Antibodies to canine pd-1

CROSS-REFERENCE TO RELATED APPLICATIONS

2This application claims priority based on U.S. Provisional Application Serial No. 61/918946 filed December 20, 2013, U.S. Provisional Application Serial Number 61/918847, filed December 20, 2013, and U.S. Provisional Application Serial No. 62/030812. filed July 30, 2014, the entire contents of which are incorporated herein by reference.

FIELD OF INVENTION

4The present invention relates to murine antibodies to canine PD-1 which contain specific sequences and have a high binding affinity for canine PD-1. The invention also relates to the use of antibodies of the present invention in a method for treating cancer in dogs.

BACKGROUND OF THE INVENTION

6An immunoinhibitory receptor that is mainly expressed on activated T and B cells, programmed cell death receptor 1, also referred to as programmed death receptor 1 (PD-1), is a member of the immunoglobulin superfamily associated with CD28 and CTLA-4. PD-1 and similar family members are type I transmembrane glycoproteins that contain an extracellular domain of the Ig variable domain type (V-type) and capable of binding ligands and a cytoplasmic tail that binds signaling molecules. The cytoplasmic tail of PD-1 contains two tyrosine-containing signaling motifs, ITIM (immunoreceptor tyrosine-containing inhibitory motif) and ITSM (immunoreceptor tyrosine-containing switching motif).

7PD-1 attenuates the T cell response when it binds to programmed cell death ligand 1, also referred to as programmed death ligand 1 (PD-L1), and / or programmed cell death ligand 2, also referred to as programmed cell death ligand 2 (PD-L2 ). The binding of any of these ligands to PD-1 has a negative effect on the antigen receptor signaling pathway. Blocking PD-L1 Binding to PD-1 Increases CD8 T Cell Mediated Tumor-Specific Immunity⁺, contributing to the destruction of tumor cells by the immune system. The three-dimensional structure of murine PD-1 as well as the co-crystal structure of the complex of murine PD-1 with human PD-L1 are described in [Zhang<i>et al.</i>, <i>Immunity</i> 20: 337-347 (2004); Lin<i>et al.</i>, <i>Proc. Natl. Acad. Sci. USA</i> 105: 3011-3016 (2008)].

8PD-L1 and PD-L2 are type I transmembrane ligands that contain both IgV and IgC-like extracellular domains along with short cytoplasmic regions lacking any known signaling motifs. Both PD-L1 and PD-L2 are either constitutively expressed or induced in different cell types, including non-hematopoietic tissues, as well as in different types of tumors. PD-L1 is expressed not only on B-, T-, myeloid and dendritic (DC) cells, but also on peripheral cells such as microvascular endothelial cells and cells of non-lymphoid organs such as heart or lung cells. PD-L2 is found only on macrophages and DC. The expression pattern of PD-1 ligands suggests that PD-1 plays an important role in maintaining peripheral resistance and, in addition, may be involved in the regulation of autoreactive T and B cell responses in peripheral tissues.

9In any event, it is now abundantly clear that PD-1 plays an important role in the development of at least some human malignant tumors, presumably by mediating an escape mechanism from immune surveillance. In accordance with the above data, it has been shown that PD-L1 is expressed on cells of a number of murine and human tumors and is induced by IFN-gamma in most PD-L1 negative tumor cell lines [Iwai<i>et al.</i>, <i>Proc. Natl. Acad. Sci. USA</i> 99: 12293-12297 (2002); Strome<i>et al.</i>, <i>Cancer Res.</i>, 63: 6501-6505 (2003)]. In addition, expression of PD-1 on tumor-infiltrating lymphocytes and / or PD-L1 on tumor cells has been identified in a number of biopsy specimens of primary human tumors. Such tumors include cancer of the lung, liver, ovary, cervix, skin, colon, glioma, bladder, breast, kidney, esophagus, stomach, oral squamous cell carcinoma, urothelial cell and pancreatic cancer, and head tumors. and neck [Brown<i>et al.</i>, <i>J. Immunol.</i> 170: 1257-1266 (2003); Dong<i>et al.</i>, <i>Nat. Med.</i>8: 793-800 (2002); Wintterle<i>et al.</i>, <i>Cancer Res.</i> 63: 7462-7467 (2003); Strome<i>et al.</i>, <i>Cancer Res.</i>63: 6501-6505 (2003); Thompson<i>et al.</i>, <i>Cancer Res</i>... 66: 3381-5 (2006); Thompson<i> et al.</i>, <i>Clin. Cancer Res</i>... 13: 1757-1761 (2007); Nomi<i>et al.</i>, <i>Clin.Cancer Res</i>... 13: 2151-2157. (2007)]. More surprisingly, PD ligand expression on tumor cells correlates with poor prognosis in human cancer patients with different types of tumors [reviewed by Okazaki and Honjo,<i>Int. Immunol.</i> 19: 813-824 (2007)].

10Also, Nomi <i>et al. [Clin. Cancer Res.</i>13: 2151-2157 (2007)] demonstrate the therapeutic efficacy of blocking PD-L1 binding to PD-1 in an aggressive pancreatic cancer mouse model by administering an antibody directed to PD-1 or PD-L1. These antibodies effectively stimulate tumor infiltration of tumor-reactive CD8 T cells⁺by providing up-regulation of antitumor effectors such as IFN-gamma, granzyme B and perforin. Likewise, blocking the binding of PD-L1 and PD-1 by antibodies leads to significant inhibition of tumor growth in a murine model of squamous cell carcinoma [Tsushima<i>et al.</i>, <i>Oral oncol</i>. 42: 268-274 (2006)].

11Other studies have found that transfection of a mouse mastocytoma line containing PD-L1 results in decreased lysis of tumor cells when co-cultured with a tumor-specific CTL clone. After the addition of anti-PD-L1 monoclonal antibody, lysis is restored [Iwai<i>et al.</i>, <i>Proc. Natl. Acad. Sci. USA</i> 99: 12293-12297 (2002)]. It has been shown that blockade of PD1 / PD-L1 interaction in vivo increases the efficiency of adoptive therapy with T-cell transfer in a mouse tumor model [Strome<i>et al.</i>, <i>Cancer Res.</i> 63: 6501-6505 (2003)]. Another evidence of the possibility of using PD-1 in a method of treating cancer is the results of experiments carried out using PD-1 knockout mice, where myeloma cells expressing PD-L1 grow only in wild-type animals (leading to tumor growth and related death of animals), but not in PD-1 deficient mice [Iwai Y. et al., Proc. Natl. Acad. Sci. USA 99: 12293-12297 (2002)]. More recently, anti-PD-1 antibodies (including humanized murine anti-human PD-1 monoclonal antibodies) have been shown to be successful, at least initially, in the treatment of human cancer [see, for example, US 8354509 B2, US 8008449 B2 and US 7595048 B2].

12Anti-PD-1 antibodies can also be used in chronic viral infections. Memory T cells CD8⁺formed after an acute viral infection are highly functional and represent an important component of protective immunity. Conversely, chronic infections are often characterized by varying degrees of functional impairment (depletion) of virus-specific T-cell responses, with these impairments being the main cause of the host's failure to eliminate the pathogen present. Although functional effector T cells develop in the early stages of infection, they gradually lose their ability to function as chronic infection progresses. Barber<i>et al.</i> [<i>Nature</i> 439: 682-687 (2006)] demonstrate that mice infected with the laboratory strain of LCMV develop chronic infection, leading to high levels of the virus in blood and other tissues. These mice initially develop a robust T-cell response, but ultimately succumb to infection as a result of T-cell depletion. Barber<i>et al.</i> found that the number and function of effector T cells in chronically infected mice can be restored by injection of an antibody that blocks the interaction between PD-1 and PD-L1.

13The citation of any reference in this specification should not be construed as an admission that such reference refers to the "prior art" of this application.

SUMMARY OF THE INVENTION

15The present invention provides antibodies to canine PD-1 which have a high binding affinity for canine PD-1 and the ability to block the binding of canine PD-1 to canine PD-L1. In specific embodiments, such anti-canine PD-1 antibodies are murine anti-canine PD-1 antibodies. In specific embodiments, anti-canine PD-1 antibodies have a high binding affinity for canine PD-1 and the ability to block the binding of canine PD-1 to canine PD-L2.

16In addition, the present invention relates to the hypervariable regions (CDRs) included in these antibodies, and the combination of such CDRs (for example, derived from mouse anti-canine PD-1 antibodies) with canine framework regions to obtain caninated (<i>caninized</i>) antibodies to PD-1 dogs. The present invention also relates to the use of said antibodies for the treatment of diseases such as cancer and / or diseases resulting from infections.

17Accordingly, the present invention provides unique CDR sets of seven exemplary murine anti-canine PD-1 antibodies. While each of the seven exemplary mouse anti-canine PD-1 antibodies each has a unique set of CDRs, namely three light chain CDRs: light chain CDR 1 (CDRL1), light chain CDR 2 (CDRL2), and light chain CDR 3 (CDRL3 ), and the three heavy chain CDRs: heavy chain CDR 1 (CDRH1), heavy chain CDR 2 (CDRH2) and heavy chain CDR 3 (CDRH3), as described below, there is significant sequence homology within each CDR group, e.g. collectively CDRL1. Thus, the present invention provides not only the amino acid sequences of six CDRs out of seven exemplary mouse anti-canine PD-1 antibodies, but also conservatively modified variants of these CDRs, as well as variants that contain the same canonical structure (e.g., have the same canonical structure), and / or bind to one or more (for example, 1-4 or all) amino acid residues of PD-1 dogs, which are part of the PD-1 epitope of dogs.

18Thus, the present invention relates to an antibody or antigen-binding fragment thereof capable of specifically binding to canine programmed death receptor 1 (canine PD-1) and containing a hypervariable light chain region 1 (VL CDR1), which contains the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and / or light chain hypervariable region 2 (VL CDR2), which contains the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and / or light chain hypervariable region 3 (VL CDR3), which contains the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26, and / or heavy chain hypervariable region 1 (VH CDR1), where CDRH1 contains the amino acid sequence of SEQ ID NO: 27, SEQ ID NO : 28, SEQ ID NO: 29 or SEQ ID NO: 30, and / or heavy chain hypervariable region 2 (VH CDR2), which contains the amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 35, and / or hypervariable region of the heavy chain 3 (VH CDR3), which contains the amino acid sequence of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 114. In certain embodiments, the antibody is a mammalian antibody ... In more specific embodiments, the antibody is a caninated antibody.

19Accordingly, the caninated antibody of the present invention, or antigen-binding fragment thereof, contains one or more of the hypervariable regions of the heavy chain 1 (VH CDR1) having the amino acid sequences SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30. In another embodiment, the heavy chain hypervariable region 2 (VH CDR2) comprises the amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35. In a further embodiment hypervariable region of the heavy chain 3 (VH CDR3) contains the amino acid sequence of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 or SEQ ID NO: 114. In a specific embodiment of this type, the caninated antibody, or antigen-binding fragment thereof, contains both VH CDR1 containing the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30, and VH CDR2 containing the amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 35. In another such embodiment, the caninated antibody, or antigen-binding fragment thereof, comprises both a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30, and a VH CDR3, containing the amino acid sequence of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 or SEQ ID NO: 114. In a further such embodiment, the caninated antibody, or antigen binding fragment thereof, comprises both a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35 and VH CDR3 containing the amino acid sequence of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 or SEQ ID NO: 114. In another such embodiment, the caninated antibody, or antigen-binding fragment thereof, comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30, VH CDR2 comprising the amino acid sequence SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 35, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 or SEQ ID NO: 114.

20In certain embodiments, the caninated antibody, or antigen-binding fragment thereof, also comprises a hypervariable light chain region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In related embodiments, the hypervariable light chain region 2 (VL CDR2) contains the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21. In another embodiment, the light chain hypervariable region 3 (VL CDR3) comprises the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26. In a specific embodiment of this type, the caninated antibody, or antigen-binding fragment thereof, contains both a VL CDR1 containing the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and a VL CDR2 containing the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

21In other such embodiments, the caninated antibody, or antigen-binding fragment thereof, comprises both a VL CDR1 containing the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 and a VL CDR3 containing the amino acid sequence of SEQ ID NO : 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26. In other such embodiments, the caninated antibody, or antigen-binding fragment thereof, comprises both a VL CDR2 containing the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 and VL CDR3 containing the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26. In other such embodiments, the caninated antibody, or antigen-binding fragment thereof, comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.

22In specific embodiments, the caninated canine anti-PD-1 antibody also contains hypervariable regions (CDRs) that have the following canonical structures: H1-1, H2-1, and H3-6, respectively, for the CDR1, CDR2, and CDR3 of the heavy chain, i.e. ... The heavy chain CDR1 has a class 1 canonical structure, the heavy chain CDR2 has a class 1 canonical structure, and the heavy chain CDR3 has a class 6 canonical structure. In more specific embodiments, the respective light chain CDRs have the following canonical structures: L1-3, L2-1, and L3-1, respectively, for the light chain CDR1, CDR2, and CDR3. In other embodiments, the caninated canine anti-PD-1 antibody also contains hypervariable regions (CDRs) that have the following canonical structures: H1-1, H2-1, and H3-11, respectively, for the heavy chain CDR1, CDR2, and CDR3. In even more specific embodiments of this type, the corresponding light chain CDRs have the following canonical structures: L1-2A, L2-1, and L3-1, respectively, for the light chain CDR1, CDR2, and CDR3. In other embodiments, the caninated canine anti-PD-1 antibody also contains hypervariable regions (CDRs) that have the following canonical structures: H1-1, H2-2A, and H3-11, respectively, for the heavy chain CDR1, CDR2, and CDR3. In even more specific embodiments of this type, the CDRs of the respective light chains have the canonical structures: L1-2A, L2-1, and L3-1, respectively, for the CDR1, CDR2, and CDR3 of the light chain. In other embodiments, the caninated canine anti-PD-1 antibody further comprises hypervariable regions (CDRs) that have the following canonical structures: H1-1, H2-2A, and H3-13, respectively, for the heavy chain CDR1, CDR2, and CDR3. In even more specific embodiments of this type, the corresponding light chain CDRs have the following canonical structures: L1-4, L2-1, and L3-1, respectively, for the light chain CDR1, CDR2, and CDR3.

23In addition, the present invention relates to antibodies to canine PD-1, for example, monoclonal antibodies, which contain the CDR variants of the present invention having the corresponding canonical structures described herein and are capable of binding to the amino acid sequence of SEQ ID NO: 103. B in specific embodiments of this type, the dissociation constant (Kd) of the canine antibody-PD-1 complex is in the range of 1 x 10^-5 up to 1 × 10^-12 M. In more specific embodiments, anti-canine PD-1 antibodies comprise the CDR variants of the present invention that have the corresponding canonical structures described herein and bind to the amino acid sequence of SEQ ID NO: 104.

24The present invention also relates to an isolated caninated antibody or antigen-binding fragment thereof capable of specifically binding the programmed death receptor 1 (PD-1) and comprising a canine IgG heavy chain and canine kappa or lambda light chain. In specific embodiments of this type of canine kappa or lambda light chain, which comprises three light chain hypervariable regions (CDRs): light chain CDR 1 (CDRL1), light chain CDR 2 (CDRL2), and light chain CDR 3 (CDRL3); and canine IgG heavy chain, which contains three heavy chain CDRs: heavy chain CDR 1 (CDRH1), heavy chain CDR 2 (CDRH2), and heavy chain CDR 3 (CDRH3), are derived from murine anti-canine PD-1 antibodies. Specific embodiments of the caninated antibodies of the present invention and antigen-binding fragments thereof bind canine PD-1 and / or block the binding of canine PD-1 to canine programmed death ligand 1 (PD-L1).

25In specific embodiments, the present invention provides an isolated mammalian antibody or antigen-binding fragment thereof that has the ability to specifically bind canine programmed death receptor 1 (canine PD-1) and contains three light chain hypervariable regions (CDRs): light chain CDR-1 (CDRL1 ), Light chain CDR 2 (CDRL2), and light chain CDR 3 (CDRL3); and three heavy chain CDRs: heavy chain CDR 1 (CDRH1), heavy chain CDR 2 (CDRH2), and heavy chain CDR 3 (CDRH3). In some embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO: 13, a variant of SEQ ID NO: 13, a conservatively modified variant of SEQ ID NO: 13, a variant of SEQ ID NO: 13 that contains the canonical structure of class 3, SEQ ID NO: 15, a variant of SEQ ID NO: 15, a conservatively modified variant of SEQ ID NO: 15, or a variant of SEQ ID NO: 15, which contains the canonical structure of class 2A; CDRL2 contains the amino acid sequence of SEQ ID NO: 16, variant SEQ ID NO: 16, conservatively modified variant of SEQ ID NO: 16, variant SEQ ID NO: 16, which contains the canonical structure of class 1, SEQ ID NO: 18, variant SEQ ID NO : 18, conservatively modified variant of SEQ ID NO: 18, variant of SEQ ID NO: 18, which contains the canonical structure of class 1, SEQ ID NO: 19, variant SEQ ID NO: 19, conservatively modified variant of SEQ ID NO: 19, variant SEQ ID NO: 19, which contains the canonical structure of class 1, SEQ ID NO: 20, variant SEQ ID NO: 20, conservatively modified variant of SEQ ID NO: 20, variant of SEQ ID NO: 20, which contains the canonical structure of class 1, SEQ ID NO: 21 , a variant of SEQ ID NO: 21, a conservatively modified variant of SEQ ID NO: 21, or a variant of SEQ ID NO: 21, which contains the canonical structure of class 1, CDRL3 contains the amino acid sequence of SEQ ID NO: 22, a variant of SEQ ID NO: 22, a conservatively modified variant of SEQ ID NO: 22, or a variant of SEQ ID NO: 22, which contains the canonical structure of class 1, SEQ ID NO: 24, variant SEQ ID NO: 24, a conservatively modified variant of SEQ ID NO: 24, variant SEQ ID NO: 24, which contains the canonical structure of class 1, SEQ ID NO: 25, the variant of SEQ ID NO: 25, the conservatively modified variant of SEQ ID NO: 25, the variant of SEQ ID NO: 25, which contains the canonical structure of class 1, SEQ ID NO: 26, variant SEQ ID NO: 26, a conservatively modified variant of SEQ ID NO: 26, or a variant of SEQ ID NO: 26, which contains the canonical structure of class 1, CDRH1 contains the amino acid sequence of SEQ ID NO: 27, variant SEQ ID NO: 27, a conservatively modified variant of SEQ ID NO: 27, a variant of SEQ ID NO: 27, which contains the canonical structure of class 1, SEQ ID NO: 29, a variant of SEQ ID NO: 29, a conservatively modified variant of SEQ ID NO: 29, a variant of SEQ ID NO: 29, which contains the canonical structure of class 1, SEQ ID NO: 30, the variant of SEQ ID NO: 30, the conservatively modified variant of SEQ ID NO: 30, or the variant of SEQ ID NO: 30, which contains the canonical structure of class 1, CDRH2 contains the amino acid sequence SEQ ID NO: 31, variant SEQ ID NO: 31, conservatively modified variant of SEQ ID NO: 31, or variant SEQ ID NO: 31, which contains the canonical structure of class 1, SEQ ID NO: 33, variant SEQ ID NO: 33, a conservatively modified variant of SEQ ID NO: 33, a variant of SEQ ID NO: 33, which contains the canonical structure of class 2A, SEQ ID NO: 34, a variant of SEQ ID NO: 34, a conservatively modified variant of SEQ ID NO: 34, a variant of SEQ ID NO: 34, which contains the canonical structure of class 1, SEQ ID NO: 35, the variant of SEQ ID NO: 35, the conservatively modified variant of SEQ ID NO: 35, or the variant of SEQ ID NO: 35, which contains the canonical structure of class 1, CDRH3 contains the amino acid sequence of SEQ ID NO: 36, variant SEQ ID NO: 36, conservatively modified variant of SEQ ID NO: 36, variant of SEQ ID NO: 35, which contains the canonical structure of class 6, SEQ ID NO: 38, variant SEQ ID NO: 38, a conservatively modified variant of SEQ ID NO: 38, or a variant of SEQ ID NO: 38, which contains the canonical structure of class 11, SEQ ID NO: 114, variant SEQ ID NO: 114, a conservatively modified variant of SEQ ID NO: 114, or a variant of SEQ ID NO: 114, which contains the canonical class 11 structure. In certain embodiments, the antibody and antigen binding fragment bind canine PD-1 and block the binding of canine PD-1 to canine ligand programmable death 1 (PD-L1). In related embodiments, the antibody also blocks the binding of canine PD-1 to canine programmed death ligand 2 (PD-L2). In certain embodiments, the isolated mammalian antibody is a caninated antibody. In more specific embodiments, when binding to canine PD-1, the antibody or antigen-binding fragment thereof binds to at least one amino acid residue contained in one or more of the following amino acid sequences: SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 99, SEQ ID NO: 100, from SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103 and / or SEQ ID NO: 104.

26In other embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO: 13, a variant of SEQ ID NO: 13, a conservatively modified variant of SEQ ID NO: 13, or a variant of SEQ ID NO: 13 that contains the canonical structure of class 3; CDRL2 contains the amino acid sequence of SEQ ID NO: 16, the variant of SEQ ID NO: 16, a conservatively modified variant of SEQ ID NO: 16, or the variant of SEQ ID NO: 16, which contains the canonical structure of class 1; CDRL3 contains the amino acid sequence of SEQ ID NO: 22, a variant of SEQ ID NO: 22, a conservatively modified variant of SEQ ID NO: 22, or a variant of SEQ ID NO: 22, which contains the canonical structure of class 1, CDRH1 contains the amino acid sequence of SEQ ID NO: 27 , a variant of SEQ ID NO: 27, a conservatively modified variant of SEQ ID NO: 27, or a variant of SEQ ID NO: 27, which contains the canonical structure of class 1; CDRH2 contains the amino acid sequence of SEQ ID NO: 31, a variant of SEQ ID NO: 31, a conservatively modified variant of SEQ ID NO: 31, and a variant of SEQ ID NO: 31, which contains the canonical structure of class 1, CDRH3 contains the amino acid sequence of SEQ ID NO: 36 , a variant of SEQ ID NO: 36, a conservatively modified variant of SEQ ID NO: 36, or a variant of SEQ ID NO: 36, which contains the canonical structure of class 6. In specific embodiments, upon binding to canine PD-1, the antibody, or antigen-binding fragment thereof, binds to at least one amino acid residue contained in one or more of the following amino acid sequences: SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103 and / or SEQ ID NO: 104. In more specific embodiments, upon binding to canine PD-1, the antibody, or antigen-binding fragment thereof, binds to at least one amino acid residue of SEQ ID NO: 102.

