Therapeutic antibodies

Abstract

The present disclosure provides a separated canine antibody, or an antigen-binding portion thereof, that binds to an epitope of an extracellular domain of canine PD-1; compositions comprising the isolated canine antibody or antigen binding portion thereof; and methods of their use.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to UK patent application number 2300904.6 filed on 20 January 2023 and UK patent application number 2310818.6 filed on 14 July 2023, the entire contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on January 17, 2024, is named P44613WO1.xml and is 299.008 bytes in size. Background Art

[0005] The present invention relates to antibodies that modulate the PD-1 signaling pathway to treat inflammatory diseases in companion animals.

[0006] Human programmed death receptor 1 (PD-1) protein is encoded by the PDCD1 gene and is expressed as a 32kDa type I transmembrane protein (Agata 1996 Int Immunol 8(5):765-72). PD-1 is a member of the immunoglobulin superfamily (Ishida 1992 EMBO 11(11):3887-95), and is an inhibitory member of the expanded CD28 / CTLA-4 T cell regulatory factor family. Other members of the family include CD28, CTLA-4, ICOS, and BTLA. PD-1 exists in a monomeric form and lacks the unpaired cysteine ​​residue characteristic of other CD28 family members (Zhang 2004 Immunity 20:337-47). Its cytoplasmic domain contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM), both of which are phosphorylated during signal transduction (Riley 2009 Immunol Rev 229(1):114-25).

[0007] PD-1 is expressed on B cells, T cells, and monocytes (Agata 1996). The role of PD-1 in maintaining immune self-tolerance has been demonstrated in PDCD1- / - mice, which develop autoimmune diseases (Nishimura 1999 Immunity 11:141-51, Nishimura 2001 Science 291(5502):319-22). Therefore, the PD-1 pathway regulates antigen responses, thereby balancing autoimmunity and tolerance.

[0008] PD-1 has two ligands that mediate its regulatory function. PD-L1 (B7-H1) is typically expressed on dendritic cells, macrophages, resting B cells, bone marrow-derived mast cells and T cells, and non-hematopoietic cell lineages (see Francisco 2010 Immunol Rev 236:219-42 for details). PD-L2 (B7-DC) is primarily expressed on dendritic cells and macrophages (Tseng 2001 J Exp Med 193(7):839-45). Ligand expression is influenced by local mediators and may be upregulated by inflammatory cytokines.

[0009] PD-1 binds to its corresponding ligand, PD-L1, and attenuates T cell responses. Antibodies that bind to PD-1 and block the binding of PD-L1 are used in human cancer therapy to enhance tumor-specific CD8+ T cell immunity and the clearance of tumor cells by the immune system. PD-1 is considered an immunosuppressive protein that negatively regulates TCR signaling. The interaction between PD-1 and PD-L1 can act as an immune checkpoint, which can lead to, for example, a decrease in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and / or immune evasion of cancer cells. Immunosuppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or PD-L2; the effects are additive when the interaction of PD-1 with both PD-L1 and PD-L2 is blocked.

[0010] As T cells are activated and co-stimulated by antigen-presenting cells (APCs), T cell expression of PD-1 is induced. Engagement of PD-1 with ligands on APCs cross-links PD-1 and causes it to aggregate into the T cell receptor (TCR) complex within the immune synapse (Yokosuka 2012 J Exp Med 209(9):1201-17). Within the T cell cytoplasm, the PD-1 signaling domains ITIM and ITSM are phosphorylated. This induces tyrosine phosphatases containing Src-homology-2 domains (SHP1 / 2), which attenuate various components of T cell receptor (TCR) signaling. T cell activation is inhibited, which leads to a decrease in cytokine response, proliferation, and cytolytic activity. This downregulation of T cell function is intended to prevent overstimulation, thereby allowing cells to develop tolerance to weakly immunogenic self-antigens.

[0011] The PD-1 pathway can be exploited in cancer or infection, whereby tumors or viruses can evade effective immune recognition and T cells display an 'exhausted' phenotype. PD-L1 has also been shown to be expressed in a variety of tumor types, including urothelial carcinoma, ovarian cancer, breast cancer, cervical cancer, colon cancer, pancreatic cancer, gastric cancer, melanoma, glioblastoma, and non-small cell lung cancer (see Callahan 2014 J Leukoc Biol 94(1):41-53). Cytokines produced by cancer stromal cells can further upregulate PD-L1 in the tumor microenvironment (He 2015 Nature Scientific Reports 5:13110). As a result, tumor-specific T cells become anergic through PD-1 signaling and are therefore unable to eliminate their targets. Regulatory T cells (T regs) have also been shown to express high levels of PD-1, and these further suppress anti-tumor responses (Lowther 2016 JCI Insight 1(5):85935).

[0012] Disruption of the PD-1:PD-L1 interaction enhances T cell activity. Anti-PD-1 monoclonal antibodies have demonstrated blockade of the interaction between PD-1 and its ligand (Wang 2014 Cancer Immunol Res 2(9):846-56). PD-1 blockade enhances T cell function in vitro, as demonstrated by improved proliferation and cytokine responses in mixed lymphocyte reactions of T cells and dendritic cells. Cytotoxic lymphocytes (CTLs) derived from human melanoma patients have also been shown to be enhanced by PD-1 blockade in vitro with the antibody OPDIVO (nivolumab) and may become resistant to Treg inhibition (Wang 2009 Int Immunol 21(9):1065-1077). This antibody has been tested in human clinical dose-escalation studies in melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), and other tumors. It has shown improved overall survival in NSCLC patients compared with chemotherapy. Another PD-1 blocking antibody, KEYTRUDA® (pembrolizumab), has demonstrated responses in human NSCLC patients who are refractory to CTLA-4 blockade. Both OPDIVO® and KEYTRUDA® functionally block the interaction of human PD-1 with its ligand.

[0013] It is possible to induce human PD-1 signaling by cross-linking on the membrane with a combination of anti-PD-1 and anti-CD3 antibodies (Bennett 2003 J Immunol 170:711-18, Keir 2005 J Immunol 175:7372-7379). This function may be detrimental during anti-tumor responses because T cell activity will be suppressed. If inhibition of T cell responses is desired, agonist anti-PD-1 antibodies or antibodies with effector function can be used to treat immune-related diseases such as rheumatoid arthritis.

[0014] Antibodies that recognize canine PD-1 are described in WO 2015 / 091910, WO 2015 / 091911, and WO 2015091914. Mice were immunized with canine PD-1 to generate monoclonal antibodies, from which CDR sequences were extracted and placed into a canine immunoglobulin framework in a process called "canization."

[0015] Caninization of antibodies is an artificial process that combines a mouse CDR library with a canine immunoglobulin backbone. The process assumes that when introduced in vivo, the mouse CDR sequence will play a comparable function in the canine sequence, that is, it assumes that when the CDR region is taken out of its naturally occurring antibody molecule, the antibody specificity of these regions will be maintained in terms of binding. In addition, the use of these chimeric antibodies assumes that the presence of mouse CDR sequences within canine molecules will not cause any anti-mouse antibody response in the dog into which the chimeric antibody is introduced. The immunogenicity of antibody therapy may cause the efficacy to weaken over time. This is especially important in chronic diseases that require repeated administration.

[0016] Igase et al. (Nature Research Scientific Reports 2020; https: / / doi.org / 10.1038 / s41598-020-75533-4) describe rat-canine chimeric and caninized anti-PD1 antibodies that have been evaluated in vitro and used in pilot studies to determine their safety profile and clinical efficacy in spontaneous canine cancers, such as advanced oral malignant melanoma. These antibodies are rat monoclonal antibodies [see also WO2016 / 006241] in which the heavy and light chain variable regions of 4F12-E6 are fused to the constant regions of canine IgG type A.

[0017] A successful anti-canine PD1 antibody must have a certain degree of efficacy in the target population, that is, the antibody helps to clear and / or reduce the size of certain types of tumors. While being able to cause tumor size reduction, the antibody must also have an acceptable safety profile. The safety profile will involve a balance between the efficacy of the antibody in tumor reduction and the severity of any off-target effects. In addition to the safety profile, the pharmacokinetic (pK) characteristics of the antibody must also enable the antibody to remain in the body long enough to remain active. Even if the above criteria are met, the antibody must be able to be scalable and produced at an economically viable cost.

[0018] To the best of the inventors' knowledge, no anti-canine PD1 antibodies have been published that demonstrate a high degree of efficacy in the target population with an acceptable safety profile and any adequate and robust statistical power. Furthermore, to date, no fully caninized anti-canine PD1 antibody has been published with a pK profile. Furthermore, no caninized anti-canine PD1 antibody has been published that can be economically scalably produced.

[0019] WO2018 / 189520 and WO2020 / 074874 describe transgenic mouse models for expressing canine antibody repertoires. Advantageously, this approach allows for the utilization of highly evolved in vivo mechanisms, such as germinal center hypermutation, to generate high-affinity antibodies with optimal biophysical properties. Canine mice respond to antigenic stimulation and produce high-affinity antibodies with a CDRH3 length similar to that of canines, exhibiting broad epitope coverage. In vivo maturation enables the generation of antibodies with favorable and desirable biophysical properties, such as increased stability and reduced aggregation propensity.

[0020] Improved treatments are needed, as are drugs that address the underlying causes of the disease rather than the symptoms. In particular, there is a need for treatments that are safe, long-lasting, and effective in a wider range of patients, particularly non-responders. Summary of the Invention

[0021] The present invention relates to an isolated canine antibody, or antigen-binding portion thereof, that binds to one of the following epitopes of the extracellular domain of canine PD-1:

[0022] i) an epitope comprising T76, Q83, E84, L128, P129, P130 according to SEQ ID NO: 2

[0023] ii) an epitope comprising D58, D61, N74, T76, Y127 according to SEQ ID NO: 2

[0024] The epitope is determined using epitope mapping technology.

[0025] The present invention also relates to an isolated canine antibody, or antigen-binding portion thereof, that binds to the extracellular domain of canine PD-1, wherein the antibody comprises:

[0026] an HC CDR1 sequence comprising or consisting of SEQ ID NO: 17 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 17; and an HC CDR2 sequence comprising or consisting of SEQ ID NO: 18 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 18; and an HC CDR3 sequence comprising or consisting of SEQ ID NO: 19 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 19; and an LC CDR1 sequence comprising or consisting of SEQ ID NO: 20 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 20; and an LC CDR2 sequence comprising or consisting of SEQ ID NO: 21 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 21. ID NO: 21 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 21; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 22 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 22; or a HC CDR1 sequence comprising or consisting of SEQ ID NO: 27 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 27; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 28 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 28; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 29 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 29 having at least 70%, 80%, 85%, 90% or 95% sequence identity; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 30 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 30; and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 31 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 31;and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 32 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 32; or a HC CDR1 sequence comprising or consisting of SEQ ID NO: 207 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 207; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 208 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 208; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 209 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 209; and a LC a CDR1 sequence comprising or consisting of SEQ ID NO: 210 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 210; and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 211 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 211; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 212 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 212; or a HC CDR1 sequence comprising or consisting of SEQ ID NO: 97 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 97; and a HC a CDR2 sequence comprising or consisting of SEQ ID NO: 98 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 98; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 99 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 99; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 100 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 100;and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 101 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 101; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 102 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 102.;

[0027] The present invention also relates to an isolated canine antibody, or antigen-binding portion thereof, that binds canine PD-1, wherein the antibody comprises:

[0028] a) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 17 or an amino acid sequence that has 1, 2, 3, 4 or 5 amino acid differences compared to SEQ ID NO: 17; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 18 or an amino acid sequence that has 1, 2, 3, 4, 5 or 6 amino acid differences compared to SEQ ID NO: 18; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 19 or an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid differences compared to SEQ ID NO: 19; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 20 or an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid differences compared to SEQ ID NO: 20; and a LC a CDR2 sequence comprising or consisting of SEQ ID NO: 21 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences from SEQ ID NO: 21; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 22 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences from SEQ ID NO: 22; or

[0029] b) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 207 or an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 207; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 208 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences from SEQ ID NO: 208; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 209 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acid differences from SEQ ID NO: 209; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 210 or an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 210; and a CDR2 sequence comprising or consisting of SEQ ID NO: 211 or an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 211; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 212 or an amino acid sequence having 1, 2, 3 or 4 amino acid differences from SEQ ID NO: 212; or

[0030] c) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 97 or an amino acid sequence that has 1, 2 or 3 amino acid differences compared to SEQ ID NO: 97; and an HC CDR2 sequence comprising or consisting of SEQ ID NO: 98 or an amino acid sequence that has 1, 2, 3 or 4 amino acid differences compared to SEQ ID NO: 98; and an HC CDR3 sequence comprising or consisting of SEQ ID NO: 99 or an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid differences compared to SEQ ID NO: 99; and an LC CDR1 sequence comprising or consisting of SEQ ID NO: 100 or an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid differences compared to SEQ ID NO: and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 101 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid differences compared to SEQ ID NO: 100; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 102 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences compared to SEQ ID NO: 102.

[0031] The present invention also relates to an antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof has:

[0032] i) a HC CDR1 sequence comprising SEQ ID NO: 17 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 18 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 19 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 20 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 21 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 22 or consisting thereof,

[0033] ii) a HC CDR1 sequence comprising SEQ ID NO: 27 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 28 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 29 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 30 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 31 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 32 or consisting thereof,

[0034] iii) a HC CDR1 sequence comprising SEQ ID NO: 7 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 8 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 9 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 10 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 11 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 12 or consisting thereof,

[0035] iv) a HC CDR1 sequence comprising SEQ ID NO: 37 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 38 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 39 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 40 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 41 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 42 or consisting thereof,

[0036] v) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 47, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 48, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 49, a LC CDR1 sequence comprising SEQ ID NO: 50, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 51, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 52,

[0037] vi) a HC CDR1 sequence comprising SEQ ID NO: 57, a HC CDR2 sequence comprising SEQ ID NO: 58, a HC CDR3 sequence comprising SEQ ID NO: 59, a LC CDR1 sequence comprising SEQ ID NO: 60, a LC CDR2 sequence comprising SEQ ID NO: 61, and a LC CDR3 sequence comprising SEQ ID NO: 62,

[0038] vii) a HC CDR1 sequence comprising SEQ ID NO: 67, a HC CDR2 sequence comprising SEQ ID NO: 68, a HC CDR3 sequence comprising SEQ ID NO: 69, a LC CDR1 sequence comprising SEQ ID NO: 70, a LC CDR2 sequence comprising SEQ ID NO: 71, and a LC CDR3 sequence comprising SEQ ID NO: 72,

[0039] viii) a HC CDR1 sequence comprising SEQ ID NO: 77, a HC CDR2 sequence comprising SEQ ID NO: 78, a HC CDR3 sequence comprising SEQ ID NO: 79, a LC CDR1 sequence comprising SEQ ID NO: 80, a LC CDR2 sequence comprising SEQ ID NO: 81, and a LC CDR3 sequence comprising SEQ ID NO: 82,

[0040] ix) a HC CDR1 sequence comprising SEQ ID NO: 87 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 88 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 89 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 90 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 91 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 92 or consisting thereof,

[0041] x) a HC CDR1 sequence comprising SEQ ID NO: 97 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 98 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 99 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 100 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 101 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 102 or consisting thereof,

[0042] xi) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 107, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 108, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 109, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 110, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 111, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 112,

[0043] xii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 117, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 118, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 119, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 120, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 12, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 122,

[0044] xiii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 127, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 128, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 129, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 130, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 131, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 132,

[0045] xiv) a HC CDR1 sequence comprising SEQ ID NO: 137 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 138 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 139 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 140 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 141 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 142 or consisting thereof,

[0046] xv) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 147, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 148, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 149, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 150, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 151, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 152,

[0047] xvi) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 157, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 158, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 159, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 160, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 161, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 162,

[0048] xvii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 167, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 168, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 169, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 170, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 171, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 172,

[0049] xviii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 177, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 178, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 179, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 180, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 181, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 182,

[0050] xix) a HC CDR1 sequence comprising SEQ ID NO: 187 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 188 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 189 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 190 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 191 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 192 or consisting thereof,

[0051] xx) a HC CDR1 sequence comprising SEQ ID NO: 197 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 198 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 199 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 200 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 201 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 202 or consisting thereof,

[0052] xxi) a HC CDR1 sequence comprising SEQ ID NO: 207 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 208 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 209 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 210 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 211 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 212 or consisting thereof,

[0053] xxii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 217, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 218, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 219, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 220, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 221, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 222,

[0054] xxiii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 227, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 228, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 229, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 230, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 231, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 232,

[0055] xxiv) a HC CDR1 sequence comprising SEQ ID NO: 237 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 238 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 239 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 240 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 241 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 242 or consisting thereof,

[0056] xxv) a HC CDR1 sequence comprising SEQ ID NO: 247 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 248 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 249 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 250 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 251 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 252 or consisting thereof,

[0057] xxvi) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 257, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 258, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 259, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 260, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 261, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 262,

[0058] xxvii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 267, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 268, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 269, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 270, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 271, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 272,

[0059] xxviii) a HC CDR1 sequence comprising SEQ ID NO: 277 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 278 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 279 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 280 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 281 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 282 or consisting thereof, or

[0060] xxix) a HC CDR1 sequence comprising SEQ ID NO: 287 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 288 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 289 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 290 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 291 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 292 or consisting of thereof.

