animals as disclosed herein.
A particular canine CTLA-4 amino acid sequence will generally be at least 90% identical to the canine CTLA-4 comprising the amino acid sequence of SEQ ID NO: 126, excluding the signal sequence. In certain cases, a canine CTLA-4, may be at least 95%, or even at least 96%, 97%, 98% or 99% identical to the canine CTLA-4 comprising the amino acid sequence of SEQ ID NO: 126, excluding the signal sequence. In certain embodiments, a canine CTLA-4 amino acid sequence will display no more than 10 amino acid differences from the canine CTLA-4 comprising the amino acid sequence of SEQ ID NO: 126, excluding the signal sequence. In certain embodiments, the canine CTLA-4 amino acid sequence may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the canine CTLA-4 comprising the amino acid sequence of SEQ ID NO: 126, excluding the signal sequence. Percent identity can be determined as described herein below.
The term “immune response” refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the mammalian body (e.g., canine body) of cancerous cells, cells or tissues infected with pathogens, or invading pathogens.
Anti-Canine CTLA-4 Antibodies
The present invention provides isolated antibodies (particularly murine anti-canine CTLA-4 antibodies and caninized antibodies thereof) or antigen binding fragments thereof that bind canine CTLA-4 and uses of such antibodies or fragments thereof. In specific embodiments murine anti-canine CTLA-4 CDRs from murine anti-canine CTLA-4 antibodies are provided that have been shown to both bind canine CTLA-4 and to block the binding of canine CTLA-4 to one or both of its ligands, canine CD86 or CD80. These CDRs can be inserted into a modified canine frame of a canine antibody to generate a caninized murine anti-canine CTLA-4 antibody.
As used herein, an “anti-canine CTLA-4 antibody” refers to an antibody that was raised against canine CTLA-4 (e.g., in a mammal such as a mouse or rabbit) and that specifically binds to canine CTLA-4. An antibody that “specifically binds to canine CTLA-4,” and in particular to canine CTLA-4, or an antibody that “specifically binds to a polypeptide comprising the amino acid sequence of canine CTLA-4”, is an antibody that exhibits preferential binding to canine CTLA-4 as compared to other canine antigens, but this specificity does not require absolute binding specificity. An anti-canine CTLA-4 antibody is considered “specific” for canine CTLA-4 if its binding is determinative of the presence of canine CTLA-4 in a sample that is limited to canine proteins, or if it is capable of altering the activity of canine CTLA-4 without unduly interfering with the activity of other molecules in a canine sample, e.g. without producing undesired results such as false positives in a diagnostic context or side effects in a therapeutic context. The degree of specificity necessary for an anti-canine CTLA-4 antibody may depend on the intended use of the antibody, and at any rate is defined by its suitability for use for an intended purpose. The antibody, or binding compound derived from the antigen-binding site of an antibody, of the contemplated method binds to its antigen, or a variant or mutein thereof, with an affinity that is at least two-fold greater, preferably at least ten-times greater, more preferably at least 20-times greater, and most preferably at least 100-times greater than the affinity with any other canine antigen.
As used herein, an antibody is said to bind specifically to a polypeptide comprising a given antigen sequence (in this case a portion of the amino acid sequence of canine CTLA-4) if it binds to polypeptides comprising the portion of the amino acid sequence of canine CTLA-4, but does not bind to other canine proteins lacking that portion of the sequence of canine CTLA-4. For example, an antibody that specifically binds to a polypeptide comprising canine CTLA-4, may bind to a FLAG®-tagged form of canine CTLA-4, but will not bind to other FLAG®-tagged canine proteins. An antibody, or binding compound derived from the antigen-binding site of an antibody, binds to its canine antigen, or a variant or mutein thereof, “with specificity” when it has an affinity for that canine antigen or a variant or mutein thereof which is at least ten-times greater, more preferably at least 20-times greater, and even more preferably at least 100-times greater than its affinity for any other canine antigen tested.
As used herein, the term “antibody” refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), canonized antibodies, fully canine antibodies, chimeric antibodies and camelized single domain antibodies. “Parental antibodies” are antibodies obtained by exposure of an immune system to an antigen prior to modification of the antibodies for an intended use, such as caninization of an antibody for use as a canine therapeutic antibody.
As used herein, unless otherwise indicated, “antibody fragment” or “antigen binding fragment” refers to antigen binding fragments of antibodies, i.e. antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g. fragments that retain 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 antibody fragments.
A “Fab fragment” is comprised of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A “Fab fragment” can be the product of papain cleavage of an antibody.
A “fragment crystallizable” (“Fc”) region contains two heavy chain fragments comprising the CH3 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
A “Fab′ fragment” contains one light chain and a portion or fragment of one heavy chain that contains the VH domain and the CH1 domain and also the region between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab′ fragments to form a F(ab′)2 molecule.
A “F(ab′)2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab′) 2 fragment thus is composed of two Fab′ fragments that are held together by a disulfide bond between the two heavy chains. An “F(ab′)2 fragment” can be the product of pepsin cleavage of an antibody.
The “Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions.
The term “single-chain Fv” or “scFv” antibody refers to antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. [See, Pluckthun, T
As used herein, an anti-canine CTLA-4 antibody or antigen-binding fragment thereof that “blocks” or is “blocking” or is “blocking the binding” of canine CTLA-4 to its binding partner (ligand) e.g., canine CD80 or canine CD 86, is an anti-canine CTLA-4 antibody or antigen-binding fragment thereof that blocks (partially or fully) the binding of canine CTLA-4 to canine CD86 and/or CD80 as determined in standard binding assays (e.g., BIACore®, ELISA, or flow cytometry). Such “blocking” is exemplified in Example 4 below, using an ELISA-based blocking assay.
As used herein, the term “canonical structure” refers to the local conformation that can be adopted by each of the hypervariable regions of the heavy and light chain of an antibody within the framework that they reside. For each hypervariable region, there are a small number of canonical structures (generally denoted by simple integers such as 1 or 2 etc.), which can be predicted with great accuracy from the amino acid sequences of the corresponding hypervariable region [particularly within the context of the amino acid sequence of its framework for the corresponding anti-canine CTLA-4 variable domains]. These canonical structures can be determinative regarding whether a modification of the amino acid sequence of a given CDR will result in the retention or loss of the ability to bind to its antigen binding partner [See, Chothia and Lesk, Canonical Structures for the hypervariable regions of immunoglobulins, J. Mol. Biol. 196:901-917(1987); Chothia et al., Conformation of immunoglobulin hypervaribale regions, Nature, 34:877-883(1989); and Al-Lazikani et al., Standard Conformations for the canonical structures of immunoglobulins, J. Mol. Biol. 273:927-948 (1997)].
A “domain antibody” is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same or different antigens.
A “bivalent antibody” comprises two antigen binding sites. In some instances, the two binding sites have the same antigen specificities. However, bivalent antibodies may be bispecific (see below).
In certain embodiments, monoclonal antibodies herein also include camelized single domain antibodies. [See, e.g., Muyldermans et al., Trends Biochem. Sci. 26:230 (2001); Reichmann et al., J. Immunol. Methods 231:25 (1999); WO 94/04678; WO 94/25591; U.S. Pat. No. 6,005,079]. In one embodiment, the present invention provides single domain antibodies comprising two VH domains with modifications such that single domain antibodies are formed.
As used herein, the term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. [See, EP 0 404 097 B1; WO 93/11161; and Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)]. For a review of engineered antibody variants [generally see Holliger and Hudson Nat. Biotechnol. 23:1126-1136 (2005)].
Typically, an antibody or antigen binding fragment of the invention retains at least 10% of its canine CTLA-4 binding activity (when compared to the parental antibody) when that activity is expressed on a molar basis. Preferably, an antibody or antigen binding fragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the canine CTLA-4 binding affinity as the parental antibody. It is also intended that an antibody or antigen binding fragment of the invention can include conservative or non-conservative amino acid substitutions (referred to as “conservative variants” or “function conserved variants” of the antibody) that do not substantially alter its biologic activity.
“Isolated antibody” refers to the purification status and in such context means the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media. Generally, the term “isolated” is not intended to refer to a complete absence of such material or to an absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with experimental or therapeutic use of the binding compound as described herein.
As used herein, a “chimeric antibody” is an antibody having the variable domain from a first antibody and the constant domain from a second antibody, where the first and second antibodies are from different species. [U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)]. Typically the variable domains are obtained from an antibody from an experimental animal (the “parental antibody”), such as a rodent, and the constant domain sequences are obtained from the animal subject antibodies, e.g., human or canine so that the resulting chimeric antibody will be less likely to elicit an adverse immune response in a human or canine subject respectively, than the parental (e.g., rodent) antibody.