27In other embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO: 13, a variant of SEQ ID NO: 13, a conservatively modified variant of SEQ ID NO: 13, or a variant of SEQ ID NO: 13 that contains the canonical structure of class 3; CDRL2 contains the amino acid sequence of SEQ ID NO: 19, the variant of SEQ ID NO: 19, a conservatively modified variant of SEQ ID NO: 19, or the variant of SEQ ID NO: 19, which contains the canonical structure of class 1; CDRL3 contains the amino acid sequence of SEQ ID NO: 25, a variant of SEQ ID NO: 25, a conservatively modified variant of SEQ ID NO: 25, or a variant of SEQ ID NO: 25, which contains the canonical structure of class 1, CDRH1 contains the amino acid sequence of SEQ ID NO: 27 , a variant of SEQ ID NO: 27, a conservatively modified variant of SEQ ID NO: 27, or a variant of SEQ ID NO: 27, which contains the canonical structure of class 1; CDRH2 contains the amino acid sequence of SEQ ID NO: 31, a variant of SEQ ID NO: 31, a conservatively modified variant of SEQ ID NO: 31, and a variant of SEQ ID NO: 31, which contains the canonical structure of class 1, CDRH3 contains the amino acid sequence of SEQ ID NO: 36 , a variant of SEQ ID NO: 36, a conservatively modified variant of SEQ ID NO: 36, or a variant of SEQ ID NO: 36, which contains the canonical structure of class 6. In specific embodiments, upon binding to canine PD-1, the antibody, or antigen-binding fragment thereof, binds to at least one amino acid residue contained in one or more of the following amino acid sequences: SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103 and / or SEQ ID NO: 104. In more specific embodiments, upon binding to canine PD-1, the antibody, or antigen-binding fragment thereof, binds to one of the amino acid residues R₇₅ and R₉₀included in SEQ ID NO: 2, or both of these amino acid residues.

28In other embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO: 13, a variant of SEQ ID NO: 13, a conservatively modified variant of SEQ ID NO: 13, or a variant of SEQ ID NO: 13 that contains the canonical structure of class 3; CDRL2 contains the amino acid sequence of SEQ ID NO: 20, the variant of SEQ ID NO: 20, a conservatively modified variant of SEQ ID NO: 20, or the variant of SEQ ID NO: 20, which contains the canonical structure of class 1; CDRL3 contains the amino acid sequence of SEQ ID NO: 25, a variant of SEQ ID NO: 25, a conservatively modified variant of SEQ ID NO: 25, or a variant of SEQ ID NO: 25, which contains the canonical structure of class 1, CDRH1 contains the amino acid sequence of SEQ ID NO: 27 , a variant of SEQ ID NO: 27, a conservatively modified variant of SEQ ID NO: 27, or a variant of SEQ ID NO: 27, which contains the canonical structure of class 1; CDRH2 contains the amino acid sequence of SEQ ID NO: 34, a variant of SEQ ID NO: 34, a conservatively modified variant of SEQ ID NO: 34, and a variant of SEQ ID NO: 34, which contains the canonical structure of class 1, CDRH3 contains the amino acid sequence of SEQ ID NO: 36 , a variant of SEQ ID NO: 36, a conservatively modified variant of SEQ ID NO: 36, or a variant of SEQ ID NO: 36, which contains the canonical structure of class 6. In specific embodiments, upon binding to canine PD-1, the antibody, or antigen-binding fragment thereof, binds to at least one amino acid residue contained in one or more of the following amino acid sequences: SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103 and / or SEQ ID NO: 104.

29In other embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO: 13, a variant of SEQ ID NO: 13, a conservatively modified variant of SEQ ID NO: 13, or a variant of SEQ ID NO: 13 that contains the canonical structure of class 3; CDRL2 contains the amino acid sequence of SEQ ID NO: 16, the variant of SEQ ID NO: 16, a conservatively modified variant of SEQ ID NO: 16, or the variant of SEQ ID NO: 16, which contains the canonical structure of class 1; CDRL3 contains the amino acid sequence of SEQ ID NO: 22, the variant of SEQ ID NO: 22, a conservatively modified variant of SEQ ID NO: 22, or the variant of SEQ ID NO: 22, which contains the canonical structure of class 1, CDRH1 contains the amino acid sequence of SEQ ID NO: 30 , a variant of SEQ ID NO: 30, a conservatively modified variant of SEQ ID NO: 30, or a variant of SEQ ID NO: 30, which contains the canonical structure of class 1; CDRH2 contains the amino acid sequence of SEQ ID NO: 31, a variant of SEQ ID NO: 31, a conservatively modified variant of SEQ ID NO: 31, and a variant of SEQ ID NO: 31, which contains the canonical structure of class 1, CDRH3 contains the amino acid sequence of SEQ ID NO: 36 , a variant of SEQ ID NO: 36, a conservatively modified variant of SEQ ID NO: 36, or a variant of SEQ ID NO: 36, which contains the canonical structure of class 6. In specific embodiments, upon binding to canine PD-1, the antibody, or antigen-binding fragment thereof, binds to at least one amino acid residue contained in one or more of the following amino acid sequences: SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103 and / or SEQ ID NO: 104.

30In other embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO: 15, a variant of SEQ ID NO: 15, a conservatively modified variant of SEQ ID NO: 15, or a variant of SEQ ID NO: 15 that contains the canonical structure of class 2A; CDRL2 contains the amino acid sequence of SEQ ID NO: 18, the variant of SEQ ID NO: 18, a conservatively modified variant of SEQ ID NO: 18, or the variant of SEQ ID NO: 18, which contains the canonical structure of class 1; CDRL3 contains the amino acid sequence of SEQ ID NO: 24, the variant of SEQ ID NO: 24, a conservatively modified variant of SEQ ID NO: 24, or the variant of SEQ ID NO: 24, which contains the canonical structure of class 1, CDRH1 contains the amino acid sequence of SEQ ID NO: 29 , a variant of SEQ ID NO: 29, a conservatively modified variant of SEQ ID NO: 29, or a variant of SEQ ID NO: 29, which contains the canonical structure of class 1; CDRH2 contains the amino acid sequence of SEQ ID NO: 33, a variant of SEQ ID NO: 33, a conservatively modified variant of SEQ ID NO: 33 and a variant of SEQ ID NO: 33, which contains the canonical structure of class 1, CDRH3 contains the amino acid sequence of SEQ ID NO: 38, a variant of SEQ ID NO: 38, a conservatively modified variant of SEQ ID NO: 38, or a variant of SEQ ID NO: 38, which contains the canonical structure of class 11. In specific embodiments, upon binding to canine PD-1, the antibody, or antigen-binding fragment thereof, binds to at least one amino acid residue contained in one or more of the following amino acid sequences: SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 99, SEQ ID NO: 100, from SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103 and / or SEQ ID NO: 104. In more specific embodiments, upon binding to canine PD-1, the antibody, or antigen-binding fragment thereof, binds to at least one amino acid residue of SEQ ID NO: 84.

31In further embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO: 15, a variant of SEQ ID NO: 15, a conservatively modified variant of SEQ ID NO: 15, or a variant of SEQ ID NO: 15 that contains the canonical structure of class 2A; CDRL2 contains the amino acid sequence of SEQ ID NO: 21, variant SEQ ID NO: 21, a conservatively modified variant of SEQ ID NO: 21, or variant SEQ ID NO: 21, which contains the canonical structure of class 1; CDRL3 contains the amino acid sequence of SEQ ID NO: 26, a variant of SEQ ID NO: 26, a conservatively modified variant of SEQ ID NO: 26, or a variant of SEQ ID NO: 26, which contains the canonical structure of class 1, CDRH1 contains the amino acid sequence of SEQ ID NO: 29 , a variant of SEQ ID NO: 29, a conservatively modified variant of SEQ ID NO: 29, or a variant of SEQ ID NO: 29, which contains the canonical structure of class 1; CDRH2 contains the amino acid sequence of SEQ ID NO: 35, a variant of SEQ ID NO: 35, a conservatively modified variant of SEQ ID NO: 35, and a variant of SEQ ID NO: 35, which contains the canonical structure of class 1, CDRH3 contains the amino acid sequence of SEQ ID NO: 114 , a variant of SEQ ID NO: 114, a conservatively modified variant of SEQ ID NO: 114, or a variant of SEQ ID NO: 114, which contains the canonical structure of class 11. In specific embodiments, upon binding to canine PD-1, the antibody, or antigen-binding fragment thereof, binds to at least one amino acid residue contained in one or more of the following amino acid sequences: SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103 and / or SEQ ID NO: 104.

32In other embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO: 14, a variant of SEQ ID NO: 14, a conservatively modified variant of SEQ ID NO: 14, or a variant of SEQ ID NO: 14, which contains the canonical structure of class 4; CDRL2 contains the amino acid sequence of SEQ ID NO: 17, the variant of SEQ ID NO: 17, a conservatively modified variant of SEQ ID NO: 17, or the variant of SEQ ID NO: 17, which contains the canonical structure of class 1; CDRL3 contains the amino acid sequence of SEQ ID NO: 23, variant SEQ ID NO: 23, conservatively modified variant of SEQ ID NO: 23, or variant SEQ ID NO: 23, which contains the canonical structure of class 1, CDRH1 contains the amino acid sequence of SEQ ID NO: 28 , a variant of SEQ ID NO: 28, a conservatively modified variant of SEQ ID NO: 28, or a variant of SEQ ID NO: 28, which contains the canonical structure of class 1; CDRH2 contains the amino acid sequence of SEQ ID NO: 32, a variant of SEQ ID NO: 32, a conservatively modified variant of SEQ ID NO: 32, and a variant of SEQ ID NO: 32, which contains the canonical structure of class 2A, CDRH3 contains the amino acid sequence of SEQ ID NO: 37 , a variant of SEQ ID NO: 37, a conservatively modified variant of SEQ ID NO: 37, or a variant of SEQ ID NO: 37, which contains the canonical structure of class 13. In specific embodiments, upon binding to canine PD-1, the antibody, or antigen-binding fragment thereof, binds to at least one amino acid residue contained in one or more of the following amino acid sequences: SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 99, SEQ ID NO: 100, from SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103 and / or SEQ ID NO: 104. In more specific embodiments, when binding to canine PD-1, the antibody, or antigen-binding fragment thereof, binds to at least one amino acid residue of SEQ ID NO: 83, SEQ ID NO: 84 and / or SEQ ID NO: 100 In more specific embodiments, upon binding to canine PD-1, the antibody, or antigen-binding fragment thereof, binds to at least one or more of the following arginine amino acid residues: R₆₂, R₆₉, R₇₂ and R₇₅included in SEQ ID NO: 2.

33The present invention provides antibodies and antigen-binding fragments capable of specifically binding canine programmed death receptor 1 (canine PD-1) such that the antibody binds to at least one amino acid residue in SEQ ID NO: 103. In certain embodiments, implementation this type of antibody and antigen binding fragments bind canine PD-1 and block the binding of canine PD-1 to canine programmed death ligand 1 (PD-L1). In more specific embodiments, the antibodies and antigen binding fragments bind canine PD-1 and also block the binding of canine PD-1 to canine programmed death ligand 2 (PD-L2).

34Accordingly, in specific embodiments, upon binding to canine PD-1, an antibody (such as an antibody containing one or more CDR variants, e.g., a variant including a conservatively modified variant and / or a variant that contains the canonical structure of a particular class) binds to at least one amino acid residue included in one or more of the following amino acid sequences: SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102 and / or SEQ ID NO: 104. In even more specific embodiments, when binding to canine PD-1, antibodies or their antigen-binding fragments bind to one or more of the following arginine residues: R₆₂, R₆₉, R₇₂, R₇₅ and R₉₀included in SEQ ID NO: 2. In even more specific embodiments, when binding to canine PD-1, antibodies or antigen binding fragments thereof bind to at least one amino acid residue in SEQ ID NO: 104. In more specific embodiments when binding to canine PD-1, antibodies or antigen-binding fragments thereof bind to one or more of the following arginine residues: R₆₂, R₆₉, R₇₂ and R₇₅included in SEQ ID NO: 2. In even more specific embodiments, when binding to canine PD-1, antibodies or antigen binding fragments thereof bind to R₇₅included in SEQ ID NO: 2.

35The present invention also relates to mammalian antibodies or antigen-binding fragments thereof capable of binding to canine PD-1 with a dissociation constant (Kd) below 1 x 10^-12M (e.g. 1 × 10^-13 M, or below). In specific embodiments, mammalian antibodies or antigen-binding fragments bind to canine PD-1 with a dissociation constant of 1 x 10^-5M up to 1 × 10^-12 M. In more specific embodiments, mammalian antibodies or antigen-binding fragments thereof bind to canine PD-1 with a dissociation constant of 1 x 10^-7M up to 1 × 10^-11 M. In even more specific embodiments, mammalian antibodies or antigen-binding fragments thereof bind to canine PD-1 with a dissociation constant of 1 x 10^-8M up to 1 × 10^-11 M. In even more specific embodiments, mammalian antibodies or antigen-binding fragments thereof bind to canine PD-1 with a dissociation constant of 1 x 10^-8M up to 1 × 10^-10 M.

36The present invention also relates to mammalian antibodies or antigen-binding fragments thereof that bind to canine PD-1 at a rate (k_on) exceeding 1 × 10⁷ M^-1With^-1... In certain embodiments, mammalian antibodies or antigen-binding fragments thereof bind to canine PD-1 at a rate ranging from 1 x 10² M^-1With^-1 up to 1 × 10⁷ M^-1With^-1... In more specific embodiments, mammalian antibodies or antigen-binding fragments thereof bind to canine PD-1 at a rate ranging from 1 x 10³ M^-1With^-1 up to 1 × 10⁶ M^-1With^-1... In even more specific embodiments, mammalian antibodies or antigen-binding fragments thereof bind to canine PD-1 at a rate ranging from 1 x 10³ M^-1With^-1 up to 1 × 10⁵ M^-1With^-1... In even more specific embodiments, mammalian antibodies or antigen-binding fragments bind to canine PD-1 at a rate ranging from 1 x 10⁴ M^-1With^-1 up to 1 × 10⁵ M^-1With^-1.

37The present invention also relates to mammalian antibodies or antigen-binding fragments thereof that bind to canine PD-1 to form complexes characterized by the rate of dissociation <b>(</b>k_off) below 1 × 10^-7 With^-1... In specific embodiments, the dissociation rate of complexes of mammalian antibodies or antigen-binding fragments thereof with canine PD-1 is in the range of 1 x 10^-3 With^-1 up to 1 × 10^-8 With^-1... In more specific embodiments, the dissociation rate of complexes of mammalian antibodies or antigen-binding fragments thereof with canine PD-1 ranges from 1 x 10^-4 With^-1 up to 1 × 10^-7 With^-1... In even more specific embodiments, the dissociation rate of complexes of mammalian antibodies or antigen-binding fragments thereof with canine PD-1 ranges from 1 x 10^-5 With^-1 up to 1 × 10^-7 With^-1.

38In related embodiments, mammalian antibodies, or antigen-binding fragments thereof, stimulate responses to a tumor or pathogen characterized by antigen-specific memory. In certain embodiments, mammalian antibodies or antigen-binding fragments thereof stimulate a humoral response<i>in vivo</i>... In other specific embodiments, mammalian antibodies or antigen-binding fragments thereof stimulate an immune response in an animal. In more specific embodiments, the animal is a dog. In a related embodiment, the animal is a feline.

39Accordingly, any of the antibodies of the present invention may have one, two, three, four, five, or all of the above properties, including the above dissociation constants of complexes with canine PD-1, the above rates of complexation with canine PD-1, the above rates dissociation of complexes of antibodies with canine PD-1, stimulation of responses to a tumor or pathogen characterized by antigen-specific memory, stimulation of a humoral response <i>in vivo</i>, and / or stimulating an immune response in an animal.

40As indicated above, the antibodies (and antigen-binding fragments thereof) according to the present invention, including the aforementioned antibodies (and their antigen-binding fragments), can be monoclonal antibodies (and their antigen-binding fragments), mammalian antibodies (and their antigen-binding fragments), for example, mouse antibodies (mouse antibodies) (and their antigen-binding fragments), caninated antibodies (and their antigen-binding fragments), including caninated mouse antibodies (and antigen binding fragments thereof), in some embodiments, the antibodies (and antigen binding fragments thereof) are isolated.

41The present invention also relates to nucleic acids (including isolated nucleic acids) that encode one of the light chains of a caninated antibody of the present invention. Likewise, the present invention relates to isolated nucleic acids that encode one of the heavy chains of a caninated antibody of the present invention. Examples of specific nucleotide sequences are provided herein.

42The present invention also relates to expression vectors that contain one or more of the nucleic acids (including isolated nucleic acids) according to the present invention. The present invention also relates to host cells that contain one or more expression vectors of the present invention.

43In specific embodiments, the antibody is a recombinant antibody or antigen-binding fragment thereof. In related embodiments, the heavy chain variable domain and the light chain variable domain are linked together by a flexible linker to form a single chain antibody.

44In specific embodiments, the antibody or antigen binding fragment thereof is a Fab fragment.

45In other embodiments, the antibody or antigen binding fragment thereof is a Fab 'fragment. In other embodiments, the antibody or antigen binding fragment thereof is a (Fab ') fragment₂... In further embodiments, the antibody or antigen binding fragment thereof is a diabody. In specific embodiments, the antibody or antigen-binding fragment thereof is a domain antibody. In certain embodiments, the antibody or antigen binding fragment thereof is a camelized single domain antibody.

46In certain embodiments, the caninated mouse anti-canine PD-1 antibody, or antigen-binding fragment thereof, enhances the immune response in the dog to be treated.

47The present invention also relates to isolated nucleic acids that encode caninated mouse anti-canine PD-1 antibodies, or fragments thereof. In related embodiments, the implementation of such antibodies or antigennegative fragments thereof can be used in a method of obtaining a medicament for the treatment of cancer in dogs. Alternatively, or in addition to the above, the present invention provides the use of any of the antibodies or antibody fragments of the present invention for diagnosis. In other embodiments, the invention relates to a kit containing one of the caninated antibodies disclosed herein, or antigen-binding fragments thereof.

48In other embodiments, the invention provides an expression vector comprising an isolated nucleic acid encoding one of the caninated mouse anti-canine PD-1 antibodies or antigen binding fragments thereof according to the present invention. The invention also relates to a host cell containing one of the expression vectors described herein. In specific embodiments, said nucleic acids, expression vectors, or polypeptides of the present invention can be used in methods for producing antibodies.

49The present invention also relates to antigenic peptides (including isolated antigenic peptides) that contain 80 or less amino acid residues constituting the amino acid sequence of SEQ ID NO: 103 and / or SEQ ID NO: 83 and / or SEQ ID NO: 84 and / or SEQ ID NO: 99 and / or SEQ ID NO: 100 and / or SEQ ID NO: 101 and / or SEQ ID NO: 102 and / or SEQ ID NO: 104. In related embodiments, antigenic peptides (including isolated peptides) contain 60 or fewer amino acid residues constituting the amino acid sequence of SEQ ID NO: 103 and / or SEQ ID NO: 83 and / or SEQ ID NO: 84 and / or SEQ ID NO: 99 and / or SEQ ID NO: 100 and / or SEQ ID NO: 101 and / or SEQ ID NO: 102 and / or SEQ ID NO: 104. In other embodiments, the antigenic peptides comprise 10 to 44 amino acid residues of the amino acid sequence of SEQ ID NO: 103. In other embodiments, the peptides comprise 15 to 45 amino acid residues of the amino acid sequence of SEQ ID NO: 103 ...

50The present invention also relates to antigenic peptides (including isolated peptides) that contain 80 or fewer amino acid residues constituting an amino acid sequence that is 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 103 and / or SEQ ID NO: 83 and / or SEQ ID NO: 84 and / or SEQ ID NO: 99 and / or SEQ ID NO: 100 and / or SEQ ID NO: 101 and / or SEQ ID NO: 102, and / or SEQ ID NO: 104, and is capable of binding to an isolated mammalian antibody or antigen-binding fragment of the present invention. In related embodiments, antigenic peptides (including isolated antigenic peptides) contain 60 or fewer amino acid residues constituting an amino acid sequence that is 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 103, and / or SEQ ID NO: 83 and / or SEQ ID NO: 84 and / or SEQ ID NO: 9 and / or SEQ ID NO: 100 and / or SEQ ID NO: 101 and / or SEQ ID NO : NO: 102, and / or SEQ ID NO: 104, and is capable of binding to an isolated mammalian antibody or antigen-binding fragment thereof. In other embodiments, the peptides comprise 10 to 44 amino acid residues of an amino acid sequence that is 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 103 and / or SEQ ID NO: 83, and / or SEQ ID NO: 84 and / or SEQ ID NO: 99 and / or SEQ ID NO: 100 and / or SEQ ID NO: 101 and / or SEQ ID NO: 102 and / or SEQ ID NO: NO: 104, and is capable of binding to an isolated mammalian antibody or antigen-binding fragment thereof. In specific embodiments, the antibody is IB5. In other embodiments, the antibody is 3B6. In other specific embodiments, the antibody is 2H9. In further embodiments, the antibody is 2G9. In other embodiments, the antibody is 1Al. In other embodiments, the antibody is 1E4.

51The present invention also relates to fusion proteins that contain one of the aforementioned antigenic peptides. In a specific embodiment, the fusion protein comprises such an antigenic peptide and the Fc region of a non-canine mammalian IgG antibody. In a more specific embodiment, the fusion protein comprises the Fc region of a non-canine mammalian IgG antibody. In some embodiments, the non-canine mammalian IgG antibody is mouse IgG. In alternative embodiments, the non-canine mammalian IgG antibody is human IgG. In other embodiments, the non-canine mammalian IgG antibody is equine IgG. In other embodiments, the non-canine mammalian IgG antibody is porcine IgG. In other embodiments, the non-canine mammalian IgG antibody is bovine IgG.

52In certain embodiments, the non-canine mammalian IgG antibody is IgG1. In other embodiments, the non-canine mammalian IgG antibody is IgG2a. In other embodiments, the non-canine mammalian IgG antibody is IgG3. In further embodiments, the non-canine mammalian IgG antibody is IgG4.