[0061] The present invention also relates to an antibody, or antigen-binding portion thereof, wherein the antibody, or antigen-binding portion thereof, comprises an HC variable region sequence comprising SEQ ID NO: 4, or a sequence having at least 45%, 50%, 60%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereof, e.g., an LC variable region sequence comprising SEQ ID NO: 6.

[0062] The present invention also relates to an antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof has:

[0063] a) an HC variable region sequence comprising SEQ ID NO: 4, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or

[0064] b) an HC variable region sequence comprising SEQ ID NO: 14, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 16, or a sequence having at least 35%, 45%, 55%, 65%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0065] c) an HC variable region sequence comprising SEQ ID NO: 24, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0066] d) an HC variable region sequence comprising SEQ ID NO: 34, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0067] e) an HC variable region sequence comprising SEQ ID NO: 44, or a sequence having at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 46, or a sequence having at least 35%, 45%, 55%, 65%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0068] f) an HC variable region sequence comprising SEQ ID NO: 54, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 56, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0069] g) an HC variable region sequence comprising SEQ ID NO: 64, or a sequence having at least 45%, 65%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 36, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0070] h) an HC variable region sequence comprising SEQ ID NO: 74, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0071] i) an HC variable region sequence comprising SEQ ID NO: 84, or a sequence having at least 40%, 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 86, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0072] j) an HC variable region sequence comprising SEQ ID NO: 94, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0073] k) an HC variable region sequence comprising SEQ ID NO: 104, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0074] 1) an HC variable region sequence comprising SEQ ID NO: 114, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or

[0075] m) an HC variable region sequence comprising SEQ ID NO: 124, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0076] n) an HC variable region sequence comprising SEQ ID NO: 134, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 136, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0077] o) a HC variable region sequence comprising SEQ ID NO: 144, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0078] p) an HC variable region sequence comprising SEQ ID NO: 154, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 156, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0079] q) an HC variable region sequence comprising SEQ ID NO: 164, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 176, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0080] r) an HC variable region sequence comprising SEQ ID NO: 174, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0081] s) an HC variable region sequence comprising SEQ ID NO: 184, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 186, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0082] t) an HC variable region sequence comprising SEQ ID NO: 194, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or

[0083] u) an HC variable region sequence comprising SEQ ID NO: 204, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 206, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0084] v) an HC variable region sequence comprising SEQ ID NO: 214, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 216, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0085] w) an HC variable region sequence comprising SEQ ID NO: 224, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 226, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0086] x) an HC variable region sequence comprising SEQ ID NO: 234, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 236, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0087] y) an HC variable region sequence comprising SEQ ID NO: 244, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 246, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0088] z) an HC variable region sequence comprising SEQ ID NO: 254, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 256, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0089] aa) an HC variable region sequence comprising SEQ ID NO: 264, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 266, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0090] bb) an HC variable region sequence comprising SEQ ID NO: 274, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 276, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0091] cc) an HC variable region sequence comprising SEQ ID NO: 284, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 286, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0092] dd) an HC variable region sequence comprising SEQ ID NO: 24, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 26, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof.

[0093] The present invention also relates to a pharmaceutical composition comprising an antibody or antigen-binding portion thereof as described herein.

[0094] The present invention also relates to an antibody or antigen-binding portion thereof as described herein or a pharmaceutical composition as described herein for use in treating a disease.

[0095] The present invention also relates to a method of treating a disease in a canine subject in need thereof, comprising administering an effective amount of an antibody or antigen-binding portion thereof as described herein or a pharmaceutical composition as described herein.

[0096] In one embodiment, the disease is cancer or a tumor.

[0097] The present invention also relates to a nucleic acid sequence encoding an antibody or an antibody antigen-binding portion thereof as described herein.

[0098] The present invention also relates to a vector comprising the nucleic acid sequence as described herein.

[0099] The present invention also relates to a host cell comprising the nucleic acid sequence as described herein or the vector as described herein.

[0100] The present invention also relates to a kit comprising an antibody or antigen-binding portion thereof as described herein or a pharmaceutical composition as described herein.

[0101] The present invention also relates to a method for preparing a canine antibody that binds to PD-1, the method comprising culturing the isolated host cell as described herein and recovering the antibody.

[0102] The present invention also relates to a method for preparing canine antibodies that bind to PD-1, the method comprising the following steps:

[0103] a) immunizing a transgenic mouse expressing a nucleic acid construct comprising a canine heavy chain V gene and a canine light chain V gene with a PD-1 antigen,

[0104] b) generating an antibody library from said mouse, and

[0105] c) isolating antibodies from said library.

[0106] The present invention also relates to a method for detecting a PD-1 protein or an extracellular domain of a PD-1 protein in a biological sample from a canine subject, the method comprising contacting the biological sample with an antibody or antigen-binding portion thereof as described herein, wherein the antibody or antigen-binding portion thereof is linked to a detectable label.

[0107] The present invention also relates to a method of inhibiting tumor growth or metastasis, which comprises contacting tumor cells with an effective amount of an antibody or antigen-binding portion thereof as described herein or a pharmaceutical composition as described herein.

[0108] The present invention also relates to a method for killing tumor cells expressing PD-1, comprising contacting the cell with an antibody as described herein or a pharmaceutical composition as described herein, so that the cell expressing PD-1 is killed.

[0109] The present invention also relates to a binding agent comprising an antibody, or antigen binding portion thereof, as described herein, wherein the antibody, or antigen binding portion thereof, is linked to a second antibody, or antigen binding portion thereof, that binds to a second target.

[0110] The present invention also relates to an immunoconjugate comprising an antibody or antigen-binding portion thereof as described herein or a binding agent as described herein.

[0111] The present invention also relates to a method of modulating an immune response, the method comprising administering an antibody or fragment thereof as described herein, or a pharmaceutical composition as described herein, or a binding agent as described herein, or an immunoconjugate as described herein.

[0112] The present invention also relates to a combination therapy comprising: an antibody or fragment thereof as described herein; or a pharmaceutical composition as described herein; or a binding agent as described herein; or an immunoconjugate as described herein, and an additional therapeutic moiety. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] The invention will be further described in the following non-limiting drawings.

[0114] Figure 1 Sections of each expression plasmid encoding PD-1 ECD-mFc, PD-1 ECD-6His, PD-L1 ECD-mFc, and PD-L2 ECD-mFc (ECD = extracellular domain) are shown. PD-1 ECD, PD-L1 ECD, and PD-L2 ECD refer to the predicted extracellular domains of PD-1, PD-L1, and PD-L2, respectively. The "-mFc" suffix refers to the Fc portion of murine IgG2a, which contains the gene annotation: hinge-constant heavy 2-constant heavy 3-constant heavy secretory (H-CH2-CH3-CHS), and "6His" refers to a hexahistidine tag.

[0115] Figure 2 Graph showing the binding strength of anti-PD-1 antibodies to full-length canine PD-1 protein embedded in the plasma membrane of HEK293 cells, measured using flow cytometry. Cells stably transfected with canine PD-1 were incubated with anti-canine PD-1 mAb and then detected with an anti-canine IgG FITC secondary antibody.

[0116] Figures 3A-3B . Figure 3A Shown is the binding of AlexaFluor 647-labeled PD-L1 ECD-mFc to PD-1-expressing HEK293 cells that were previously incubated with 2 µg / mL anti-PD-1 antibody in a flow cytometry-based assay. Figure 3BShown is the binding of AlexaFluor 647-labeled PD-L2ECD-mFc to PD-1-expressing HEK293 cells that were previously incubated with 2µg / mL anti-PD-1 antibody in a flow cytometry-based assay.

[0117] Figures 4A-4B FIG4A is a table showing SPR-based affinity and kinetics of anti-PD-1 antibodies performed as a multi-cycle kinetic experiment in either monovalent format (PD-1 analyte) or bivalent format (mAb analyte). Figure 4B A table showing the apparent affinities of anti-PD-1 antibodies using flow cytometry-based affinity determination is shown. The apparent affinities were determined by fitting the data to a "[agonist] vs. response" 4-parameter logistic curve in Prism 8.3.0 (GraphPad) and the concentration at which the reporter signal reached 50% of the maximum value.

[0118] Figures 5A-5C . Figure 5A 5 B shows a correlation matrix of the binding profiles of each mAb with the spectra of all other mAbs. To construct this table, the Pearson product-moment correlation coefficient between the relative binding values ​​for each mAb pair was calculated. Pearson product-moment correlation coefficients ≥ 0.97 were highlighted. Figure 5C A visual representation of the resulting epitope partitioning is shown.

[0119] Figure 6 Figure 2 shows the interaction between anti-PD-1 antibody and concanavalin A-activated CD5 + Figure 3. PBMC Fc receptors were blocked with canine immunoglobulin and anti-PD-1 mAb was detected by flow cytometry using anti-canine IgG FITC.

[0120] Figure 7 Figure 1 shows a functional in vitro assay for IFN-γ secretion using supernatants collected from PBMCs stimulated with Concanavalin A in the presence of 10 µg / mL anti-PD-1 mAb or an isotype control mAb. After 72 hours of incubation with each anti-PD-1 antibody, IFN-γ secretion was assessed by sandwich ELISA using a canine IFN-γ ELISA.

[0121] Figures 8A-8B . Figure 8A Shown is PD-1 / PD-L1 receptor blockade in a luciferase reporter assay using 3 µg / mL anti-PD-1 mAb. Figure 8B Receptor blockade of selected anti-PD-1 mAbs in a luciferase reporter assay using a concentration range spanning 666 nM to 0.03 nM is shown. 50 Concentrations are expressed in nM. Reporter Jurkat cells express chimeric canine (extracellular domain) and human (transmembrane and intracellular domains) PD-1, a TCR specific for a known MHC-peptide complex, and transcribe luciferase from an NFAT-regulated promoter. Raji antigen-presenting cells express the MHC-peptide complex bound by the Jurkat TCR and full-length canine PD-L1. In the absence of inhibition, TCR signaling is inhibited by PD-1 signaling, resulting in low levels of luciferase transcription. High levels of PD-1 receptor blockade result in undiminished TCR signaling, resulting in high levels of luciferase transcription.

[0122] Figure 9 A-9B. Figure 9 A shows a schematic structural representation of the residue positions identified in the PMX-126 binding epitope on PD-1 using homology models of the canine PD-1 and PD-L1 extracellular domains (PD-1 ECD and PD-L1 ECD, respectively). Figure 9 B. Sequence-based representation of the PD-1 extracellular domain with secondary structural features highlighted. Residue positions identified in the PMX-126 binding epitope on PD-1 are shown in grey.

[0123] Figure 10 A-10B. Figure 10 A shows a schematic structure diagram highlighting in black the residue positions identified in the PMX-127 binding epitope on PD-1 using homology models of the canine PD-1 and PD-L1 extracellular domains (PD-1 ECD and PD-L1 ECD, respectively). Figure 10 B. Sequence-based representation of the PD-1 extracellular domain with secondary structural features highlighted. Residue positions identified in the PMX-127 binding epitope on PD-1 are shown in grey.

[0124] Figure 11 A sequence representation of the full-length canine PD-1 is shown. The predicted extracellular domain (ECD) is highlighted in grey.

[0125] Figures 12A-12B . Figure 12AShown are flow cytometry and histograms demonstrating binding of the PMX-127 antibody to the PD-1 receptor expressed on the cell surface of T cells from lymphoma-bearing dogs (n=2). Live single CD5+ T cells were pre-gated. Figure 12B Flow cytometry and histograms demonstrating binding of the PMX-127 antibody to the PD-1 receptor expressed on the cell surface of T cells from dogs with transitional cell carcinoma (n=2). Live single CD5+ T cells were pre-gated.

[0126] Figure 13 Shown is an increase in IFN-γ production in PBMCs from cancer-bearing dogs following in vitro treatment with the canine anti-PD-1 antibodies PMX-126 and PMX-127. Patient PBMC samples from dogs with rectal adenocarcinoma and lymphoma were stimulated with ConA in the presence of 10 μg / ml anti-PD-1 mAb or the Ritux-def2 isotype control. Supernatants were collected after 96 hours, and the amount of canine IFN-γ was measured in triplicate using a canine IFN-γ ELISA.

[0127] Figures 14A-14C . Figure 14A Shown is a pharmacokinetic analysis showing the linear antibody concentration-time profile of canine anti-PD-1 antibodies in healthy beagle dogs following single intravenous administration of low and high doses of PMX-126 (mean ± SD, n = 3). The presence of antibodies in plasma was assessed by direct ELISA using purified canine PD-1 Fc-tagged protein. Figure 14B Pharmacokinetic analysis showing the antibody concentration-time profiles of canine anti-PD-1 antibodies in healthy beagle dogs following single intravenous administration of low and high doses of PMX-127 (mean ± SD, n = 3). The presence of antibodies in plasma was assessed by direct ELISA using purified canine PD-1 Fc-tagged protein. Figure 14C The results of non-compartmental analysis of PMX-126 or PMX-127 antibodies are shown in the table. These antibodies showed similar half-lives of 5 days. VD(L): Volume of distribution. Cmax: Maximum observed plasma concentration. AUC: Area under the plasma concentration vs. time curve from 0 to 28 days.

[0128] Figure 15 The following is a summary table showing potential adverse events following administration of a single intravenous dose of canine PD-1 PMX-126 and PMX-127 antibodies to laboratory beagle dogs. These changes were mild, transient, and likely not specific to the antibody treatment.

[0129] Figures 16A-16B . Figure 16AShown is a graph of an SPR-based assay demonstrating that the anti-canine mAbs PMX-126 and PMX-127 have no cross-reactivity with recombinant human PD-1 protein at a concentration of 900 nM. Figure 16B ELISA assay graph showing that PMX-126 and PMX-127 do not cross-react with human PD-1 protein at concentrations up to 100 µg / mL. The anti-human PD-1 mAb pembrolizumab was included as a control to confirm the activity of the recombinant human PD-1 protein.

[0130] Figure 17 Figure 2 is a receptor occupancy graph demonstrating that canine anti-PD-1 antibodies PMX-126 and PMX-127 remain bound to circulating CD5+ T cells for up to 21 days after a single intravenous dose in experimental dogs (n=3 dogs per group). Data are shown as mean ± SD.

[0131] Figures 18A-18B . Figure 18A This is a volcano plot showing differentially expressed genes at day 7 in experimental dogs treated with the canine anti-PD-1 antibody PMX-126 compared to pre-treatment. Highlighted genes meet the significance criteria: log2 fold change > 1.5 and p-value < 0.05. The horizontal line on the graph depicts statistical significance; therefore, differentially expressed (DE) genes with high statistical significance are displayed from bottom to top. Highly DE genes are located at the horizontal ends of the graph, so the further to the right, the greater the gene expression at day 7 compared to pre-treatment. Figure 18B This is a volcano plot showing differentially expressed genes at day 7 after treatment with the canine anti-PD-1 antibody PMX-127 in experimental dogs compared to before treatment. Highlighted genes meet the significance criteria: log2 fold change > 1.5 and p-value < 0.05. The horizontal line on the graph depicts statistical significance; therefore, differentially expressed (DE) genes with high statistical significance are displayed from bottom to top. Highly DE genes are located at the horizontal ends of the graph, so the further to the right, the greater the gene expression at day 7 compared to before treatment.