As used herein, the term “caninized antibody” refers to forms of antibodies that contain sequences from both canine and non-canine (e.g., murine) antibodies. In general, the caninized antibody will comprise substantially all of at least one or more typically, two variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-canine immunoglobulin (e.g., comprising 6 murine anti-canine CTLA-4 CDRs as exemplified below), and all or substantially all of the framework (FR) regions (and typically all or substantially all of the remaining frame) are those of a canine immunoglobulin sequence. As exemplified herein, a caninized antibody comprises both the three heavy chain CDRs and the three light chain CDRS from a murine anti-canine CTLA-4 antibody together with a canine frame or a modified canine frame. A modified canine frame comprises one or more amino acids changes as exemplified herein that further optimize the effectiveness of the caninized antibody, e.g., to increase its binding to canine CTLA-4 and/or its ability to block the binding of canine CTLA-4 to canine CD86 and/or CD80.
The term “fully canine antibody” refers to an antibody that comprises canine immunoglobulin protein sequences only. A fully canine antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, “mouse antibody” refers to an antibody that comprises mouse immunoglobulin sequences only. Alternatively, a fully canine antibody may contain rat carbohydrate chains if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell. Similarly, “rat antibody” refers to an antibody that comprises rat immunoglobulin sequences only.
There are four known IgG heavy chain subtypes of dog IgG and they are referred to as IgG-A, IgG-B, IgG-C, and IgG-D. The two known light chain subtypes are referred to as lambda and kappa.
The variable regions of each light/heavy chain pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are, in general, the same.
Typically, the variable domains of both the heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FR). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. In general, from N-terminal to C-terminal, both light and heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat, Adv. Prot. Chem. 32:1-75 (1978); Kabat, et al., J. Biol. Chem. 252:6609-6616 (1977); Chothia, et al., J. Mol. Biol. 196:901-917 (1987) or Chothia, et al., Nature 342:878-883 (1989)].
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 a “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), defining the CDR regions of an antibody by sequence; see also Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987) defining the CDR regions of an antibody by structure]. As used herein, the term “framework” or “FR” residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues.
In specific embodiments of the invention, besides binding and activating of canine immune cells, a canine or caninized antibody against CTLA-4 optimally has two attributes:
None of the naturally occurring canine IgG isotypes satisfy both criteria. For example, IgG-B can be purified using protein A, but has high level of ADCC activity. On the other hand, IgG-A binds weakly to protein A, but also displays ADCC activity. Moreover, neither IgG-C nor IgG-D can be purified on protein A columns, although IgG-D displays no ADCC activity. (IgG-C has considerable ADCC activity). One way the present invention addresses these issues is by providing modified canine IgG-B antibodies specific to CTLA-4 that lack the effector functions such as ADCC and can be easily of purified using industry standard protein A chromatography.
In alternative embodiments of the present invention, the canine IgG-B or IgG-C antibodies specific to CTLA-4 are purposely not modified to remove/substantially diminish the effector functions such as ADCC, and therefore retain the effector functions such as ADCC.
“Homology” refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences when they are optimally aligned. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology is the number of homologous positions shared by the two sequences divided by the total number of positions compared×100. For example, if 6 of 10 of the positions in two sequences are matched or homologous when the sequences are optimally aligned then the two sequences are 60% homologous. Generally, the comparison is made when two sequences are aligned to give maximum percent homology. “Isolated nucleic acid molecule” means a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin or some combination thereof which is not associated with all or a portion 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 purposes of this disclosure, it should be understood that “a nucleic acid molecule comprising” a particular nucleotide sequence does not encompass intact chromosomes. Isolated nucleic acid molecules “comprising” specified nucleic acid sequences may include, in addition to the specified sequences, coding sequences for up to ten or even up to twenty or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences, and/or may include vector sequences.
The phrase “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.
A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
As used herein, the expressions “cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny. Thus, the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that not all progeny will have precisely identical DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
As used herein, “germline sequence” refers to a sequence of unrearranged immunoglobulin DNA sequences. Any suitable source of unrearranged immunoglobulin sequences may be used. Human germline sequences may be obtained, for example, from JOINSOLVER® germline databases on the website for the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the United States National Institutes of Health. Mouse germline sequences may be obtained, for example, as described in Giudicelli et al. [Nucleic Acids Res. 33:D256-D261 (2005)].
The present invention provides isolated murine anti-canine CTLA-4 antibodies and caninized antibodies thereof, methods of use of the antibodies or antigen binding fragments thereof in the treatment of disease e.g., the treatment of cancer in canines. In canine, there are four IgG heavy chains referred to as A, B, C, and D. These heavy chains represent four different subclasses of dog IgG, which are referred to as IgGA, IgGB, IgGC and IgGD. Each of the two heavy chains consists of one variable domain (VH) and three constant domains referred to as CH-1, CH-2, and CH-3. The CH-1 domain is connected to the CH-2 domain via an amino acid sequence referred to as the “hinge” or alternatively as the “hinge region”.
The DNA and amino acid sequences of these four heavy chains were first identified by Tang et al. [Vet. Immunol. Immunopathol. 80: 259-270 (2001)]. The amino acid and DNA sequences for these heavy chains are also available from the GenBank data bases. For example, the amino acid sequence of IgGA heavy chain has accession number AAL35301.1, IgGB has accession number AAL35302.1, IgGC has accession number AAL35303.1, and IgGD has accession number (AAL35304.1). Canine antibodies also contain two types of light chains, kappa and lambda. The DNA and amino acid sequence of these light chains can be obtained from GenBank Databases. For example the kappa light chain amino acid sequence has accession number ABY 57289.1 and the lambda light chain has accession number ABY 55569.1.
In the present invention, the amino acid sequence for each of the four canine IgG Fc fragments is based on the identified boundary of CH1 and CH2 domains as determined by Tang et al, supra. Caninized murine anti-canine CTLA-4 antibodies that bind canine CTLA-4 include, but are not limited to: antibodies that comprise canine IgG-A, IgG-B, IgG-C, and IgG-D heavy chains and/or canine kappa light chains together with murine anti-canine CTLA-4 CDRs. Accordingly, the present invention provides isolated murine anti-canine CTLA-4 and/or caninized murine anti-canine CTLA-4 antibodies or antigen binding fragments thereof that bind to canine CTLA-4 and block the binding of canine CTLA-4 to canine CD86 and/or canine CD80.
The present invention further provides full length canine heavy chains that can be matched with corresponding light chains to make a caninized antibody. Accordingly, the present invention further provides caninized murine anti-canine antigen antibodies (including isolated caninized murine anti-canine CTLA-4 antibodies) and methods of use of the antibodies or antigen binding fragments thereof in the treatment of disease e.g., the treatment of cancer in canines.
The present invention also provides caninized murine anti-canine-CTLA-4 antibodies that comprise a canine fragment crystallizable region (cFc region) in which the cFc has been genetically modified to augment, decrease, or eliminate one or more effector functions. In one aspect of the present invention, the genetically modified cFc decreases or eliminates one or more effector functions. In another aspect of the invention the genetically modified cFc augments one or more effector function. In certain embodiments, the genetically modified cFc region is a genetically modified canine IgGB Fc region. In another such embodiment, the genetically modified cFc region is a genetically modified canine IgGC Fc region. In a particular embodiment the effector function is antibody-dependent cytotoxicity (ADCC) that is augmented, decreased, or eliminated. In another embodiment the effector function is complement-dependent cytotoxicity (CDC) that is augmented, decreased, or eliminated. In yet another embodiment, the cFc region has been genetically modified to augment, decrease, or eliminate both the ADCC and the CDC.
In order to generate variants of canine IgG that lack effector functions, a number of mutant canine IgGB heavy chains were generated. These variants may include one or more of the following single or combined substitutions in the Fc portion of the heavy chain amino acid sequence: P4A, D31A, N63A, G64P, T65A, A93G, and P95A. Variant heavy chains (i.e., containing such amino acid substitutions) were cloned into expression plasmids and transfected into HEK 293 cells along with a plasmid containing the gene encoding a light chain. Intact antibodies expressed and purified from HEK 293 cells were evaluated for binding to FcγRI and C1q to assess their potential for mediation of immune effector functions. [See, U.S. Pat. No. 10,106,607 B2, the contents of which are hereby incorporated by reference in its entirety.]
The present invention also provides modified canine IgGDs which in place of its natural IgGD hinge region they comprise a hinge region from:
Alternatively, the IgGD hinge region can be genetically modified by replacing a serine residue with a proline residue, i.e., PKESTCKCIPPCPVPES, SEQ ID NO: 131 (with the proline residue (P) underlined and in bold substituting for the naturally occurring serine residue). Such modifications can lead to a canine IgGD lacking fab arm exchange. The modified canine IgGDs can be constructed using standard methods of recombinant DNA technology [e.g., Maniatis et al., Molecular Cloning, A Laboratory Manual (1982)]. In order to construct these variants, the nucleic acids encoding the amino acid sequence of canine IgGD can be modified so that it encodes the modified IgGDs. The modified nucleic acid sequences are then cloned into expression plasmids for protein expression.