53In other embodiments, the fusion protein comprises one of the aforementioned antigenic peptides and a maltose binding protein. In other embodiments, implementation of the present invention, the fusion protein contains one of the above antigenic peptides and beta-galactosidase. In other embodiments, the fusion protein comprises one of the aforementioned antigenic peptides and glutathione S-transferase. In other embodiments, the fusion protein comprises one of the above antigenic peptides and thioredoxin. In further embodiments, the fusion protein comprises one of the aforementioned antigenic peptides and a Gro EL. In other embodiments, the fusion protein comprises one of the aforementioned antigenic peptides and NusA.

54The present invention also relates to nucleic acids (including isolated nucleic acids) that encode antigenic peptides and corresponding fusion proteins of the present invention. The present invention also relates to expression vectors that contain said nucleic acids.

55In addition, the present invention relates to pharmaceutical compositions containing antibodies to PD-1 dogs or antigen-binding fragments thereof according to the present invention, antigenic peptides (including isolated antigenic peptides) from PD-1 dogs, fusion proteins containing antigenic peptides from PD- 1 dogs according to the present invention, nucleic acids (including isolated nucleic acids), encoding antigenic fragments and / or fusion proteins according to the present invention, expression vectors containing such nucleic acids, or any combination thereof, as well as a pharmaceutically acceptable carrier or diluent.

56In addition, the present invention relates to methods for enhancing the activity of an immune cell, comprising administering to a patient a therapeutically effective amount of such pharmaceutical compositions. In some embodiments, the method is used to treat cancer. In other embodiments, the method is used to treat an infection or infectious disease. In other embodiments, a caninated antibody of the present invention, or an antigen binding fragment thereof, is used as an adjuvant vaccine.

57The following brief description of the drawings and detailed description of the invention are provided to facilitate understanding of these and other aspects of the present invention.

BRIEF DESCRIPTION OF DRAWINGS

59Figure 1 shows the reactivity of murine mAbs against the extracellular domain of canine PD-1. Various murine mAbs are tested for binding to the extracellular domain of canine PD-1 by ELISA. The tested mAbs are designated as ♦ 3B6, ■ 5F3, - 5G5, × 4D12, * 2H9, • 2C12, + 2G9, ▲ nonspecific mAb.

60Figure 2 shows the reactivity of murine mAbs against cell surface expressed canine PD-1. Various murine mAbs are tested for their ability to bind to canine PD-1 expressed on CHO cells by CELISA. Antibodies are designated as follows:<img file="RU2761663C2_D0001.tif" /><img file="RU2761663C2_D0002.tif" />

61Figure 3A shows ligand blockade by mouse anti-canine PD-1 mAbs. Various murine mAbs are tested for their ability to inhibit the binding of PD-1 expressed on CHO cells to PD-L1. Antibodies are designated as follows:<img file="RU2761663C2_D0003.tif" /><img file="RU2761663C2_D0004.tif" />.

62Figure 3B shows ligand blockade by mouse anti-canine PD-1 mAbs. Various murine mAbs are tested for their ability to inhibit the binding of PD-1 expressed on CHO cells to PD-L1. Antibodies are designated as follows:<img file="RU2761663C2_D0005.tif" /><img file="RU2761663C2_D0006.tif" />.

63Figure 3C shows ligand blockade by mouse anti-canine PD-1 mAbs. Various murine mAbs are tested for their ability to inhibit the binding of PD-1 expressed on CHO cells to PD-L1. Antibodies are designated as follows:<img file="RU2761663C2_D0007.tif" /><img file="RU2761663C2_D0008.tif" />.

64Figure 4 shows binding of murine mAbs to canine PD-1 on CD T cells⁺ in PBMCs of healthy dogs. Various murine mAbs tested for the ability to bind to canine PD-1 expressed on CD T cells⁺ in PBMCs of healthy dogs. Antibodies are tested using 2-fold dilutions starting in the 0.156-20 μg / ml range.

65Figure 5 shows binding of murine mAbs to canine PD-1 on CD T cells⁺ in PBMCs of dogs suffering from cancer. These murine mAbs are tested for the ability to bind to canine PD-1 expressed on CD T cells⁸⁺ dogs suffering from cancer (sarcoma). Antibodies are tested at concentrations of 2.5 and 5 μg / ml.

66Figure 6 shows cytokine secretion induced by mouse anti-canine PD-1 mAbs. Various murine mAbs are tested for their ability to induce cytokine secretion from PBMCs from healthy dogs.

67Figure 7 shows cytokine secretion induced by mouse anti-canine PD-1 mAbs. Various murine mAbs are tested for their ability to induce the secretion of cytokines from PBMCs of dogs suffering from cancer (hemangiosarcoma).

68Figure 8 shows the alignment results for the heavy chain (CH) constant regions of canine IgGB that have lost ADCC function. Shown are canine wild-type IgB [cIgGB wild-type], canine IgGB (+) A-hinge [cIgGB (+) A-hinge], canine IgGB (+) D-hinge [cIgGB (+) D-hinge] and canine IgGB ( -) ADCC [cIgGB (-) ADCC]. (+) The A-hinge is obtained by substitution for an IgG-A hinge region containing amino acid substitutions for lysine and asparagine, as shown; The (+) D-hinge is obtained by substitution for an IgG-D hinge region containing amino acid substitutions for lysine and asparagine, as shown. (-) ADCC means lysine and asparagine amino acid substitution.

69Figure 9A depicts the interaction surface of canine PD-1 and caninated 2G9 antibody. The amino acid positions correspond to positions in the PD-1 amino acid sequence lacking the signal sequence, i.e. SEQ ID NO: 2. Determination by chemical crosslinking, high mass MALDI mass spectrometry and nLC-Orbitrap mass spectrometry.

70Figure 9B depicts the interaction surface of canine PD-1 and caninated 3B6 antibody. The amino acid positions correspond to positions in the PD-1 amino acid sequence lacking the signal sequence, i.e. SEQ ID NO: 2. Determination by chemical crosslinking, high mass MALDI mass spectrometry and nLC-Orbitrap mass spectrometry.

DETAILED DESCRIPTION OF THE INVENTION

ABBREVIATIONS

73In the detailed description and examples of the present invention, the following abbreviations are used:

74ADCC - Antibody Dependent Cellular Cytotoxicity

75CDC - Complement Dependent Cytotoxicity

76CDR - hypervariable region of the variable domain of immunoglobulin, determined using the Kabat numbering system

77CHO - Chinese hamster ovary cells

78EC50 - concentration providing 50% efficiency or 50% binding

79ELISA - enzyme-linked immunosorbent assay

80FR - antibody framework: immunoglobulin variable domain excluding CDR regions

81HRP - horseradish peroxidase

82IFN - interferon

83IC50 - concentration providing 50% inhibition

84IgG - immunoglobulin G

85Kabat is an immunoglobulin alignment and numbering system pioneered by Elvin A. Kabat [Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)]

86mAb - monoclonal antibody (also referred to as Mab or MAb)

87MES - 2- (N-morpholino) ethanesulfonic acid

88MOA - Mechanism of Action

89NHS - Normal Human Serum

90PCR - polymerase chain reaction

91PK - pharmacokinetics

92SEB - staphylococcal enetrotoxin B

93TT - tetanus toxoid

94V region - a segment of the IgG chain, the sequence of which varies among different antibodies. It extends from residue 109 of the light chain to residue 113 of the heavy chain, according to Kabat numbering

95VH - variable region of the heavy chain of immunoglobulin

96VK - variable region of the light chain of the kappa immunoglobulin

DEFINITIONS

98To facilitate an understanding of the present invention, specific definitions of certain technical and scientific terms are provided below. Unless otherwise specifically indicated herein, all other technical and scientific terms used herein have conventional meanings known to those of ordinary skill in the art to which the present invention belongs.

99As used herein, unless the context clearly indicates otherwise, singular terms include corresponding plural references.

100The term "activation" as used in reference to cells or receptors refers to the activation of a cell or receptor, or to the treatment of cells or receptors with a ligand, unless the context explicitly indicates otherwise. The term "ligand" encompasses natural and synthetic ligands, for example, cytokines, variants, analogs, cytokine muteins, and binding compounds derived from antibodies. The term "ligand" also encompasses small molecules such as peptide mimetics of cytokines and peptide mimetics of antibodies. The term "activation" can refer to the activation of cells by internal mechanisms as well as external or environmental factors.

101The term "activity" as applied to a molecule can refer to the binding of a molecule to a ligand or to a receptor, catalytic activity; the ability to stimulate gene expression or cellular signaling pathway, differentiation or maturation of cells; antigenic activity, modulation of the activity of other molecules, etc. The term "activity" as applied to a molecule can also refer to the ability to modulate or maintain cell-cell interactions, such as adhesion, or the ability to maintain a cell's structure, for example, cell membranes or the cytoskeleton. “Activity” can also include specific activity such as [catalytic activity] / [mg protein] or [immunological activity] / [mg protein], accumulation in a biological compartment, and the like. "Activity" can include the ability to modulate components of the innate or adaptive immune system.

102The terms "administration" and "treatment" as used in application to an animal, such as an experimental dog, as well as to cells, tissues, organs or body fluids of a dog, includes bringing exogenous pharmaceutical, therapeutic, diagnostic agents or compositions into contact with the animal. , for example, with a dog's body, cell, tissue, organ or body fluid. Cell processing involves bringing the reagent into contact with the cell, and also bringing the reagent into contact with a liquid if the liquid is in contact with the cell. The terms "introduction" and "processing" also include processing<i>in vitro</i> and <i>ex vivo</i>, for example, a cell, a reagent, a diagnostic agent, a binding compound, or another cell. The term "individual" includes any organism, preferably an animal, more preferably a mammal (such as a dog, cat, or human), and most preferably a dog.

103As used herein, the term "substitution of an amino acid residue" with another amino acid residue in the amino acid sequence of an antibody, for example, is equivalent to the term "substitution of an amino acid residue" with another amino acid residue, and means that a particular amino acid residue at a particular position in the amino acid sequence is replaced (or substituted) by another amino acid residue ... Such substitutions can be deliberately planned, for example, it is possible to purposefully replace alanine with serine at a specific position in the amino acid sequence, using, for example, recombinant DNA technology. Alternatively, a particular amino acid residue or sequence of amino acid residues of an antibody can be replaced with one or more amino acid residues as a result of more natural selection processes, for example, allowing an antibody produced by a cell to bind to a given site on a specific antigen, for example, on an antigen containing an epitope , or part of it, and / or providing the production of an antibody containing a specific CDR, which has the same canonical structure as the substituted CDR. Such substitutions / substitutions can lead to the formation of "variant" CDRs and / or variant antibodies.

104The term "treat" or "treatment" refers to the internal or external administration of a therapeutic agent, such as a composition containing an antibody or antigen-binding fragment of the present invention, to a dog or patient having one or more symptoms of a disease, or susceptible to a disease against which the therapeutic activity is directed. the specified funds.

105Typically, the agent is administered in an amount effective to alleviate and / or ameliorate one or more symptoms of the disease in the subject to be treated, or the population to be treated, by inducing regression or inhibiting the development of such symptom (s) to a clinically significant extent. The amount of a therapeutic agent effective to alleviate any particular symptom of a disease (also called a "therapeutically effective amount") may vary depending on factors such as the disease state, the age and weight of the patient (eg, dog), and the ability of the pharmaceutical composition elicit the desired response from the individual. Relief or improvement of a symptom of a disease can be assessed using any clinical analysis commonly used by veterinarians or other medical professionals to assess the severity or extent of a given symptom. While an embodiment of the present invention (e.g., a treatment method or manufactured product) may not provide effective relief of the target disease symptom (s) in each subject, it should alleviate the target disease symptom (s) in a statistically significant number of individuals as determined by any statistical a test known in the art, such as Student's t-test, chi test², Mann and Whitney U-test, Kruskal-Wallis test (H-test), Jonkhier-Terpstra test and Wilcoxon test.

106The term "treatment" as used in application to a human, animal (such as a dog), or experimental subject, refers to therapeutic treatment as well as research and diagnostic use. The term "treatment" as used in application to a human, animal (such as a dog) or experimental subject, or to a cell, tissue or organ, includes bringing antibodies or antigen-binding fragments of the present invention into contact with the body of a dog or other animal, with a cell , tissue, physiological compartment, or physiological fluid.

107Unless otherwise indicated, the term "dog" as used herein includes domestic dogs, Canis lupus familiaris or Canis Familiaris.

108As used herein, the term "cat" refers to any member of the feline family. Members of this family include wild, zoo-dwelling and domestic animals such as members of the Felinae subfamilies such as cats, lions, tigers, cougars, jaguars, leopards, snow leopards, panthers, North American mountain lions, cheetahs, lynxes, red lynxes , caracals or any of their crosses. Cats also include domestic cats, purebred and / or outbred companion cats, show cats, laboratory cats, cloned cats, and wild or stray cats.

109As used herein, the term "canine framework" refers to the amino acid sequence of the heavy chain and light chain of a canine antibody, excluding hypervariable region residues, defined herein as CDR residues. With respect to the caninated antibody, in most embodiments, the amino acid sequences of the native canine CDRs in both strands are replaced with corresponding foreign CDRs (eg, derived from a murine antibody). If necessary, the heavy and / or light chains of a canine antibody may contain some foreign residues other than CDRs, for example, allowing the conformation of the foreign CDRs in the canine antibody to be retained and / or modifying Fc functions, as described below.

110Canine PD-1 is found to comprise the amino acid sequence of SEQ ID NO: 2. In a specific embodiment, canine PD-1 is encoded by a nucleic acid that comprises the nucleotide sequence of SEQ ID NO: 1. The sequences of canine PD-1 may differ, for example, they may contain conservative differences in non-conserved regions, however, the resulting canine PD-1 should perform substantially the same biological function as canine PD-1 containing the amino acid sequence of SEQ ID NO: 2. For example, the biological function of PD-1 includes weakening the T cell response by binding to PD-L1 and / or PD-L2. That is, PD-1 can be considered a negative regulator. It should be noted that the cytoplasmic tail of PD-1 contains two tyrosine-based signaling motifs, ITIM (immunoreceptor tyrosine-containing inhibitory motif) and ITSM (immunoreceptor tyrosine-containing switching motif). In addition, the biological function of canine PD-1 may include, for example, specific binding of an antibody of the present invention via an epitope in the extracellular domain.

111Canine PD-L1 is found to comprise the amino acid sequence of SEQ ID NO: 8. In a specific embodiment, canine PD-L1 is encoded by a nucleotide sequence comprising SEQ ID NO: 7. The sequences of canine PD-L1 may differ, for example, they may contain conservative differences in non-conserved regions, however, the resulting canine PD-L1 should perform essentially the same biological function as canine PD-L1 containing the amino acid sequence of SEQ ID NO: 8 For example, one of the biological functions of PD-L1 includes weakening the T cell response by binding to PD-1.

112The particular amino acid sequence of canine PD-1 or PD-L1 is generally at least 90% identical to the amino acid sequence of SEQ ID NO: 2 to PD-1, or to the amino acid sequence of SEQ ID NO: 8 from canine PD-L1, respectively. In some cases, canine PD-1 or PD-L1 may be identical, respectively, to canine PD-1 containing the amino acid sequence of SEQ ID NO: 2, or canine PD-L1 containing the amino acid sequence of SEQ ID NO: 8 by at least 95 %, or even at least 96%, 97%, 98% or 99%. In some embodiments, the amino acid sequence of canine PD-1 or PD-L1 may be up to 10 differences compared to canine PD-1 comprising the amino acid sequence of SEQ ID NO: 2, or canine PD-L1 comprising the amino acid sequence of SEQ ID NO: 8, respectively. In some embodiments, the amino acid sequence of canine PD-1 or PD-L1 may contain no more than 5 differences, or even no more than 4, 3, 2, or 1 differences, compared to canine PD-1 comprising the amino acid sequence of SEQ ID NO: 2, or canine PD-L1 comprising the amino acid sequence of SEQ ID NO: 8, respectively. The percentage of identity can be determined using the method described below.

113The term "immune response" refers to the function of, for example, lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by said cells or liver (such as antibodies, cytokines, and complement), resulting in selective damage, destruction or removal from the body mammalian (eg, from a dog) cancer cells, cells or tissues infected with pathogens, or invasive pathogens.

114<u>Antibodies </u>to PD-1 dogs

115The present invention provides isolated antibodies (such as murine anti-canine PD-1 antibodies and their corresponding caninated antibodies) or antigen-binding fragments thereof capable of binding to canine PD-1, as well as the use of such antibodies or fragments thereof. In specific embodiments, the invention provides mouse anti-canine PD-1 CDRs derived from mouse anti-canine PD-1 antibodies that have been shown to bind canine PD-1 and block the binding of canine PD-1 to its ligand, PD-L1 dogs. These CDRs can be inserted into a modified canine canine antibody framework to produce a caninated mouse anti-canine PD-1 antibody.

116As used herein, the term "anti-canine PD-1 antibody" refers to an antibody produced against canine PD-1 (eg, in a mammal such as a mouse or rabbit) and is capable of specifically binding to canine PD-1. An antibody capable of “specifically binding to canine PD-1”, in particular canine PD-1, or an antibody capable of “specifically binding to a polypeptide comprising the amino acid sequence of canine PD-1” is an antibody capable of selectively binding PD- 1 dogs among other antigens, however this specificity is not an absolute binding specificity. An anti-canine PD-1 antibody is considered "specific" for canine PD-1 if it can detect the presence of canine PD-1 in a sample, or if it can alter the activity of canine PD-1 without interfering with the activity of other molecules in the sample. in a canine sample, for example, in the absence of unwanted results, such as false positives in a diagnostic context, or side effects in a therapeutic context. The degree of specificity required for an anti-canine PD-1 antibody may depend on the intended use of the antibody and, in any event, is determined by its suitability for its intended use. An antibody or binding compound derived from an antigen-binding portion of an antibody, intended for use in an intended method, binds to its antigen, or a variant or mutein thereof, with an affinity that is at least twice the affinity for any other antigen, preferably at least ten times, more preferably at least 20 times, and most preferably at least 100 times.

117In accordance with this description, an antibody is considered to specifically bind to a polypeptide containing a particular antigenic sequence (in this case, a fragment of the amino acid sequence of canine PD-1) if it binds to polypeptides containing a fragment of the amino acid sequence of PD-1 from dogs, but does not bind to other canine proteins not containing the indicated canine PD-1 sequence fragment. For example, an antibody capable of specifically binding to a polypeptide containing canine PD-1 can bind to FLAG^®-Labeled PD-1 in dogs, but not with other FLAGs^®-labeled canine proteins. An antibody or binding compound derived from an antigen-binding site of an antibody is capable of "specifically" binding to a canine antigen, or a variant or mutein thereof, if its affinity for said canine antigen, or a variant or mutein thereof, exceeds its affinity for any other tested canine antigen, according to at least ten times, more preferably at least 20 times, and even more preferably at least 100 times.

118As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. Thus, this term is used in the broadest sense and specifically includes, without limitation, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies), caninated antibodies, fully canine antibodies, chimeric antibodies and camelized single domain antibodies. "Parental antibodies" are antibodies obtained by exposing the immune system to an antigen before modifying the antibodies for their intended use, such as caninizing an antibody for use as a canine therapeutic antibody.

119Unless otherwise indicated, as used herein, the term "antibody fragment" or "antigen-binding fragment" includes antigen-binding antibody fragments, that is, antibody fragments that retain the ability to specifically bind to an antigen bound by a full-length antibody, for example, 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 such as SC-Fv; nanobodies and polyspecific antibodies obtained from antibody fragments.

120A "Fab fragment" contains one light chain and a C_H1, as well as variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with the heavy chain of another molecule. A "Fab fragment" can be a papain cleavage product of an antibody.

121A "crystallizing fragment" ("Fc") is a region that contains two heavy chain fragments containing domains of antibody C_Hone and C_N2. Two fragments of heavy chains are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domains.

122A "Fab 'fragment" contains one light chain and a portion or fragment of one heavy chain containing the V domain_H and domain C_H1, as well as the section between domains C_Hone and C_H2, so that a disulfide bond can be formed between the two heavy chains of the two Fab 'fragments to give the F (ab')₂.

123"Fragment F (ab ')₂"contains two light chains and two heavy chains containing part of the constant region between domains C_Hone and C_H2, so that an interchain disulfide bond is formed between the two heavy chains. Thus, the fragment F (ab ')₂ consists of two Fab 'fragments that are held together by a disulfide bond formed between two heavy chains. "Fragment F (ab ')₂"may be a pepsin cleavage product of an antibody.

124The "Fv region" contains the variable regions of the heavy and light chains, but does not contain the constant regions.

125The term "single chain Fv" or antibody "scFv" refers to antibody fragments comprising the V domains_H and V_L antibodies present on a single polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the V domains_H and V_L, which allows ScFv to form the structure necessary for antigen binding. [cm. Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113 Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); WO 88/01649; US 4946778 and US 5260203].

126As used herein, the term "canonical structure" refers to the local conformation that each of the hypervariable regions of the heavy and light chains of an antibody can acquire within the framework in which they are located. For each hypervariable region, there are a small number of canonical structures (usually denoted by simple integers such as 1, 2, etc.) that can be predicted with a high degree of accuracy based on the amino acid sequences of the corresponding hypervariable region (in particular, with considering the amino acid sequence of its framework region, as described below for the corresponding variable domains to PD-1 dogs). Using these canonical structures, it is possible to determine whether modification of the amino acid sequence of a given CDR results in the maintenance or loss of the ability to bind to the antigen as a binding partner [cf. <i>Chothia and Lesk, Canonical Structures for the hypervariable regions of immunoglobulins, J. Mol. Biol.</i>196: 901-917(1987)<i>;</i> Chothia <i>et al.</i>, <i>Conformation of immunoglobulin hypervaribale regions</i>, <i>Nature, </i>34: 877-883(1989)<i>; </i>and Al-Lazikani <i>et al.</i>,<i> Standard Conformations for the canonical structures of immunoglobulins</i>, <i>J. Mol. Biol.</i>273: 927-948 (1997)].

127A "domain antibody" is an immunologically functional immunoglobulin fragment containing only a heavy chain variable region or a light chain variable region. In some cases, two or more V_H covalently linked with a peptide linker to obtain a bivalent domain antibody. Two plots V_H bivalent domain antibodies can be specific for the same antigen, or for different antigens.

128A "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites have the same antigenic specificity. However, bivalent antibodies can be bispecific (see below).