[0132] Table 1. Examples of amino acid residues and conservative amino acid substitutions

[0133] Table 2. Sequence IDs for the amino acid and nucleotide sequences of each anti-PD-1 antibody included in the specification. DETAILED DESCRIPTION

[0134] Now will further describe the embodiment of the present invention.In the following, describe different embodiments.Unless explicitly indicated to the contrary, otherwise each aspect defined in this way can be combined with any one or more other aspects.

[0135] In general, the term list and technology used in cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization combination as described herein are well known in the art and commonly used term lists and technology. Unless otherwise indicated, the methods and technologies of the present disclosure are generally carried out according to conventional methods well known in the art and the methods described in each general and more specific reference as quoted and discussed throughout this specification. See, for example, Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012); Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (editor), Wiley, (2009); and Antibody Engineering, 2nd edition, Volumes 1 and 2, Ontermann and Dubel, eds., Springer-Verlag, Heidelberg (2010).

[0136] Enzyme reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art or as described herein. The glossary and laboratory procedures and techniques used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0137] The inventors have developed fully canine antibodies that specifically bind to canine PD-1. These antibodies are produced in transgenic rodents expressing canine V, D, and J genes. Therefore, compared to caninized or chimeric antibodies, these antibodies are less likely to elicit immunogenicity when administered to canine subjects. Furthermore, because these antibodies can be used directly without further modification of their variable regions, there is no risk of reduced affinity or other impairment of the antibody. Other techniques risk introducing development or efficacy deficiencies by ex vivo combining canine-derived antibody sequences with antibody sequences from other species, typically rodents. Therefore, the present invention preferably relates to an isolated canine antibody, or antigen-binding portion thereof, that binds to canine PD-1.

[0138] Thus, the present invention provides isolated antibodies that bind to canine PD-1 and block or reduce the interaction of PD-1 with PD-L1 and / or the interaction of PD-1 with PD-L2, pharmaceutical compositions comprising such binding molecules, and isolated nucleic acids, isolated recombinant expression vectors, and isolated host cells for preparing such binding proteins. Also provided are methods for detecting canine PD-1 and methods for treating diseases using the antibodies disclosed herein. In another aspect, the present invention provides binding molecules comprising antibodies or antigen-binding portions that bind to canine PD-1 and block the interaction of PD-1 with PD-L1 and / or the interaction of PD-1 with PD-L2, as described herein. The properties of the antibodies and antigen-binding portions thereof of the present invention can be developed for use in therapeutic methods and uses and pharmaceutical formulations as described herein.

[0139] In one embodiment of the present invention, an isolated canine antibody or antigen-binding fragment thereof that binds to canine PD-1 is provided, wherein the antibody comprises: an HC CDR1 sequence comprising or consisting of SEQ ID NO: 17 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences compared to SEQ ID NO: 17; and an HC CDR2 sequence comprising or consisting of SEQ ID NO: 18 or an amino acid sequence having 1, 2, 3, 4, 5 or 6 amino acid differences compared to SEQ ID NO: 18; and an HC CDR3 sequence comprising or consisting of SEQ ID NO: 19 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid differences compared to SEQ ID NO: 19; and an LC CDR1 sequence comprising or consisting of SEQ ID NO: 20 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid differences compared to SEQ ID NO: NO: 207 or an amino acid sequence having 1 or 2 amino acid differences compared to SEQ ID NO: 207; and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 21 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences compared to SEQ ID NO: 21; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 22 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences compared to SEQ ID NO: 22; or a HC CDR1 sequence comprising or consisting of SEQ ID NO: 207 or an amino acid sequence having 1 or 2 amino acid differences compared to SEQ ID NO: 207; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 208 or an amino acid sequence having 1 or 2 amino acid differences compared to SEQ ID NO: and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 211 or an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 211;and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 212 or an amino acid sequence having 1, 2, 3 or 4 amino acid differences compared to SEQ ID NO: 212; or a HC CDR1 sequence comprising or consisting of SEQ ID NO: 97 or an amino acid sequence having 1, 2 or 3 amino acid differences compared to SEQ ID NO: 97; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 98 or an amino acid sequence having 1, 2, 3 or 4 amino acid differences compared to SEQ ID NO: 98; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 99 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid differences compared to SEQ ID NO: 99; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 100 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid differences compared to SEQ ID NO: 100; and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 101 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences compared to SEQ ID NO: 101; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 102 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences compared to SEQ ID NO: 102.

[0140] In one embodiment of the present invention, the amino acid differences are selected from conservative amino acid substitutions and / or non-conservative amino acid substitutions.

[0141] In another related embodiment of the present invention, an isolated canine antibody or antigen-binding fragment thereof that binds to canine PD-1 is provided, wherein the antibody or antigen-binding fragment thereof has: i) a HC CDR1 sequence comprising SEQ ID NO: 17 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 18 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 19 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 20 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 21 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 22 or consisting thereof; ii) a HC CDR1 sequence comprising SEQ ID NO: 47 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 48 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 49 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 50 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 51 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 52 or consisting of the same; iii) a HC CDR1 sequence comprising SEQ ID NO: 57 or consisting of SEQ ID NO: 58 or consisting of SEQ ID NO: 59 or consisting of SEQ ID NO: 60 or consisting of LC CDR1 sequence, a LC CDR2 sequence comprising SEQ ID NO: 61 or consisting of SEQ ID NO: 62 or consisting of LC CDR3 sequence; iv) a HC CDR1 sequence comprising SEQ ID NO: 37 or consisting of SEQ ID NO: 38 or consisting of SEQ ID NO: 39 or consisting of SEQ ID NO: 40 or consisting of LC CDR1 sequence, a LC CDR2 sequence comprising SEQ ID NO: 41 or consisting of SEQ ID NO: 42 or consisting of LC CDR3 sequence;v) a HC CDR1 sequence comprising SEQ ID NO: 87 or consisting of SEQ ID NO: 88 or consisting of SEQ ID NO: 89 or consisting of SEQ ID NO: 90, a LC CDR2 sequence comprising SEQ ID NO: 91 or consisting of SEQ ID NO: 92 or consisting of LC CDR3 sequence; vi) a HC CDR1 sequence comprising SEQ ID NO: 137, a HC CDR2 sequence comprising SEQ ID NO: 138, a HC CDR3 sequence comprising SEQ ID NO: 139, a LC CDR1 sequence comprising SEQ ID NO: 141, a LC CDR2 sequence comprising SEQ ID NO: 142, and a LC CDR3 sequence comprising SEQ ID NO: 143; vii) a HC CDR1 sequence comprising SEQ ID NO: 157, a HC CDR2 sequence comprising SEQ ID NO: 158, a LC CDR1 sequence comprising SEQ ID NO: 159, a LC CDR2 sequence comprising SEQ ID NO: 160, a LC CDR3 sequence comprising SEQ ID NO: 161; : 182; ix) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 187, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 188, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 189, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 190, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 191, and a LC CDR3 sequence comprising SEQ ID NO: 192. 191 or a LC CDR2 sequence consisting thereof, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 153;x) a HC CDR1 sequence comprising SEQ ID NO: 207 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 208 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 209 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 210 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 211 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 212 or consisting thereof; xi) a HC CDR1 sequence comprising SEQ ID NO: 217 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 218 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 219 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 220 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 221 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 222 or consisting thereof; xii) a HC CDR1 sequence comprising SEQ ID NO: 224 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 225 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 226 or consisting thereof; : xiii) a HC CDR1 sequence comprising SEQ ID NO: 237 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 238 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 239 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 240 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 241 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 242 or consisting of thereof; xiv) a HC CDR1 sequence comprising SEQ ID NO: 247 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 248 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 249 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 250 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 251 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 252 or consisting of thereof; a CDR1 sequence, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 248, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 249, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 250, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 251, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 252;xv) a HC CDR1 sequence comprising SEQ ID NO: 257 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 258 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 259 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 260 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 261 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 262 or consisting thereof; xvi) a HC CDR1 sequence comprising SEQ ID NO: 267 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 268 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 269 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 270 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 271 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 271 or consisting thereof; xvii) a HC CDR1 sequence comprising SEQ ID NO: 271 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 272 or consisting thereof, a LC CDR3 sequence comprising SEQ ID NO: 273 or consisting thereof 117 or consisting of a HC CDR1 sequence comprising SEQ ID NO: 118 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 119 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 120 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 121 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 122 or consisting of thereof; xviii) a HC CDR1 sequence comprising SEQ ID NO: 127 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 128 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 129 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 130 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 131 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 132 or consisting of thereof; xviiii) a HC CDR1 sequence comprising SEQ ID NO: 167 or consisting of thereof a CDR1 sequence, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 168, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 169, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 170, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 171, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 172;xx) a HC CDR1 sequence comprising SEQ ID NO: 197 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 198 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 199 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 200 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 201 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 202 or consisting of thereof; xxi) a HC CDR1 sequence comprising SEQ ID NO: 97 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 98 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 99 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 100 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 101 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 102 or consisting of thereof; xxii) a HC CDR1 sequence comprising SEQ ID NO: 97 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 98 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 99 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 100 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 101 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 102 or consisting of thereof; 277 or consisting of a HC CDR1 sequence, a HC CDR2 sequence, a HC CDR3 sequence, a LC CDR1 sequence, a LC CDR2 sequence, a LC CDR3 sequence, a LC CDR2 sequence, a LC CDR3 sequence, a LC CDR1 sequence, a LC CDR2 sequence, a LC CDR3 sequence, a LC CDR2 sequence, a LC CDR3 sequence, a LC CDR1 sequence, a LC CDR2 sequence, a LC CDR2 sequence, a LC CDR3 sequence, a LC CDR3 sequence, a LC CDR1 sequence, a LC CDR2 sequence, a LC CDR2 sequence, a LC CDR3 sequence, a LC CDR3 sequence, a LC CDR2 sequence, a LC CDR3 sequence, a LC CDR1 sequence, a LC CDR2 sequence, a LC CDR2 sequence, a LC CDR3 sequence, a LC CDR3 sequence, a LC CDR2 sequence, a LC CDR3 sequence, a LC CDR1 sequence, a LC CDR2 sequence, a LC CDR3 ... a CDR1 sequence, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 108, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 109, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 110, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 111, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 112;xxv) a HC CDR1 sequence comprising SEQ ID NO: 147 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 148 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 149 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 150 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 151 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 152 or consisting thereof; xxvi) a HC CDR1 sequence comprising SEQ ID NO: 7 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 8 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 9 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 10 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 11 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 12 or consisting thereof; xxvii) a HC CDR1 sequence comprising SEQ ID NO: 27 or consisting thereof : xxviii) a HC CDR1 sequence comprising SEQ ID NO: 67 or consisting of SEQ ID NO: 68 or consisting of SEQ ID NO: 69 or consisting of SEQ ID NO: 70 or consisting of SEQ ID NO: 71 or consisting of SEQ ID NO: 72 or consisting of SEQ ID NO: 73; or xxix) a HC CDR1 sequence comprising SEQ ID NO: 77 or consisting of SEQ ID NO: 78 or consisting of SEQ ID NO: 79 or consisting of SEQ ID NO: 80 or consisting of SEQ ID NO: 81 or consisting of SEQ ID NO: 82 or consisting of SEQ ID NO: 83; CDR2 sequence, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 79, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 80, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 81, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 82.

[0142] Also within the scope of the present invention is an isolated canine antibody or antigen-binding fragment thereof that binds to canine PD-1, wherein the antibody or antigen-binding fragment thereof has VH and LC CDRs that have at least 90% or 95% sequence identity with the CDRs shown in i) to xxviii) above.

[0143] In an embodiment of the invention, an antibody, or antigen-binding portion thereof, that binds to canine PD-1 comprises an HC variable region sequence comprising SEQ ID NO: 4, or a sequence having at least 45%, 50%, 60%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto, e.g., an LC variable region sequence comprising SEQ ID NO: 6.

[0144] In another related embodiment of the present invention, an antibody or antigen-binding portion thereof that binds to canine PD-1 is provided, the antibody or antigen-binding portion thereof having: a) an HC variable region sequence comprising SEQ ID NO: 14 or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereto and a LC variable region sequence comprising SEQ ID NO: 16 or a sequence having at least 35%, 45%, 55%, 65%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or b) an HC variable region sequence comprising SEQ ID NO: 44 or a sequence having at least 40%, 50%, 60%, 70%, 75%, 80%, 85% or 90% sequence identity thereto and a LC variable region sequence comprising SEQ ID NO: 46, or a sequence having at least 35%, 45%, 55%, 65%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or c) a HC variable region sequence comprising SEQ ID NO: 54, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 56, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or d) a HC variable region sequence comprising SEQ ID NO: 34, or a sequence having at least 45%, 65%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 36, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or e) a HC variable region sequence comprising SEQ ID NO: 84, or a sequence having at least 40%, 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 86, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or f) a HC variable region sequence comprising SEQ ID NO: 134, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 136, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto;or g) a HC variable region sequence comprising SEQ ID NO: 154, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 156, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or h) a HC variable region sequence comprising SEQ ID NO: 174, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 176, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or i) a HC variable region sequence comprising SEQ ID NO: 184 or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, the LC variable region sequence comprising SEQ ID NO: 186 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or j) a HC variable region sequence comprising SEQ ID NO: 204 or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 206 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or k) a HC variable region sequence comprising SEQ ID NO: : 1) a HC variable region sequence comprising SEQ ID NO: 224 or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 226 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or 2) a HC variable region sequence comprising SEQ ID NO: 224 or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 226 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto;or m) a HC variable region sequence comprising SEQ ID NO: 234, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 236, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or n) a HC variable region sequence comprising SEQ ID NO: 244, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 246, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or o) a HC variable region sequence comprising SEQ ID NO: 254 or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, the LC variable region sequence comprising SEQ ID NO: 256 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or p) a HC variable region sequence comprising SEQ ID NO: 264 or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereto and a LC variable region sequence comprising SEQ ID NO: 266 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or q) a HC variable region sequence comprising SEQ ID NO: 114, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, the LC variable region sequence comprising SEQ ID NO: 116, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or r) a HC variable region sequence comprising SEQ ID NO: 124, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 126, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto;or s) a HC variable region sequence comprising SEQ ID NO: 164, or a sequence having at least 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 166, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or t) a HC variable region sequence comprising SEQ ID NO: 194, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 196, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or u) a HC variable region sequence comprising SEQ ID NO: NO: 94 or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, the LC variable region sequence comprising SEQ ID NO: 96 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or v) a HC variable region sequence comprising SEQ ID NO: 274 or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 276 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or w) a HC variable region sequence comprising SEQ ID NO: 284 or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: NO: 286, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or x) a HC variable region sequence comprising SEQ ID NO: 104, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 106, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto;or y) a HC variable region sequence comprising SEQ ID NO: 144, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 146, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or z) a HC variable region sequence comprising SEQ ID NO: 4, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or aa) a HC variable region sequence comprising SEQ ID NO: NO: 24 or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, the LC variable region sequence comprising SEQ ID NO: 26 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or bb) a HC variable region sequence comprising SEQ ID NO: 64 or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and the LC variable region sequence comprising SEQ ID NO: 66 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or cc) a HC variable region sequence comprising SEQ ID NO: 74 or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and the LC variable region sequence comprising SEQ ID NO: 76 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto. ;

[0145] The antibodies or antigen-binding portions thereof according to the present invention may bind to one of the following epitopes of the extracellular domain of canine PD-1:

[0146] i) T76, Q83, E84, L128, P129, P130, or

[0147] ii) D58, D61, N74, T76, Y127

[0148] Wherein the epitope is determined using epitope mapping technology. The epitope residues are provided with reference to the canine PD-1 sequence. SEQ ID NO: 2 provides the sequence of canine PD-1, and Figure 11 Residue numbering for canine PD-1 is shown.