The antibody or antigen binding fragment thereof that binds canine CTLA-4 can comprise three, four, five, or six of the complementarity determining regions (CDRs) of a murine anti-canine antibody, as described herein. The three, four, five, or six CDRs may be independently selected from the CDR sequences of those provided below. In a further embodiment, the isolated antibody or antigen-binding fragment thereof that binds canine CTLA-4 comprises a canine antibody kappa or lambda light chain comprising a murine light chain CDR-1, CDR-2 and/or CDR-3 and a canine antibody heavy chain IgG comprising a murine heavy chain CDR-1, CDR-2 and/or CDR-3.
In other embodiments, the invention provides antibodies or antigen binding fragments thereof that specifically bind canine CTLA-4 and have canine antibody kappa or lambda light chains comprising a given set of three CDRs comprising at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the amino acid sequences of SEQ ID NOs: 92, 94, and 96 for the VLCDR-1, VLCDR-2 and VLCDR-3, respectively, and canine antibody heavy chain IgG comprising given set of three CDRs comprising at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the amino acid sequences of SEQ ID NOs: 86, 88, and 90 for the VHCDR-1, VHCDR-2 and VHCDR-3, respectively; or canine antibody kappa or lambda light chains comprising a given set of three CDRs comprising at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the amino acid sequences of SEQ ID NOs: 104, 106, and 108, for the VLCDR-1, VLCDR-2 and VLCDR-3, respectively, and canine antibody heavy chain IgG comprising a set of different CDRs comprising at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the amino acid sequences of SEQ ID NOs: 98, 100, and 102 for the VHCDR-1, VHCDR-2 and VHCDR-3, respectively; or canine antibody kappa or lambda light chains comprising a given set of three CDRs comprising at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the amino acid sequences of SEQ ID NOs: 117, 94, and 96, for the VLCDR-1, VLCDR-2 and VLCDR-3, respectively, and canine antibody heavy chain IgG comprising a set of different CDRs comprising at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the amino acid sequences of SEQ ID NOs: 86, 88, and 113 for the VHCDR-1, VHCDR-2 and VHCDR-3, respectively; or canine antibody kappa or lambda light chains comprising a given set of three CDRs comprising at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the amino acid sequences of SEQ ID NOs: 119, 122, and 96 for the VLCDR-1, VLCDR-2 and VLCDR-3, respectively, and canine antibody heavy chain IgG comprising a set of different CDRs comprising at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the amino acid sequences of SEQ ID NOs: 86, 88, and 115 for the VHCDR-1, VHCDR-2 and VHCDR-3, respectively; while still exhibiting the desired binding and functional properties. In another embodiment the antibody or antigen binding fragment of the present invention comprises a canine frame comprising a combination of IgG heavy chain sequence with a kappa or lambda light chain having one or more of the above-mentioned set of three light chain CDRs and three heavy chain CDRs with 0, 1, 2, 3, 4, or 5 conservative or non-conservative amino acid substitutions, while still exhibiting the desired binding and functional properties.
Sequence identity refers to the degree to which the amino acids of two polypeptides are the same at equivalent positions when the two sequences are optimally aligned. As used herein one amino acid sequence is 100% “identical” to a second amino acid sequence when the amino acid residues of both sequences are identical. Accordingly, 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. The sequence comparison is performed over a contiguous block of amino acid residues comprised by a given protein, e.g., a protein, or a portion of the polypeptide being compared. In particular embodiments selected deletions or insertions that could otherwise alter the correspondence between the two amino acid sequences are taken into account.
Sequence similarity includes identical residues and nonidentical, biochemically related amino acids. Biochemically related amino acids that share similar properties and may be interchangeable are discussed
“Conservatively modified variants” or “conservative substitution” refers to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g. charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that the changes can frequently be made without altering the biological activity of the protein. Those of skill in this 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 structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary conservative substitutions are set forth in Table A directly below.
Function-conservative variants of the antibodies of the invention are also contemplated by the present invention. “Function-conservative variants,” as used herein, refers to antibodies or fragments in which one or more amino acid residues have been changed without altering a desired property, such an antigen affinity and/or specificity. Such variants include, but are not limited to, replacement of an amino acid with one having similar properties, such as the conservative amino acid substitutions of Table A above.
The present invention further comprises the nucleic acids encoding the immunoglobulin chains of murine anti-canine CTLA-4 and/or caninized murine anti-canine CTLA-4 antibodies and antigen binding fragments thereof disclosed herein (see e.g., Examples below).
Also included in the present invention are nucleic acids that encode immunoglobulin polypeptides comprising amino acid sequences that are 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 sequences of the caninized antibodies provided herein when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences. The present invention further provides nucleic acids that encode immunoglobulin polypeptides comprising amino acid sequences that are at least about 70% similar, preferably at least about 80% similar, more preferably at least about 90% similar and most preferably at least about 95% similar (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to any of the reference amino acid sequences when the comparison is performed with a BLAST algorithm, wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences, are also included in the present invention.
As used herein, nucleotide and amino acid sequence percent identity can be determined using C, MacVector (MacVector, Inc. Cary, N.C. 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996) and the Clustal W algorithm with the alignment default parameters, and default parameters for identity. These commercially available programs can also be used to determine sequence similarity using the same or analogous default parameters. Alternatively, an Advanced Blast search under the default filter conditions can be used, e.g., using the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program using the default parameters.
The following references relate to BLAST algorithms often used for sequence analysis: BLAST ALGORITHMS: Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1990); Gish, W., et al., Nature Genet. 3:266-272 (1993); Madden, T. L., et al., Meth. Enzymol. 266:131-141(1996); Altschul, S. F., et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang, J., et al., Genome Res. 7:649-656 (1997); Wootton, J. C., et al., Comput. Chem. 17:149-163 (1993); Hancock, J. M. et al., Comput. Appl. Biosci. 10:67-70 (1994); ALIGNMENT SCORING SYSTEMS: Dayhoff, M O., et al., “A model of evolutionary change in proteins.” in Atlas of Protein Sequence and Structure, vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp. 345-352, (1978); Natl. Biomed. Res. Found., Washington, D.C.; Schwartz, R. M., et al., “Matrices for detecting distant relationships.” in Atlas of Protein Sequence and Structure, vol. 5, suppl. 3.” (1978), M. O. Dayhoff (ed.), pp. 353-358 (1978), Natl. Biomed. Res. Found., Washington, D.C.; Altschul, S. F., J. Mol. Biol. 219:555-565 (1991); States, D. J., et al., Methods 3:66-70(1991); Henikoff, S., et al., Proc. Natl. Acad. Sci. USA 89:10915-10919 (1992); Altschul, S. F., et al., J. Mol. Evol. 36:290-300 (1993); ALIGNMENT STATISTICS: Karlin, S., et al., Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990); Karlin, S., et al., Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); Dembo, A., et al., Ann. Prob. 22:2022-2039 (1994); and Altschul, S. F. “Evaluating the statistical significance of multiple distinct local alignments.” in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), pp. 1-14, Plenum, New York (1997).
This present invention also provides expression vectors comprising the nucleic acids of the invention, in which the nucleic acid is operably linked to control sequences that are recognized by a host cell when the host cell is transfected with the vector. Also provided are host cells comprising an expression vector of the present invention and methods for producing the antibody or antigen binding fragment thereof disclosed herein comprising culturing a host cell harboring an expression vector encoding the antibody or antigen binding fragment in culture medium and isolating the antigen or antigen binding fragment thereof from the host cell or culture medium.
A caninized murine anti-canine CTLA-4 antibody can be produced recombinantly by methods that are known in the field. Mammalian cell lines available as hosts for expression of the antibodies or fragments disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells. When recombinant expression vectors encoding the heavy chain or antigen-binding portion or fragment thereof, the light chain and/or antigen-binding fragment thereof are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.
Antibodies can be recovered from the culture medium using standard protein purification methods. Further, expression of antibodies of the invention (or other moieties therefrom) from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 and European Patent Application No. 89303964.4.
In general, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic for glycoproteins produced in the cell line or transgenic animal. Therefore, the particular glycosylation pattern of an antibody will depend on the particular cell line or transgenic animal used to produce the antibody. However, all antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein, comprise the instant invention, independent of the glycosylation pattern that the antibodies may have. Similarly, in particular embodiments, antibodies with a glycosylation pattern comprising only non-fucosylated N-glycans may be advantageous, because these antibodies have been shown to typically exhibit more potent efficacy than their fucosylated counterparts both in vitro and in vivo [See for example, Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Pat. Nos. 6,946,292 and 7,214,775].