129In some embodiments, the implementation of the monoclonal antibodies of the present invention also include camelized single domain antibodies. [see, for example, Muyldermans<i>et al</i>., <i>Trends Biochem. Sci</i>... 26: 230 (2001); Reichmann<i>et al.,</i><i>J. Immunol. Methods</i> 231: 25 (1999); WO 94/04678; WO 94/25591; US 6005079]. In one embodiment, the present invention provides single domain antibodies comprising two V domains_Hmodified to form single-domain antibodies.

130As used herein, the term "diabodies" refers to small antibody fragments containing two antigen binding sites, where the fragments contain a heavy chain variable domain (V_H) linked to the light chain variable domain (V_L) in one polypeptide chain (V_H-V_L or V_L-V_H). When using a linker that is too short to allow pairing of two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain, forming two antigen-binding sites. [See, EP 0404097B1; WO 93/11161; and Holliger<i>et al.,</i><i>Proc. Natl. Acad. Sci. USA</i> 90: 6444-6448 (1993)]. An overview of recombinant antibody variants can be found in [Holliger and Hudson<i>Nat. Biotechnol</i>. 23:1126-1136 (2005)].

131Typically, an antibody or antigen binding fragment of the present invention retains at least 10% of the canine PD-1 binding activity (compared to the parent antibody) when expressed in moles. Preferably, the binding affinity of the antibody or antigen binding fragment of the present invention for canine PD-1 is at least 20%, 50%, 70%, 80%, 90%, 95%, 100% or more of the binding affinity of the parent antibody for PD-1 dogs. It is also contemplated that the antibody or antigennegative fragment of the present invention may contain conservative or non-conservative amino acid substitutions (so-called "conservative variants" or "functionally conservative variants" of the antibody), which practically do not affect biological activity.

132The term "isolated antibody" refers to the degree of purification and, in this context, means that the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other materials such as cellular debris and culture media. Typically, the term "isolated" does not mean the complete absence of such materials, or the absence of water, buffers or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compound described herein.

133As used herein, a "chimeric antibody" is an antibody comprising a variable domain of a first antibody and a constant domain of a second antibody, wherein the first and second antibodies are derived from different species. [US 4816567; and Morrison<i>et al</i>., <i>Proc. Natl. Acad</i>. <i>Sci. USA</i> 81: 6851-6855 (1984)]. Typically, the variable domain is derived from an antibody ("parent antibody") of an experimental animal, such as a rodent, and constant domain sequences are derived from the antibody of a patient animal, such as a human or canine, so that the resulting chimeric antibody is less likely to cause adverse immune responses. responses in humans or dogs, respectively, than the parent antibody (eg, rodent antibody).

134As used herein, the term "caninated antibody" refers to forms of antibodies that contain both canine and non-canine (eg, murine) antibody sequences. Typically, a caninated antibody contains substantially all of at least one or, more often, two variable domains, in which all or almost all of the hypervariable loops are derived from a non-canine immunoglobulin (e.g., containing 6 mouse anti-canine PD-1 CDRs, such as described below), and all or almost all of the framework regions (FR) (and usually all or almost all of the remaining framework) are derived from the canine immunoglobulin sequence. As described herein, the caninated antibody contains three heavy chain CDRs and three light chain CDRs derived from a murine anti-canine PD-1 antibody, as well as a canine framework or a modified canine framework. The modified canine framework contains one or more amino acid substitutions, examples of which are described herein, which further optimize the efficacy of the caninated antibody, for example, increase its ability to bind canine PD-1 and / or block the binding of canine PD-1 to canine PD-L1.

135The term "fully canine antibody" refers to an antibody that contains only canine immunoglobulin protein sequences. A fully canine antibody may contain murine carbohydrate chains if produced in a mouse, by a mouse cell, or a hybridoma derived from a mouse cell. Likewise, a "murine antibody" is an antibody that contains only murine immunoglobulin sequences. Alternatively, a fully canine antibody may contain rat carbohydrate chains if produced in a rat by a rat cell, or a rat cell derived hybridoma. Likewise, a "rat antibody" is an antibody that contains only rat immunoglobulin sequences.

136There are four known subtypes of canine IgG heavy chains, which are designated IgG-A, IgG-B, IgG-C and IgG-D. Two well-known subtypes of light chains are called lambda and kappa.

137The variable regions of each light chain / heavy chain pair form an antibody binding region. Thus, an intact antibody usually contains two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites are generally the same.

138Typically, the variable domains of both heavy and light chains contain three hypervariable regions, also called complementarity determining regions (CDRs), which are located in relatively conserved framework regions (FR). The framework regions are responsible for the correct orientation of the CDR, allowing for binding to a specific epitope. Typically, from the N-terminus to the C-terminus, the variable domains of both light and heavy chains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain is usually carried out in accordance with the rules described in <i>Sequences of Proteins of Immunological Interest</i>, Kabat, <i>et al</i>.; National Institutes of Health, Bethesda, Md .; 5^th ed .; NIH Publ. No. 91-3242 (1991); Kabat,<i>Adv. Prot. Chem.</i> 32: 1-75 (1978); Kabat,<i>et al</i>., <i>J. Biol. Chem.</i>252: 6609-6616 (1977); Chothia,<i>et al</i>., <i>J. Mol. Biol.</i>196: 901-917 (1987) or Chothia, <i>et al</i>., <i>Nature</i> 342: 878-883 (1989)].

139As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region contains amino acid residues from the "complementarity determining region" or "CDR" (ie, CDRL1, CDRL2 and CDRL3 in the variable domain of the light chain and CDRH1, CDRH2 and CDRH3 in the variable domain of the heavy chain). [Determination of antibody CDRs by sequence is described in Kabat<i>et al.</i><i>Sequences of Proteins of Immunological Interest</i>, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Determination of antibody CDRs by structure can also be found in Chothia and Lesk.<i>J. Mol. Biol</i>... 196: 901-917 (1987)]. As used herein, the term "framework" or "FR" residues refers to residues other than the hypervariable region residues, defined herein as CDR residues.

140In addition to binding and activating canine immune cells, a canine or caninated anti-PD-1 antibody should ideally have the following two characteristics:

141one. lack of effector functions such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and

1422. the ability to be easily purified on a large scale using standard industrial techniques such as protein A.

143None of the naturally occurring canine IgG isotypes meet both criteria. For example, IgG-B can be purified using Protein A, but has a high level of ADCC activity. IgG-A, on the other hand, weakly binds to protein A, but exhibits undesirable ADCC activity. In addition, neither IgG-C nor IgG-D can be purified on Protein A columns, although IgG-D does not exhibit ADCC activity. (IgG-C has significant ADCC activity). The present invention overcomes this problem by providing mutant canine IgG-B antibodies specific for PD-1; such antibodies lack effector functions such as ADCC and can be readily purified by standard industrial chromatography using protein A.

144The term "homology" refers to the similarity between two polynucleotide sequences or two polypeptide sequences, as determined by their optimal alignment. If at a certain position of both compared sequences are the same bases or amino acid monomeric subunits, for example, if adenine is located at a particular position of each of the two DNA molecules, then the molecules are homologous at this position. Percent homology is calculated as the number of homologous positions in the two sequences divided by the total number of positions and x 100. For example, if 6 out of 10 positions in two sequences are the same or homologous with optimal sequence alignment, then the two sequences are 60% homologous. Typically, comparisons are made by aligning the two sequences to obtain the maximum percent homology.

145The term "isolated nucleic acid molecule" refers to a DNA or RNA molecule of genomic or synthetic origin, mRNA, cDNA, or some combination thereof, which is not associated with the entire polynucleotide in which the isolated polynucleotide molecule occurs in nature, or with a portion thereof, or associated with a polynucleotide with which it is not associated in nature. It should be understood that for purposes of this description, the term "nucleic acid molecule containing" a particular nucleotide sequence does not encompass intact chromosomes. Isolated nucleic acid molecules "containing" said nucleotide sequences may include, in addition to said sequences, sequences encoding up to ten or even up to twenty or more other proteins, or parts or fragments thereof, or they may functionally include linked regulatory sequences that control the expression of the coding region of said nucleotide sequences, and / or they can include vector sequences.

146The expression "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences 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.

147A nucleic acid is "operably linked" if it is functionally dependent on another nucleotide sequence. For example, DNA encoding a presequence or secretory leader sequence is operably linked to DNA encoding a polypeptide if it is expressed as a precursor 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 the ribosome binding site is operably linked to the coding sequence if its location allows translation. Typically, the term "operably linked" means that the functionally linked DNA sequences are contiguous, and in the case of a secretory leader, they are contiguous and in the same reading frame. However, enhancers are not necessarily in an adjacent position. Linking is carried out by ligation at suitable restriction sites. If such regions are absent, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

148As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably and all include progeny. Thus, the terms "transformants" and "transformed cells" include the primary test cell and cultures derived therefrom, regardless of the number of passages. It should also be understood that not all offspring will have exactly the same DNA composition due to deliberate or accidental mutations. Mutant progeny that have the same function or biological activity as screened for originally transformed cells are within the scope of the present invention. Where different designations are used, the context determines the specific application.

149As used herein, the term "germ sequence" refers to an unrearranged immunoglobulin DNA sequence. Any suitable source of unrearranged immunoglobulin sequences can be used. Human germline sequences can be obtained, for example, from the JOINSOLVER germline database^® on the website of the National Institute of Arthritis, Musculoskeletal, and Skin Diseases of the US National Institutes of Health. Mouse germline sequences can be obtained, for example, according to the method described in Giudicelli<i>et al</i>. [<i>Nucleic Acids Res. </i>33: D256-D261 (2005)].

150<u>Characteristics of murine antibodies </u>to PD-1 dogs<u> and caninated mouse antibodies </u>to PD-1 dogs

151The present invention provides isolated murine anti-canine PD-1 antibodies and caninated antibodies derived therefrom, as well as methods of using such antibodies or antigen-binding fragments thereof for treating a disease, for example, for treating cancer in dogs. Four IgG heavy chains are found in dogs, designated A, B, C and D. These heavy chains correspond to four different subclasses of canine IgG, designated IgGA, IgGB, IgGC and IgGD. The amino acid sequences of these four heavy chains and their encoding DNA sequences were first identified by Tang <i>et al.</i> [<i>Vet. Immunol. Immunopathol.</i> 80: 259-270 (2001)]. The amino acid sequences of these heavy chains and their coding DNA sequences can also be obtained from the GenBank database. For example, the heavy chain amino acid sequence of IgGA 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 amino acid sequences of these light chains and their coding DNA sequences can be obtained from the GenBank database. For example, the amino acid sequence of the kappa light chain has accession number ABY 57289.1, and the sequence of the light chain lambda has accession number ABY 55569.1. In the present invention, the amino acid sequences of the Fc fragments of all four canine IgGs are determined using the identified boundary between the CH1 and CH2 domains as described in Tang <i>et al</i>, above. Caninized murine anti-canine PD-1 antibodies capable of binding canine PD-1 include, but are not limited to: antibodies that contain canine IgG-A, IgG-B, and IgG-D heavy chains and / or canine kappa light chains along with murine CDRs to PD-1 dogs. Accordingly, the present invention provides isolated mouse anti-canine PD-1 antibodies and / or caninated mouse anti-canine PD-1 antibodies or antigen-binding fragments thereof capable of binding canine PD-1 and blocking the binding of canine PD-1 to canine PD-L1 ...

152The present invention also provides full-length canine heavy chains that can pair with the corresponding light chains to form a caninated antibody. Accordingly, the present invention also provides caninated mouse anti-canine antigen antibodies (including isolated caninated canine anti-PD-1 mouse antibodies), as well as methods of using such antibodies, or antigen-binding fragments thereof, for the treatment of diseases, for example, for the treatment of cancer in dogs. ...

153An isolated antibody, or antigen-binding fragment thereof, capable of binding canine PD-1, may contain one, two, three, four, five, or six mouse anti-canine antigen hypervariable regions (CDRs) as described herein. One, two, three, four, five, or six CDRs can be independently selected from the CDR sequences below. In another embodiment, an isolated antibody, or antigen-binding fragment thereof, capable of binding canine PD-1 comprises a canine kappa light chain comprising mouse CDR-1, CDR-2 and / or CDR-3 and a canine IgG heavy chain containing CDR-1, CDR-2 and / or CDR-3 mouse heavy chain.

154In other embodiments, the invention provides antibodies, or antigen-binding fragments thereof, capable of specifically binding PD-1, which contain canine kappa light chains containing from one to six different CDRs by at least 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequences of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and / or 26, and canine IgG antibody heavy chains containing from one to six different CDRs on at least 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequences of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 and / or 114, while having the desired binding characteristics and functional properties. In another embodiment, the antibody or antigen binding fragment of the present invention comprises a canine framework containing a combination of an IgG heavy chain sequence with a kappa light chain containing one or more of the aforementioned CDR amino acid sequences differing by 0, 1, 2, 3, 4, or 5 conservative or non-conservative amino acid substitutions, while having the desired binding characteristics and functional properties.

155The term "sequence identity" refers to the degree of amino acid match of two polypeptides at equivalent positions in an optimal sequence alignment. As used herein, one amino acid sequence is 100% "identical" to a second amino acid sequence if the amino acid residues of both sequences are identical. Accordingly, an amino acid sequence is 50% "identical" to a second amino acid sequence if 50% of the amino acid residues of the two amino acid sequences are identical. The sequence comparison is carried out over a continuous block of amino acid residues that make up a particular protein, for example, a protein to be compared, or a fragment of a polypeptide. In a particular embodiment, allowance is made for individual deletions or insertions that would otherwise lead to a change in the correspondence of the two amino acid sequences.

156The term "sequence similarity" refers to the presence of identical residues as well as non-identical residues of biochemically related amino acids. Biochemically related amino acids are taken into account, which have similar properties and can be used interchangeably.

157The term "conservatively modified variants" or "conservative substitution" refers to substitutions of amino acids in a protein for other amino acids having similar characteristics (e.g. charge, side chain size, hydrophobicity / hydrophilicity, skeletal conformation, rigidity, etc.), which are often not lead to a change in the biological activity of the protein. It is known to those skilled in the art that single amino acid substitutions in regions of a polypeptide that do not play an important role in maintaining its function do not significantly affect biological activity [see, for example, Watson <i>et al., Molecular Biology of the Gene</i>, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed .; 1987)]. In addition, substitutions for structurally or functionally similar amino acids are less likely to disrupt biological activity. Examples of conservative substitutions are shown in Table 1 below.

158<tables num="1"><table frame="all"><tgroup align="center" rowsep="1" colsep="1" cols="2"><colspec colname="c1" colwidth="72mm" /><colspec colname="c2" colwidth="92mm" /><tbody><row><entry namest="c1" nameend="c2" align="right" rowsep="1" colsep="0">Table 1<br />Examples of Conservative Amino Acid Substitutions</entry></row><row><entry align="center" rowsep="1" colsep="1">Original residue</entry><entry align="center" rowsep="1" colsep="0">Conservative substitution</entry></row><row><entry align="center" rowsep="1" colsep="1">Ala (A)</entry><entry align="center" rowsep="1" colsep="0">Gly; Ser;</entry></row><row><entry align="center" rowsep="1" colsep="1">Arg (R)</entry><entry align="center" rowsep="1" colsep="0">Lys; His</entry></row><row><entry align="center" rowsep="1" colsep="1">Asn (N)</entry><entry align="center" rowsep="1" colsep="0">Gln; His</entry></row><row><entry align="center" rowsep="1" colsep="1">Asp (D)</entry><entry align="center" rowsep="1" colsep="0">Glu; Asn</entry></row><row><entry align="center" rowsep="1" colsep="1">Cys (C)</entry><entry align="center" rowsep="1" colsep="0">Ser; Ala</entry></row><row><entry align="center" rowsep="1" colsep="1">Gln (Q)</entry><entry align="center" rowsep="1" colsep="0">Asn</entry></row><row><entry align="center" rowsep="1" colsep="1">Glu (E)</entry><entry align="center" rowsep="1" colsep="0">Asp; Gln</entry></row><row><entry align="center" rowsep="1" colsep="1">Gly (G)</entry><entry align="center" rowsep="1" colsep="0">Ala</entry></row><row><entry align="center" rowsep="1" colsep="1">His (H)</entry><entry align="center" rowsep="1" colsep="0">Asn; Gln</entry></row><row><entry align="center" rowsep="1" colsep="1">Ile (I)</entry><entry align="center" rowsep="1" colsep="0">Leu; Val</entry></row><row><entry align="center" rowsep="1" colsep="1">Leu (L)</entry><entry align="center" rowsep="1" colsep="0">Ile; Val</entry></row><row><entry align="center" rowsep="1" colsep="1">Lys (K)</entry><entry align="center" rowsep="1" colsep="0">Arg; His</entry></row><row><entry align="center" rowsep="1" colsep="1">Met (M)</entry><entry align="center" rowsep="1" colsep="0">Leu; Ile; Tyr</entry></row><row><entry align="center" rowsep="1" colsep="1">Phe (F)</entry><entry align="center" rowsep="1" colsep="0">Tyr; Met; Leu</entry></row><row><entry align="center" rowsep="1" colsep="1">Pro (P)</entry><entry align="center" rowsep="1" colsep="0">Ala; Gly</entry></row><row><entry align="center" rowsep="1" colsep="1">Ser (S)</entry><entry align="center" rowsep="1" colsep="0">Thr</entry></row><row><entry align="center" rowsep="1" colsep="1">Thr (T)</entry><entry align="center" rowsep="1" colsep="0">Ser</entry></row><row><entry align="center" rowsep="1" colsep="1">Trp (W)</entry><entry align="center" rowsep="1" colsep="0">Tyr; Phe</entry></row><row><entry align="center" rowsep="1" colsep="1">Tyr (Y)</entry><entry align="center" rowsep="1" colsep="0">Trp; Phe</entry></row><row><entry align="center" rowsep="0" colsep="1">Val (V)</entry><entry align="center" rowsep="0" colsep="0">Ile; Leu</entry></row></tbody></tgroup></table></tables>

159Functionally conservative variants of the antibodies of the present invention are also within the scope of the present invention. As used herein, the term "functionally conserved variants" refers to antibodies or fragments that contain substitutions of one or more amino acid residues that do not alter the desired property, such as affinity and / or specificity for an antigen. Such variants include, without limitation, molecules resulting from the substitution of an amino acid with an amino acid having similar properties, for example, as a result of the conservative amino acid substitutions listed above in Table 1.

160<u>Nucleic acids</u>

161The present invention also provides nucleic acids encoding the immunoglobulin chains of mouse anti-canine PD-1 antibodies and / or caninated mouse anti-canine PD-1 antibodies and antigen binding fragments thereof described herein (see the Examples section below).

162In addition, the present invention encompasses nucleic acids that encode immunoglobulin polypeptides containing amino acid sequences that are at least about 70%, preferably at least about 80%, more preferably at least about 90 %, and most preferably at least about 95% (e.g., 95%, 96%, 97%, 98%, 99%, 100%) identical to the CDR amino acid sequences and antibodies described herein, where the comparison is carried out using the BLAST algorithm, the parameters of which are chosen so as to obtain the greatest match of the corresponding sequences over the entire length of the corresponding standard sequences. The present invention also provides nucleic acids that encode immunoglobulin polypeptides containing amino acid sequences that are at least about 70%, preferably at least about 80%, more preferably at least about 90%, and most preferably at least about 95% (e.g., 95%, 96%, 97%, 98%, 99%, 100%) similar to any of the standard amino acid sequences, where the comparison is carried out using the BLAST algorithm, the parameters of which are chosen so as to obtain the greatest match of the corresponding sequences over the entire length of the corresponding standard sequences.

163As used herein, percent nucleotide and amino acid sequence identity can be determined using algorithms C, MacVector (MacVector, Inc. Cary, NC 27519), Vector NTI (InforMax, Inc. MD), Oxford Molecular Group PLC (1996) and Clustal W using the default alignment and identity options. With these commercially available programs, it is also possible to determine the sequence similarity using the same or similar defaults. Alternatively, you can use the advanced Blast search under default filtering conditions, such as with the GCG (Genetics Computer Group, GCG Package Programming Guide, Version 7, Madison, Wisconsin) overlay program using the default parameters.

164The following references related to BLAST algorithms are often used for sequence analysis: BLAST ALGORITHMS: Altschul, SF, <i>et al</i>., <i>J. Mol. Biol.</i> 215: 403-410 (1990); Gish, W.,<i>et al</i>., <i>Nature genet</i>... 3: 266-272 (1993); Madden, TL,<i>et al.</i>, <i>Meth. Enzymol.</i> 266: 131-141 (1996); Altschul, SF,<i>et al.</i>, <i>Nucleic Acids Res.</i> 25: 3389-3402 (1997); Zhang, J.,<i>et al.</i>, <i>Genome Res.</i> 7: 649-656 (1997); Wootton, JC,<i>et al.</i>, <i>Comput. Chem.</i> 17: 149-163 (1993); Hancock, JM<i>et al.</i>, <i>Comput. Appl. Biosci.</i> 10: 67-70 (1994); ALIGNMENT SCORING SYSTEMS: Dayhoff, MO,<i>et al.</i>, "A model of evolutionary change in proteins" in <i>Atlas of Protein Sequence and Structure</i>, vol. 5, suppl. 3. MO Dayhoff (ed.), Pp. 345-352, (1978);<i>Natl. Biomed. Res. Found</i>., Washington, DC; Schwartz, RM,<i>et al.</i>, "Matrices for detecting distant relationships" in Atlas of Protein Sequence and Structure, vol. 5, suppl. 3. "(1978), MO Dayhoff (ed.), Pp. 353-358 (1978),<i>Natl. Biomed. Res. Found.</i>, Washington, DC; Altschul, SF,<i>J. Mol. Biol.</i> 219: 555-565 (1991); States, DJ,<i>et al.</i>, <i>Methods</i> 3: 66-70 (1991); Henikoff, S.,<i>et al.</i>, <i>Proc. Natl. Acad. Sci.</i>USA 89: 10915-10919 (1992); Altschul, SF,<i>et al.</i>, <i>J. Mol. Evol.</i> 36: 290-300 (1993); ALIGNMENT STATISTICS: Karlin, S.,<i>et al.</i>, <i>Proc. Natl. Acad. Sci.</i> USA 87: 2264-2268 (1990); Karlin, S.,<i>et al.</i>, <i>Proc. Natl. Acad. Sci.</i> USA 90: 5873-5877 (1993); Dembo, A.,<i>et al.</i>, <i>Ann. Prob.</i> 22: 2022-2039 (1994); and Altschul, SF "Evaluating the statistical significance of multiple distinct local alignments" in<i>Theoretical and Computational Methods in Genome Research</i> (S. Suhai, ed.), Pp. 1-14, Plenum, New York (1997).