[0149] The antibodies or antigen-binding portions thereof according to the present invention may bind to one of the following epitopes of the extracellular domain of canine PD-1:

[0150] i) T76, Q83, E84, L128, P129, P130 according to SEQ ID NO: 2

[0151] ii) D58, D61, N74, T76, Y127 according to SEQ ID NO: 2

[0152] The epitope is determined using epitope mapping technology.

[0153] Epitope mapping techniques that can be used to determine whether an antibody binds to a specific epitope include site-directed mutagenesis epitope mapping, shotgun mutagenesis epitope mapping, oligopeptide scanning (e.g., peptide scanning analysis), hydrogen-deuterium exchange (optionally combined with mass spectrometry), cross-linking mass spectrometry, X-ray crystallography, electron microscopy, NMR analysis, computer simulation methods (e.g., using computer-generated structure predictions), or a combination of the above techniques. In an embodiment, the epitope mapping technique is site-directed mutagenesis epitope mapping. In one embodiment, the epitope mapping technique is alanine scanning mutagenesis epitope mapping. In one embodiment, the epitope mapping technique is site-directed mutagenesis epitope mapping, such as the techniques described in Example 17 herein.

[0154] In one embodiment of the present invention, an isolated canine antibody or antigen-binding fragment thereof that binds to canine PD-1 is provided, wherein the antibody binds to one of the following epitopes of the extracellular domain of canine PD-1:

[0155] i) T76, Q83, E84, L128, P129, P130 according to SEQ ID NO: 2

[0156] ii) D58, D61, N74, T76, Y127 according to SEQ ID NO: 2

[0157] wherein the epitope is determined using epitope mapping technology,

[0158] and wherein the antibody comprises: an HC CDR1 sequence comprising or consisting of SEQ ID NO: 17 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 17; and an HC CDR2 sequence comprising or consisting of SEQ ID NO: 18 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 18; and an HC CDR3 sequence comprising or consisting of SEQ ID NO: 19 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 19; and an LC CDR1 sequence comprising or consisting of SEQ ID NO: 20 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 20; and an LC a CDR2 sequence comprising or consisting of SEQ ID NO: 21 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 21; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 22 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 22; or a HC CDR1 sequence comprising or consisting of SEQ ID NO: 27 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 27; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 28 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 28; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 29 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 29; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 30 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 30; and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 31 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 31;and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 32 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 32; or a HC CDR1 sequence comprising or consisting of SEQ ID NO: 207 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 207; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 208 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 208; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 209 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 209; and a LC a CDR1 sequence comprising or consisting of SEQ ID NO: 210 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 210; and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 211 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 211; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 212 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 212; or a HC CDR1 sequence comprising or consisting of SEQ ID NO: 97 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 97; and a HC a CDR2 sequence comprising or consisting of SEQ ID NO: 98 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 98; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 99 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 99; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 100 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 100;and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 101 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 101; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 102 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 102.;

[0159] In one embodiment of the present invention, an isolated canine antibody or antigen-binding fragment thereof that binds to canine PD-1 is provided, wherein the antibody binds to one of the following epitopes of the extracellular domain of canine PD-1:

[0160] i) T76, Q83, E84, L128, P129, P130 according to SEQ ID NO: 2

[0161] ii) D58, D61, N74, T76, Y127 according to SEQ ID NO: 2

[0162] wherein the epitope is determined using epitope mapping technology,

[0163] and wherein the antibody comprises: an HC CDR1 sequence comprising or consisting of SEQ ID NO: 17 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid difference(s) from SEQ ID NO: 17; and an HC CDR2 sequence comprising or consisting of SEQ ID NO: 18 or an amino acid sequence having 1, 2, 3, 4, 5 or 6 amino acid difference(s) from SEQ ID NO: 18; and an HC CDR3 sequence comprising or consisting of SEQ ID NO: 19 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid difference(s) from SEQ ID NO: 19; and an LC CDR1 sequence comprising or consisting of SEQ ID NO: 20 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid difference(s) from SEQ ID NO: 20; and an LC a CDR2 sequence comprising or consisting of SEQ ID NO: 21 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences compared to SEQ ID NO: 21; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 22 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences compared to SEQ ID NO: 22; or a HC CDR1 sequence comprising or consisting of SEQ ID NO: 207 or an amino acid sequence having 1 or 2 amino acid differences compared to SEQ ID NO: 207; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 208 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences compared to SEQ ID NO: 208; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 209 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences compared to SEQ ID NO: and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 211 or an amino acid sequence having 1 or 2 amino acid differences compared to SEQ ID NO: 211;and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 212 or an amino acid sequence having 1, 2, 3 or 4 amino acid differences compared to SEQ ID NO: 212; or a HC CDR1 sequence comprising or consisting of SEQ ID NO: 97 or an amino acid sequence having 1, 2 or 3 amino acid differences compared to SEQ ID NO: 97; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 98 or an amino acid sequence having 1, 2, 3 or 4 amino acid differences compared to SEQ ID NO: 98; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 99 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid differences compared to SEQ ID NO: 99; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 100 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid differences compared to SEQ ID NO: 100; and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 101 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences compared to SEQ ID NO: 101; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 102 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences compared to SEQ ID NO: 102.

[0164] In another related embodiment of the present invention, an isolated canine antibody or antigen-binding fragment thereof that binds to canine PD-1 is provided, wherein the antibody binds to one of the following epitopes of the extracellular domain of canine PD-1:

[0165] i) T76, Q83, E84, L128, P129, P130 according to SEQ ID NO: 2

[0166] ii) D58, D61, N74, T76, Y127 according to SEQ ID NO: 2

[0167] wherein the epitope is determined using epitope mapping technology,

[0168] And wherein the antibody or antigen-binding fragment has: i) a HC CDR1 sequence comprising SEQ ID NO: 17 or consisting of it, a HC CDR2 sequence comprising SEQ ID NO: 18 or consisting of it, a HC CDR3 sequence comprising SEQ ID NO: 19 or consisting of it, a LC CDR1 sequence comprising SEQ ID NO: 20 or consisting of it, a LC CDR2 sequence comprising SEQ ID NO: 21 or consisting of it, and a LC CDR3 sequence comprising SEQ ID NO: 22 or consisting of it; ii) a HC CDR1 sequence comprising SEQ ID NO: 47 or consisting of it, a HC CDR2 sequence comprising SEQ ID NO: 48 or consisting of it, a HC CDR3 sequence comprising SEQ ID NO: 49 or consisting of it, a LC CDR1 sequence comprising SEQ ID NO: 50 or consisting of it, a LC CDR2 sequence comprising SEQ ID NO: 51 or consisting of it, and a LC CDR3 sequence comprising SEQ ID NO: 52 or consisting of it; iii) a HC CDR1 sequence comprising SEQ ID NO: 57 or consisting of it : the HC CDR1 sequence comprising SEQ ID NO: 58 or consisting of thereof, the HC CDR2 sequence comprising SEQ ID NO: 59 or consisting of thereof, the LC CDR1 sequence comprising SEQ ID NO: 60 or consisting of thereof, the LC CDR2 sequence comprising SEQ ID NO: 61 or consisting of thereof, and the LC CDR3 sequence comprising SEQ ID NO: 62 or consisting of thereof; iv) a HC CDR1 sequence comprising SEQ ID NO: 37 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 38 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 39 or consisting of thereof, the LC CDR1 sequence comprising SEQ ID NO: 40 or consisting of thereof, the LC CDR2 sequence comprising SEQ ID NO: 41 or consisting of thereof, and the LC CDR3 sequence comprising SEQ ID NO: 42 or consisting of thereof; v) a HC CDR1 sequence comprising SEQ ID NO: 87 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 88 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 89 or consisting of thereof, NO: 89 or a HC CDR3 sequence consisting of SEQ ID NO: 89, a LC CDR1 sequence consisting of SEQ ID NO: 90 or a LC CDR2 sequence consisting of SEQ ID NO: 91 or a LC CDR3 sequence consisting of SEQ ID NO: 92 or a LC CDR3 sequence consisting of SEQ ID NO: 93;vi) a HC CDR1 sequence comprising SEQ ID NO: 137 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 138 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 139 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 141 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 142 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 143 or consisting of thereof; vii) a HC CDR1 sequence comprising SEQ ID NO: 157 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 158 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 159 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 160 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 161 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 162 or consisting of thereof; viii) a HC CDR1 sequence comprising SEQ ID NO: 157 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 158 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 159 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 160 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 161 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 162 or consisting of thereof; 177 or consisting of a HC CDR1 sequence comprising SEQ ID NO: 178 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 179 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 180 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 181 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 182 or consisting of thereof; ix) a HC CDR1 sequence comprising SEQ ID NO: 187 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 188 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 189 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 190 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 191 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 153 or consisting of thereof; x) a HC CDR1 sequence comprising SEQ ID NO: 207 or consisting of thereof a CDR1 sequence, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 208, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 209, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 210, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 211, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 212;xi) a HC CDR1 sequence comprising SEQ ID NO: 217 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 218 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 219 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 220 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 221 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 222 or consisting of thereof; xii) a HC CDR1 sequence comprising SEQ ID NO: 227 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 228 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 229 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 230 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 231 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 232 or consisting of thereof; xiii) a HC CDR1 sequence comprising SEQ ID NO: 234 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 235 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 236 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 237 or consisting of thereof : xiv) a HC CDR1 sequence comprising SEQ ID NO: 247 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 248 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 249 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 250 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 251 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 252 or consisting of thereof; xv) a HC CDR1 sequence comprising SEQ ID NO: 257 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 258 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 259 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 260 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 261 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 262 or consisting of thereof; xv) a HC CDR1 sequence comprising SEQ ID NO: 267 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 268 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 269 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 270 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 271 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 272 or consisting of thereof; a CDR1 sequence, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 258, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 259, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 260, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 261, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 262;xvi) a HC CDR1 sequence comprising SEQ ID NO: 267 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 268 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 269 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 270 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 271 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 271 or consisting of thereof; xvii) a HC CDR1 sequence comprising SEQ ID NO: 117 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 118 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 119 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 120 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 121 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 122 or consisting of thereof; xviii) a HC CDR1 sequence comprising SEQ ID NO: 117 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 118 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 119 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 120 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 121 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 122 or consisting of thereof; : xviiii) a HC CDR1 sequence comprising SEQ ID NO: 167 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 168 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 169 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 170 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 171 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 172 or consisting of thereof; xx) a HC CDR1 sequence comprising SEQ ID NO: 197 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 198 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 199 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 190 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 191 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 192 or consisting of thereof; a CDR1 sequence, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 198, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 199, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 200, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 201, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 202;xxi) a HC CDR1 sequence comprising SEQ ID NO: 97 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 98 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 99 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 100 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 101 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 102 or consisting thereof; xxii) a HC CDR1 sequence comprising SEQ ID NO: 277 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 278 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 279 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 280 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 281 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 282 or consisting thereof; xxiii) a HC CDR1 sequence comprising SEQ ID NO: 277 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 278 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 279 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 280 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 281 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 282 or consisting thereof; 287 or consisting of a HC CDR1 sequence comprising SEQ ID NO: 288 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 289 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 290 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 291 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 292 or consisting of thereof; xxiv) a HC CDR1 sequence comprising SEQ ID NO: 107 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 108 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 109 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 110 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 111 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 112 or consisting of thereof; xxv) a HC CDR1 sequence comprising SEQ ID NO: 147 or consisting of thereof a CDR1 sequence, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 148, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 149, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 150, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 151, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 152;xxvi) a HC CDR1 sequence comprising SEQ ID NO: 7 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 8 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 9 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 10 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 11 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 12 or consisting of thereof; xxvii) a HC CDR1 sequence comprising SEQ ID NO: 27 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 28 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 29 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 30 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 31 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 32 or consisting of thereof; xxviii) a HC CDR1 sequence comprising SEQ ID NO: 67 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 68 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 69 or consisting of thereof NO: 68 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 69 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 70 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 71 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 72 or consisting thereof; or xxix) a HC CDR1 sequence comprising SEQ ID NO: 77 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 78 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 79 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 80 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 81 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 82 or consisting thereof.;

[0169] In an embodiment of the present invention, one of the following epitopes of the extracellular domain of canine PD-1:

[0170] i) T76, Q83, E84, L128, P129, P130 according to SEQ ID NO: 2

[0171] ii) D58, D61, N74, T76, Y127 according to SEQ ID NO: 2

[0172] wherein the epitope is determined using epitope mapping techniques and the binding antibody, or antigen binding portion thereof, comprises a HC variable region sequence comprising SEQ ID NO: 4, or a sequence having at least 45%, 50%, 60%, 70%, 75%, 80%, 85% or 90% sequence identity thereof and a LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto, e.g., a LC variable region sequence comprising SEQ ID NO: 6.

[0173] In another related embodiment of the present invention, an antibody or antigen-binding portion thereof is provided that binds to one of the following epitopes of the extracellular domain of canine PD-1:

[0174] i) an epitope comprising T76, Q83, E84, L128, P129, P130 according to SEQ ID NO: 2

[0175] ii) an epitope comprising D58, D61, N74, T76, Y127 according to SEQ ID NO: 2

[0176] wherein the epitope is determined using epitope mapping techniques, the antibody, or antigen-binding portion thereof, has: a) a HC variable region sequence comprising SEQ ID NO: 14, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 16, or a sequence having at least 35%, 45%, 55%, 65%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or b) a HC variable region sequence comprising SEQ ID NO: 44, or a sequence having at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 46, or a sequence having at least 35%, 45%, 55%, 65%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or c) a HC variable region sequence comprising SEQ ID NO: 54, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 56, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or d) a HC variable region sequence comprising SEQ ID NO: 34, or a sequence having at least 45%, 65%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 36, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or e) a HC variable region sequence comprising SEQ ID NO: 84, or a sequence having at least 40%, 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 86, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or f) a HC variable region sequence comprising SEQ ID NO: 134, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 136, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto;or g) a HC variable region sequence comprising SEQ ID NO: 154, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 156, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or h) a HC variable region sequence comprising SEQ ID NO: 174, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 176, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or i) a HC variable region sequence comprising SEQ ID NO: 184 or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, the LC variable region sequence comprising SEQ ID NO: 186 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or j) a HC variable region sequence comprising SEQ ID NO: 204 or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 206 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or k) a HC variable region sequence comprising SEQ ID NO: 214, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, the LC variable region sequence comprising SEQ ID NO: 216, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or 1) a HC variable region sequence comprising SEQ ID NO: 224, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 226, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto;or m) a HC variable region sequence comprising SEQ ID NO: 234, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 236, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or n) a HC variable region sequence comprising SEQ ID NO: 244, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 246, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or o) a HC variable region sequence comprising SEQ ID NO: NO: 254 or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, the LC variable region sequence comprising SEQ ID NO: 256 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or p) a HC variable region sequence comprising SEQ ID NO: 264 or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereto and a LC variable region sequence comprising SEQ ID NO: 266 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or q) a HC variable region sequence comprising SEQ ID NO: 114, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, the LC variable region sequence comprising SEQ ID NO: 116, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or r) a HC variable region sequence comprising SEQ ID NO: 124, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 126, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto;or s) a HC variable region sequence comprising SEQ ID NO: 164, or a sequence having at least 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 166, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or t) a HC variable region sequence comprising SEQ ID NO: 194, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 196, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or u) a HC variable region sequence comprising SEQ ID NO: 94, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, the LC variable region sequence comprising SEQ ID NO: 96, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or v) a HC variable region sequence comprising SEQ ID NO: 274, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 276, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or w) a HC variable region sequence comprising SEQ ID NO: 284, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 284, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, ID NO: 286, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or x) a HC variable region sequence comprising SEQ ID NO: 104, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 106, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto;or y) a HC variable region sequence comprising SEQ ID NO: 144, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 146, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or z) a HC variable region sequence comprising SEQ ID NO: 4, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or aa) a HC variable region sequence comprising SEQ ID NO: NO: 24 or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, the LC variable region sequence comprising SEQ ID NO: 26 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or bb) a HC variable region sequence comprising SEQ ID NO: 64 or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and the LC variable region sequence comprising SEQ ID NO: 66 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto; or cc) a HC variable region sequence comprising SEQ ID NO: 74 or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereto, and the LC variable region sequence comprising SEQ ID NO: NO:76 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereto. ;

[0177] In one embodiment, the antibody, or antigen-binding portion thereof, binds to an epitope of the extracellular domain of a canine PD-1 extracellular domain comprising D58, D61, N74, T76, Y127 according to SEQ ID NO: 2, wherein the antibody comprises: a HC CDR1 sequence comprising or consisting of SEQ ID NO: 17 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 17; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 18 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 18; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 19 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 19; and a CDR1 sequence comprising or consisting of SEQ ID NO: 20 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 20; and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 21 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 21; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 22 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 22. Optionally, the antibody or antigen binding portion may comprise an HC variable region sequence comprising SEQ ID NO: 14, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 16, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof.