The present invention further includes antibody fragments of the murine anti-canine CTLA-4 antibodies disclosed herein. The antibody fragments include F(ab)2 fragments, which may be produced by enzymatic cleavage of an IgG by, for example, pepsin. Fab fragments may be produced by, for example, reduction of F(ab)2 with dithiothreitol or mercaptoethylamine. A Fab fragment is a VL-CL chain appended to a VH-CH1 chain by a disulfide bridge. A F(ab)2 fragment is two Fab fragments which, in turn, are appended by two disulfide bridges. The Fab portion of an F(ab)2 molecule includes a portion of the Fc region between which disulfide bridges are located. An Fv fragment is a VL or VH region.
In one embodiment, the antibody or antigen binding fragment comprises a heavy chain constant region, e.g., a canine constant region, such as IgGA, IgGB, IgGC and IgGD canine heavy chain constant region or a variant thereof. In another embodiment, the antibody or antigen binding fragment comprises a light chain constant region, e.g., a canine light chain constant region, such as lambda or kappa canine light chain region or variant thereof. By way of example, and not limitation, the canine heavy chain constant region can be from IgG-B and the canine light chain constant region can be from kappa.
Antibody Engineering
Caninized murine anti-canine CTLA-4 antibodies of the present invention can be engineered to include modifications to canine framework and/or canine frame residues within the variable domains of a parental (i.e., canine) monoclonal antibody, e.g. to improve the properties of the antibody.
Epitope Binding and Binding Affinity
The present invention further provides antibodies or antigen binding fragments thereof that bind to amino acid residues of the same epitope of canine CTLA-4 as the murine anti-canine CTLA-4 antibodies disclosed herein. In particular embodiments the murine anti-canine CTLA-4 antibodies or antigen binding fragments thereof also are capable of inhibiting/blocking the binding of canine CTLA-4 to canine CD86 and/or CD80. In related embodiments the caninized murine anti-canine CTLA-4 antibodies or antigen binding fragments thereof also are capable of inhibiting/blocking the binding of canine CTLA-4 to canine CD86 and/or CD80.
Experimental and Diagnostic Uses
Murine anti-canine CTLA-4 and/or caninized murine anti-canine CTLA-4 antibodies or antigen-binding fragments thereof of the present invention may also be useful in diagnostic assays for canine CTLA-4 protein, e.g., detecting its expression in conjunction with and/or relation to cancer for example.
For example, such a method comprises the following steps:
In a further embodiment, the labeled antibody is labeled with peroxidase which react with ABTS [e.g., 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)] or 3,3′,5,5′-Tetramethylbenzidine (TMB) to produce a color change which is detectable. Alternatively, the labeled antibody is labeled with a detectable radioisotope (e.g., 41) which can be detected by scintillation counter in the presence of a scintillant. Murine anti-canine CTLA-4 antibodies of the invention may be used in a Western blot or immuno protein blot procedure.
Such a procedure forms part of the present invention and includes for example:
Detection of the bound antibody or antigen-binding fragment may be by binding the antibody or antigen-binding fragment with a secondary antibody (an anti-immunoglobulin antibody) which is detectably labeled and, then, detecting the presence of the secondary antibody.
The murine anti-canine CTLA-4 antibodies, the caninized murine anti-canine CTLA-4 antibodies, and/or the antigen-binding fragments thereof disclosed herein may also be used for immunohistochemistry. Such a method forms part of the present invention and comprises, e.g., (1) contacting a cell to be tested for the presence of canine CTLA-4 with e.g., a murine anti-canine CTLA-4 antibody or antigen-binding fragment thereof of the present invention; and (2) detecting the antibody or fragment on or in the cell. If the antibody or antigen-binding fragment itself is detectably labeled, it can be directly detected. Alternatively, the antibody or antigen-binding fragment may be bound by a detectably labeled secondary antibody which is detected.
Imaging techniques include SPECT imaging (single photon emission computed tomography) or PET imaging (positron emission tomography). Labels include e.g., iodine-123 (123I) and technetium-99m (99mTc), e.g., in conjunction with SPECT imaging or 11C, 13N, 15O or 18F, e.g., in conjunction with PET imaging or Indium-111 [See e.g., Gordon et al., International Rev. Neurobiol. 67:385-440 (2005)].
Cross-Blocking Antibodies
Furthermore, an anti-canine CTLA-4 antibody or antigen-binding fragment thereof of the present invention includes any antibody or antigen-binding fragment thereof that binds to the same epitope in canine CTLA-4 to which the antibodies and fragments discussed herein bind and any antibody or antigen-binding fragment that cross-blocks (partially or fully) or is cross-blocked (partially or fully) by an antibody or fragment discussed herein for canine CTLA-4 binding; as well as any variant thereof.
The cross-blocking antibodies and antigen-binding fragments thereof discussed herein can be identified based on their ability to cross-compete with the antibodies disclosed herein (on the basis of the CDRs as provided below in Example 5), i.e., 45A9, 27G12, 22A11, 110E3; and more particularly, 12B3 and/or 39A11 in standard binding assays (e.g., BIACore®, ELISA, as exemplified below, or flow cytometry). For example, standard ELISA assays can be used in which a recombinant canine CTLA-4 protein is immobilized on the plate, one of the antibodies is fluorescently labeled and the ability of non-labeled antibodies to compete off the binding of the labeled antibody is evaluated. Additionally or alternatively, BIAcore® analysis can be used to assess the ability of the antibodies to cross-compete. The ability of a test antibody to inhibit the binding of, for example, 27G12, 45A9, 110E3 and/or 22A11; and even more particularly 12B3 and/or 39A11, to canine CTLA-4 demonstrates that the test antibody can compete with 27G12, 45A9, 110E3 and/or 22A11, and/or 12B3 and/or 39A11 for binding to canine CTLA-4 and thus, may, in some cases, bind to the same epitope on canine CTLA-4 as 27G12, 45A9, 110E3 and/or 22A11, and/or 12B3 and/or 39A11. As stated above, antibodies and fragments that bind to the same epitope as any of the anti-canine CTLA-4 antibodies or fragments of the present invention also form part of the present invention.
Pharmaceutical Compositions and Administration
To prepare pharmaceutical or sterile compositions of a caninized murine anti-canine CTLA-4 antibody or antigen binding fragment thereof it can be admixed 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)].
Formulations of therapeutic and diagnostic agents may be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions [see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; 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, N.Y.]. In one embodiment, anti-CTLA-4 antibodies of the present invention are diluted to an appropriate concentration in a sodium acetate solution pH 5-6, and NaCl or sucrose is added for tonicity. Additional agents, such as polysorbate 20 or polysorbate 80, may be added to enhance stability.
Toxicity and therapeutic efficacy of the antibody compositions, administered alone or in combination with another agent, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (LD50/ED50). In particular aspects, antibodies exhibiting high therapeutic indices are desirable. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in canines. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration.
The mode of administration can vary. Suitable routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intra-arterial. In particular embodiments, the caninized murine anti-canine CTLA-4 antibody or antigen binding fragment thereof can be administered by an invasive route such as by injection. In further embodiments of the invention, a caninized murine anti-canine CTLA-4 antibody or antigen binding fragment thereof, or pharmaceutical composition thereof, is administered intravenously, subcutaneously, intramuscularly, intraarterially, or by inhalation, aerosol delivery. Administration by non-invasive routes (e.g., orally; for example, in a pill, capsule or tablet) is also within the scope of the present invention.
Compositions can be administered with medical devices known in the art. For example, a pharmaceutical composition of the invention can be administered by injection with a hypodermic needle, including, e.g., a prefilled syringe or autoinjector. The pharmaceutical compositions disclosed herein may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Pat. 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.
The pharmaceutical compositions disclosed herein may also be administered by infusion. Examples of well-known implants and modules form administering pharmaceutical compositions include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments. Many other such implants, delivery systems, and modules are well known to those skilled in the art.
Alternately, one may administer a murine anti-canine or a caninized murine anti-canine CTLA-4 antibody in a local rather than systemic manner, for example, via injection of the antibody directly into an arthritic joint or pathogen-induced lesion characterized by immunopathology, often in a depot or sustained release formulation. Furthermore, one may administer the antibody in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody, targeting, for example, arthritic joint or pathogen-induced lesion characterized by immunopathology. The liposomes will be targeted to and taken up selectively by the afflicted tissue.