165The present invention also provides expression vectors containing the isolated nucleic acids of the present invention, in which the nucleic acid is operably linked to regulatory sequences that are recognized by a host cell transfected with the vector. The invention also provides host cells containing an expression vector of the present invention, and methods for producing an antibody or antigen binding fragment thereof disclosed herein, comprising culturing a host cell carrying an expression vector encoding an antibody or antigen binding fragment thereof in a culture medium and isolating an antigen or an antigen-binding fragment thereof from a host cell or culture medium.

166<u>Epitope Binding and Binding Affinity</u>

167The present invention also provides antibodies or antigen-binding fragments thereof capable of binding to amino acid residues of a canine PD-1 epitope, such as the murine anti-canine PD-1 antibodies disclosed herein. In specific embodiments, murine anti-canine PD-1 antibodies, or antigen-binding fragments thereof, are also capable of inhibiting / blocking the binding of canine PD-1 to canine PD-L1.

168Caninized murine anti-canine PD-1 antibody can be prepared using recombinant techniques known in the art. Mammalian cell lines that can be used as hosts for expressing the antibodies or fragments disclosed herein are well known in the art and include numerous immortalized cell lines available from the American Type Culture Collection (ATCC). Such cells include but are not limited to Chinese hamster ovary (CHO) cells, NSO cells, 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 cells that can be used as hosts include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells. Highly expressed cell lines are particularly preferred. Other suitable cell lines include insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. If recombinant expression vectors encoding a heavy chain or antigen-binding region or fragment thereof, light chain and / or antigen-binding fragment thereof are introduced into mammalian host cells, antibodies are obtained by culturing the host cells for a period of time sufficient to induce antibody expression in the cells -hosts, or, more preferably, the secretion of the antibody into the culture medium in which the host cells are grown.

169Antibodies can be isolated from the culture medium using standard protein purification methods. In addition, the expression of antibodies of the present invention (or fragments thereof) by producing cell lines can be increased using a number of known methods. For example, the glutamine synthetase gene expression system (GS system) is a common way to enhance expression under certain conditions. The GS system is described in whole or in part in European Patent Nos. 0216846, 0256055 and 0323997 and European Patent Application 89303964.4.

170Typically, glycoproteins produced in a particular cell line or in a particular transgenic animal determine the glycosylation pattern specific to glycoproteins produced in a cell line or in a transgenic animal. Thus, the specific glycosylation pattern of an antibody depends on the particular cell line or transgenic animal used to produce the antibody. However, all antibodies encoded by the nucleic acid molecules described herein, or containing the amino acid sequences described herein, are within the scope of the present invention, regardless of the glycosylation pattern of the antibodies. Likewise, in specific embodiments, antibodies with a glycosylation pattern that includes only non-fucosylated N-glycans are preferred because these antibodies are generally shown to be more effective than their fucosylated counterparts, such as <i>in vitro</i>and <i>in vivo</i> [see for example Shinkawa <i>et al.</i>, <i>J. Biol. Chem.</i> 278: 3466-3473 (2003); US patents No. 6946292 and 7214775].

171The present invention also provides fragments of the murine anti-canine PD-1 antibodies disclosed herein. Antibody fragments include F (ab) fragments₂, which can be obtained by enzymatic cleavage of IgG, for example, with pepsin. Fragments of Fab can be obtained, for example, by reducing F (ab)₂ dithiothreitol or mercaptoethylamine. The Fab fragment is a V chain_L-C_Lconnected to the chain V_H-C_H1 through a disulfide bridge. Fragment F (ab)₂ consists of two Fab fragments, which, in turn, are connected by two disulfide bridges. Fab fragments of the F (ab) molecule₂ contain fragments of Fc regions, between which disulfide bridges are formed. The Fv fragment is the V region_L or V_H.

172In one embodiment, the antibody or antigen binding fragment thereof comprises a heavy chain constant region, for example, a canine constant region, such as a canine IgG-A, IgG-B, IgG-C, and IgG-D heavy chain constant region, or a variant thereof. In another embodiment, the antibody or antigen binding fragment thereof comprises a light chain constant region, for example, a canine light chain constant region, such as a lambda or kappa canine light chain region, or a variant thereof. As a non-limiting example, the canine heavy chain constant region can be derived from IgG-B, and the canine light chain constant region can be derived from kappa light chain.

173<u>Antibody Construction by Recombinant Methods</u>

174Using recombinant techniques, caninated mouse anti-canine PD-1 antibodies of the present invention can be obtained that contain modifications in the canine framework and / or among the canine framework residues of the variable domains of the parent (i.e., canine) monoclonal antibody, for example, to improve properties antibodies.

175<u>Experimental and diagnostic applications</u>

176Mouse anti-canine PD-1 antibodies and / or caninated mouse anti-canine PD-1 antibodies of the present invention or antigen-binding fragments thereof can also be used in diagnostic assays for canine PD-1 protein, for example, by detecting its expression in specific tumor cells, tissues, or serum. Such diagnostic methods can be used to diagnose a variety of diseases, especially certain types of cancer in dogs.

177For example, this method includes the following steps:

178(a) coating a substrate (such as the surface of a well of a microtiter plate, eg, a plastic plate) with a mouse anti-canine PD-1 antibody, or an antigen-binding fragment thereof;

179(b) applying to a substrate a sample to be tested for the presence of PD-1 dogs;

180(c) washing the plate to remove unbound substances;

181(d) applying antibodies containing a detectable label (eg, enzyme-linked antibodies) and specific for the PD-1 antigen;

182(e) washing the support to remove unbound labeled antibodies;

183(f) if the labeled antibodies are linked to an enzyme, adding a chemical capable of being converted by the enzyme to generate a fluorescent signal; and

184(g) detecting the presence of a labeled antibody.

185In another embodiment, the antibody is labeled with a peroxidase that reacts with ABTS [2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)] or 3,3 ', 5,5'-tetramethylbenzidine, causing a color change that can be detected. Alternatively, the antibody is labeled with a detectable radioisotope (e.g.³H), which can be detected by scintillation counting with a scintillator. The murine anti-canine PD-1 antibody of the present invention can be used in Western blotting or protein blot immunoassay procedures.

186Such a procedure is part of the present invention and includes, for example:

187(i) contacting a membrane or other solid support, tested for bound canine PD-1 or a fragment thereof, with a murine anti-canine PD-1 antibody or antigen binding fragment of the present invention. Such a membrane can be a nitrocellulose or vinyl [e.g., polyvinylidene fluoride (PVDF)] membrane, onto which proteins are transferred (e.g. after gel electrophoretic separation) to be tested for canine PD-1 by non-denaturing PAGE (polyacrylamide gel electrophoresis). or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Before contacting with a murine anti-canine PD-1 antibody or antigen-binding fragment thereof, the membrane is optionally blocked, for example, with skim milk powder or the like, to block non-specific protein binding sites on the membrane;

188(ii) washing the membrane one or more times to remove unbound mouse anti-canine PD-1 antibody or antigen binding fragment thereof and other unbound substances; and

189(iii) detection of bound mouse anti-canine PD-1 antibody or antigen-binding fragment thereof.

190Detection of bound antibody or antigen-binding fragment thereof can be carried out by binding the antibody or antigen-binding fragment thereof to a secondary antibody (anti-immunoglobulin antibody) containing a detectable label, followed by detection of the secondary antibody.

191The murine anti-canine PD-1 antibodies and antigen-binding fragments thereof described herein can also be used for immunohistochemical analysis. Such an assay is within the scope of the present invention and includes, for example, (1) contacting a cell to be tested for the presence of canine PD-1 with a murine anti-canine PD-1 antibody or antigen binding fragment of the present invention; and (2) detecting an antibody or a fragment thereof on a cell, or in a cell. If an antibody or antigen-binding fragment thereof contains a detectable label, it can be detected directly. Alternatively, the antibody, or antigen-binding fragment thereof, can be bound to a secondary antibody containing a detectable label and subsequently detected.

192Certain murine anti-canine PD-1 antibodies and antigen binding fragments thereof described herein can be used for tumor imaging <i>in vivo</i>... Such a method may include administering radiolabelled mouse anti-canine PD-1 antibodies, or antigen-binding fragments thereof, to a dog to be tested for a tumor associated with canine PD-1 expression, followed by nuclear imaging of the patient to detect the presence of a labeled antibody or antigen-binding fragment thereof, for example, in loci containing a high concentration of an antibody or its antigen-binding fragment, associated with a tumor.

193Imaging techniques include SPECT (single photon emission computed tomography) or PET (positron emission tomography) imaging. As labels, you can use, for example, iodine-123 (¹²³I) and technetium-99m (^99mTc), for example, in combination with SPECT tomography, or ¹¹C, ¹³N, ¹⁵O or ¹⁸F, for example, in combination with PET, or indium-111 [see, for example, Gordon <i>et al</i>., <i>International Rev. Neurobiol.</i> 67:385-440 (2005)].

194<u>Cross-blocking antibodies</u>

195In addition, the anti-canine PD-1 antibody or antigen-binding fragment of the present invention includes any antibody or antigen-binding fragment thereof capable of binding to the same canine PD-1 epitope to which the antibodies and fragments described herein bind, and any antibody or antigen-binding fragment thereof. a fragment capable of cross-blocking (partially or completely) the binding of an antibody described herein or a fragment thereof to canine PD-1, or the binding of which to canine PD-1 is cross-blocked (partially or completely) by an antibody or fragment thereof described herein; as well as any of their options.

196The cross-blocking antibodies and antigen-binding fragments thereof described herein can be identified by their ability to cross-compete with any of IB5, 3B6, 4D12, 7C9, 2H9, 5G5, and / or 2G9 in standard binding assays (such as BIACore^®, ELISA, examples of which are given below, or flow cytometry). For example, standard ELISA assays can be used that involve immobilizing a recombinant canine PD-1 protein on a plate, attaching a fluorescent label to one of the antibodies, and determining the ability of an unlabeled antibody to compete with the labeled antibody for binding. Additionally or alternatively, BIAcore analysis^® can be used to assess the ability of antibodies to cross-compete. The ability of a test antibody to inhibit the binding of, for example, IB5, 3B6, 4D12, 7C9, 2H9, 5G5 and / or 2G9 to canine PD-1 indicates that the test antibody can compete with IB5, 3B6, 4D12, 7C9, 2H9, 5G5 and / or 2G9 for binding to canine PD-1 and therefore in some cases may bind to the same epitope of canine PD-1 as IB5, 3B6, 4D12, 7C9, 2H9, 5G5 and / or 2G9. As indicated above, antibodies and fragments capable of binding to the same epitope as any of the anti-canine PD-1 antibodies or fragments thereof described herein also form part of the present invention.

197<u>Pharmaceutical compositions and routes of administration</u>

198Pharmaceutical or sterile compositions of caninated mouse anti-canine PD-1 antibody or antigen binding fragment thereof may be prepared by admixing them with a pharmaceutically acceptable carrier or excipient. [see, for example,<i>Remington</i>'<i>s</i><i>Pharmaceutical Sciences</i> and <i>US Pharmacopeia: National Formulary</i>, Mack Publishing Company, Easton, PA (1984)].

199Compositions of therapeutic and diagnostic agents can be obtained by mixing them with suitable carriers, excipients or stabilizers, for example, in the form of lyophilized powders, suspensions, aqueous solutions or suspensions [see, for example, Hardman, <i>et al</i>. (2001) <i>Goodman and gilman</i>'<i>s The Pharmacological Basis of Therapeutics</i>, McGraw-Hill, New York, NY; Gennaro (2000)<i>Remington: The Science and Practice of Pharmacy</i>, Lippincott, Williams, and Wilkins, New York, NY; Avis,<i>et al</i>... (eds.) (1993)<i>Pharmaceutical Dosage Forms: Parenteral Medications</i>, Marcel Dekker, NY; Lieberman,<i>et al</i>... (eds.) (1990)<i>Pharmaceutical Dosage Forms: Tablets</i>, Marcel Dekker, NY; Lieberman,<i>et al</i>... (eds.) (1990)<i>Pharmaceutical Dosage Forms: Disperse Systems</i>, Marcel Dekker, NY; Weiner and Kotkoskie (2000)<i>Excipient Toxicity and Safety</i>, Marcel Dekker, Inc., New York, NY]. In one embodiment, the anti-PD-1 antibodies of the present invention are diluted to a suitable concentration with sodium acetate solution, pH 5-6, and NaCl or sucrose is added to maintain tonicity. To improve stability, auxiliary agents such as Polysorbate 20 or Polysorbate 80 can be added.

200The toxicity and therapeutic efficacy of antibody compositions, administered alone or in combination with another agent, can be determined using standard pharmaceutical procedures in cell cultures or experimental animals, for example, by measuring LD₅₀ (dose lethal for 50% of the population) and ED₅₀ (dose therapeutically effective for 50% of the population). The ratio of the toxic and therapeutic effects of the dose is called the therapeutic index (LD₅₀/ ED₅₀). In specific aspects, antibodies with high therapeutic indices are desirable. The results obtained from these cell culture assays and animal studies can be used to determine a dosage range suitable for dogs. The dose of such compounds is preferably in the range to provide circulating concentrations including ED₅₀ at low toxicity, or in the absence of toxicity. The dose can vary within the specified range depending on the dosage form used and the route of administration.

201You can use different routes of administration. Suitable routes of administration include oral administration, rectal administration, transmucosal administration, parenteral administration; intramuscular administration, subcutaneous administration, intradermal administration, intramedullary administration, intrathecal administration, direct intraventricular administration, intravenous administration, intraperitoneal administration, intranasal administration, intraocular administration, administration by inhalation, by insufflation, local administration, dermal administration, transdermal administration, or intraarterial administration.

202In specific embodiments, the murine anti-canine PD-1 antibody, or antigen-binding fragment thereof, can be administered invasively, eg, by injection. In other embodiments, the murine anti-canine PD-1 antibody, or antigen-binding fragment thereof, or a pharmaceutical composition thereof, is administered intravenously, subcutaneously, intramuscularly, intraarterially, intratumorally, or by inhalation, aerosol delivery. Administration by non-invasive means (eg, oral administration of the composition in the form of pills, capsules or tablets) is also within the scope of the present invention.

203The compositions can be administered using medical devices known in the art. For example, the pharmaceutical composition of the present invention can be administered using a hypodermic needle, including, for example, using a pre-filled syringe or autoinjector. The pharmaceutical compositions described herein can also be administered using a hypodermic needleless device; including the devices disclosed in US patent No. 6620135; 6096002; 5399163; 5383851; 5312335; 5064413; 4941880; 4790824 or 4596556.

204The pharmaceutical compositions described herein can also be administered by infusion. Examples of administration of pharmaceutical compositions using well known implants and modular forms are described, for example, in US Pat. No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; US Pat. No. 4,447,233, which discloses an infusion pump for delivering a drug at a precise infusion rate; US Pat. No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; US patent No. 4439196, which discloses an osmotic multi-chamber drug delivery system. Many other such implants, delivery systems and modules are well known to those skilled in the art.

205Alternatively, the mouse anti-canine PD-1 antibody or caninated mouse anti-canine PD-1 antibody can be administered topically rather than systemically, for example, by injecting the antibody directly into an arthritic joint or an area of ​​pathogen-induced damage characterized by immunopathology, often as a depot or composition with slow release. In addition, the antibody can be administered using a targeted drug delivery system, such as liposomes coated with tissue-specific antibody, for delivery, for example, to an arthritic joint or an immunopathological pathogen-induced injury site. Such liposomes are selectively targeted and absorbed by diseased tissue.

206The mode of administration depends on several factors, including the rate of conversion of the therapeutic antibody in serum or tissue, the severity of symptoms, the immunogenicity of the therapeutic antibody, and the availability of target cells in the biological environment. Preferably, the mode of administration provides an amount of therapeutic antibody sufficient to improve the target disease state while minimizing unwanted side effects. Accordingly, the amount of biological agent delivered depends in part on the particular therapeutic antibody and the severity of the condition being treated. There is guidance on the selection of appropriate doses of therapeutic antibodies [see, for example, Wawrzynczak<i>Antibody Therapy</i>, Bios Scientific Pub. Ltd, Oxfordshire, UK (1996); Kresina (ed.)<i>Monoclonal Antibodies, Cytokines and Arthritis</i>, Marcel Dekker, New York, NY (1991); Bach (ed.)<i>Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases</i>, Marcel Dekker, New York, NY (1993); Baert,<i>et al</i>. <i>New Engl. J. Med.</i> 348: 601-608 (2003); Milgrom<i>et al</i>. <i>New Engl. J. Med.</i> 341: 1966-1973 (1999); Slamon<i>et al</i>. <i>New Engl. J. Med.</i> 344: 783-792 (2001); Beniaminovitz<i>et al</i>. <i>New Engl. J. Med.</i> 342: 613-619 (2000); Ghosh<i>et al</i>. <i>New Engl. J. Med.</i>348: 24-32 (2003); Lipsky<i>et al</i>. <i>New Engl. J. Med.</i> 343:1594-1602 (2000)].

207Determining the appropriate dose is up to the veterinarian, for example, taking into account parameters or factors that are known or contemplated in the art to influence treatment. Typically, they start with a dose that is slightly less than the optimal dose and then increase it in small increments until the desired or optimal effect is achieved, taking into account any negative side effects. Important diagnostic indicators include symptoms such as inflammation or the level of produced inflammatory cytokines.

208The antibodies or antigen-binding fragments thereof described herein are mono delivered by continuous infusion, or by dosing, for example, daily, 1-7 times a week, once a week, once every two weeks, once a month, twice a month, once a quarter , once every six months, once a year, etc. Doses can be administered, for example, intravenously, subcutaneously, topically, orally, intranasally, rectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. The total weekly dose is preferably at least 0.05 μg / kg body weight, more preferably at least 0.2 μg / kg, 0.5 mg / kg, 1 μg / kg, 10 μg kg, 100 μg / kg, 0.25 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 5.0 mg / ml, 10 mg / kg, 25 mg / kg, 50 mg / kg or more [see ., for example, Yang, <i>et al.</i><i>New Engl. J. Med.</i>349: 427-434 (2003); Herold,<i>et al.</i><i>New Engl. J. Med.</i> 346: 1692-1698 (2002); Liu,<i>et al.</i><i>J.</i><i>Neurol. Neurosurg. Psych.</i> 67: 451-456 (1999); Portielji,<i>et al</i>. <i>Cancer Immunol. Immunother.</i> 52: 133-144 (2003)]. Doses can also be used to achieve a predetermined concentration of caninated mouse anti-PD-1 antibody in dogs in the serum of an individual, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg / ml or more. In other embodiments, the caninated canine anti-PD-1 mouse antibody of the present invention is administered subcutaneously or intravenously, weekly, biweekly, "every 4 weeks," once a month, twice a month, or quarterly at a dose of 10, 20 , 50, 80, 100, 200, 500, 1000, or 2500 mg / individual.

209The antigenic peptides recognized by the anti-canine PD-1 and PDL-1 mAbs can also be used as vaccines that elicit antibodies that block the binding of PD-1 to PDL-1, resulting in T cell activation and enhanced immune response. These vaccines can be used as therapeutic vaccines against diseases such as cancer, or to enhance the immune response to other vaccines. To use these antigenic peptides as vaccines, one or more of these peptides can be chemically or using recombinant DNA techniques to another carrier protein in order to enhance the immunogenicity of these peptides and induce peptide-specific antibodies. Methods for attaching peptides to carrier proteins are known to those skilled in the art. Peptide vaccines can be used to vaccinate animals by i.m., s.c., oral, aerosol, or<i>in ovo</i> ways. Peptide vaccines can be used as subunit proteins expressed by bacterial, viral, yeast, or baculovirus systems. Alternatively, such peptide vaccines can be delivered by administering a variety of viral or bacterial vectors expressing such peptide vaccines using methods known to those of skill in the art. Peptide vaccines, which can be administered in doses ranging from 1-1000 μg, may optionally contain an adjuvant and an acceptable pharmaceutical carrier.

210As used herein, the term "inhibit" or "treat" or "treatment" includes delaying the onset of symptoms associated with a disorder and / or decreasing the severity of symptoms of such a disorder. These terms also include amelioration of existing uncontrolled or undesirable symptoms, prevention of the onset of other symptoms, and amelioration or prevention of the causes of such symptoms. Thus, these terms mean the achievement of a positive result in a vertebrate suffering from a disorder, disease or symptom, or at risk of developing such a disorder, disease or symptom.

211As used herein, the terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to the amount of caninated mouse anti-PD-1 antibody from dogs of the present invention, or antigen-binding fragment thereof, which, when said antibody or fragment thereof is administered separately or in combination with another therapeutic agent, into a cell, tissue or organism of an individual, is capable of effectively causing a measurable improvement in one or more symptoms of a disease or condition, or preventing the development of such a disease or condition. A therapeutically effective dose also refers to an amount of a binding compound sufficient to achieve at least partial relief of symptoms, for example, to treat, heal, prevent, or improve a related medical condition, or to increase the rate of treatment, healing, prevention, or amelioration of such conditions. When applied to a single active ingredient in its pure form, the therapeutically effective dose relates only to that ingredient. When applied to a combination, the therapeutically effective dose refers to the total amount of active ingredients that produces a therapeutic effect, whether the combination is administered sequentially or simultaneously. An effective amount of a therapeutic agent results in an improvement in the diagnostic index or parameter by at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably at least 50%. An effective amount can also lead to an improvement in the subjective score if subjective scores are used to assess the severity of the disease.

212<u>Other combined treatments</u>

213As indicated previously, the caninated mouse anti-canine PD-1 antibody, or antigen-binding fragment and / or antigenic peptide of the present invention, can be administered in combination with one or more other therapeutic agents (such as a chemotherapeutic agent). The antibody can be associated with such an agent (to form an immunocomplex), or it can be administered separately from the specified agent. In the latter case (separate administration), the antibody can be administered before, after, or simultaneously with the specified agent, or it can be administered in conjunction with other known therapeutic agents.

214<u>Sets</u>

215In addition, the invention provides kits containing one or more components, which include, but are not limited to, an antibody or antigen binding fragment thereof as described herein capable of specifically binding to PD-1 (e.g., caninated mouse anti-canine PD-1 antibody or antigen binding fragment), along with one or more other components, including, but not limited to, a pharmaceutically acceptable carrier and / or chemotherapeutic agent, as indicated in the present description. The binding composition and / or chemotherapeutic agent can be used alone or in combination with a pharmaceutically acceptable carrier in a pharmaceutical composition.