[0178] In one embodiment, the antibody, or antigen binding portion thereof, binds to an epitope of the extracellular domain of the canine PD-1 extracellular domain comprising D58, D61, N74, T76, Y127 according to SEQ ID NO: 2, wherein the antibody comprises: a HC CDR1 sequence comprising or consisting of SEQ ID NO: 17 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences compared to SEQ ID NO: 17; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 18 or an amino acid sequence having 1, 2, 3, 4, 5 or 6 amino acid differences compared to SEQ ID NO: 18; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 19 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid differences compared to SEQ ID NO: 19; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: : SEQ ID NO: 20 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid difference(s) compared to SEQ ID NO: 20; and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 21 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid difference(s) compared to SEQ ID NO: 21; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 22 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid difference(s) compared to SEQ ID NO: 22;

[0179] In one embodiment, the antibody, or antigen binding portion thereof, binds to an epitope of the extracellular domain of the canine PD-1 extracellular domain comprising T76, Q83, E84, L128, P129, P130 according to SEQ ID NO: 2, and the antibody, or antigen binding portion thereof, comprises: a HC CDR1 sequence comprising or consisting of SEQ ID NO: 27 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 27; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 28 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 28; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 29 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 29 having at least 70%, 80%, 85%, 90% or 95% sequence identity; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 30 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 30; and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 31 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 31; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 32 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 32. Optionally, the antibody or antigen binding portion may comprise an HC variable region sequence comprising SEQ ID NO: 24, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 26, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof.

[0180] In one embodiment, the antibody, or antigen binding portion thereof, binds to an epitope of the extracellular domain of the canine PD-1 extracellular domain comprising T76, Q83, E84, L128, P129, P130 according to SEQ ID NO: 2, and the antibody, or antigen binding portion thereof, comprises: a HC CDR1 sequence comprising or consisting of SEQ ID NO: 27 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences from SEQ ID NO: 27; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 28 or an amino acid sequence having 1, 2, 3, 4, 5 or 6 amino acid differences from SEQ ID NO: 28; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 29 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid differences from SEQ ID NO: 29; and a LC a CDR1 sequence comprising or consisting of SEQ ID NO: 30 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid difference(s) compared to SEQ ID NO: 30; and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 31 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid difference(s) compared to SEQ ID NO: 31; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 32 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 32, an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid difference(s) compared to SEQ ID NO: 32.

[0181] The term PD-1 refers to a protein also known as CD279 (cluster of differentiation 279), which is found on the surface of T and B cells. Antibodies and antigen-binding portions thereof specifically bind to wild-type canine PD-1 as defined in SEQ ID NO: 1 (nucleotide sequence) and SEQ ID NO: 2 (amino acid sequence). Unless otherwise indicated, as used herein, the term PD-1 refers to canine PD-1 or the T and B cell antigen PD-1. In humans and dogs, PD-1 is encoded by the PDCD1 gene.

[0182] Unless otherwise indicated, as used herein, the term PD-1 refers to canine PD-1. The terms "programmed death 1," "programmed cell death 1," "protein PD-1," "PD-1," "PD1," "PDCD1," "hPD-1," and "hPD-1" are used interchangeably and include variants, isoforms, and species homologs of canine PD-1.

[0183] The terms "PD-1 binding molecule / protein / polypeptide / agent / portion," "PD-1 antigen binding molecule / protein / polypeptide / agent / portion," "anti-PD-1 antibody," and "anti-PD-1 antibody or antigen-binding portion thereof" refer to molecules that specifically bind to canine PD-1 antigen. Binding can be demonstrated by standard methods (e.g., by reference to negative control assays using antibodies of unrelated specificity). The term "PD-1 binding molecule / agent" includes PD-1 binding proteins.

[0184] An antibody or antigen-binding portion thereof of the invention (including the multispecific (e.g., bispecific or trispecific) binding agents described herein) that "binds" or is "capable of binding" to an antigen of interest (i.e., canine PD-1) is one that binds the antigen with sufficient affinity such that the antibody, antigen-binding domain, or antigen-binding portion thereof can be used as a therapeutic agent targeted to cells or tissues expressing the antigen PD-1 as described herein.

[0185] The binding molecules of the present invention, including the antibodies and multivalent or multispecific binding agents described herein, specifically bind to canine PD-1. In other words, the binding to the PD-1 antigen is measurably distinct from non-specific interactions. As demonstrated in the Examples, the antibodies of the present invention do not cross-react with mouse PD-1. Further, as demonstrated in the Examples, the antibodies of the present invention do not cross-react with human PD-1. Preferably, the antibodies of the present invention bind to canine PD-1.

[0186] As used herein, the term "specifically binds to a particular polypeptide or an epitope on a particular polypeptide target" or "specifically binds to a particular polypeptide or an epitope on a particular polypeptide target" or "specific for a particular polypeptide or an epitope on a particular polypeptide target" can be, for example, defined as having at least about 10 -6 M, alternatively at least about 10 - 7 M, alternatively at least about 10 -8 M, alternatively at least about 10 -9 M, alternatively at least about 10 -10 M, alternatively at least about 10 -11 M, alternatively at least about 10 -12 M, alternatively at least about 10 -13In one embodiment, as used herein, the term "specific binding" can be represented by a molecule that has a KD of at least about 10 M or less for the target. -8 M, alternatively at least about 10 -9 M, alternatively at least about 10 -10 M, alternatively at least about 10 -11 M, alternatively at least about 10 -12 M, alternatively at least about 10 -13 In one embodiment, the term "specific binding" refers to binding in which a molecule binds to a specific polypeptide or an epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope. In an embodiment, the KD of a binding molecule of the invention for a target is determined using monovalent and / or bivalent binding assays (e.g., the monovalent and bivalent binding assays described in Example 9). In one embodiment, when determined by a bivalent binding assay, as used herein, the term "specific binding" can be determined by having at least about 10 -8 M, alternatively at least about 10 -9 M, alternatively at least about 10 -10 M, alternatively at least about 10 -11 M, alternatively at least about 10 -12 M, alternatively at least about 10 -13 The KD of a molecule for a target may be between about 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M, 10 -10 M to 10 -13 M, 10 -11 M to 10 -13 M, 10 -12 M to 10 -13 In embodiments, the KD of a binding molecule for a target may be between about 10 M and 20 M, when determined by a bivalent binding assay. -10 M to 10 -13 In one embodiment, when determined by a monovalent binding assay, the term "specific binding" as used herein can be determined by having at least about 10 -8 M, alternatively at least about 10 -9 M, alternatively at least about 10 -10 M, alternatively at least about 10 -11M, alternatively at least about 10 -12 M, alternatively at least about 10 -13 The KD of a molecule for a target may be between about 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M, 10 -10 M to 10 -13 M, 10 -11 M to 10 -13 M, 10 -12 M to 10 -13 In embodiments, the KD of a binding molecule for a target may be between about 10 M and 20 M, as determined by a monovalent binding assay. -8 M to 10 -11 Between M.

[0187] As used herein, the term "antibody" broadly refers to any immunoglobulin (Ig) molecule or its antigen-binding portion (composed of four polypeptide chains (two heavy chains (H) and two light chains (L))), or any functional fragment, mutant, variant or derivative thereof that retains the basic epitope-binding characteristics of the Ig molecule. Such mutant, variant or derivative antibody forms are known in the art.

[0188] In a full-length antibody, each heavy chain is composed of a heavy chain variable region or domain (abbreviated herein as HCVR) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region or domain (abbreviated herein as LCVR) and a light chain constant region. The light chain constant region comprises one domain, CL.

[0189] The heavy and light chain variable regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each heavy and light chain variable region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0190] Immunoglobulin molecules can generally be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2), or any subclass.

[0191] In canines, there are four IgG heavy chains, designated A, B, C, and D. These heavy chains represent the four different subclasses of dog IgG, designated IgG-A, IgG-B, IgG-C, and IgG-D. The DNA and amino acid sequences of these four heavy chains were originally described by Tang et al. (Vet. Immunol. Immunopathol. 80: 259-270 (2001)). The amino acid and DNA sequences of these heavy chains are also available from the GenBank database (IgGA: Accession No. AAL35301.1; IgGB: Accession No. AAL35302.1; IgGC: Accession No. AAL35303.1; IgGD: Accession No. AAL35304.1). Canine antibodies also contain two types of light chains, kappa (κ) and lambda (λ) (GenBank Accession No. κ light chain amino acid sequence ABY 57289.1, GenBank Accession No. ABY 55569.1). The antibodies herein may have either a lambda or a kappa light chain. In one embodiment, the light chain is a lambda light chain.

[0192] The term "CDR" refers to the complementarity determining region within an antibody variable sequence. Three CDRs are present in each of the heavy and light chain variable regions, each designated CDR1, CDR2, and CDR3. The term "CDR set" refers to a group of three CDRs present in a single variable region that are capable of binding an antigen. The exact boundaries of these CDRs can be defined differently according to various systems known in the art.

[0193] The Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). Chothia refers to the position of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). When referring to residues in the variable domain (approximately residues 1 to 107 of the light chain and residues 1 to 113 of the heavy chain), the Kabat numbering system is generally used. Another system is the ImMunoGeneTics (IMGT) numbering scheme. The IMGT numbering scheme is described in Lefranc et al., Dev. Comp. Immunol., 29, 185-203 (2005). Unless otherwise stated, the IMGT numbering scheme is used in this article.

[0194] In another embodiment, the invention relates to an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region that is the product of or derived from a canine IGHV3-5 VH gene, a canine IGHV3-19 VH gene, or a canine IGHV4-1 VH gene, wherein the antibody specifically binds to canine PD- 1. In another embodiment, the invention relates to an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a light chain variable region that is the product of or derived from a canine IGKV2-9 VK gene or a canine IGKV2-5 VK gene; or a canine IGLV3-3 VL gene or a canine IGLV3-11 VL gene, wherein the antibody specifically binds to canine PD- 1.

[0195] The anti-PD-1 antibody according to the present invention may be a canine, humanized, chimeric, feline or caninized antibody.

[0196] A "chimeric antibody" is a recombinant protein that contains the variable domains (including the complementarity determining regions (CDRs)) of an antibody derived from one species, preferably a rodent or human antibody, while the constant domains of the antibody molecule are derived from the constant domains of a canine antibody.

[0197] As used herein, the term "caninized antibody" refers to a form of recombinant antibody that contains sequences from both canine and non-canine (e.g., murine) antibodies. Generally, a caninized antibody will comprise substantially all of at least one or more (typically two) variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-canine immunoglobulin, and all or substantially all of the framework (FR) regions (and typically all or substantially all of the remaining frames) are those of a canine immunoglobulin sequence. A caninized antibody may comprise three heavy chain CDRs and three light chain CDRs from a murine or human antibody, along with a canine frame or a modified canine frame. The modified canine frame may contain one or more amino acid changes that further optimize the effectiveness of the caninized antibody, for example, to enhance its binding to its target. The non-canine sequence (e.g., the sequence of the hypervariable loops) may be further compared to the canine sequence, and as many residues as possible may be altered to make it as similar to the authentic canine sequence as possible.

[0198] As used herein, the term "felinized antibody" refers to a form of recombinant antibody that contains sequences from both feline and non-feline (e.g., canine) antibodies. Generally, a felinized antibody will comprise substantially all of at least one or more (typically two) variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-feline immunoglobulin, and all or substantially all of the framework (FR) regions (and typically all or substantially all of the remaining frames) are those of a feline immunoglobulin sequence. A felinized antibody may comprise three heavy chain CDRs and three light chain CDRs from a murine or human antibody, as well as a feline frame or a modified feline frame. The modified feline frame may comprise one or more amino acid changes that further optimize the effectiveness of the felinized antibody, for example, to enhance its binding to its target. The non-feline sequence (e.g., the sequence of the hypervariable loops) may be further compared to the feline sequence, and as many residues as possible may be altered to make it as similar to the authentic feline sequence as possible.

[0199] "Specialized" antibodies (e.g., humanized, caninized, chimeric, feline) are antibodies that have been engineered to resemble antibodies from a target species. In one embodiment, a "specific" antibody is greater than about 80%, 85%, or 90% similar to an antibody from the target species.

[0200] In contrast, the fully canine antibodies of the present invention can have canine variable regions and do not contain complete or partial CDRs or FRs from other species. Advantageously, the fully canine antibodies described herein are obtained from transgenic mice containing canine immunoglobulin sequences. The antibodies produced in these immunized mice are generated through in vivo B cell signaling and development to achieve natural affinity maturation, including in vivo V(D)J recombination, in vivo junction diversification, in vivo pairing of heavy and light chains, and in vivo hypermutation. Fully canine antibodies produced in this manner generate antibodies with optimal developability properties, minimizing lengthy lead optimization before large-scale production. Advantageously, such fully canine antibodies may have the lowest immunogenicity risk when introduced into patient animals, which in turn facilitates repeated dosing regimens. Given the risks of introducing development defects, immunogenicity, and reduced affinity associated with ex vivo mAb engineering (as described above), the fully canine antibodies of the present invention have the greatest potential to become effective therapies in clinical settings.

[0201] In one embodiment, the antibody or antibody fragment is canine. In one embodiment, the antibody is fully canine.

[0202] In one embodiment, the antibody or antibody fragment is cross-reactive with another species. In one embodiment, the canine antibody cross-reacts with feline PD-1 (GenBank ID 100135770). In another embodiment, the canine antibody cross-reacts with canine PD-1 is feline. The antibody or antibody fragment cross-reacts with feline PD-1 may have any of the characteristics described herein. In embodiments, the antibody or antibody fragment that cross-reacts with feline PD-1 comprises: an HC CDR1 sequence comprising or consisting of SEQ ID NO: 207 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 207; and an HC CDR2 sequence comprising or consisting of SEQ ID NO: 208 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 208; and an HC CDR3 sequence comprising or consisting of SEQ ID NO: 209 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 209; and an LC CDR1 sequence comprising or consisting of SEQ ID NO: 210 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 210 having an amino acid sequence with at least 70%, 80%, 85%, 90% or 95% sequence identity; and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 211 or an amino acid sequence with at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 211; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 212 or an amino acid sequence with at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 212.

[0203] The term "antigen binding site" refers to the portion of an antibody or antibody fragment that contains the region that specifically binds to an antigen. The antigen binding site can be provided by one or more antibody variable domains. Preferably, the antigen binding site is contained within the relevant VH and VL of the antibody or antibody fragment.

[0204] As used herein, the terms antigen-binding portion, antigen-binding fragment, or portion or fragment of an antibody are used interchangeably. In an embodiment of the present invention, an antibody or antigen-binding portion thereof that binds to canine PD-1 is provided, wherein the antigen-binding portion thereof is a scFv, Fv, heavy chain, or single domain antibody, such as a VH single domain antibody.

[0205] Antibody fragments are functional portions of full-length antibodies, such as F(ab')2, Fab, Fv, and sFv. Functional fragments of full-length antibodies retain the target specificity of the full-length antibody. Therefore, recombinant functional antibody fragments, such as Fab (fragment, antibody), scFv (single-chain variable chain fragment), and single-domain antibodies (dAbs), have been used to develop therapeutic agents as an alternative to mAb-based therapeutics.

[0206] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight, non-covalent association. The folding of these two domains produces six hypervariable loops (three loops each from the H and L chains) that donate the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only the three HVRs specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site. "Single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment that comprises the VH and VL antibody domains linked into a single polypeptide chain.

[0207] scFv fragments (approximately 25 kDa) are composed of two variable domains, VH and VL. Naturally, the VH and VL domains associate non-covalently via hydrophobic interactions and tend to dissociate. However, stable fragments can be engineered by linking the domains with a hydrophilic, flexible linker to create a single-chain Fv (scFv).