The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of the target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic antibody to effect improvement in the target disease state, while simultaneously minimizing undesired side effects. Accordingly, the amount of biologic delivered depends in part on the particular therapeutic antibody and the severity of the condition being treated. Guidance in selecting appropriate doses of therapeutic antibodies is available [see, e.g., Wawrzynczak Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, U K (1996); Kresina (ed.) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y. (1991); Bach (ed.) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y. (1993); Baert, et al. New Engl. J. Med. 348:601-608 (2003); Milgrom et al. New Engl. J. Med. 341:1966-1973 (1999); Slamon et al. New Engl. J. Med. 344:783-792 (2001); Beniaminovitz et al. New Engl. J. Med. 342:613-619 (2000); Ghosh et al. New Engl. J. Med. 348:24-32 (2003); Lipsky et al. New Engl. J. Med. 343:1594-1602 (2000)].
Determination of the appropriate dose is made by the veterinarian, e.g., using parameters or factors known or suspected in the art to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., tumor size.
Antibodies or antigen binding fragments thereof disclosed herein may be provided by continuous infusion, or by doses administered, e.g., daily, 1-7 times per week, weekly, bi-weekly, monthly, bimonthly, quarterly, semiannually, annually etc. Doses may be provided, e.g., intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, intraspinally, or by inhalation. A total weekly dose is generally at least 0.05 μg/kg body weight, more generally at least 0.2 μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 0.25 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 5.0 mg/ml, 10 mg/kg, 25 mg/kg, 50 mg/kg or more [see, e.g., Yang, et al. New Engl. J. Med. 349:427-434 (2003); Herold, et al. New Engl. J. Med. 346:1692-1698 (2002); Liu, et al. J. Neurol. Neurosurg. Psych. 67:451-456 (1999); Portielji, et al. Cancer Immunol. Immunother. 52:133-144 (2003)]. Doses may also be provided to achieve a pre-determined target concentration of a caninized murine anti-canine CTLA-4 antibody in the subject's serum, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg/ml or more. In other embodiments, a caninized murine anti-canine CTLA-4 antibody of the present invention is administered subcutaneously or intravenously, on a weekly, biweekly, “every 4 weeks,” monthly, bimonthly, or quarterly basis at 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg/subject.
Antigenic peptides (e.g., peptides comprising epitopes or portions thereof from CTLA-4) that are recognized by anti-canine CTLA-4 mAbs also may be used as vaccines to elicit antibodies that block the binding of canine CTLA-4 to canine CD80 and/or CD86. Such vaccines may be useful as therapeutic vaccines for diseases such as cancer. In order to use these antigenic peptides as vaccines, one or more of these peptides may be coupled chemically or through the techniques of recombinant DNA technology to another carrier protein in order to enhance the immunogenicity of these peptides and elicit peptide-specific antibodies. Techniques for coupling peptides to carrier proteins are known to those skilled in the art. Peptide vaccines may be used to vaccinate animals by IM, S/C, oral, spray or in ovo routes. Peptide vaccines may be used as subunit proteins expressed from bacterial, viral, yeast or baculovirus virus systems. Alternatively such peptide vaccines may be delivered following administration of a variety of viral or bacterial vectors that express such peptide vaccines as can be practiced by methods known to those skilled in the art. The peptide vaccines may be administered in doses from 1-1000 μg and may optionally contain an adjuvant and an acceptable pharmaceutical carrier.
As used herein, “inhibit” or “treat” or “treatment” includes a postponement of development of the symptoms associated with a disorder and/or a reduction in the severity of the symptoms of such disorder. The terms further include ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result has been conferred on a vertebrate subject (e.g., a canine) with a disorder, disease or symptom, or with the potential to develop such a disorder, disease or symptom.
As used herein, the terms “therapeutically effective amount”, “therapeutically effective dose” and “effective amount” refer to an amount of a caninized murine anti-canine CTLA-4 antibody or antigen binding fragment thereof of the present invention that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to cause a measurable improvement in one or more symptoms of a disease or condition or the progression of such disease or condition. A therapeutically effective dose further refers to that amount of the binding compound sufficient to result in at least partial amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously. An effective amount of a therapeutic will result in an improvement of a diagnostic measure or parameter by at least 10%; usually by at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably by at least 50%. An effective amount can also result in an improvement in a subjective measure in cases where subjective measures are used to assess disease severity.
Other Combination Therapies
As previously described, a caninized murine anti-canine CTLA-4 antibody or antigen binding fragment thereof and/or an antigenic peptide of the present invention may be coadministered with one or other more therapeutic agents (such as an inhibitor as discussed in the next paragraph) and/or a caninized murine anti-canine PD-1 antibody [see e.g., U.S. Pat. No. 9,944,704 B2 and U.S. Pat. No. 10,106,107 B2, the contents of both of which are hereby incorporated by reference in their entireties] and/or a caninized murine anti-canine PD-L1 antibody [see e.g., U.S. 20180237535 A1, the contents of which are hereby incorporated by reference in their entireties]. The antibod(ies) may be linked to the agent (as an immunocomplex) and/or can be administered separately from the agent or other antibody. In the latter case (separate administration), the antibodies can be administered before, after or concurrently with the agent or can be coadministered with other known therapies.
Kits
Further provided are kits comprising one or more components that include, but are not limited to, an antibody or antigen binding fragment, as discussed herein, which specifically binds CTLA-4 (e.g., a caninized murine anti-canine CTLA-4 antibody or antigen binding fragment thereof) in association with one or more additional components including, a caninized murine anti-canine PD-1 antibody and/or a caninized murine anti-canine PD-L1 antibody. The binding compositions as described directly above, can be formulated as a pure composition or in combination with a pharmaceutically acceptable carrier, in a pharmaceutical composition.
In one embodiment, the kit includes a binding composition of the present invention (e.g., a caninized murine anti-canine CTLA-4 or a pharmaceutical composition thereof in one container (e.g., in a sterile glass or plastic vial), a caninized murine anti-canine PD-1 antibody, and/or a caninized murine anti-canine PD-L1 antibody or pharmaceutical composition(s) thereof in another container (e.g., in a sterile glass or plastic vial).
If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can also include a device for performing such administration. For example, the kit can include one or more hypodermic needles or other injection devices as discussed above. The kit can also include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids pet owners and veterinarians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the invention may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer/distributor information and patent information.
As a matter of convenience, an antibody or specific binding agent disclosed herein can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic or detection assay. Where the antibody or antibodies is/are labeled with an enzyme, the kit will include substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides the detectable chromophore or fluorophore). In addition, other additives may be included such as stabilizers, buffers (e.g., a block buffer or lysis buffer) and the like. The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay. Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.
Mouse monoclonal antibodies were generated using mouse hybridoma technology with the canine CTLA-4 (cCTLA-4) recombinant protein as the immunogen. Positive hybridoma clones were selected based on the antibody reactivity with cCTLA-4 and the blocking of the interaction of canine CD86 or CD80 with cCTLA-4 (blocking activity) by ELISA and FACS assays. Selected hybridoma clones were sequenced by rapid amplification of cDNA ends (RACE) for antibody fragments of VH and VL sequence. The six monoclonal antibodies selected are denoted as: 12B3, 27G12, 39A11, 45A9, 110E3 and 22A11, respectively. The amino acid sequences of the six antibodies are SEQ ID NOs: 2, 4, 6, 8, 10, and 12 for the heavy chain variable region respectively, and SEQ ID NOs: 14, 16, 18, 20, 22, and 24 for light chain variable region, respectively. The CDRs are underlined in the sequences provided below [see also, Table 1 below]. The corresponding nucleotide sequences that encode the above-identified amino acid sequences are listed as SEQ ID NOs: 1, 3, 5, 7, 9, and 11 for heavy chain variable region, respectively and SEQ ID NOs: 13, 15, 17, 19, 21, and 23 for light chain variable region, respectively. The nucleotide sequences of the heavy chain variable regions were fused to the nucleotide sequence of a modified canine constant heavy chain (CH1-Hinge-CH2-CH3), respectively, to produce a chimeric mouse-canine heavy chain nucleotide sequence designated as SEQ ID NOs: 25, 27, 29, 31, 33, and 35. The variable regions are in bold. The nucleotide sequences of the light chain variable region were fused to the nucleotide sequence of the canine constant kappa light chain domain, respectively to produce a chimeric mouse-canine light chain nucleotide sequence designated as SEQ ID NOs: 37, 39, 41, 43, 45, and 47. The variable regions are in bold. The amino acid sequences encoded by the chimeric mouse-canine heavy chain nucleotide sequences were designated as SEQ ID NOs: 26, 28, 30, 32, 34, and 36. The amino acid sequences encoded by the chimeric mouse-canine light chain nucleotide sequences were designated as SEQ ID NOs: 38, 40, 42, 44, 46, and 48. The variable regions are in bold and the CDRs are underlined. The chimeric human-canine heavy and light chains were cloned into separate expression plasmids using standard molecular biology techniques. Plasmids containing heavy and light chain genes were transfected into HEK 293 cells and the expressed antibody was purified from HEK 293 cell supernatant using protein A.
The CDRs from mouse anti-canine CTLA-4 monoclonal antibodies are listed in Table 1 below.