216In one embodiment, the kit contains a binding composition of the present invention (e.g., caninated mouse anti-canine PD-1 antibody, or a pharmaceutical composition thereof, in one container (e.g., in a sterile beaker or plastic bottle)) and a pharmaceutical composition and / or chemotherapeutic agent thereof in another container (for example, in a sterile glass or plastic bottle).

217If the kit contains a pharmaceutical composition for parenteral administration to an individual, it may also contain a device for such administration. For example, the kit may contain one or more hypodermic needles or other injection devices described above. The kit may also include a package insert containing information regarding the pharmaceutical compositions and dosage forms present in the kit. Typically, such information will assist pet owners and veterinarians in the efficient and safe use of the pharmaceutical compositions and dosage forms included in the kit. For example, the insert may contain the following information about the combination of the present invention: pharmacokinetics, pharmacodynamics, clinical trial results, efficacy parameters, indications and methods of use, contraindications, warnings, precautions, adverse reactions, overdose, appropriate doses and routes of administration, dosage forms, appropriate storage conditions, links, manufacturer / distributor information and patent information.

218For convenience, the kit may contain an antibody or specific binding agent as described herein, i. E. a packaged combination of reagents in predetermined quantities with instructions for performing a diagnostic or detection analysis. When an enzyme-labeled antibody is used, the kit may contain substrates and cofactors necessary for the enzyme to function (eg, a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives such as stabilizers, buffers (eg, blocking buffer or lysis buffer), and the like may be included in the kit. The relative amounts of different reagents, which can vary widely, provide reagent concentrations in solution that can significantly optimize the sensitivity of the assay. In particular, reagents including excipients can be provided as dry powders, usually lyophilized, which can be dissolved to provide a reagent solution having a suitable concentration.

EXAMPLES

220<u>EXAMPLE 1</u>

221<u>PD-1 DOG and PD-L1</u>

222<u>Identification and cloning of PD-1 dogs:</u>

223Nucleic acid encoding full length canine PD-1 (cPD-1) was identified by searching the NCBI genebank database (accession number XM_543338.4, SEQ ID NO: 1). The translated amino acid sequence of SEQ ID NO: 2 (accession number XP-543338.3), which corresponds to the putative canine PD-1 protein, was also identified by searching the gene bank protein database (NCBI), followed by alignment of the identified amino acid sequence with the amino acid sequences of mouse, feline and human PD-1. A DNA sequence corresponding to the full length canine PD-1 gene and codon optimized for CHO cells was synthesized and cloned into the plasmid designated p96793. Comparison of the DNA and protein sequences of a putative canine PD-1 with known DNA and PD-1 protein sequences allows the identification of DNA sequences encoding the extracellular domain (ECD) of canine PD-1 (SEQ ID NO: 3) and the amino acid sequence of PD-1 ECD 1 dogs (SEQ ID NO: 4).

224A DNA sequence encoding canine PD-1 ECD, along with a GT linker and 8 histidine residues, was synthesized and then cloned into a plasmid designated LPD2726. A nucleotide sequence (SEQ ID NO: 5) corresponding to a canine PD-1 ECD containing the GT linker and the Fc region of the human IgG1 Fc gene was chemically synthesized and cloned into a plasmid designated LPD2727. The canine ECD PD-1 and the human IgG1 Fc region contain the amino acid sequence of SEQ ID NO: 6.

225<u>Canine PD-L1 Identification and Cloning:</u>

226A nucleic acid encoding a full-length canine PD-L1 was identified by searching the NCBI genebank database (accession number XM_541302.4, SEQ ID NO: 7). The translated amino acid sequence (accession number XP-541302.4; SEQ ID NO: 8), which corresponds to the putative canine PD-L1 protein, is identified by searching the gene bank protein database (NCBI), followed by an alignment of the identified amino acid sequence with known murine and human sequences. PD-L1.

227Comparison of the DNA encoding canine PD-L1 with the known PD-L1 sequences allows the identification of the DNA sequence corresponding to the ECD domain of canine PD-L1 (SEQ ID NO: 9; codon-optimized for CHO cells). The predicted amino acid sequence of canine PD-L1 ECD is described in SEQ ID NO: 10. DNA encoding PD-L1 ECD along with a GT linker and 8 histidine residues was synthesized and then cloned into the plasmid designated LPD2695.

228A DNA sequence encoding the amino acid sequence of canine PD-L1 ECD containing a GT linker and a human IgG1 Fc region (SEQ ID NO: 11) was chemically synthesized and cloned into a plasmid designated LPD2697. Canine PD-L1 ECD plus GT linker and human IgG1 Fc region contain the amino acid sequence of SEQ ID NO: 12. Table 2 provides a description of the above expression plasmids.

229<tables num="1"><table frame="all"><tgroup align="center" rowsep="1" colsep="1" cols="2"><colspec colname="c1" colwidth="54mm" /><colspec colname="c2" colwidth="119mm" /><tbody><row><entry namest="c1" nameend="c2" align="right" rowsep="1" colsep="0">table 2<br />Plasmids containing DNA encoding PD-1 or PD-L1</entry></row><row><entry align="center" rowsep="1" colsep="1">NAME OF PLASMID</entry><entry align="center" rowsep="1" colsep="0">EXPRESSED GENE</entry></row><row><entry align="center" rowsep="1" colsep="1">P96793</entry><entry align="center" rowsep="1" colsep="0">PD-1 dogs</entry></row><row><entry align="center" rowsep="1" colsep="1">LPD2726</entry><entry align="center" rowsep="1" colsep="0">ECD PD-1 Dogs-8HIS</entry></row><row><entry align="center" rowsep="1" colsep="1">LPD2727</entry><entry align="center" rowsep="1" colsep="0">Canine ECD PD-1 / Human IgG1 Fc</entry></row><row><entry align="center" rowsep="1" colsep="1">LPD2695</entry><entry align="center" rowsep="1" colsep="0">ECD canine PD-L1-8HIS</entry></row><row><entry align="center" rowsep="0" colsep="1">LPD2697</entry><entry align="center" rowsep="0" colsep="0">Canine PD-L1 ECD / Human IgG1 Fc</entry></row></tbody></tgroup></table></tables>

230<u>Expression of PD-1 and PD-L1 proteins:</u>

231Expression plasmids encoding the proteins PD-1ECD-HIS, PD-1ECD-Fc, PDL-1ECD-HIS and PD-L1ECD-Fc are transfected into HEK 293 cells, after which the proteins are isolated from the supernatant of the transfected cells by chromatography on a column containing the protein A in the case of Fc-fusion proteins, or nickel (Ni) in the case of HIS-labeled proteins. The purified proteins are used for ELISA or binding assays described below. The expressed proteins are analyzed by SDS-PAGE.

232Full length canine PD-1 DNA sequence: signal sequence underlined and highlighted in bold

233In SEQ ID NO: 1, there is no signal sequence; in SEQ ID NO: 105, the signal sequence is present.

234<b></b><b><u>atggggagccggcgggggccctggccgctcgtctgggccgtgctgcagctgggctggtggccaggatggctc</u></b><b></b>ctagactcccctgacaggccctggagcccgctcaccttctccccggcgcagctcacggtgcaggagggagagaacgccacgttcacctgcagcctggccgacatccccgacagcttcgtgctcaactggtaccgcctgagcccccgcaaccagacggacaagctggccgccttccaggaggaccgcatcgagccgggccgggacaggcgcttccgcgtcatgcggctgcccaacgggcgggacttccacatgagcatcgtcgctgcgcgcctcaacgacagcggcatctacctgtgcggggccatctacctgccccccaacacacagatcaacgagagtccccgcgcagagctctccgtgacggagagaaccctggagccccccacacagagccccagccccccacccagactcagcggccagttgcaggggctggtcatcggcgtcacgagcgtgctggtgggtgtcctgctactgctgctgctgacctgggtcctggccgctgtcttccccagggccacccgaggtgcctgtgtgtgcgggagcgaggacgagcctctgaaggagggccccgatgcagcgcccgtcttcaccctggactacggggagctggacttccagtggcgagagaagacgccggagcccccggcgccctgtgccccggagcagaccgagtatgccaccatcgtcttcccgggcaggccggcgtccccgggccgcagggcctcggccagcagcctgcagggagcccagcctccgagccccgaggacggacccggcctgtggcccctctga

235Amino acid sequence of canine full length PD-1: signal sequence underlined and in bold

236In SEQ ID NO: 2, there is no signal sequence; in SEQ ID NO: 106 the signal sequence is present.

237<b></b><b><u>MGSRRGPWPLVWAVLQLGWWPGWL</u></b><b></b>LDSPDRPWSPLTFSPAQLTVQEGENATFTCSLADIPDSFVLNWYRLSPRNQTDKLAAFQEDRIEPGRDRRFRVMRLPNGRDFHMSIVAARLNDSGIYLCGAIYLPPNTQINESPRAELSVTERTLEPPTQSPSPPPRLSGQLQGLVIGVTSVLVGVLLLLLLTWVLAAVFPRATRGACVCGSEDEPLKEGPDAAPVFTLDYGELDFQWREKTPEPPAPCAPEQTEYATIVFPGRPASPGRRASASSLQGAQPPSPEDGPGLWPL

238DNA sequence of the extracellular domain of the downstream PD-1: SEQ ID NO: 3 (codon-optimized for expression in CHO cells)

239ctggattcccccgacagaccctggagccctctcaccttctcccctgcccagctgaccgtccaggaaggcgagaatgccaccttcacctgcagcctcgccgacatccccgacagcttcgtgctgaactggtacagactgagccccaggaaccagaccgacaagctggccgctttccaggaggacaggatcgaacccggcagggacaggaggtttagggtcatgaggctgcccaacggcagggacttccacatgtccatcgtggccgccagactgaacgactccggcatctacctgtgcggcgctatctacctgccccccaacacccagatcaacgagagccccagggccgaactgagcgtgacagagagaaccctggaacctcccacccagagcccttcccctcctcctagactgagcggacagctgcagggcctggtg

240Canine PD-1 Extracellular Domain: SEQ ID NO: 4:

241LDSPDRPWSPLTFSPAQLTVQEGENATFTCSLADIPDSFVLNWYRLSPRNQTDKLAAFQEDRIEPGRDRRFRVMRLPNGRDFHMSIVAARLNDSGIYLCGAIYLPPNTQINESPRAELSVTERTLEPPLVSGPSPPQL

242DNA sequence of the extracellular domain of human IgG1 downstream PD-1-Fc: SEQ ID NO: 5 (codon-optimized for expression in HEK-293 cells)

243ctggattcccccgacagaccctggagccctctcaccttctcccctgcccagctgaccgtccaggaaggcgagaatgccaccttcacctgcagcctcgccgacatccccgacagcttcgtgctgaactggtacagactgagccccaggaaccagaccgacaagctggccgctttccaggaggacaggatcgaacccggcagggacaggaggtttagggtcatgaggctgcccaacggcagggacttccacatgtccatcgtggccgccagactgaacgactccggcatctacctgtgcggcgctatctacctgccccccaacacccagatcaacgagagccccagggccgaactgagcgtgacagagagaaccctggaacctcccacccagagcccttcccctcctcctagactgagcggacagctgcagggcctggtgggtaccgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

244Fusion protein containing canine PD-1 extracellular domain and human IgG1 Fc: signal sequence underlined and highlighted in bold:

245In SEQ ID NO: 6, there is no signal sequence; in SEQ ID NO: 113, the signal sequence is present.

246<b></b><b><u>MNFLLSWVHWSLALLLYLHHAKWSQA</u></b><b></b>LDSPDRPWSPLTFSPAQLTVQEGENATFTCSLADIPDSFVLNWYRLSPRNQTDKLAAFQEDRIEPGRDRRFRVMRLPNGRDFHMSIVAARLNDSGIYLCGAIYLPPNTQINESPRAELSVTERTLEPPTQSPSPPPRLSGQLQGLVGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

247Full length canine PD-L1 DNA sequence: signal sequence underlined and in bold

248In SEQ ID NO: 7, there is no signal sequence; in SEQ ID NO: 107, the signal sequence is present.

249<b></b><b><u>atgagaatgtttagtgtctttacattcatggcctactgccatttgctaaaagca</u></b><b></b>tttacgatcacagtttctaaggacctgtatgtggtagagtatggtggcaatgtgacaatggaatgcaaattcccggtggaaaaacagttaaacttgtttgcactaatcgtctactgggaaatggaggataaaaaaattatacaatttgtgaatggaaaggaagacctgaaagttcagcacagcagctacagccagagggctcagctattgaaggaccagctcttcttggggaaggctgcgcttcagatcacagatgtgagattgcaggatgcaggggtttactgctgcttgatcggctatggcggtgctgactacaagcggattactttgaaagttcatgccccgtaccgcaacatcagccaaagaatttctgtggatcctgtcacctctgaacatgaactaatgtgtcaggctgagggttaccctgaggctgaagtcatctggacaagcagtgaccaccgagtcctgagtggcaaaaccaccatcactaattccaatagggaagagaagcttttcaatgtgaccagcacgctgaacatcaatgcaacagctaatgagattttctactgcacttttcaaagatcaggtcctgaggaaaacaatactgccgagttggtcatcccagaacgactgcccgttccagcaagtgagaggactcatttcatgattctgggacctttcctgttgcttcttggtgtagtcctggcagtcactttctgtctaaaaaaacatgggagaatgatggatgtggaaaaatgttgcacccgagataggaactcaaagaaacgaaatgatatacaatttgaagagacataa

250Full-size canine PD-L1: signal sequence underlined and in bold

251In SEQ ID NO: 8, there is no signal sequence; in SEQ ID NO: 108 the signal sequence is present.

252<b></b><b><u>MRMFSVFTFMAYCHLLKA</u></b><b></b>FTITVSKDLYVVEYGGNVTMECKFPVEKQLNLFALIVYWEMEDKKIIQFVNGKEDLKVQHSSYSQRAQLLKDQLFLGKAALQITDVRLQDAGVYCCLIGYGGADYKRITLKVHAPYRNISQRISVDPVTSEHELMCQAEGYPEAEVIWTSSDHRVLSGKTTITNSNREEKLFNVTSTLNINATANEIFYCTFQRSGPEENNTAELVIPERLPVPASERTHFMILGPFLLLLGVVLAVTFCLKKHGRMMDVEKCCTRDRNSKKRNDIQFEET

253Canine PD-L1 Extracellular Domain DNA Sequence: SEQ ID NO: 9 (Codon-Optimized for Expression in CHO Cells)

254tttaccatcaccgtgtccaaggacctgtacgtggtcgagtacggcggcaatgtgaccatggagtgcaagttccccgtggagaagcagctgaacctgttcgccctcatcgtgtactgggagatggaggacaagaagatcatccagttcgtgaacggcaaggaggacctgaaggtgcagcactccagctactcccagagagcccagctgctgaaggaccagctgttcctgggcaaggccgccctgcagatcaccgacgtgagactgcaggacgccggcgtgtattgctgcctgatcggctacggaggcgccgactacaagaggatcaccctgaaggtgcatgcaccctacaggaacatcagccagaggatcagcgtcgatcccgtgaccagcgagcacgagctgatgtgccaagccgagggctatcccgaggccgaagtgatctggaccagcagcgaccacagggtcctgagcggcaagaccaccatcaccaacagcaacagggaggagaagctgttcaacgtgaccagcaccctcaacatcaacgccaccgccaacgagatcttctactgcaccttccagaggagcggccccgaagagaacaacaccgccgagctggtgatccccgagagactgcctgtgcctgccagcgagaggacccac

255Canine PD-L1 Extracellular Domain Protein Sequence: SEQ ID NO: 10

256FTITVSKDLYVVEYGGNVTMECKFPVEKQLNLFALIVYWEMEDKKIIQFVNGKEDLKVQHSSYSQRAQLLKDQLFLGKAALQITDVRLQDAGVYCCLIGYGGADYKRITLKVHAPYRNISQRISVDPVTSEHELMCQAEGYPEAEVIWTSSDHRVLSGKTTITNSNREEKLFNVTSTLNINATANEIFYCTFQRSGPEENNTAELVIPERLPVPASERTH

257Canine PD-L1-Fc Human IgG1 Extracellular Domain DNA Sequence: SEQ ID NO: 11 (codon-optimized for expression in HEK-293 cells)

258tttaccatcaccgtgtccaaggacctgtacgtggtcgagtacggcggcaatgtgaccatggagtgcaagttccccgtggagaagcagctgaacctgttcgccctcatcgtgtactgggagatggaggacaagaagatcatccagttcgtgaacggcaaggaggacctgaaggtgcagcactccagctactcccagagagcccagctgctgaaggaccagctgttcctgggcaaggccgccctgcagatcaccgacgtgagactgcaggacgccggcgtgtattgctgcctgatcggctacggaggcgccgactacaagaggatcaccctgaaggtgcatgcaccctacaggaacatcagccagaggatcagcgtcgatcccgtgaccagcgagcacgagctgatgtgccaagccgagggctatcccgaggccgaagtgatctggaccagcagcgaccacagggtcctgagcggcaagaccaccatcaccaacagcaacagggaggagaagctgttcaacgtgaccagcaccctcaacatcaacgccaccgccaacgagatcttctactgcaccttccagaggagcggccccgaagagaacaacaccgccgagctggtgatccccgagagactgcctgtgcctgccagcgagaggacccacggtaccgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

259Fusion protein containing canine PD-L1 extracellular domain and human IgG1 Fc: SEQ ID NO: 12

260FTITVSKDLYVVEYGGNVTMECKFPVEKQLNLFALIVYWEMEDKKIIQFVNGKEDLKVQHSSYSQRAQLLKDQLFLGKAALQITDVRLQDAGVYCCLIGYGGADYKRITLKVHAPYRNISQRISVDPVTSEHELMCQAEGYPEAEVIWTSSDHRVLSGKTTITNSNREEKLFNVTSTLNINATANEIFYCTFQRSGPEENNTAELVIPERLPVPASERTHGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

261<u>EXAMPLE 2</u>

262<u>ANTIBODIES </u>to PD-1 dogs

263<u>Obtaining monoclonal antibodies against canine PD1:</u>

264A total of three Balb / c mice were immunized multiple times (10 μg each time) for 17 days. Canine-Fc ECD PD-1 fusion protein was used as an immunizing antigen. After immunization, sera are collected from each mouse and assayed for reactivity with the HIS-labeled dog PD-1 ECD protein. Mouse spleen cells with the highest serum titer against PD-1 ECD-HIS hybridized to the myeloma cell line P3X63Ag8.653. Approximately 2 weeks after hybridization, the supernatant of the putative hybridoma cells was analyzed by ELISA for reactivity with the labeled PD-1 ECD-HIS protein. Hybridomas producing strong positive signals in ELISA are subcloned by serial dilution and re-assayed for reactivity with labeled dog PD-1 protein ECD-HIS.

265<u>Confirmation of the Reactivity of Mouse Monoclonal Antibodies to Dog PD-1</u>:

266The reactivity of antibodies secreted by the hybridomas against canine ECD PD-1 was confirmed by ELISA. Hybridoma cells are cultured using CELLine bioreactors (Integra-biosciences) for 10-30 days. Cells are first cultured in DMEM containing 4 mM L-glutamine and 10% ultra-low IgG fetal bovine serum (FBS), Gibco. Hybridoma cells are seeded into the cell chambers of the CELLine bioreactor at a cell density of approximately 2 × 10⁶ cells / ml in 15 ml of the same medium with FBS concentration increased to 20%. The outer chamber is filled with 1 L of growth medium (DMEM containing 4 mM L-glutamine and 2% standard FBS). Hybridoma cells in a cell chamber are grown to a density of about 2.5 x 10⁷ cells / ml for 3-7 days. Then, 10 ml of cell suspension is collected from the cell chamber and replaced with fresh medium to allow new cell growth and subsequent collection. This procedure is repeated, as necessary, the required number of times to obtain a sufficient amount of mAbs from each hybridoma clone. The collected cell suspensions are centrifuged and the supernatants are filtered through 0.2 μm filter membranes. To isolate antibodies, the supernatant of each clone was purified using a 5 ml fast flow column containing Sepharose 4-Protein G (GE Healthcare) with gravity flow. After washing with Tris-EDTA (TE) buffer, pH 8.0, the bound antibodies are eluted with a buffer containing 0.1M glycine, pH 2.7, after which the pH is neutralized with 1M Tris, pH 8.0. Antibodies are concentrated and the buffer is exchanged for phosphate buffered saline (PBS) using Centriprep YM-10.10 kDa NMWL centrifuge filters (Millipore). The concentration of antibodies is quantitatively determined by spectrophotometry.

267Purified anti-canine PD-1 mAbs are tested for reactivity to the HIS-tagged canine PD-1 ECD domain by ELISA as follows: HIS-tagged canine PD-1 ECD protein is diluted to 10 μg / ml with coating buffer (carbonate / bicarbonate, pH 9.0) and plated at 100 μl / well in 96 well flat bottom ELISA plates (Nunc). The plates are incubated at 4 ° C overnight. The plates are then washed three times with phosphate buffered saline containing 0.05% Tween-20 (PBST). Then, 200 μl of blocking buffer (5% skim milk in PBST) was added to each well and the plates were incubated at 37 ° C for 60 minutes. The plates are then washed three times with PBST. To the first wells of the respective columns, add 100 μl of the test mAbs diluted with blocking buffer. The mAbs to be tested are diluted in half in the respective positions of the plate. After incubation at 37 ° C for 60 minutes, the plates are washed with PBST three times. Then add to the plates 100 μl per well of horseradish peroxidase conjugated with goat anti-mouse IgG (KPL) antibody at a dilution of 1: 2000 and incubated at 37 ° C for 60 minutes. Plates are washed three times with PBST and 100 μl / well of 3,3 ', 5,5'-tetramethylbenzidine (TMB) substrate (KPL) is added. The color reaction is allowed to develop for 5-20 minutes at 37 ° C and then the absorbance is measured at 650 nm.