[0208] The smallest antigen-binding fragment is a single variable fragment, namely the variable heavy chain (VH) or variable light chain (VL) domain. The VH and VL domains, respectively, are capable of binding to the antigen. Target binding does not require binding to the light / heavy chain partners, respectively, or indeed, the presence of the remaining portions of the intact antibody. Antibody antigen-binding entities reduced to a single domain (corresponding to the VH or VL domain) are often referred to as "single-domain antibodies" or "immunoglobulin single variable domains." Thus, single-domain antibodies (approximately 12 to 15 kDa) have either a VH or VL domain.

[0209] In one aspect, the invention relates to an isolated single domain antibody, an isolated variable single domain or an isolated immunoglobulin single variable domain, wherein the isolated single domain antibody, isolated variable single domain or isolated immunoglobulin single variable domain binds to canine PD-1 and blocks the interaction of PD-1 with PD-L1 and / or PD-L2.

[0210] The terms "single domain antibody, variable single domain, or immunoglobulin single variable domain (ISV)" are all well known in the art and describe a single variable fragment of an antibody that binds to a target antigen. These terms are used interchangeably herein. As described below, embodiments of various aspects of the present invention relate to single heavy chain variable domain antibodies / immunoglobulin heavy chain single variable domains that bind to the PD-1 antigen in the absence of a light chain. Thus, canine heavy chain single variable domain antibodies are also within the scope of the present invention.

[0211] Thus, in some embodiments, the isolated binding agents / molecules of the invention comprise or consist of at least one single domain antibody, wherein the domain is a canine heavy chain variable domain. Thus, in one aspect, the binding agents of the invention comprise or consist of at least one canine immunoglobulin single variable heavy chain domain; they do not contain a VL domain.

[0212] The term "isolated" single domain antibody refers to a single domain antibody that is substantially free of other single domain antibodies, antibodies with different antigenic specificities, or antibody fragments. In addition, an isolated single domain antibody may be substantially free of other cellular materials and / or chemicals.

[0213] In an embodiment of the present invention, an isolated canine antibody or antigen-binding portion thereof that binds to PD-1 is provided, wherein the antibody or antigen-binding portion thereof competes with PD-L1 and / or PD-L2.

[0214] In a related embodiment of the present invention, an isolated canine antibody or antigen-binding portion thereof that binds to PD-1 is provided, wherein the antibody or antigen-binding portion thereof blocks the interaction of PD-1 with PD-L1 and / or PD-L2 and / or prevents a cellular response associated with the interaction of PD-1 with PD-L1 and / or PD-L2.

[0215] As used herein, a "blocking antibody or antibodies" or "neutralizing antibody or antibodies" refers to an antibody that, upon binding to PD-1, results in inhibition of at least one biological activity of PD-1. For example, the antibodies of the present invention can prevent or block the binding of PD-1 to PD-L1 and / or PD-L2. In one embodiment, the antibodies of the present invention block the binding of PD-1 to PD-L1. In one embodiment, the antibodies of the present invention block the binding of PD-1 to PD-L2.

[0216] Each single VH domain antibody comprises three CDRs and four FRs, which are arranged from amino-terminus to carboxyl-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Thus, in one embodiment of the invention, the domain is a human variable heavy chain (VH) domain having the following formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0217] The C-terminal or N-terminal VH framework sequences of the antibodies of the invention may be modified to improve their properties. For example, the VH domain may comprise a C-terminal or N-terminal extension or deletion.

[0218] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies contained in the antibody population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., heteromerization, amidation, carbohydrate addition) that may be present in small amounts. Such monoclonal antibodies can be derived from a single B cell or plasma cell. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Compared to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to its specificity, monoclonal antibodies have the advantage of being synthesized by hybridoma culture and thus not contaminated by other immunoglobulins.

[0219] The term "antigen binding site" refers to the portion of an antibody or antibody fragment that contains the region that specifically binds to an antigen. The antigen binding site can be provided by one or more antibody variable domains. The antigen binding site is typically contained within the associated VH and VL of an antibody or antibody fragment.

[0220] The term "epitope" or "antigenic determinant" refers to a site on the surface of an antigen that specifically binds to an immunoglobulin, antibody, or antibody fragment. Generally, an antigen has multiple or many different epitopes and reacts with many different antibodies. The term specifically includes linear epitopes and conformational epitopes. Epitopes within a protein antigen can be formed by both contiguous amino acids (usually linear epitopes) or non-contiguous amino acids juxtaposed by the protein's tertiary folding (usually conformational epitopes). Epitopes formed by contiguous amino acids are typically, but not always, retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining which epitopes a given antibody or antibody fragment binds (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which overlapping or contiguous peptides are tested for reactivity with a given antibody or antibody fragment.

[0221] Two antibodies bind to "substantially the same epitope" when they recognize the same or spatially overlapping epitope as a reference antibody. The most widely used and rapid method to determine whether two epitopes bind to the same or spatially overlapping epitope is a competitive assay, which can be configured in different formats, using labeled antigens or labeled antibodies. The epitope may or may not be a three-dimensional surface feature of the antigen. In some embodiments, the antibody can, for example, bind to a monomer / single subunit and block the formation of the active form. Suitably, the antibody binds to the extracellular domain of PD-1.

[0222] Proteolytic digestion of antibodies releases different fragments, termed Fv (variable fragment), Fab (fragment antigen binding), and Fc (fragment crystallization). The Fc fragment contains the carboxyl-terminal portions of two H chains held together by disulfides. The constant domains of the Fc fragment are responsible for mediating the antibody's effector functions.

[0223] The present invention extends to the antigen-binding portion or antigen-binding fragment of an antibody. The terms "binding portion" and "fragment" are used interchangeably herein. An antibody fragment is a portion of an antibody, such as F(ab')2, Fab, Fv, scFv, heavy chain, light chain, variable heavy chain (VH) domain, variable light chain (VL) domain, etc. Functional fragments of a full-length antibody retain the target specificity of the intact antibody. Therefore, recombinant functional antibody fragments, such as Fab (fragment, antibody), scFv (single-chain variable chain fragment), and single-domain antibodies (dAbs), have been used to develop therapeutic agents as an alternative to mAb-based therapeutics.

[0224] The invention also extends to antibody mimetics comprising the sequences as described herein.

[0225] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight, non-covalent association. Folding of these two domains produces six hypervariable loops (three loops each from the H and L chains) that contribute the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only the three HVRs specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

[0226] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment (approximately 25 kDa) composed of two variable domains, VH and VL, linked into a single polypeptide chain. Naturally, the VH and VL domains associate non-covalently via hydrophobic interactions and tend to dissociate. However, stable fragments can be engineered by linking the domains with a hydrophilic, flexible linker to create a single-chain Fv (scFv).

[0227] The smallest antigen-binding fragment is a single variable fragment, namely the variable heavy (VH) or variable light (VL) chain domain. The VH and VL domains, respectively, are capable of binding to an antigen. Target binding does not require binding to a light / heavy chain partner, respectively, or indeed, the presence of the rest of the intact antibody. Antibody antigen-binding entities reduced to a single domain (corresponding to the VH or VL domain) are often referred to as "single-domain antibodies" or "immunoglobulin single variable domains." Single-domain antibodies (approximately 12 to 15 kDa) possess only a VH or VL domain and lack the rest of the intact antibody. The term "dAb," or "domain antibody," generally refers to a single immunoglobulin variable domain (VH, VHH, or VL) polypeptide that specifically binds to an antigen.

[0228] The term "isolated" refers to a portion that is separated from its natural environment. For example, the term "isolated" refers to an antibody or fragment thereof that is substantially free of other antibodies, antibodies, or antibody fragments. Additionally, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0229] As used herein, the term "homology" or "identity" generally refers to the percentage of amino acid residues in a sequence that are identical to the residues in the reference polypeptide being compared, after alignment of the sequences, and in some embodiments, after introducing gaps where necessary to achieve the maximum homology percentage, and any conservative substitutions are not considered part of the sequence identity. Thus, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. N-terminal or C-terminal extensions, tags, or insertions should not be construed as reducing identity or homology. Methods and computer programs for comparison are well known. The percent identity between two amino acid sequences can be determined using a well-known mathematical algorithm.

[0230] As used herein, "amino acid" means one of the 20 naturally occurring amino acids, or any non-natural analogs that may be present at a specific, defined position. Amino acids encompass both naturally occurring amino acids and synthetic amino acids. Although in most cases, when proteins are produced by recombinant means, only naturally occurring amino acids are used.

[0231] As used herein, "substituting an amino acid residue" with another amino acid residue in the amino acid sequence of a heterodimeric protein or polypeptide (e.g., an antibody) as shown herein is equivalent to "substituting an amino acid residue" with another amino acid residue, and means that a specific amino acid residue at a specific position in the original (e.g., wild-type / germline) amino acid sequence has been replaced (or substituted) with a different amino acid residue. This can be achieved using standard techniques available to those skilled in the art (e.g., using recombinant DNA technology). Amino acids have changed relative to the native (wild-type / germline) sequence (as found naturally in wild-type (wt)), but may also change in IgG molecules containing other changes relative to the native sequence. "Wild-type" or "WT" or "native" herein means an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A WT protein, polypeptide, antibody, or immunoglobulin has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[0232] The antibodies or antigen-binding portions thereof according to the present invention have one or more of the following properties:

[0233] a) binds to canine PD-1;

[0234] b) blocking or reducing the functional interaction between PD-1 and PD-L1 and / or PD-L2;

[0235] c) exhibiting PD-1 blockade as demonstrated in the Examples;

[0236] d) increasing the secretion of IFN-γ from canine PBMCs as shown in the Examples;

[0237] e) binds to canine PD-1 on activated canine T cells as shown in the Examples;

[0238] f) capable of binding to cells expressing canine PD-1;

[0239] g) provides good stability as shown in the examples;

[0240] h) does not cross-react with human PD-1;

[0241] i) half-life between 1 and 20 days;

[0242] j) capable of increasing or upregulating T cell activation; and / or

[0243] k) is capable of upregulating one or more genes involved in T cell proliferation, activation and / or differentiation, such as IFGGC1, CD3D, ICOS, CXCL10, CD80, CCR5, IL-7 and / or INF-γ, and / or GZMB, PRF1, CD28, CXCR3, CTLA-4, CD8A, CD8B, TBX21, GATA3, IL2RA, CD40LG and / or IRF1.

[0244] These properties can be measured by methods known in the art, such as those disclosed in the Examples.

[0245] For example, blocking the functional interaction of PD-1 with PD-L1 and / or PD-L2 means that the antibody reduces or eliminates the binding of PD-1 to PD-L1 and / or PD-L2, thereby eliminating or reducing signaling through these pathways.

[0246] The antibodies or antigen-binding portions thereof according to the present invention have a favorable half-life. In some embodiments, the antibodies, or antigen binding portions thereof, exhibit a half-life of 1 to 20 days, 1 to 19 days, 1 to 18 days, 1 to 17 days, 1 to 16 days, 1 to 15 days, 1 to 14 days, 1 to 13 days, 1 to 12 days, 1 to 11 days, 1 to 10 days, 2 to 10 days, 3 to 10 days, 4 to 10 days, 5 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 5 to 20 days, 5 to 19 days, 5 to 18 days, 5 to 17 days, 5 to 16 days, 5 to 15 days, 5 to 14 days, 5 to 13 days, 5 to 12 days, 5 to 11 days, 5 to 10 days, 10 to 20 days, 10 to 19 days, 10 to 18 days, 10 to 17 days, 10 to 16 days, 10 to 15 days. In some embodiments, the antibody, or antigen-binding portion thereof, exhibits a half-life in the range of 10 to 15 days, e.g., 10 days, 11 days, 12 days, 13 days, 14 days, 15 days.

[0247] The increase or upregulation of T cell activation can be assessed by comparing the level of T cell activation with a reference value. The reference value can be the T cell activation level of a subject before treatment with the antibody of the present invention. The reference value can be the T cell activation level of a healthy subject. The reference value can be the T cell activation level of a diseased subject. The diseased subject may or may not have received therapy.

[0248] T cell activation can be measured using any suitable method known in the art. Examples of suitable methods for determining T cell activation are provided herein in Examples 12 and 20. In embodiments, increased IFN-ɣ secretion may indicate increased T cell activation. In embodiments, T cell activation can be determined by upregulation of certain genes associated with T cell activation, such as upregulation of one or more of IFGGC1, CD3D, ICOS, CXCL10, CD80, CCR5, IL-7, and / or INF-γ.

[0249] The upregulation of the gene can be assessed by comparing the level (e.g., expression level) of one or more genes selected from IFGGC1, CD3D, ICOS, CXCL10, CD80, CCR5, IL-7, and / or INF-γ with a reference value. The reference value can be the level of one or more of the genes from a subject before treatment with the antibody of the present invention. The reference value can be the level of one or more of the genes from a healthy subject. The reference value can be the level of one or more of the genes from a diseased subject. The diseased subject may or may not have received therapy.

[0250] The term "IFGGC1" refers to the gene encoding an interferon-inducible GTPase. The term "CD3D" refers to the gene encoding the CD3 delta subunit of the T-cell receptor complex. The term "ICOS" refers to the gene encoding the inducible T-cell co-stimulatory receptor. The term "CXCL10" refers to the gene encoding the CXC motif chemokine ligand 10. The term "CD80" refers to the gene encoding cluster of differentiation 80. The term "CCR5" refers to the gene encoding the CC chemokine receptor type 5.

[0251] In one embodiment, the antibody is PMX126, PMX127, PMX128, PMX129, PMX130, PMX131, PMX132, PMX133, PMX138, PMX140, PMX142, PMX143, PMX145, PMX146, PMX147, PMX148, PMX149, PMX150, PMX151, PMX136, PMX137, PMX141, PMX144, PMX134, PMX152, PMX153, PMX135, PMX139, PMX125, or a fragment thereof, as shown in the Examples and sequence information. The VH CDR amino acid sequences, VLCDR amino acid sequences, VH amino acid sequences, and VL amino acid sequences of the PMX molecules shown in Table 2 are specifically within the scope of the invention.

[0252] In one embodiment, the antibody, or antigen-binding portion thereof, comprises an Fc region, eg, a canine Fc region, eg, a canine IgGB Fc region.

[0253] Variants of the antibodies and antigen-binding portions described above are also within the scope of the invention.

[0254] Variants of antibodies or antigen-binding portions thereof (e.g., VH or VL) as described herein have at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the non-variant molecule. In one embodiment, the sequence identity is at least 95%. In one embodiment, the modification is a conservative sequence modification.

[0255] As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention, or their antigen-binding portions, by standard techniques known in the art (such as site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR region of an antibody of the present invention can be replaced with other amino acid residues from the same side chain family, and the functional assays described herein can be used to test whether the altered antibody retains function (i.e., PD-1 binding).

[0256] The amino acid residues of polypeptides and peptides described herein are abbreviated as "abbreviated amino acid residues" and are conservatively substituted for the amino acid residues of the polypeptides and peptides described herein.

[0257] Table 1. Amino acid residues and examples of conservative amino acid substitutions.

[0258]

[0259] In certain embodiments, the present invention provides an antibody or antigen-binding portion thereof, which is a variant of an antibody or antigen-binding portion thereof compared to a sequence selected from any one of the amino acid sequences of SEQ ID NO: 3 to SEQ ID NO: 292, the variant comprising one or more sequence modifications, and compared to an unmodified antibody or fragment thereof, there is improvement in one or more of the properties of binding affinity, specificity, thermal stability, expression level, effector function, glycosylation, reduced immunogenicity or solubility. Modifications for altered effector function activity are described in, for example, WO2023012496 and WO2021165417.