The individual canonical structure assignments for the six CDRs of each of the six antibodies are provided in Table 2 below.
A chimeric antibody usually possesses the same reactivity as its parental mouse antibody. To confirm the reactivity of the six antibodies with cCTLA-4, the mouse-canine chimeric antibodies were produced and tested for their reactivities with cCTLA-4 by ELISA as follows:
The ELISA results indicate that the chimeric antibodies can bind to cCTLA-4 [see,
To investigate the blocking activity of the chimeric antibodies, an ELISA-based blocking assay was conducted as follows:
The chimeric antibodies were found to block the interaction of cCTLA-4 with CD86
A CHO-K1 cell line stably expressing cCTLA-4 was generated. The cells were used to test antibody binding and blocking activity in FACS flow assay. To test cCTLA-4 binding activity of the chimeric antibodies, the FACS assay was conducted as follows:
The FACS results show that the chimeric antibody can bind to the CHO-cCTLA-4 cells [see,
Isolation of Canine Peripheral Blood Mononuclear Cells
Cell Proliferation Assay for Canine Peripheral Blood Mononuclear Cells
IFNγELISA
The results demonstrate that the selected antibodies, including 12B3, can activate canine T cells to produce IFNγ [see,
With their strong binding affinity for cCTLA-4 and their blocking activity on cCTLA-4 with its ligands, CD86 and CD80, murine antibodies 12B3 and 39A11 were selected for making the initial caninized antibodies. To execute the process of caninization, the DNA sequence that encodes the heavy and light chains of canine IgG were determined. The DNA and protein sequence of the canine heavy and light chains are known in the art and can be obtained by searching of the NCBI gene and protein databases. There are four known IgG subtypes of dog IgG and they are referred to as IgGA, IgGB, IgGC, and IgGD. Like human IgG1, canine IgGB has strong effector function. To knock out the effector function of IgGB, a modified IgGB was constructed (IgGBm) removing the native ADCC and CDC functions [see, U.S. Pat. No. 10,106,107 B2, hereby incorporated by reference in its entirety]. There are two types of light chains in canine antibodies referred to as kappa and lambda. Without being bound by any specific approach, the overall process of producing caninized heavy and light chains that can be mixed in different combinations to produce caninized anti-canine CTLA-4 mAbs may involve the following protocol:
The nucleotide and amino acid sequences of the CDRs of 12B3 and 39A11 are listed in Table 3 below.
A set of caninized Light and Heavy chain sequences were constructed. Their Sequence Identification Numbers are provided in Tables 4-6 below.
1The CDRs are underlined;
2the variable regions are in bold in the sequences that follow.
1The CDRs are underlined;
2the variable regions are in bold in the sequences that follow.
1The CDRs are underlined;
2the variable regions are in bold in the sequences that follow.
The present invention provides the caninized antibodies of 12B3 and 39A11 formed by the combination of caninized heavy and light chains of each antibody listed in the tables above; such antibodies demonstrate a particularly tight binding with cCTLA-4. As indicated in
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gatctagtcagagcattgtatatagtaatggaaacacctatttagaatggtacctgcagaaaccaggcca
gtctccaaagctcctgatctacaaagtttccaaccgattttctggggtcccagacaggttcagtggcagt
ggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttattactgct
ttcaaggttcacatgttccgtggacgttcggtggaggcaccaagctggaaatcaaacgcaacgatgcgca
DVLMTQTPLSLPVSLGDQASISC
WYLQKPGQSPKLLIY
GVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYC
FGGGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCL
gatgttttgatgacccagactccactctccctgcctgtcagtcttggagatcacgcctccatctcttgca
aatctagtcagagcattgtatatattaatggaaacacctatttagaatggtacctgcagaagccaggcca
gtctccaaagctcctgatctacaaagtttccaaacgattttctggggtcccagacaggttcagtggcagt
ggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttattactgct
ttcaaggttcacatgttccgtggacgttcggtggaggcaccaagctggaaatcaaacgcaacgatgcgca
DVLMTQTPLSLPVSLGDHASISC
WYLQKPGQSPKLLIY
GVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYC
FGGGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCL
gaaaatgtgctcatccagtctccagcaatcatgtctgcttctccaggggaaaaggtcaccatgacctgca
gggccagctcaagtgtaagttccagttacttgcactggtaccagcagaagtcaggtgcctcccccaaact
ctggatttttagcacatccaacttggcttctggagtccctgctcgcttcagtggcagtgggtctgggacc
tcttattctctcacaatcaacagtgtggaggctgaagatgctgccacttattactgccagcagtacagtg
gtctcccactcacgttcggaggggggaccaagctggaaataaaacgcaacgatgcgcagccggcggtgta
ENVLIQSPAIMSASPGEKVTMTC
WYQQKSGASPKLWIF
GVPARFSGSGSGT
SYSLTINSVEAEDAATYYC
FGGGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSF
gatgttttgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgca
gatctagtcagagtattgtatatagtcatggaaacacctatttagaatggtacctgcagaaaccaggcca
gtctccaaaggtcctgatctacaaagtttccaaccgattttctggggtcccagacaggttcagtggcagt
ggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttattactgct
ttcaaggttcacatgttccgtggacgttcggtggaggcaccaagctggaaatcaaacgcaacgatgcgca
DVLMTQTPLSLPVSLGDQASISC
WYLQKPGQSPKVLIY
GVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYC
FGGGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCL
gatgttttgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgca
gatctagtcagagcattgtatatattagtggaagcacctatttagaatggtatctgcagaaaccaggcca
gtctccaaagctcctgatctacaaagtttccagtcgattttctggggtcccagacaggttcagtggcagt
ggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttattactgct
ttcaaggttcacatgttccgtggacgttcggtggaggcaccaagctggaaatcaaacgcaacgatgcgca
DVLMTQTPLSLPVSLGDQASISC
WYLQKPGQSPKLLIY
GVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYC
FGGGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCL
gacatccagatgaaccagtctccatccagtctgtctgcatcccttggagacacaattaccatcacttgcc
atgccagtcagaacattaatgtttggttaagctggtaccagcagaaaccaggaaatattcctaaactttt
gatctataagtcttccaacttgcacacaggcgtcccatcaaggtttagtggcagtggatctggaacaggt
ttcacattaaccatcagcagcctgcagcctgaagacattgccacttactactgtcaacagggtcaaagtt
atccgtggacgttcggtggaggcaccaagctggaaatcaaacgcaacgatgcgcagccggcggtgtatct
DIQMNQSPSSLSASLGDTITITC
WYQQKPGNIPKLLIY
GVPSRFSGSGSGTG
FTLTISSLQPEDIATYYC
FGGGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFY
gatattgtgatgacccagaccccgctgagcctgagcgtgagcccgggcgaaccggcgagcattagctgcc
gcagcagccagagcattgtgtatagcaacggcaacacctatctggaatggtttcagcagaaaccgggcca
gagcccgcagcgcctgatttataaagtgagcaaccgctttagcggcgtgccggatcgctttagcggcagc