268<u>CHO cells expressing canine PD-1 protein:</u>

269The dog full length PD-1 gene was cloned into plasmid p96793. In this plasmid, the expression of the PD-1 protein is regulated by the HCMV promoter. CHO DXB11 (dhfr-) cells are maintained in MEM-alpha medium (Gibco) containing 10% fetal bovine serum. Transfection of CHO cells with plasmid p96793 is carried out in flasks with a volume of 75 cm²containing about 6 × 10⁶ cells by liposome-mediated gene delivery using lipofectamine (Invitrogen). After 48 hours, the cells are transplanted into nucleoside-free MEM-alpha medium supplemented with 10% FBS and 400 μg / ml hygromycin B (selective medium). Cloning by the serial dilution method is performed on a pool of dhfr + hygromycin-resistant cells. Clones are tested for canine PD-1 expression by immunofluorescence assay. In short, cell monolayers are fixed in 96 well plates with 80% acetone. The fixed and dried cell monolayers are then incubated for 1 hour with a polyclonal goat anti-human PD-1 antibody (R&amp;D Systems). Plates are washed with PBS and then incubated for 1 hour with fluorescein-labeled rabbit anti-goat IgG (KPL). The plates were washed with PBS. Clones generating fluorescence are cultured and stockpiled.

270<u>Reactivity of mouse mAbs to canine PD-1 proteins expressed on CHO cells:</u>

271The reactivity of mouse anti-canine PD-1 mAb to canine PD-1 on CHO cells is determined by cellular assay using CHO cells that express PD-1. In short, canine PD-1 expressing CHO cells are cultured to 80-100% confluence in 50 μl medium (DMEM / HAM's F12, 10% FBS). Then add 50 μl of medium containing different concentrations of purified mAbs, and incubate for 1 hour at 37 ° C. After three washes with PBS-Tween, 100 μl of a goat anti-mouse immunoglobulin-horseradish peroxidase (HRP) -conjugated solution diluted 1: 1000 with culture medium is added and incubated for one hour at 37 ° C. Washed three more times with PBS-Tween and then the bound mAbs were visualized with peroxidase substrate (TMB). The increase in light absorption associated with peroxidase activity is measured at 450 nm using a microplate reader.

272<u>Murine mAb Binding Assay </u>to PD-1 dogs<u> and caninated mouse mAbs </u>to PD-1 dogs<u> with canine PD-1</u>

273About 70 Resonance Units (RU) of canine PD-1 antigen are immobilized directly by amine coupling. Measurement of affinity is carried out by surface plasmon resonance in the absence of labels (for example, Biacore^® T200) with an association time of 300 seconds, a dissociation time of 1200 seconds, at concentrations of 50, 100, 200 (× 2) 400 and 800 nanomoles (nmol). An appropriate 1: 1 binding model is used. Antigen (canine PD-1) is immobilized on the sensor chip by amine coupling, and then the four antibodies indicated in Table 3 below, used as analytes, are passed over the surface containing the antigen. The results demonstrate that the antibodies of the present invention to canine PD-1 have a high affinity for the canine PD-1 antigen, which is characterized by a dissociation constant (Kd) in the nanomolar and even subnanomolar range. In addition, the mouse anti-canine PD-1 monoclonal antibody and the corresponding caninated mouse anti-canine PD-1 antibody from the same clone gave strikingly similar Kd values ​​(see Table 3 below).

274<tables num="1"><table frame="all"><tgroup rowsep="1" colsep="1" cols="6"><colspec colname="c1" colwidth="34mm" /><colspec colname="c2" colwidth="24mm" /><colspec colname="c3" colwidth="27mm" /><colspec colname="c4" colwidth="28mm" /><colspec colname="c5" colwidth="19mm" /><colspec colname="c6" colwidth="43mm" /><tbody><row><entry namest="c1" nameend="c6" align="right" rowsep="1" colsep="0">Table 3<br />Defining a binding constant</entry></row><row><entry valign="middle" align="center" rowsep="1" colsep="1">Antibody</entry><entry valign="middle" align="center" rowsep="1" colsep="1">k_on(k¹)<br />M^-1With^-1</entry><entry valign="middle" align="center" rowsep="1" colsep="1">k_off(k^-1)<br />With^-1</entry><entry valign="middle" align="center" rowsep="1" colsep="1">Kd<br />M</entry><entry valign="middle" align="center" rowsep="1" colsep="1">Chi²<br />(RU²)</entry><entry valign="middle" align="center" rowsep="1" colsep="0">Rmax (RU)</entry></row><row><entry valign="middle" align="center" rowsep="1" colsep="1">Mouse 2H9</entry><entry valign="middle" align="center" rowsep="1" colsep="1">2,3×10⁴</entry><entry valign="middle" align="center" rowsep="1" colsep="1">≤5×10^-6#</entry><entry valign="middle" align="center" rowsep="1" colsep="1">≤2,0×10^-10#</entry><entry valign="middle" align="center" rowsep="1" colsep="1">0,19</entry><entry valign="middle" align="center" rowsep="1" colsep="0">25,6</entry></row><row><entry valign="middle" align="center" rowsep="1" colsep="1">Caninized 2H9</entry><entry valign="middle" align="center" rowsep="1" colsep="1">1,0×10⁴</entry><entry valign="middle" align="center" rowsep="1" colsep="1">5,9×10^-6</entry><entry valign="middle" align="center" rowsep="1" colsep="1">5,9×10^-10</entry><entry valign="middle" align="center" rowsep="1" colsep="1">0,10</entry><entry valign="middle" align="center" rowsep="1" colsep="0">27,7</entry></row><row><entry valign="middle" align="center" rowsep="1" colsep="1">Mouse 3B6</entry><entry valign="middle" align="center" rowsep="1" colsep="1">1,8×10⁴</entry><entry valign="middle" align="center" rowsep="1" colsep="1">3,4×10^-5</entry><entry valign="middle" align="center" rowsep="1" colsep="1">2,0×10^-9</entry><entry valign="middle" align="center" rowsep="1" colsep="1">0,13</entry><entry valign="middle" align="center" rowsep="1" colsep="0">48,7</entry></row><row><entry valign="middle" align="center" rowsep="1" colsep="1">Caninized 3B6</entry><entry valign="middle" align="center" rowsep="1" colsep="1">1,6×10⁴</entry><entry valign="middle" align="center" rowsep="1" colsep="1">4,7×10^-5</entry><entry valign="middle" align="center" rowsep="1" colsep="1">2,9×10^-9</entry><entry valign="middle" align="center" rowsep="1" colsep="1">0,07</entry><entry valign="middle" align="center" rowsep="1" colsep="0">49,9</entry></row><row><entry namest="c1" nameend="c6" align="center" rowsep="0" colsep="0">^#The dissociation rate is below the detection limit of the instrument being used.</entry></row></tbody></tgroup></table></tables>

275<u>Ligand Blockade with Mouse mAbs </u>to PD-1 dogs<u>:</u>

276A cell-based ELISA (CELISA) assay, performed on a CHO cell line expressing canine PD-1, is used to identify murine mAbs capable of interacting with canine PD-1 (cPD-1). Ligand blockade was confirmed by this assay using biotinylated cPD-L1 / Fc protein. In short, cPD-1 CHO cells are seeded in 96-well plates at 4x10⁴ cells per well and incubated at 37 ° C for 18-24 hours until 95-100% confluence is reached. The cell culture medium is aspirated and the plates are washed 3 times with PBS + 0.05% Tween20 and 1 × CHO medium. Three-fold serial dilutions of the anti-cPD1 mAb are prepared in CHO medium, starting at 30 μg / ml, and each antibody dilution is added to the plate in a volume of 50 μl / well. Incubation was carried out at 37 ° C, 5% CO₂shaking continuously for 30 min. Add 50 μl / well of cPD-L1-Fc-biotin (2 μg / ml in CHO stock) and incubate at 37 ° C, 5% CO₂, continuing to shake, for 45 min. Plates are washed 6 times with PBS + 0.05% Tween 20. Add 100 μl / well streptavidin-HRP (1: 2000) in CHO medium and incubate for 30-60 min at 37 ° C, 5% CO₂... Plates are washed 5 times with PBS + 0.05% Tween 20 and then 100 µl / well of TMB developing substrate color is added. Color development is stopped by the addition of 50 μl / well of 1M phosphoric acid. Measure the optical density (OD) at A450-A620 using an ELISA plate reader.

277<u>Reactivity of mouse mAbs to PD-1 expressed on PBMCs obtained from healthy and cancer patients:</u>

278PBMC are prepared from EDTA-treated blood samples obtained from healthy and cancer-affected dogs by Ficoll separation. PBMCs resuspended in FACS buffer (PBS, 1% FBS and 0.1% sodium azide) are added at a concentration of 2.5 × 10⁵ cells per well and incubated with test monoclonal antibodies (mAbs) at different concentrations. The cells are incubated for 30 min at room temperature and then washed twice. The cells are then resuspended and incubated with a donkey anti-mouse IgG antibody (H + L chains) conjugated to Alexa-488 for 30 min at room temperature and then washed twice. The cells are then incubated with PB- and PE-conjugated antibodies against canine CD4 and CD8 for 30 min and washed. The washed cells are resuspended in FACS buffer and analyzed by flow cytometry to determine the percentage of CD4 or CD8 T cells that bind to anti-PD-1 monoclonal antibodies. Cells incubated only with secondary antibodies or with mAbs having a different specificity but belonging to the same isotype are used as controls.

279<u>Release of cytokines from PBMCs obtained from healthy and cancer patients:</u>

280PBMC are prepared from EDTA-treated blood samples obtained from healthy and cancer-affected dogs by Ficoll separation. Cells are washed 3 times, resuspended in complete tissue culture medium at a concentration of 2.5 x 10⁵ cells per well and plated into 96-well plates in triplicate. Cells are activated with Concanavalin A, 1 μg / ml. The test antibodies are added at various concentrations and the cultures are incubated for 96 hours. Cells incubated with conA in the absence of antibody or with conA and an antibody having a different specificity but belonging to the same isotype were used as controls. After culturing for 96 hours, supernatants are harvested and assayed for IFN-gamma release using a commercial canine IFN-gamma ELISA kit (R&amp;D Systems).

281<u>Cloning and identification of DNA sequences corresponding to variable regions of mouse mAbs:</u>

282The DNA sequence of the murine VH and VL strands and the DNA sequences encoding their CDRs are identified after the isolation of mRNA from each hybridoma using standard molecular biology techniques. The SEQ ID NO of the predicted CDR amino acid sequences derived from these hybridomas are listed below:

283It should be noted that there is a significant degree of homology between the amino acid sequences of the CDRs of each of the seven murine anti-canine PD-1 antibodies, examples of which are provided below.

284Table 4

285<u>CDR amino acid sequences</u>

286<img file="RU2761663C2_D0009.tif" />

287<img file="RU2761663C2_D0010.tif" />

288<u>Table 5A</u>

289<u>Canonical structures (classes) CDR VH-chain:</u>

290<img file="RU2761663C2_D0011.tif" />

291<u>Table 5B</u>

292<u>Canonical structures (classes) CDR VL-chain:</u>

293<img file="RU2761663C2_D0012.tif" />

294<u>EXAMPLE 3</u>

295<u>MUTANT DOG ANTIBODIES IgG-B SPECIFIC TO PD-1</u>

296There are four known canine IgG heavy chain subtypes designated IgG-A, IgG-B, IgG-C and IgG-D. Two well-known light chain subtypes are called lambda and kappa. However, in addition to binding and activating canine immune cells, a canine or caninated anti-PD-1 antibody should ideally have the following two characteristics:

297one. lack of effector functions such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and

2982. the ability to be easily purified on a large scale using standard industrial techniques such as protein A.

299None of the naturally occurring canine IgG isotypes meet both criteria. For example, IgG-B can be purified using Protein A, but has a high level of ADCC activity. IgG-C also has significant ADCC activity. IgG-A, on the other hand, weakly binds to protein A, but exhibits undesirable ADCC activity. In addition, neither IgG-C nor IgG-D can be purified on Protein A columns, although IgG-D does not exhibit ADCC activity. The present invention overcomes this problem by providing mutant canine IgG-B antibodies specific for PD-1; such antibodies lack effector functions such as ADCC and can be readily purified by standard industrial chromatography using protein A. Specific modifications are shown in Figure 8.

300Described IgG-B variants with reduced effector functions include the first IgG-B variant, which is obtained by replacing each of a lysine residue (D 277) and an asparagine residue (N 325) with an alanine residue [cIgGB (-) ADCC], the second variant , which is obtained by replacing the IgG-B hinge region with an IgG-D hinge region [cIgGB (+) D-hinge], the third variant, which is obtained by replacing the IgG-B hinge region with an IgG-A hinge region [cIgGB (+) A -hinge]. In addition, the second and third variants also contain the replacement of the same lysine and asparagine residues as in the first variant with an alanine residue. The numbering of the lysine and asparagine residues substituted in this invention follows the numbering scheme described for the heavy chains of canine IgG in Tang<i>et al.</i>, [<i>Vet Immunol and Immunopathol, 80: 259-270 </i>(2001)].

301<img file="RU2761663C2_D0013.tif" />

302<img file="RU2761663C2_D0014.tif" />

303<img file="RU2761663C2_D0015.tif" />

304<img file="RU2761663C2_D0016.tif" />

305<u>EXAMPLE 4</u>

306<u>ANTIBODY SEQUENCE INFORMATION</u>

307<u>(from EXAMPLE 2 above)</u>

308Leadership consistency is emphasized; CDR sequences are in bold; and the sequence of the wireframe is not underlined or in bold.

309mAb<b></b>1B5: Heavy chain variable region DNA (SEQ ID NO: 43)

310<img file="RU2761663C2_D0017.tif" />

311mAb<b></b>1B5: heavy chain variable region, protein (SEQ ID NO: 44)

312<img file="RU2761663C2_D0018.tif" />

313mAb<b></b>1B5: light chain variable region DNA (SEQ ID NO: 45)

314<img file="RU2761663C2_D0019.tif" />

315mAb<b></b>1B5: light chain variable region, protein (SEQ ID NO: 46)

316<img file="RU2761663C2_D0020.tif" />

317mAb<b></b>2G9: heavy chain variable region DNA (SEQ ID NO: 47)

318<img file="RU2761663C2_D0021.tif" /><img file="RU2761663C2_D0022.tif" />

319mAb<b></b>2G9: heavy chain variable region, protein (SEQ ID NO: 48)

320<img file="RU2761663C2_D0023.tif" />

321mAb<b></b>2G9: light chain variable region DNA (SEQ ID NO: 49)

322<img file="RU2761663C2_D0024.tif" />

323mAb<b></b>2G9: light chain variable region, protein (SEQ ID NO: 50)

324<img file="RU2761663C2_D0025.tif" />

325mAb<b></b>2H9: heavy chain variable region DNA (SEQ ID NO: 51)

326<img file="RU2761663C2_D0026.tif" /><img file="RU2761663C2_D0027.tif" />

327mAb<b></b>2H9: heavy chain variable region, protein (SEQ ID NO: 52)

328<img file="RU2761663C2_D0028.tif" />

329mAb<b></b>2H9: light chain variable region DNA (SEQ ID NO: 53)

330<img file="RU2761663C2_D0029.tif" />

331mAb<b></b>2H9: light chain variable region, protein (SEQ ID NO: 54)

332<img file="RU2761663C2_D0030.tif" />

333mAb<b></b>3B6: heavy chain variable region DNA (SEQ ID NO: 55)

334<img file="RU2761663C2_D0031.tif" />

335gcaccactctcacagtctcctca

336mAb<b></b>3B6: heavy chain variable region, protein (SEQ ID NO: 56)

337<img file="RU2761663C2_D0032.tif" />

338mAb<b></b>3B6: light chain variable region DNA (SEQ ID NO: 57)

339<img file="RU2761663C2_D0033.tif" />

340mAb<b></b>3B6: light chain variable region, protein (SEQ ID NO: 58)

341<img file="RU2761663C2_D0034.tif" />

342mAb 4D12: heavy chain variable region, DNA (SEQ ID NO: 59)

343<img file="RU2761663C2_D0035.tif" />

344mAb<b></b>4D12: heavy chain variable region, protein (SEQ ID NO: 60)

345<img file="RU2761663C2_D0036.tif" />

346mAb<b></b>4D12: light chain variable region DNA (SEQ ID NO: 61)

347<img file="RU2761663C2_D0037.tif" />

348mAb<b></b>4D12: light chain variable region, protein (SEQ ID NO: 62)

349<img file="RU2761663C2_D0038.tif" />

350mAb<b></b>5G5: heavy chain variable region DNA (SEQ ID NO 63)

351<img file="RU2761663C2_D0039.tif" />

352mAb<b></b>5G5: heavy chain variable region, protein (SEQ ID NO: 64)

353<img file="RU2761663C2_D0040.tif" />

354mAb<b></b>5G5: light chain variable region DNA (SEQ ID NO: 65)

355<img file="RU2761663C2_D0041.tif" />

356mAb<b></b>5G5: light chain variable region, protein (SEQ ID NO: 66)

357<img file="RU2761663C2_D0042.tif" />

358mAb<b></b>7C9: heavy chain variable region DNA (SEQ ID NO: 67)

359<img file="RU2761663C2_D0043.tif" />

360mAb<b></b>7C9: heavy chain variable region, protein (SEQ ID NO: 68)

361<img file="RU2761663C2_D0044.tif" />

362<img file="RU2761663C2_D0045.tif" />

363mAb<b></b>7C9: light chain variable region DNA (SEQ ID NO: 69)

364<img file="RU2761663C2_D0046.tif" />

365mAb<b></b>7C9: light chain variable region, protein (SEQ ID NO: 70)

366<img file="RU2761663C2_D0047.tif" />

367mAb<b></b>1E4: heavy chain variable region DNA (SEQ ID NO: 109)

368<img file="RU2761663C2_D0048.tif" />

369mAb<b></b>1E4: heavy chain variable region, protein (SEQ ID NO: 110)

370<img file="RU2761663C2_D0049.tif" />

371mAb<b></b>1E4: light chain variable region, DNA (SEQ ID NO: 111)

372<img file="RU2761663C2_D0050.tif" /><img file="RU2761663C2_D0051.tif" />

373mAb<b></b>1E4: light chain variable region, protein (SEQ ID NO: 112)

374<img file="RU2761663C2_D0052.tif" />

375<u>EXAMPLE 5</u>

376<u>EPITOPE MAPPING OF ANTIBODIES </u>to PD-1 dogs

377<u>Introduction</u>

378The interaction of antibodies with the protein antigens recognized by them is carried out through the binding of specific amino acids (paratopes) of the antibody to specific amino acids (epitopes) of the target antigens. An epitope is an antigenic determinant capable of interacting specifically with immunoglobulin. It consists of a group of amino acids on the surface of an antigen.

379A protein of interest may contain several epitopes that are recognized by different antibodies. Epitopes recognized by antibodies are classified as linear or conformational epitopes. Linear epitopes are formed by a continuous chain of amino acids that make up a protein, while conformational epitopes are composed of amino acids that are not adjacent (for example, far from each other) in the primary amino acid sequence, but converge as a result of the three-dimensional folding of the protein.

380Epitope mapping is the process of identifying amino acid sequences (i.e., epitopes) that are recognized by antibodies on target antigens. The identification of epitopes recognized by monoclonal antibodies (mAbs) on target antigens is important. For example, it can contribute to the development of new therapeutics, diagnostics and vaccines. Epitope mapping can also be used to select optimized therapeutic monoclonal antibodies and elucidate their mechanisms of action. The epitope information can also be used to identify unique cancer epitopes and to determine the protective or pathogenic effects of vaccines.

381Epitope mapping can be performed using polyclonal or monoclonal antibodies, using different methods to identify epitopes, depending on the intended nature of the epitope (ie, linear or conformational). Linear epitope mapping is simpler and relatively easy to perform. Peptide scans are often used by commercial services to map linear epitopes. In this case, a plurality of overlapping short peptide sequences of the target protein are prepared by chemical synthesis and tested for their ability to bind antibodies of interest. This strategy is fast, relatively inexpensive, and has high throughput. On the other hand, discontinuous epitope mapping is technically more difficult and requires more specialized techniques such as X-ray co-crystallography of the monoclonal antibody with the target protein, hydrogen-deuterium (H / D) exchange, and / or mass spectroscopy in combination with enzymatic splitting.

382<u>PD-1 epitope mapping using the ProImmune microchip</u>^®<u>:</u>

383To identify the amino acids that form the epitopes for the anti-PD1 mAb, a total of 28 peptides are chemically synthesized, which are 15 amino acids in length and overlap in 10 amino acids. Such a library of overlapping peptides spans the entire length of the canine PD-1 protein. The sequences of these peptides are shown in Table 6 below. Determination of peptide-antibody binding is carried out by attaching the antibody samples to the ProArray Ultra peptide microchip^®, followed by incubation with a secondary antibody containing a fluorescent label. All peptides are synthesized separately and then attached to the ProArray Ultra work surface^® along with mouse IgG ProImmune^®used as a control. This optimized method ensures that the peptides present on the chip accurately mimic the properties of the corresponding region of the protein, avoids the physicochemical variations inherent in free peptides, and obtains a compatible, combined peptide and protein matrix platform. Test analytes (peptides) are dispensed onto ProArray Ultra plates^® at individual points and then the corresponding gal-files ensure an exact match of the obtained parameters of the chip with the applied analytes. ProArray Ultra Plates^® blocked with a validated blocking buffer to reduce nonspecific mAb binding. Thereafter, they are incubated with mAb samples and then with a specific fluorescently labeled secondary antibody. After several washing steps, the ProArray Ultra chips^® dried and scanned with a high resolution fluorescent microarray scanning system. After scanning fluorescently labeled ProArray Ultra plates^®, the scanner records an image that is analyzed by image analysis software that interprets and quantifies the fluorescence intensity levels in each fluorescent spot on the microchip plate being scanned. The results of this experiment indicate that some of the canine PD-1 peptides are recognized by the individual mAbs analyzed. The specific mAbs and the amino acid sequences they recognize are shown in Table 7. The results of this study demonstrate that mAb 2H9 recognizes an epitope located in the extracellular domain of canine PD-1 and contains the amino acid sequence described in SEQ ID NO: 84, and mAb 1A1 recognizes an epitope containing the amino acid sequence described in SEQ ID NO: 84. and the overlapping amino acid sequence described in SEQ ID NO: 83.