[0260] In certain embodiments, the invention provides an antibody or its antigen-binding portion thereof, it is selected from SEQ ID NO: 3 to SEQ ID NO: The variant of antibody or its antigen-binding portion thereof compared to the sequence of any one of 292 amino acid sequences, the variant includes one or more sequence modifications, and compared with unmodified antibody or its antigen-binding portion thereof, there is improvement in half-life. Suitable sequence modifications for improving half-life are known in the art, and are described in, for example, WO2018073185, WO 2020082048, WO2020116560, WO2020142625, WO2021212081, WO2021212081 and WO2022067233. Where the antibody or antigen binding portion thereof comprises a sequence modification that extends half-life, the half-life of the molecule may exhibit 1 to 20 days, 1 to 19 days, 1 to 18 days, 1 to 17 days, 1 to 16 days, 1 to 15 days, 1 to 14 days, 1 to 13 days, 1 to 12 days, 1 to 11 days, 1 to 10 days, 2 to 10 days, 3 to 10 days, 4 to 10 days, 5 to 10 days, 1 to 9 days. , 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 5 to 20 days, 5 to 19 days, 5 to 18 days, 5 to 17 days, 5 to 16 days, 5 to 15 days, 5 to 14 days, 5 to 13 days, 5 to 12 days, 5 to 11 days, 5 to 10 days, 10 to 20 days, 10 to 19 days, 10 to 18 days, 10 to 17 days, 10 to 16 days, 10 to 15 days half-life

[0261] Suitable methods for measuring properties that may indicate that the antibody can be successfully developed on a large scale include initial purification using chromatography, such as affinity chromatography (Protein A: MabSelect Sure LX), anion exchange chromatography (Capto Q), cation exchange chromatography (Capto S), and buffer exchange (G-25 Fine), followed by assessment of whether the antibody remains intact (e.g., using SDS PAGE analysis to determine molecular weight, HPLC-SEC to calculate % monomer), assessment of aggregation, and thermal stability (Tm) studies.

[0262] In an embodiment of the present invention, an antibody or antigen-binding portion thereof that binds to PD-1 is provided, wherein the antibody or antigen-binding portion thereof is conjugated to a therapeutic moiety, such as a drug, enzyme, or toxin. In one embodiment, the therapeutic moiety is a toxin, such as a cytotoxic radionuclide, a chemical toxin, or a protein toxin. In another related embodiment of the present invention, an antibody or antigen-binding portion thereof that binds to PD-1 is provided, wherein the therapeutic moiety is a second antibody or antigen-binding portion thereof. In related embodiments of the present invention, the second antibody or antigen-binding portion thereof binds to a different target. In further related embodiments, the different targets can be one or more of the following: LAG-3, OX40L, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand, CD3, CD8, CD28, CD4 or ICAM-1.

[0263] In another embodiment of the present invention, an antibody or antigen-binding portion thereof that binds to PD-1 is provided, wherein the antibody or antigen-binding portion thereof is conjugated to an additional moiety selected from the group consisting of: a half-life extending moiety, a label, a cytotoxin, a liposome, a nanoparticle, or a radioisotope. In related embodiments of the present invention, the half-life extending moiety is selected from the group consisting of: an albumin binding moiety, a transferrin binding moiety, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human serum albumin, a fragment of human serum albumin, an albumin binding peptide, or a single domain antibody that binds to human serum albumin. It will be understood by those skilled in the art that other half-life extending moieties may be conjugated to the antibody or antigen-binding portion thereof according to the present invention. Where the antibody or antigen binding portion thereof comprises a half-life extending moiety, the molecule may exhibit a half-life of 1 to 20 days, 1 to 19 days, 1 to 18 days, 1 to 17 days, 1 to 16 days, 1 to 15 days, 1 to 14 days, 1 to 13 days, 1 to 12 days, 1 to 11 days, 1 to 10 days, 2 to 10 days, 3 to 10 days, 4 to 10 days, 5 to 10 days, 1 to 9 days, 1 to 10 days, In some embodiments, the half-life of the molecule may be in the range of 10 to 15 days, for example, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 5 to 16 days, 5 to 15 days, 5 to 14 days, 5 to 13 days, 5 to 12 days, 5 to 11 days, 5 to 10 days, 10 to 20 days, 10 to 19 days, 10 to 18 days, 10 to 17 days, 10 to 16 days, 10 to 15 days. In some embodiments, the half-life of the molecule may be in the range of 10 to 15 days, for example, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days.

[0264] In another aspect of the present invention, a pharmaceutical composition is provided, comprising the antibody or antigen-binding portion thereof according to any of the preceding embodiments. The pharmaceutical composition can be used to treat a disease. In one embodiment, the disease is cancer or a tumor.

[0265] The pharmaceutical composition according to the present invention may comprise an antibody or antigen-binding portion thereof as described herein and optionally a pharmaceutically acceptable carrier. As used herein, the term pharmaceutical composition refers to a composition for treating companion animals, i.e., a composition for veterinary use, i.e., a veterinary composition. In a preferred embodiment, the animal being treated is a dog.

[0266] The pharmaceutical composition may optionally comprise a pharmaceutically acceptable carrier. The antibody, protein or construct or pharmaceutical composition may be administered by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation or topically (particularly to the ear, nose, eye or skin) or by inhalation.

[0267] Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration.Preferably, the composition is administered parenterally.

[0268] Pharmaceutically acceptable carriers or vehicles can be granular, such that the composition is, for example, in tablet or powder form. The term "carrier" refers to a diluent, adjuvant, or excipient used together with the drug-antibody conjugate of the present invention. Such pharmaceutical carriers can be liquids, such as water and oils, including oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. The carrier can be salt, gum arabic, gelatin, starch paste, talc, keratin, colloidal silicon dioxide, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants, and colorants can be used. In one embodiment, when administered to an animal, the antigen-binding domains or antibodies or compositions of the present invention and pharmaceutically acceptable carriers are sterile. When the drug-antibody conjugate of the present invention is administered intravenously, water is a preferred carrier. Saline solutions and dextrose and glycerol aqueous solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene glycol, water, ethanol, etc. If desired, the composition of the present invention may also contain a small amount of a wetting agent or emulsifier or a pH buffering agent.

[0269] The pharmaceutical compositions of the present invention may be in liquid form, such as solutions, emulsions, or suspensions. The liquid may be delivered by injection, infusion (e.g., intravenous infusion), or subcutaneous administration. When intended for oral administration, the composition is preferably in solid or liquid form, with semi-solid, semi-liquid, suspension, and gel forms being included within the forms considered herein as solid or liquid.

[0270] As a solid composition for oral administration, the composition can be formulated into a powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, and the like. Such solid compositions typically contain one or more inert diluents. Furthermore, one or more of the following may be present: a binder such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; an excipient such as starch, lactose, or dextrin; a disintegrant such as alginic acid, sodium alginate, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; a flavoring such as mint, methyl salicylate, or orange flavoring; and a coloring agent. When the composition is in capsule form (e.g., a gelatin capsule), in addition to the aforementioned types of materials, it may also contain a liquid carrier such as polyethylene glycol, cyclodextran, or a fatty oil.

[0271] The composition can be in liquid form, such as an elixir, syrup, solution, emulsion, or suspension. The liquid can be used for oral administration or for delivery by injection. When intended for oral administration, the composition can include one or more of a sweetener, a preservative, a dye / colorant, and a flavor enhancer. In compositions for administration by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer, and an isotonic agent can also be included.

[0272] The composition may be in the form of one or more dosage units. In particular embodiments, it may be desirable to administer the composition topically to the area in need of treatment, or by intravenous injection or infusion.

[0273] In another aspect of the present invention, a method for treating a disease in a canine subject in need thereof is provided, the method comprising administering an effective amount of an antibody or antigen-binding portion thereof as described in any of the preceding embodiments of the present invention. The method can be used to treat the disease. In one embodiment, the disease is cancer or a tumor.

[0274] In another aspect, the invention relates to an antibody or antigen binding portion thereof as described herein for use in treating a disease in a canine subject. In one embodiment, the disease is cancer or a tumor.

[0275] In another aspect, the invention relates to the use of an antibody or antigen-binding portion thereof as described herein in the manufacture of a medicament for treating cancer or a tumor.

[0276] Cancers that can be treated by the antibodies, or antigen-binding portions thereof, pharmaceutical compositions and / or methods according to the present invention are selected from the group consisting of bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, oral melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, breast cancer, brain cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, kidney cancer, soft tissue sarcoma, urethral cancer, bladder cancer, renal cancer, lung cancer, non-small cell lung cancer, thymoma, urothelial carcinoma, leukemia, prostate cancer, mesothelioma, adrenocortical carcinoma, lymphoma (such as Hodgkin's disease, non-Hodgkin's disease), gastric cancer and multiple myeloma. Canine cancers that can be targeted by the antibodies, or antigen-binding portions thereof, pharmaceutical compositions, and / or methods according to the invention are selected from the group consisting of mammary cancer (cancer of the tissue lining internal organs), osteosarcoma (bone cancer), melanoma (skin cancer), and angiosarcoma (cancer of the lining of blood vessels).

[0277] In other related embodiments of the present invention, antibodies or antigen-binding portions thereof, pharmaceutical compositions or methods as described in the foregoing embodiments are provided, wherein these embodiments further include administering another therapeutic agent to the subject alone. In related embodiments, another therapeutic agent is a radiotoxic agent, an immunosuppressant, an immunomodulator, or an antibody or antibody fragment thereof. In related embodiments, the immunomodulator is a cytokine, a chemokine or an anticancer therapy. The therapeutic agent is a compound or molecule that can be used to treat a disease. Examples of therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptotic agents, anti-angiogenic agents, boron compounds, photosensitizers or dyes, and radioisotopes. Antibody molecules include complete antibodies or fragments thereof (e.g., Fab, F(ab')2, Fv, single-chain Fv fragments (scFv) or single-domain antibodies, such as VH domains or antibody mimetic proteins). Anticancer therapy can include therapeutic agents or radiotherapy, and includes gene therapy, virotherapy, RNA therapy, bone marrow transplantation, nanotherapy, targeted anticancer therapy or oncolytic drugs. Examples of other therapeutic agents include other checkpoint inhibitors, anti-tumor drugs, immunogenic drugs, attenuated cancer cells, tumor antigens, antigen-presenting cells (such as dendritic cells pulsed with tumor-derived antigens or nucleic acids), immunostimulatory cytokines (e.g., IL-2, IFNa2, GM-CSF), targeted small molecules and biomolecules (such as components of signal transduction pathways, such as tyrosine kinase regulators and receptor tyrosine inhibitors, and drugs that bind to tumor-specific antigens, including EGFR antagonists), anti-inflammatory agents, cytotoxic agents, radiotoxic agents or immunosuppressive agents, and cells transfected with genes encoding immunostimulatory cytokines (e.g., GM-CSF), chemotherapy. In one embodiment, the antibody is used in combination with surgery.

[0278] In one embodiment, the antibody or pharmaceutical composition of the invention is administered together with an immunomodulator, a checkpoint regulator, an agent involved in T cell activation, a tumor microenvironment regulator (TME), or a tumor-specific target. For example, the immunomodulator can be an inhibitor of an immune checkpoint molecule selected from an inhibitor of one or more of the following: PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, CEACAM, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR beta, OX40, OX40L, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand, CD3, CD8, CD28, CD4 or ICAM-1. In another embodiment, the immunomodulator can be an activator of a co-stimulatory molecule selected from an agonist of one or more of the following: LAG-3, OX40, OX40L, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand, CD3, CD8, CD28, CD4 or ICAM-1.

[0279] In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody selected from Nivolumab®, Pembrolizumab®, Pidilizumab®, or Gilvetmab®.

[0280] In one embodiment of the present invention, the composition is administered concurrently with a chemotherapeutic agent or radiation therapy. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered before or after the administration of the composition of the present invention, preferably at least one hour, five hours, 12 hours, one day, one week, one month, more preferably several months (e.g., up to three months) before or after the administration of the composition of the present invention.

[0281] The antibodies or antigen-binding portions thereof or pharmaceutical compositions of the present invention can be administered at the same time or at different times with other therapies or therapeutic compounds or therapies, for example, simultaneously, separately or sequentially. In one embodiment, the other therapy can be any additional PD-1, PD-L1 or PD-L2 therapy.

[0282] The amount of the therapeutic agent effective / active in treating a particular disease or condition will depend on the nature of the disease or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be used to help identify the optimal dose range. The precise dosage to be adopted in the composition will also depend on the route of administration and the severity of the disease or condition, and should be determined according to the practitioner's judgment and the situation of each patient. Factors such as age, body weight, sex, diet, time of administration, excretion rate, host condition, drug combination, reaction sensitivity and disease severity should be considered.

[0283] Typically, the amount of the antibody, antigen-binding domain or fragment thereof of the invention is at least about 0.01% by weight of the composition. When intended for oral administration, this amount may vary within the range of about 0.1% to about 80% by weight of the composition. Preferred oral compositions may contain about 4% to about 50% by weight of the composition of the antibody or fragment thereof of the invention.

[0284] In embodiments, the antibody, or antigen binding portion thereof, is provided at a dose of 0.1 to 50 mg / kg, 0.1 to 40 mg / kg, 0.1 to 30 mg / kg, 0.1 to 20 mg / kg, 0.1 to 10 mg / kg, 0.1 to 5 mg / kg, 0.2 to 50 mg / kg, 0.3 to 50 mg / kg, 0.4 to 50 mg / kg, 0.5 to 50 mg / kg, 0.6 to 50 mg / kg, 0.2 to 40 mg / kg, 0.3 to 40 mg / kg, 0.4 to 40 mg / kg, 0.5 to 40 mg / kg, 0.6 to 40 mg / kg, 0.2 to 30 mg / kg, 0.3 to 30 mg / kg, 0.4 to 30 mg / kg, 0.5 to 30 mg / kg, 0.6 to 30 mg / kg, 0.2 to 20 mg / kg, 0.3 to 20 In some embodiments, the antibody or antigen-binding portion thereof is provided at a dose of about 3 mg / kg. In some embodiments, the antibody or antigen-binding portion thereof is provided at a dose of about 0.6 mg / kg.

[0285] Preferred compositions of the invention are prepared so that a parenteral dosage unit contains from about 0.01% to about 2% by weight of an antibody, antigen-binding domain or fragment thereof of the invention.

[0286] For administration by injection, such as intravenous or subcutaneous injection, the composition can contain about, typically about 0.01 mg / kg to about 250 mg / kg, for example, 0.1 mg / kg to about 250 mg / kg of the subject's body weight, for example, between about 0.1 mg / kg and about 20 mg / kg of the animal's body weight, and more preferably about 1 mg / kg to about 10 mg / kg of the animal's body weight, although less than 0.1 mg / kg is also contemplated. In one embodiment, the composition is administered at a dose of about 0.5 to 30 mg / kg (e.g., about 5 to 25 mg / kg, about 10 to 20 mg / kg, about 0.5 to 5 mg / kg, about 0.5 to 2.5 mg / kg, about 0.5 to 2.0 mg / kg, or about 2 or 3 mg / kg). In one embodiment, the composition is administered at a dose of 2 to 50 mg / ml. In one embodiment, the composition is administered at a dose of 0.5 mg / ml to 2.5 mg / ml or 0.5 mg / ml to 5 mg / ml. The dosing schedule can vary, for example, from once a week to once every 2, 3, or 4 weeks, or with an interval of up to 8 weeks between doses. In one embodiment, the composition is administered at a dose of 0.5 mg / ml to 2.5 mg / ml every three to four weeks (e.g., 0.5 mg / ml or 2.5 mg / ml every three to four weeks). In some embodiments, the dose is selected to provide prolonged depletion of PD-1 positive cells, thereby allowing for a three to four week interval between doses. Multiple doses may be administered, suitably up to about 6 or more repeated doses.

[0287] In one embodiment, after treatment, the subject has a disease-free period of at least 7 days, or at least 14 days, or at least 21 days, or at least 28 days, or at least 40 days, or at least 50 days, or at least 60 days. In one embodiment, after treatment, the subject has a disease-free period of at least 7 days, or at least 14 days, or at least 21 days, or at least 28 days, or at least 40 days, or at least 50 days, or at least 60 days.

[0288] In one embodiment, the number of survival days, disease-free days, or disease progression-free days is at least 2 months, or at least 3 months, or at least 4 months, for example at least 5 months, such as at least 6 months.