ggcagcggcaccgattttaccctgcgcattagccgcgtggaagcggatgatgcgggcgtgtattattgct
ttcagggcagccatgtgccgtggacctttggcggcggcaccaaactggaaattaaaaggaacgacgctca
DIVMTQTPLSLSVSPGEPASISC
WFQQKPGQSPQRLIY
GVPDRFSGS
GSGTDFTLRISRVEADDAGVYYC
FGGGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCL
gatattgtgatgacccagaccccgctgagcctgagcgtgagcccgggcgaaccggcgagcattagctgcc
gcagcagccagagcattgtgtatagcaacggcaacacctatctggaatggtatcagcagaaaccgggcca
gagcccgaaactgctgatttataaagtgagcaaccgctttagcggcgtgccggatcgctttagcggcagc
ggcagcggcaccgattttaccctgcgcattagccgcgtggaagcggatgatgcgggcgtgtattattgct
ttcagggcagccatgtgccgtggacctttggcggcggcaccaaactggaaattaaaaggaacgacgctca
DIVMTQTPLSLSVSPGEPASISC
WYQQKPGQSPKLLIY
GVPDRFSGS
GSGTDFTLRISRVEADDAGVYYC
FGGGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCL
gatgtgctgatgacccagaccccgctgagcctgagcgtgagcccgggcgaaccggcgagcattagctgcc
gcagcagccagagcattgtgtatagcaacggcaacacctatctggaatggtatctgcagaaaccgggcca
gagcccgaaactgctgatttataaagtgagcaaccgctttagcggcgtgccggatcgctttagcggcagc
ggcagcggcaccgattttaccctgcgcattagccgcgtggaagcggatgatgcgggcgtgtattattgct
ttcagggcagccatgtgccgtggacctttggcggcggcaccaaactggaactgaaaaggaacgacgctca
DVLMTQTPLSLSVSPGEPASISC
WYLQKPGQSPKLLIY
GVPDRFSGS
GSGTDFTLRISRVEADDAGVYYC
FGGGTKLELKRNDAQPAVYLFQPSPDQLHTGSASVVCL
gaaattgtgatgacccagagcccggcgagcctgagcctgagccaggaagaaaaagtgaccattacctgcc
gcgcgagcagcagcgtgagcagcagctatctgcattggtatcagcagaaaccgggccaggcgccgaaact
gctgatttatagcaccagcaacctggcgagcggcgtgccgagccgctttagcggcagcggcagcggcacc
gattttagctttaccattagcagcctggaaccggaagatgtggcggtgtattattgccagcagtatagcg
gcctgccgctgacctttggcggcggcaccaaactggaaattaaaaggaacgacgctcagccagccgtgta
WYQQKPGQAPKLLIY
GVPSRFSGSGSGT
FGGGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSF
gaaaacgtgctgacccagagcccggcgagcctgagcctgagccaggaagaaaaagtgaccattacctgcc
gcgcgagcagcagcgtgagcagcagctatctgcattggtatcagcagaaaccgggccaggcgccgaaact
gtggatttttagcaccagcaacctggcgagcggcgtgccgagccgctttagcggcagcggcagcggcacc
gattatagctttaccattagcagcctggaaccggaagatgtggcggtgtattattgccagcagtatagcg
gcctgccgctgacctttggcggcggcaccaaactggaactgaaaaggaacgacgctcagccagccgtgta
ENVLTQSPASLSLSQEEKVTITC
WYQQKPGQAPKLWIF
GVPSRFSGSGSGT
DYSETISSLEPEDVAVYYC
FGGGTKLELKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSF
gaaaacgtgctgacccagagcccggcgagcctgagcctgagcccgggcgaaaaagtgaccattacctgcc
gcgcgagcagcagcgtgagcagcagctatctgcattggtatcagcagaaaccgggccagagcccgaaact
gtggatttttagcaccagcaacctggcgagcggcgtgccgagccgctttagcggcagcggcagcggcacc
agctatagctttaccattagcagcctggaaccggaagatgtggcggtgtattattgccagcagtatagcg
gcctgccgctgacctttggcggcggcaccaaactggaactgaaaaggaacgacgctcagccagccgtgta
ENVLTQSPASLSLSPGEKVTITC
WYQQKPGQSPKLWIF
GVPSRFSGSGSGT
SYSETISSLEPEDVAVYYC
FGGGTKLELKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSF
gaagtgcagctggtggaaagcggcggcgatctggtgaaaccgggcggcagcctgcgcctgagctgcgtgg
cgagcggctatacctttaccaactatggcatgaactgggtgcgccaggcgccgggcaaaggcctgcagtg
ggtggcgtggattaacacctataccggcgaaccgacctatgcggatgattttaaaggccgctttaccatt
agccgcgataacgcgaaaaacaccctgtatctgcagatgaacagcctgcgcgcggaagataccgcggtgt
attattgcgcgcgccgcagcatttattatccgtattggggccagggcaccaccctgaccgtgagcagcgc
EVQLVESGGDLVKPGGSLRLSCVASGYTFT
WVRQAPGKGLQWVA
RFTI
SRDNAKNTLYLQMNSLRAEDTAVYYCAR
WGQGTTLTVSSASTTAPSVFPLAPSCGSTSGSTVA
gaaattcagctggtgcagagcggcggcgatctggtgaaaccgggcggcagcctgcgcctgagctgcaaag
cgagcggctatacctttaccaactatggcatgaactgggtgcgccaggcgccgggcaaaggcctgcagtg
gatgggctggattaacacctataccggcgaaccgacctatgcggatgattttaaaggccgctttaccttt
agcctggataacgcgaaaaacaccctgtatctgcagatgaacagcctgcgcgcggaagataccgcggtgt
atttttgcgcgcgccgcagcatttattatccgtattggggccagggcaccaccctgaccgtgagcagcgc
ETQLVQSGGDLVKPGGSLRLSCKASGYTFT
WVRQAPGKGLQWMG
RFTF
SLDNAKNTLYLQMNSLRAEDTAVYFCAR
WGQGTTLTVSSASTTAPSVFPLAPSCGSTSGSTVA
gaaattcagctggtgcagagcggcggcgatctggtgaaaccgggcggcagcgtgcgcctgagctgcaaag
cgagcggctatacctttaccaactatggcatgaactgggtgaaacaggcgccgggcaaaggcctgcagtg
gatgggctggattaacacctataccggcgaaccgacctatgcggatgattttaaaggccgctttaccttt
agcctggataacgcgaaaaacaccgcgtatctgcagattaacagcctgcgcgcggaagataccgcggtgt
atttttgcgcgcgccgcagcatttattatccgtattggggccagggcaccaccctgaccgtgagcagcgc
EIQLVQSGGDLVKPGGSVRLSCKASGYTFT
WVKQAPGKGLQWMG
RFTF
SLDNAKNTAYLQINSLRAEDTAVYFCAR
WGQGTTLTVSSASTTAPSVFPLAPSCGSTSGSTVA
gaagtgcagctggtggaaagcggcggcgatctggtgaaaccgggcggcagcctgcgcctgagctgcgtgg
cgagcggctttacctttagcgattattatatgagctgggtgcgccaggcgccgggcaaaggcctggaatg
ggtggcgtttattcgcaacaaagcgaacggctataccaccgaatatagcgcgagcctgaaaggccgcttt
accattagccgcgataacgcgaaaaacatggcgtatctgcagatgaacagcctgcgcgcggaagataccg
cggtgtattattgcgcgagctttggcctgatgtattattttgattattggggccagggcaccaccctgac
cgtgagcagcgcttccacaaccgcgccatcagtctttccgttggccccatcatgcgggtcgacgagcgga
EVQLVESGGDLVKPGGSLRLSCVASGETFS
WVRQAPGKGLEWVA
RF
TISRDNAKNMAYLQMNSLRAEDTAVYYCAS
WGQGTTLTVSSASTTAPSVFPLAPSCGSTSG
gaagtgcagctggtggaaagcggcggcgatctggtgaaaccgggcggcagcctgcgcctgagctgcgcga
ccagcggctttacctttagcgattattatatgagctgggtgcgccaggcgccgggcaaaggcctggaatg
gatgggctttattcgcaacaaagcgaacggctataccaccgaatatagcgcgagcctgaaaggccgcttt
accattagccgcgataacgcgaaaaacatggcgtatctgcagatgaacagcctgcgcgcggaagataccg
cggtgtattattgcgtgcgctttggcctgatgtattattttgattattggggccagggcaccaccctgac
cgtgagcagcgcttccacaaccgcgccatcagtctttccgttggccccatcatgcgggtcgacgagcgga
EVQLVESGGDLVKPGGSLRLSCATSGETFS
WVRQAPGKGLEWMG
RF
TISRDNAKNMAYLQMNSLRAEDTAVYYCVR
WGQGTTLTVSSASTTAPSVFPLAPSCGSTSG
gaagtgaaactggtggaaagcggcggcgatctggtgaaaccgggcggcagcctgcgcctgagctgcgcga
ccagcggctttacctttagcgattattatatgagctgggtgcgccaggcgccgggcaaagcgctggaatg
gatgggctttattcgcaacaaagcgaacggctataccaccgaatatagcgcgagcctgaaaggccgcttt
accattagccgcgataacgcgaaaaacatgctgtatctgcagatgaacagcctgcgcgcggaagataccg
cggtgtattattgcgtgcgctttggcctgatgtattattttgattattggggccagggcaccaccctgac
cgtgagcagcgcttccacaaccgcgccatcagtctttccgttggccccatcatgcgggtcgacgagcgga
EVKLVESGGDLVKPGGSLRLSCATSGETFS
WVRQAPGKALEWMG
RF
TISRDNAKNMLYLQMNSLRAEDTAVYYCVR
WGQGTTLTVSSASTTAPSVFPLAPSCGSTSG
gaagtgcagctggtggaaagcggcggcgatctggtgaaaccgggcggcagcctgcgcctgagctgcgtgg
cgagcggctatacctttaccaactatggcatgaactgggtgcgccaggcgccgggcaaaggcctgcagtg
ggtggcgtggattaacacctataccggcgaaccgacctatgcggatgattttaaaggccgctttaccatt
agccgcgataacgcgaaaaacaccctgtatctgcagatgaacagcctgcgcgcggaagataccgcggtgt
attattgcgcgcgccgcagcatttattatccgtattggggccagggcaccaccctgaccgtgagcagcgc
EVQLVESGGDLVKPGGSLRLSCVASGYTFT
WVRQAPGKGLQWVA
RFTI
SRDNAKNTLYLQMNSLRAEDTAVYYCAR
WGQGTTLTVSSASTTAPSVFPLAPSCGSTSGSTVA
gaaattcagctggtgcagagcggcggcgatctggtgaaaccgggcggcagcctgcgcctgagctgcaaag
cgagcggctatacctttaccaactatggcatgaactgggtgcgccaggcgccgggcaaaggcctgcagtg
gatgggctggattaacacctataccggcgaaccgacctatgcggatgattttaaaggccgctttaccttt
agcctggataacgcgaaaaacaccctgtatctgcagatgaacagcctgcgcgcggaagataccgcggtgt