384<u>PD-1 epitope mapping by mass spectrometry:</u>

385To identify the potentially discontinuous epitopes recognized by the anti-canine PD-1 antibody, a method is used that includes chemical crosslinking and detection by mass spectrometry (CovalX^® Instrument Incorporated). The use of this technology to map canine PD-1 epitopes has identified at least a portion of the epitopes recognized by the mAbs listed in Table 8. As can be seen from Table 8, mAb 3B6 recognizes at least a portion of an epitope located in the extracellular domain of PD -1 dogs, in the amino acid sequence of SEQ ID NO: 99, and the mAb 2G9 recognizes at least part of the epitope located in the amino acid sequence of SEQ ID NO: 100. On the other hand, mAb 1E4 and mAb 1B5 recognize at least a portion of an epitope in the amino acid sequence of SEQ ID NO: 101 and the amino acid sequence of SEQ ID NO: 102, respectively.

386As shown in Figure 9a, the results of a study using chemical crosslinking, MALDI mass spectrometry for high mass particle analysis and nLC-Orbitrap mass spectrometry indicate that the epitope on canine PD-1 recognized by caninated 2G9 antibody includes yourself R₆₂, R₆₉, R₇₂ and R₇₅ from SEQ ID NO: 2. Results from a similar analysis demonstrate that the epitope on canine PD-1 recognized by caninated antibody 3B6 includes R₇₅ and R₉₀ from the sequence SEQ ID NO: 2. Accordingly, R₇₅ is a particularly important amino acid residue found in one or more epitopes of canine PD-1. Interestingly, after performing these analyzes, the amino acid sequences of CDR 1A1 were found to be identical to the amino acid sequences of CDR 2G9. The concurrent results obtained using these two very different methods for the PD-1 region to which 2G9 binds and for the region to which 1A1 binds, indicate that this region contains amino acid residues that are part of the PD-1 epitope, which is recognized by caninated anti-PD-1 antibodies (see tables 7 and 8 below).

387In addition, the region of the PD-1 amino acid sequence that is recognized by the test blocking antibodies of the present invention is located in the extracellular domain of canine PD-1. The recognition site is included in the peptide below (see tables 7 and 8 below).

388NQTDKLAAFQE<b>D</b><b><u>R</u></b><b>IEPGRD</b><b><u>R</u></b><b>RF</b><b><u>R</u></b><b>VM</b>^<b>*</b><b><u>R</u></b><b>LPNGR</b>DFHMSIVAA<b><u>R</u></b>LNDS (SEQ ID NO: 103)

389Within a given peptide, there is a short peptide that is shown in bold. The specified short peptide is recognized by the ProImmune microchip^® (see table 7).

390<b>D</b><b><u>R</u></b><b>IEPGRD</b><b><u>R</u></b><b>RF</b><b><u>R</u></b><b>VM</b>^<b>*</b><b><u>R</u></b><b>LPNGR </b>(SEQ ID NO: 104)

391It should be noted that all R₆₂, R₆₉, R₇₂ and R₇₅ from the sequence SEQ ID NO: 2 are included in both the longer (SEQ ID NO: 103) and the shorter peptide (SEQ ID NO: 104), while R₉₀ from SEQ ID NO: 2 is formulated as a longer peptide. These five arginine residues appear to be important amino acid residues in one or more epitopes of canine PD-1. As indicated in Tables 6-8, the methionine residue (*) marked with an asterisk is also described as a threonine residue.

392<tables num="1"><table frame="all"><tgroup align="center" rowsep="1" colsep="1" cols="4"><colspec colname="c1" colwidth="29mm" /><colspec colname="c2" colwidth="52mm" /><colspec colname="c3" colwidth="30mm" /><colspec colname="c4" colwidth="57mm" /><tbody><row><entry namest="c1" nameend="c4" align="right" rowsep="1" colsep="0">TABLE 6<br />PEPTIDES USED FOR EPITOPE MAPPING WITH THE PROImmune Microchip^®</entry></row><row><entry align="center" rowsep="1" colsep="1">SEQ ID NO:</entry><entry align="center" rowsep="1" colsep="1">ANTIGENIC PEPTIDE</entry><entry align="center" rowsep="1" colsep="1">SEQ ID NO:</entry><entry align="center" rowsep="1" colsep="0">ANTIGENIC PEPTIDE</entry></row><row><entry align="center" rowsep="1" colsep="1">71</entry><entry align="center" rowsep="1" colsep="1">LDSPDRPWSPLTFSP</entry><entry align="center" rowsep="1" colsep="1">85</entry><entry align="center" rowsep="1" colsep="0">FRVM^*RLPNGRDFHMS</entry></row><row><entry align="center" rowsep="1" colsep="1">72</entry><entry align="center" rowsep="1" colsep="1">RPWSPLTFSPAQLTV</entry><entry align="center" rowsep="1" colsep="1">86</entry><entry align="center" rowsep="1" colsep="0">LPNGRDFHMSIVAAR</entry></row><row><entry align="center" rowsep="1" colsep="1">73</entry><entry align="center" rowsep="1" colsep="1">LTFSPAQLTVQEGEN</entry><entry align="center" rowsep="1" colsep="1">87</entry><entry align="center" rowsep="1" colsep="0">DFHMSIVAARLNDSG</entry></row><row><entry align="center" rowsep="1" colsep="1">74</entry><entry align="center" rowsep="1" colsep="1">AQLTVQEGENATFTC</entry><entry align="center" rowsep="1" colsep="1">88</entry><entry align="center" rowsep="1" colsep="0">IVAARLNDSGIYLCG</entry></row><row><entry align="center" rowsep="1" colsep="1">75</entry><entry align="center" rowsep="1" colsep="1">QEGENATFTCSLADI</entry><entry align="center" rowsep="1" colsep="1">89</entry><entry align="center" rowsep="1" colsep="0">LNDSGIYLCGAIYLP</entry></row><row><entry align="center" rowsep="1" colsep="1">76</entry><entry align="center" rowsep="1" colsep="1">ATFTCSLADIPDSFV</entry><entry align="center" rowsep="1" colsep="1">90</entry><entry align="center" rowsep="1" colsep="0">IYLCGAIYLPPNTQI</entry></row><row><entry align="center" rowsep="1" colsep="1">77</entry><entry align="center" rowsep="1" colsep="1">SLADIPDSFVLNWYR</entry><entry align="center" rowsep="1" colsep="1">91</entry><entry align="center" rowsep="1" colsep="0">AIYLPPNTQINESPR</entry></row><row><entry align="center" rowsep="1" colsep="1">78</entry><entry align="center" rowsep="1" colsep="1">PDSFVLNWYRLSPRN</entry><entry align="center" rowsep="1" colsep="1">92</entry><entry align="center" rowsep="1" colsep="0">PNTQINESPRAELSV</entry></row><row><entry align="center" rowsep="1" colsep="1">79</entry><entry align="center" rowsep="1" colsep="1">LNWYRLSPRNQTDKL</entry><entry align="center" rowsep="1" colsep="1">93</entry><entry align="center" rowsep="1" colsep="0">NESPRAELSVTERTL</entry></row><row><entry align="center" rowsep="1" colsep="1">80</entry><entry align="center" rowsep="1" colsep="1">LSPRNQTDKLAAFQE</entry><entry align="center" rowsep="1" colsep="1">94</entry><entry align="center" rowsep="1" colsep="0">AELSVTERTLEPPTQ</entry></row><row><entry align="center" rowsep="1" colsep="1">81</entry><entry align="center" rowsep="1" colsep="1">QTDKLAAFQEDRIEP</entry><entry align="center" rowsep="1" colsep="1">95</entry><entry align="center" rowsep="1" colsep="0">TERTLEPPTQSPSPP</entry></row><row><entry align="center" rowsep="1" colsep="1">82</entry><entry align="center" rowsep="1" colsep="1">AAFQEDRIEPGRDRR</entry><entry align="center" rowsep="1" colsep="1">96</entry><entry align="center" rowsep="1" colsep="0">EPPTQSPSPPPRLSG</entry></row><row><entry align="center" rowsep="1" colsep="1">83</entry><entry align="center" rowsep="1" colsep="1">DRIEPGRDRRFRVM^*R</entry><entry align="center" rowsep="1" colsep="1">97</entry><entry align="center" rowsep="1" colsep="0">SPSPPPRLSGQLQGL</entry></row><row><entry align="center" rowsep="1" colsep="1">84</entry><entry align="center" rowsep="1" colsep="1">GRDRRFRVM^*RLPNGR</entry><entry align="center" rowsep="1" colsep="1">98</entry><entry align="center" rowsep="1" colsep="0">PSPPPRLSGQLQGLV</entry></row><row><entry namest="c1" nameend="c4" align="center" rowsep="0" colsep="0">* This methionine residue is also referred to as a threonine residue.</entry></row></tbody></tgroup></table></tables>

393<tables num="1"><table frame="all"><tgroup align="center" rowsep="1" colsep="1" cols="3"><colspec colname="c1" colwidth="49mm" /><colspec colname="c2" colwidth="55mm" /><colspec colname="c3" colwidth="64mm" /><tbody><row><entry namest="c1" nameend="c3" align="right" rowsep="1" colsep="0">TABLE 7<br />EPITOPES OF PD-1 RECOGNIZED BY CANINE PD-1 mAb IDENTIFIED BY PROImmune MICROCHIP^®</entry></row><row><entry align="center" rowsep="1" colsep="1">ANTIBODY</entry><entry align="center" rowsep="1" colsep="1">ANTIGENIC PEPTIDE</entry><entry align="center" rowsep="1" colsep="0">SEQ ID NO:</entry></row><row><entry align="center" rowsep="1" colsep="1">2H9</entry><entry align="center" rowsep="1" colsep="1">GRDRRFRVM^*RLPNGR</entry><entry align="center" rowsep="1" colsep="0">84</entry></row><row><entry align="center" rowsep="1" colsep="1">1A1^#</entry><entry align="center" rowsep="1" colsep="1">DRIEPGRDRRFRVM^*R</entry><entry align="center" rowsep="1" colsep="0">83</entry></row><row><entry align="center" rowsep="1" colsep="1">1A1</entry><entry align="center" rowsep="1" colsep="1">GRDRRFRVM^*RLPNGR</entry><entry align="center" rowsep="1" colsep="0">84</entry></row><row><entry namest="c1" nameend="c3" align="center" rowsep="0" colsep="0">* This methionine residue is also referred to as a threonine residue.<br /># CDRs of antibody 1A1 are identical to those of antibody 2G9.</entry></row></tbody></tgroup></table></tables>

394<tables num="1"><table frame="all"><tgroup align="center" rowsep="1" colsep="1" cols="3"><colspec colname="c1" colwidth="31mm" /><colspec colname="c2" colwidth="94mm" /><colspec colname="c3" colwidth="42mm" /><tbody><row><entry namest="c1" nameend="c3" align="right" rowsep="1" colsep="0">TABLE 8<br />EPITOPES OF PD-1 RECOGNIZED BY CANINE PD-1 mAb IDENTIFIED BY MASS SPECTROMETRY</entry></row><row><entry align="center" rowsep="1" colsep="1">ANTIBODY</entry><entry align="center" rowsep="1" colsep="1">ANTIGENIC PEPTIDE</entry><entry align="center" rowsep="1" colsep="0">SEQ ID NO:</entry></row><row><entry align="center" rowsep="1" colsep="1">3B6</entry><entry align="center" rowsep="1" colsep="1">RFRVM^*RLPNGRDFHMSIVAARLNDS</entry><entry align="center" rowsep="1" colsep="0">99</entry></row><row><entry align="center" rowsep="1" colsep="1">2G9</entry><entry align="center" rowsep="1" colsep="1">LAAFQEDRIEPGRDRRFRVM^*RLPNGR</entry><entry align="center" rowsep="1" colsep="0">100</entry></row><row><entry align="center" rowsep="1" colsep="1">1E4</entry><entry align="center" rowsep="1" colsep="1">EDRIEPGRDRRFRVM^*RLPNGRDFHMSIVAAR</entry><entry align="center" rowsep="1" colsep="0">101</entry></row><row><entry align="center" rowsep="1" colsep="1">1B5</entry><entry align="center" rowsep="1" colsep="1">NQTDKLAAFQEDRIEPGRDRRFRVM^*RLPNGR</entry><entry align="center" rowsep="1" colsep="0">102</entry></row><row><entry namest="c1" nameend="c3" align="center" rowsep="0" colsep="0">* This methionine residue is also referred to as a threonine residue.</entry></row></tbody></tgroup></table></tables>

395<tables num="1"><table frame="all"><tgroup align="center" rowsep="1" colsep="1" cols="8"><colspec colname="c1" colwidth="21mm" /><colspec colname="c2" colwidth="14mm" /><colspec colname="c3" colwidth="10mm" /><colspec colname="c4" colwidth="44mm" /><colspec colname="c5" colwidth="15mm" /><colspec colname="c6" colwidth="14mm" /><colspec colname="c7" colwidth="10mm" /><colspec colname="c8" colwidth="42mm" /><tbody><row><entry namest="c1" nameend="c8" valign="middle" align="right" rowsep="1" colsep="0">TABLE 9<br />DESCRIPTION OF SEQUENCES PD-1 DOG and DOG PD-L1</entry></row><row><entry valign="middle" align="center" rowsep="1" colsep="1">SEQ ID NO</entry><entry valign="middle" align="center" rowsep="1" colsep="1">N.P.</entry><entry valign="middle" align="center" rowsep="1" colsep="1">A.P.</entry><entry valign="middle" align="center" rowsep="1" colsep="1">Description</entry><entry valign="middle" align="center" rowsep="1" colsep="1">SEQ ID NO</entry><entry valign="middle" align="center" rowsep="1" colsep="1">N.P.</entry><entry valign="middle" align="center" rowsep="1" colsep="1">A.P.</entry><entry valign="middle" align="center" rowsep="1" colsep="0">Description</entry></row><row><entry valign="middle" align="center" rowsep="1" colsep="1">1</entry><entry valign="middle" align="center" rowsep="1" colsep="1">√</entry><entry valign="middle" rowsep="1" colsep="1" /><entry valign="middle" align="center" rowsep="1" colsep="1">Full size PD-1 dogs</entry><entry valign="middle" align="center" rowsep="1" colsep="1">7</entry><entry valign="middle" align="center" rowsep="1" colsep="1">√</entry><entry valign="middle" rowsep="1" colsep="1" /><entry valign="middle" align="center" rowsep="1" colsep="0">Full Size Canine PD-L1</entry></row><row><entry valign="middle" align="center" rowsep="1" colsep="1">2</entry><entry valign="middle" rowsep="1" colsep="1" /><entry valign="middle" align="center" rowsep="1" colsep="1">√</entry><entry valign="middle" align="center" rowsep="1" colsep="1">Full size PD-1 dogs</entry><entry valign="middle" align="center" rowsep="1" colsep="1">8</entry><entry valign="middle" rowsep="1" colsep="1" /><entry valign="middle" align="center" rowsep="1" colsep="1">√</entry><entry valign="middle" align="center" rowsep="1" colsep="0">Full Size Canine PD-L1</entry></row><row><entry valign="middle" align="center" rowsep="1" colsep="1">3</entry><entry valign="middle" align="center" rowsep="1" colsep="1">√</entry><entry valign="middle" rowsep="1" colsep="1" /><entry valign="middle" align="center" rowsep="1" colsep="1">ECD PD-1 dogs</entry><entry valign="middle" align="center" rowsep="1" colsep="1">9</entry><entry valign="middle" align="center" rowsep="1" colsep="1">√</entry><entry valign="middle" rowsep="1" colsep="1" /><entry valign="middle" align="center" rowsep="1" colsep="0">ECD Canine PD-L1</entry></row><row><entry valign="middle" align="center" rowsep="1" colsep="1">4</entry><entry valign="middle" rowsep="1" colsep="1" /><entry valign="middle" align="center" rowsep="1" colsep="1">√</entry><entry valign="middle" align="center" rowsep="1" colsep="1">ECD PD-1 dogs</entry><entry valign="middle" align="center" rowsep="1" colsep="1">10</entry><entry valign="middle" rowsep="1" colsep="1" /><entry valign="middle" align="center" rowsep="1" colsep="1">√</entry><entry valign="middle" align="center" rowsep="1" colsep="0">ECD Canine PD-L1</entry></row><row><entry valign="middle" align="center" rowsep="1" colsep="1">5</entry><entry valign="middle" align="center" rowsep="1" colsep="1">√</entry><entry valign="middle" rowsep="1" colsep="1" /><entry valign="middle" align="center" rowsep="1" colsep="1">Canine PD-1 - Human IgG1</entry><entry valign="middle" align="center" rowsep="1" colsep="1">11</entry><entry valign="middle" align="center" rowsep="1" colsep="1">√</entry><entry valign="middle" rowsep="1" colsep="1" /><entry valign="middle" align="center" rowsep="1" colsep="0">Canine PD-L1 - Human IgG1</entry></row><row><entry valign="middle" align="center" rowsep="1" colsep="1">6</entry><entry valign="middle" rowsep="1" colsep="1" /><entry valign="middle" align="center" rowsep="1" colsep="1">√</entry><entry valign="middle" align="center" rowsep="1" colsep="1">Canine PD-1 - Human IgG1</entry><entry valign="middle" align="center" rowsep="1" colsep="1">12</entry><entry valign="middle" rowsep="1" colsep="1" /><entry valign="middle" align="center" rowsep="1" colsep="1">√</entry><entry valign="middle" align="center" rowsep="1" colsep="0">Canine PD-L1 - Human IgG1</entry></row><row><entry valign="middle" align="center" rowsep="1" colsep="1">105</entry><entry valign="middle" align="center" rowsep="1" colsep="1">√</entry><entry valign="middle" rowsep="1" colsep="1" /><entry valign="middle" align="center" rowsep="1" colsep="1">Dogs Full Size PD-1 + Signal Sequence</entry><entry valign="middle" align="center" rowsep="1" colsep="1">107</entry><entry valign="middle" align="center" rowsep="1" colsep="1">√</entry><entry valign="middle" rowsep="1" colsep="1" /><entry valign="middle" align="center" rowsep="1" colsep="0">Full Size Canine PD-L1 + Signal Sequence</entry></row><row><entry valign="middle" align="center" rowsep="1" colsep="1">106</entry><entry valign="middle" rowsep="1" colsep="1" /><entry valign="middle" align="center" rowsep="1" colsep="1">√</entry><entry valign="middle" align="center" rowsep="1" colsep="1">Dogs Full Size PD-1 + Signal Sequence</entry><entry valign="middle" align="center" rowsep="1" colsep="1">108</entry><entry valign="middle" rowsep="1" colsep="1" /><entry valign="middle" align="center" rowsep="1" colsep="1">√</entry><entry valign="middle" align="center" rowsep="1" colsep="0">Full Size Canine PD-L1 +<br />signal sequence</entry></row><row><entry valign="middle" align="center" rowsep="0" colsep="1">113</entry><entry valign="middle" rowsep="0" colsep="1" /><entry valign="middle" align="center" rowsep="0" colsep="1">√</entry><entry valign="middle" align="center" rowsep="0" colsep="1">Canine PD-1 - Human IgG1 + Signal Sequence</entry><entry valign="middle" rowsep="0" colsep="1" /><entry valign="middle" rowsep="0" colsep="1" /><entry valign="middle" rowsep="0" colsep="1" /><entry valign="middle" rowsep="0" colsep="0" /></row></tbody></tgroup></table></tables>

396<tables num="1"><table frame="all"><tgroup align="center" rowsep="1" colsep="1" cols="8"><colspec colname="c1" colwidth="17mm" /><colspec colname="c2" colwidth="16mm" /><colspec colname="c3" colwidth="17mm" /><colspec colname="c4" colwidth="35mm" /><colspec colname="c5" colwidth="13mm" /><colspec colname="c6" colwidth="14mm" /><colspec colname="c7" colwidth="16mm" /><colspec colname="c8" colwidth="38mm" /><tbody><row><entry namest="c1" nameend="c8" align="right" rowsep="1" colsep="0">TABLE 10<br />DESCRIPTION OF DOG IgGB SEQUENCE MODIFICATIONS</entry></row><row><entry align="center" rowsep="1" colsep="1">№</entry><entry align="center" rowsep="1" colsep="1">N.P.</entry><entry align="center" rowsep="1" colsep="1">A.P.</entry><entry align="center" rowsep="1" colsep="1">Description</entry><entry align="center" rowsep="1" colsep="1">№</entry><entry align="center" rowsep="1" colsep="1">N.P.</entry><entry align="center" rowsep="1" colsep="1">A.P.</entry><entry align="center" rowsep="1" colsep="0">Description</entry></row><row><entry align="center" rowsep="1" colsep="1">39</entry><entry rowsep="1" colsep="1" /><entry align="center" rowsep="1" colsep="1">√</entry><entry align="center" rowsep="1" colsep="1">cIgGB wild type</entry><entry align="center" rowsep="1" colsep="1">41</entry><entry rowsep="1" colsep="1" /><entry align="center" rowsep="1" colsep="1">√</entry><entry align="center" rowsep="1" colsep="0">cIgGB (+) D-joint</entry></row><row><entry align="center" rowsep="0" colsep="1">40</entry><entry rowsep="0" colsep="1" /><entry align="center" rowsep="0" colsep="1">√</entry><entry align="center" rowsep="0" colsep="1">cIgGB (+) A-hinge</entry><entry align="center" rowsep="0" colsep="1">42</entry><entry rowsep="0" colsep="1" /><entry align="center" rowsep="0" colsep="1">√</entry><entry align="center" rowsep="0" colsep="0">cIgGB (-) ADCC</entry></row></tbody></tgroup></table></tables>

397All references cited herein are incorporated herein by reference to the extent that each individual reference including publication, database component (such as GenBank sequence or GeneID value), patent application or patent application, would be specifically and separately indicated as incorporated by reference. Applicants have cited this statement of incorporation by reference in accordance with 37 CFR §1.57 (b) (1) in relation to all and each of the individual references including publication, database component (such as GenBank sequence or GeneID value), patent application or patent, each of which is clearly defined in accordance with 37 CFR §1.57 (b) (2), even if such quotation is not in close proximity to the stated statement of inclusion by reference. Mention of the specified statements of inclusion by reference, if any, within the description does not in any way weaken the general statement of inclusion by reference. The citation of links in this document should not be construed as an admission that the link is related to the prior art, or as an admission regarding the content or date of publication of the said documents.

398The scope of the present invention should not be limited to the specific embodiments described herein. In fact, those skilled in the art will be able to make various modifications of the invention other than those described herein based on the above description and the accompanying drawings. Such modifications are also within the scope of the appended claims.

399The above description is considered sufficient to enable a person skilled in the art to carry out the present invention. Based on the above description, those skilled in the art may make various modifications of the invention other than those shown and described herein, which fall within the scope of the appended claims.