atttttgcgcgcgccgcagcatttattatccgtattggggccagggcaccaccctgaccgtgagcagcgc
ETQLVQSGGDLVKPGGSLRLSCKASGYTFT
WVRQAPGKGLQWMG
RFTF
SLDNAKNTLYLQMNSLRAEDTAVYFCAR
WGQGTTLTVSSASTTAPSVFPLAPSCGSTSGSTVA
gaaattcagctggtgcagagcggcggcgatctggtgaaaccgggcggcagcgtgcgcctgagctgcaaag
cgagcggctatacctttaccaactatggcatgaactgggtgaaacaggcgccgggcaaaggcctgcagtg
gatgggctggattaacacctataccggcgaaccgacctatgcggatgattttaaaggccgctttaccttt
agcctggataacgcgaaaaacaccgcgtatctgcagattaacagcctgcgcgcggaagataccgcggtgt
atttttgcgcgcgccgcagcatttattatccgtattggggccagggcaccaccctgaccgtgagcagcgc
ETQLVQSGGDLVKPGGSVRLSCKASGYTFT
WVKQAPGKGLQWMG
RFTF
SLDNAKNTAYLQINSLRAEDTAVYFCAR
WGQGTTLTVSSASTTAPSVFPLAPSCGSTSGSTVA
gaagtgcagctggtggaaagcggcggcgatctggtgaaaccgggcggcagcctgcgcctgagctgcgtgg
cgagcggctttacctttagcgattattatatgagctgggtgcgccaggcgccgggcaaaggcctggaatg
ggtggcgtttattcgcaacaaagcgaacggctataccaccgaatatagcgcgagcctgaaaggccgcttt
accattagccgcgataacgcgaaaaacatggcgtatctgcagatgaacagcctgcgcgcggaagataccg
cggtgtattattgcgcgagctttggcctgatgtattattttgattattggggccagggcaccaccctgac
cgtgagcagcgcttccacaaccgcgccatcagtctttccgttggccccatcatgcgggtcgacgagcgga
EVQLVESGGDLVKPGGSLRLSCVASGETFS
WVRQAPGKGLEWVA
RF
TISRDNAKNMAYLQMNSLRAEDTAVYYCAS
WGQGTTLTVSSASTTAPSVFPLAPSCGSTSG
gaagtgcagctggtggaaagcggcggcgatctggtgaaaccgggcggcagcctgcgcctgagctgcgcga
ccagcggctttacctttagcgattattatatgagctgggtgcgccaggcgccgggcaaaggcctggaatg
gatgggctttattcgcaacaaagcgaacggctataccaccgaatatagcgcgagcctgaaaggccgcttt
accattagccgcgataacgcgaaaaacatggcgtatctgcagatgaacagcctgcgcgcggaagataccg
cggtgtattattgcgtgcgctttggcctgatgtattattttgattattggggccagggcaccaccctgac
cgtgagcagcgcttccacaaccgcgccatcagtctttccgttggccccatcatgcgggtcgacgagcgga
EVQLVESGGDLVKPGGSLRLSCATSGETFS
WVRQAPGKGLEWMG
RF
TISRDNAKNMAYLQMNSLRAEDTAVYYCVR
WGQGTTLTVSSASTTAPSVFPLAPSCGSTSG
gaagtgaaactggtggaaagcggcggcgatctggtgaaaccgggcggcagcctgcgcctgagctgcgcga
ccagcggctttacctttagcgattattatatgagctgggtgcgccaggcgccgggcaaagcgctggaatg
gatgggctttattcgcaacaaagcgaacggctataccaccgaatatagcgcgagcctgaaaggccgcttt
accattagccgcgataacgcgaaaaacatgctgtatctgcagatgaacagcctgcgcgcggaagataccg
cggtgtattattgcgtgcgctttggcctgatgtattattttgattattggggccagggcaccaccctgac
cgtgagcagcgcttccacaaccgcgccatcagtctttccgttggccccatcatgcgggtcgacgagcgga
EVKLVESGGDLVKPGGSLRLSCATSGETFS
WVRQAPGKALEWMG
RF
TISRDNAKNMLYLQMNSLRAEDTAVYYCVR
WGQGTTLTVSSASTTAPSVFPLAPSCGSTSG
VTVLRQAGSQMTEVCAATYTVEDELAFLDDSTCTGTSSGNKVNLTIQGLRAMDTGLYICKVELMYPPPYY
VGMGNGTQTYVIDPEPCPDSDELLWILAAVSSGLFFYSFLITAVSLSKMLKKRSPLTTGVYVKMPPTEPE
CEKQFQPYFIPIN
The interaction of antibodies with their cognate protein antigens is mediated through the binding of specific amino acids of the antibodies (paratopes) with specific amino acids (epitopes) of target antigens. An epitope is an antigenic determinant that causes a specific reaction by an immunoglobulin. An epitope consists of a group of amino acids on the surface of the antigen. A protein of interest may contain several epitopes that are recognized by different antibodies. The epitopes recognized by antibodies are classified as linear or conformational epitopes. Linear epitopes are formed by a stretch of a continuous sequence of amino acids in a protein, while conformational epitopes are composed of amino acids that are discontinuous (e.g., far apart) in the primary amino acid sequence, but are brought together upon three-dimensional protein folding.
Epitope mapping refers to the process of identifying the amino acid sequences (i.e., epitopes) that are recognized by antibodies on their target antigens. Identification of epitopes recognized by monoclonal antibodies (mAbs) on target antigens has important applications. For example, it can aid in the development of new therapeutics, diagnostics, and vaccines. Epitope mapping can also aid in the selection of optimized therapeutic mAbs and help elucidate their mechanisms of action. Epitope information on canine CTLA-4 can also elucidate unique epitopes, and define the protective or pathogenic effects of vaccines. Epitope identification also can lead to development of subunit vaccines based on chemical or genetic coupling of the identified peptide epitope to a carrier protein or other immunostimulating agents.
Epitope mapping can be carried out using polyclonal or monoclonal antibodies and several methods are employed for epitope identification depending on the suspected nature of the epitope (i.e., linear versus conformational). Mapping linear epitopes is more straightforward and relatively, easier to perform. For this purpose, commercial services for linear epitope mapping often employ peptide scanning. In this case, an overlapping set of short peptide sequences of the target protein are chemically synthesized and tested for their ability to bind antibodies of interest. The strategy is rapid, high-throughput, and relatively inexpensive to perform. On the other hand, mapping of a discontinuous epitope is more technically challenging and requires more specialized techniques such as x-ray co-crystallography of a monoclonal antibody together with its target protein, Hydrogen-Deuterium (H/D) exchange, Mass Spectrometry coupled with enzymatic digestion as well as several other methods known to those skilled in the art.
Mapping of Canine CTLA-4 Receptor Alpha Epitopes Using Mass Spectroscopy:
In order to determine the epitope for caninized 12B3 (exemplified by 12B3L2H3) and 39A11 (exemplified by 39A11L3H3) on canine CTLA-4, each of the complexes of cCTLA-4/c12B3L2H3 and cCTLA-4/c39A11L2H3 was incubated with deuterated cross-linkers and subjected to multi-enzymatic cleavage. After enrichment of the cross-linked peptides, the samples were analyzed by high resolution mass spectrometry (nLC-LTQ-Orbitrap MS) and the data generated were analyzed using XQuest and Stavrox software.
The analysis indicates that c12B3L2H3 interacts with the amino acid residues at position 35, 38, 51, 53, 90, 93, 98 and 102 on cCTLA-4 comprising the amino acid sequence of SEQ ID NO: 138 (
This application claims priority under 35 U.S.C. § 119(e) of provisional applications U.S. Ser. No. 62/874,287, filed on Jul. 15, 2019, U.S. Ser. No. 62/926,047, filed on Oct. 25, 2019, and U.S. Ser. No. 63/048,873 filed on Jul. 7, 2020, the contents of U.S. Ser. No. 62/926,047 and U.S. Ser. No. 63/048,873 are hereby incorporated by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/069923 | 7/15/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62874287 | Jul 2019 | US | |
| 62926047 | Oct 2019 | US | |
| 63048873 | Jul 2020 | US |