[0289] In one embodiment, the number of survival days, disease-free days, or disease-progression-free days is at least 9 months, 200 days, 300 days, or 3 years or more. In one embodiment, it is at least one year, two years, three years, or more. The present invention provides a method for treating or preventing a PD-1-mediated disease or condition in a companion animal (e.g., a dog), comprising administering to the animal in need thereof an effective amount of an antibody, antigen-binding domain, or fragment of the present invention.

[0290] As used herein, "treat," "treating," or "treatment" means to inhibit or alleviate a disease or disorder. For example, treatment can include delaying the development of symptoms associated with a disease or disorder, and / or reducing the severity of such symptoms that would accompany or be expected to accompany the progression of the disease. These terms include improving existing symptoms, preventing additional symptoms, and improving or preventing the underlying causes of such symptoms. Thus, these terms indicate that at least some of the mammals (e.g., canine patients) undergoing treatment achieve a beneficial result. Many medical treatments are effective for some, but not all, of the patients treated. For example, in the treatment of B-cell lymphoma, improvement in symptoms can be assessed by measuring the treated lymph nodes and observing a decrease in lymph node size as an indicator of treatment success.

[0291] The term "subject" or "patient" refers to a dog that is the object of treatment, observation or experiment. For the avoidance of doubt, treatment of humans is not included.

[0292] In another aspect, the present invention provides a nucleic acid sequence encoding an antibody or an antibody antigen-binding portion thereof according to any of the preceding embodiments of the present invention. Such a DNA sequence may be selected from the group consisting of SEQ ID NOs. 3 and 5, or SEQ ID NOs. 13 and 15, or SEQ ID NOs. 23 and 25, or SEQ ID NOs 33 and 35, or SEQ ID NOs. 43 and 45, or SEQ ID NOs. 53 and 55, or SEQ ID NOs. 63 and 65, or SEQ ID NOs. 73 and 75, or SEQ ID NOs. 83 and 85, or SEQ ID NOs. 93 and 95, or SEQ ID NOs. 103 and 105, or SEQ ID NOs. 113 and 115, or SEQ ID NOs. 123 and 125, or SEQ ID NOs. 133 and 135, or SEQ ID NOs. 143 and 145, or SEQ ID NOs. 153 and 155, or SEQ ID NOs. 163 and 165, or SEQ ID NOs. 173 and 175, or SEQ ID NOs. NO.183 and 185, or 193 and 195, or 203 and 205, or SEQ ID NO.213 and 215, or SEQ ID NO.223 and 225, or SEQ ID NO.233 and 235, or SEQ ID NO.243 and 245, or SEQ ID NO.253 and 255, or SEQ ID NO.263 and 265, or SEQ ID NO.273 and 275, or SEQ ID NO.283 and 285, or a sequence having at least 75%, 80%, 90% or 95% sequence identity thereto.

[0293] In another aspect, the present invention provides a vector comprising the nucleic acid sequence described above.

[0294] In another aspect, the present invention provides a host cell comprising the nucleic acid according to any preceding embodiment or the vector as described above.

[0295] In another aspect, the present invention provides a kit comprising the antibody or antigen-binding portion thereof according to any of the preceding embodiments, or the pharmaceutical composition according to the preceding embodiments. In related embodiments, the kit further comprises a reagent for detecting the antibody or antigen-binding portion thereof as described above.

[0296] In another aspect, the present invention provides a kit for detecting PD-1 for diagnosis, prognosis, or monitoring of a disease, the kit comprising an antibody or antigen-binding portion thereof of the present invention. Such a kit may contain other components, packaging, instructions, or materials to aid in the detection of PD-1 protein. The kit may comprise a labeled antibody or antigen-binding portion thereof of the present invention as described above and one or more compounds for detecting the label.

[0297] In another aspect, the invention provides an antibody of the invention, or antigen-binding portion thereof, packaged in lyophilized form or in an aqueous medium.

[0298] In another aspect, the present invention provides a method for preparing a canine antibody that binds to PD-1, the method comprising culturing the isolated host cell of the present invention and recovering the antibody.

[0299] In another aspect, the present invention provides a method for preparing a canine antibody that binds to PD-1, the method comprising the steps of:

[0300] a) immunizing a transgenic mouse expressing a nucleic acid construct comprising a canine heavy chain V gene and a canine light chain V gene with a PD-1 antigen,

[0301] b) generating an antibody library from said mouse, and

[0302] c) isolating antibodies from said library.

[0303] In another further embodiment, the present invention provides a method for detecting a PD-1 protein or an extracellular domain of a PD-1 protein in a biological sample from a canine subject, the method comprising contacting the biological sample with an antibody or antigen-binding portion thereof of the present invention, wherein the antibody or antigen-binding portion thereof is linked to a detectable label. The biological sample is one or more of a biopsy sample, a tissue sample, a blood sample, a serum sample, a plasma sample, or a lymph fluid sample. The method can be performed in vivo, in vitro, or ex vivo.

[0304] In another aspect, the present invention provides a method for inhibiting tumor growth or metastasis, which comprises contacting tumor cells with an effective amount of an antibody or antigen-binding portion thereof according to the present invention or a pharmaceutical composition according to the present invention.

[0305] In another aspect, the present invention provides a method for killing tumor cells that express PD-1, the method comprising contacting the cell with an antibody or antigen-binding portion thereof of the present invention or a pharmaceutical composition according to the present invention, such that killing of the cell that expresses PD-1 occurs.

[0306] In one embodiment, the tumor cells are canine tumor cells.

[0307] In another embodiment, the invention provides a binding agent comprising an antibody or antigen binding portion thereof according to the invention, wherein the antibody or antigen binding portion thereof is linked to a second antibody or antigen binding portion thereof that binds to a second target. In another embodiment, the binding agent is selected from any one of OX40L, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand, CD3, CD8, CD28, CD4 or ICAM-1.

[0308] The present invention also relates to the following non-limiting provisions:

[0309] 1. An isolated canine antibody, or antigen-binding portion thereof, that binds to one of the following epitopes of the extracellular domain of canine PD-1:

[0310] i) an epitope comprising T76, Q83, E84, L128, P129, P130 according to SEQ ID NO: 2

[0311] ii) an epitope comprising D58, D61, N74, T76, Y127 according to SEQ ID NO: 2

[0312] The epitope is determined using epitope mapping technology.

[0313] 2. The antibody, or antigen-binding portion thereof, of clause 1, wherein the antibody comprises:

[0314] a) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 17 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 17; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 18 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 18; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 19 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 19; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 20 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 20; and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 21 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 21. ID NO: 21, or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 21; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 22, or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 22; or

[0315] b) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 27 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 27; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 28 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 28; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 29 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 29; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 30 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 30; and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 31 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 31. ID NO: 31, or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 31; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 32, or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 32; or

[0316] c) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 207 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 207; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 208 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 208; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 209 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 209; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 210 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 210; and a CDR2 sequence comprising or consisting of SEQ ID NO: 211, or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 211; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 212, or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 212; or

[0317] d) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 97 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 97; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 98 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 98; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 99 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 99; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 100 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 100; and a CDR2 sequence comprising or consisting of SEQ ID NO: 101 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 101; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 102 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 102.

[0318] 3. The antibody, or antigen-binding portion thereof, of clause 1 or 2, wherein the antibody comprises:

[0319] a) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 17 or an amino acid sequence that has 1, 2, 3, 4 or 5 amino acid differences compared to SEQ ID NO: 17; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 18 or an amino acid sequence that has 1, 2, 3, 4, 5 or 6 amino acid differences compared to SEQ ID NO: 18; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 19 or an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid differences compared to SEQ ID NO: 19; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 20 or an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid differences compared to SEQ ID NO: 20; and a LC a CDR2 sequence comprising or consisting of SEQ ID NO: 21 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences from SEQ ID NO: 21; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 22 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences from SEQ ID NO: 22; or

[0320] b) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 207 or an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 207; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 208 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences from SEQ ID NO: 208; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 209 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acid differences from SEQ ID NO: 209; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 210 or an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 210; and a CDR2 sequence comprising or consisting of SEQ ID NO: 211 or an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 211; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 212 or an amino acid sequence having 1, 2, 3 or 4 amino acid differences from SEQ ID NO: 212; or

[0321] c) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 97 or an amino acid sequence that has 1, 2 or 3 amino acid differences compared to SEQ ID NO: 97; and an HC CDR2 sequence comprising or consisting of SEQ ID NO: 98 or an amino acid sequence that has 1, 2, 3 or 4 amino acid differences compared to SEQ ID NO: 98; and an HC CDR3 sequence comprising or consisting of SEQ ID NO: 99 or an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid differences compared to SEQ ID NO: 99; and an LC CDR1 sequence comprising or consisting of SEQ ID NO: 100 or an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid differences compared to SEQ ID NO: and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 101 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid differences compared to SEQ ID NO: 100; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 102 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences compared to SEQ ID NO: 102.

[0322] 4. An isolated canine antibody, or antigen-binding portion thereof, that binds canine PD-1, wherein the antibody comprises:

[0323] a) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 17 or an amino acid sequence that has 1, 2, 3, 4 or 5 amino acid differences compared to SEQ ID NO: 17; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 18 or an amino acid sequence that has 1, 2, 3, 4, 5 or 6 amino acid differences compared to SEQ ID NO: 18; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 19 or an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid differences compared to SEQ ID NO: 19; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 20 or an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid differences compared to SEQ ID NO: 20; and a LC a CDR2 sequence comprising or consisting of SEQ ID NO: 21 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences from SEQ ID NO: 21; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 22 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences from SEQ ID NO: 22; or

[0324] b) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 207 or an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 207; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 208 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences from SEQ ID NO: 208; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 209 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acid differences from SEQ ID NO: 209; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 210 or an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 210; and a CDR2 sequence comprising or consisting of SEQ ID NO: 211 or an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 211; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 212 or an amino acid sequence having 1, 2, 3 or 4 amino acid differences from SEQ ID NO: 212; or

[0325] c) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 97 or an amino acid sequence that has 1, 2 or 3 amino acid differences compared to SEQ ID NO: 97; and an HC CDR2 sequence comprising or consisting of SEQ ID NO: 98 or an amino acid sequence that has 1, 2, 3 or 4 amino acid differences compared to SEQ ID NO: 98; and an HC CDR3 sequence comprising or consisting of SEQ ID NO: 99 or an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid differences compared to SEQ ID NO: 99; and an LC CDR1 sequence comprising or consisting of SEQ ID NO: 100 or an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid differences compared to SEQ ID NO: and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 101 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid differences compared to SEQ ID NO: 100; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 102 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences compared to SEQ ID NO: 102.

[0326] 5. The antibody, or antigen-binding portion thereof, of any preceding clause, wherein the antibody, or antigen-binding portion thereof, has:

[0327] i) a HC CDR1 sequence comprising SEQ ID NO: 17 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 18 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 19 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 20 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 21 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 22 or consisting thereof,

[0328] ii) a HC CDR1 sequence comprising SEQ ID NO: 27 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 28 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 29 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 30 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 31 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 32 or consisting thereof,

[0329] iii) a HC CDR1 sequence comprising SEQ ID NO: 7 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 8 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 9 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 10 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 11 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 12 or consisting thereof,

[0330] iv) a HC CDR1 sequence comprising SEQ ID NO: 37 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 38 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 39 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 40 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 41 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 42 or consisting thereof,

[0331] v) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 47, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 48, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 49, a LC CDR1 sequence comprising SEQ ID NO: 50, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 51, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 52,

[0332] vi) a HC CDR1 sequence comprising SEQ ID NO: 57, a HC CDR2 sequence comprising SEQ ID NO: 58, a HC CDR3 sequence comprising SEQ ID NO: 59, a LC CDR1 sequence comprising SEQ ID NO: 60, a LC CDR2 sequence comprising SEQ ID NO: 61, and a LC CDR3 sequence comprising SEQ ID NO: 62,

[0333] vii) a HC CDR1 sequence comprising SEQ ID NO: 67, a HC CDR2 sequence comprising SEQ ID NO: 68, a HC CDR3 sequence comprising SEQ ID NO: 69, a LC CDR1 sequence comprising SEQ ID NO: 70, a LC CDR2 sequence comprising SEQ ID NO: 71, and a LC CDR3 sequence comprising SEQ ID NO: 72,

[0334] viii) a HC CDR1 sequence comprising SEQ ID NO: 77, a HC CDR2 sequence comprising SEQ ID NO: 78, a HC CDR3 sequence comprising SEQ ID NO: 79, a LC CDR1 sequence comprising SEQ ID NO: 80, a LC CDR2 sequence comprising SEQ ID NO: 81, and a LC CDR3 sequence comprising SEQ ID NO: 82,

[0335] ix) a HC CDR1 sequence comprising SEQ ID NO: 87 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 88 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 89 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 90 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 91 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 92 or consisting thereof,

[0336] x) a HC CDR1 sequence comprising SEQ ID NO: 97 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 98 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 99 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 100 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 101 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 102 or consisting thereof,

[0337] xi) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 107, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 108, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 109, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 110, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 111, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 112,

[0338] xii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 117, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 118, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 119, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 120, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 12, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 122,

[0339] xiii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 127, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 128, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 129, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 130, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 131, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 132,

[0340] xiv) a HC CDR1 sequence comprising SEQ ID NO: 137 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 138 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 139 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 140 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 141 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 142 or consisting thereof,

[0341] xv) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 147, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 148, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 149, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 150, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 151, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 152,

[0342] xvi) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 157, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 158, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 159, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 160, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 161, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 162,

[0343] xvii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 167, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 168, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 169, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 170, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 171, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 172,

[0344] xviii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 177, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 178, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 179, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 180, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 181, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 182,

[0345] xix) a HC CDR1 sequence comprising SEQ ID NO: 187 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 188 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 189 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 190 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 191 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 192 or consisting thereof,

[0346] xx) a HC CDR1 sequence comprising SEQ ID NO: 197 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 198 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 199 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 200 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 201 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 202 or consisting thereof,

[0347] xxi) a HC CDR1 sequence comprising SEQ ID NO: 207 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 208 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 209 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 210 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 211 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 212 or consisting thereof,

[0348] xxii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 217, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 218, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 219, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 220, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 221, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 222,

[0349] xxiii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 227, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 228, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 229, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 230, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 231, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 232,

[0350] xxiv) a HC CDR1 sequence comprising SEQ ID NO: 237 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 238 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 239 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 240 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 241 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 242 or consisting thereof,

[0351] xxv) a HC CDR1 sequence comprising SEQ ID NO: 247 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 248 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 249 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 250 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 251 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 252 or consisting thereof,

[0352] xxvi) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 257, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 258, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 259, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 260, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 261, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 262,

[0353] xxvii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 267, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 268, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 269, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 270, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 271, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 272,

[0354] xxviii) a HC CDR1 sequence comprising SEQ ID NO: 277 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 278 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 279 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 280 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 281 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 282 or consisting thereof, or

[0355] xxix) a HC CDR1 sequence comprising SEQ ID NO: 287 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 288 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 289 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 290 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 291 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 292 or consisting of thereof.

[0356] 6. The antibody or antigen-binding portion thereof of the preceding clause, wherein the antibody or antigen-binding portion thereof comprises a HC variable region sequence comprising SEQ ID NO: 4, or a sequence having at least 45%, 50%, 60%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto, e.g., a LC variable region sequence comprising SEQ ID NO: 6.

[0357] 7. The antibody, or antigen-binding portion thereof, of the preceding clause, wherein the antibody, or antigen-binding portion thereof, has:

[0358] a) an HC variable region sequence comprising SEQ ID NO: 4, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or

[0359] b) an HC variable region sequence comprising SEQ ID NO: 14, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 16, or a sequence having at least 35%, 45%, 55%, 65%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0360] c) an HC variable region sequence comprising SEQ ID NO: 24, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0361] d) an HC variable region sequence comprising SEQ ID NO: 34, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0362] e) an HC variable region sequence comprising SEQ ID NO: 44, or a sequence having at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 46, or a sequence having at least 35%, 45%, 55%, 65%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0363] f) an HC variable region sequence comprising SEQ ID NO: 54, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 56, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0364] g) an HC variable region sequence comprising SEQ ID NO: 64, or a sequence having at least 45%, 65%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 36, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0365] h) an HC variable region sequence comprising SEQ ID NO: 74, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0366] i) an HC variable region sequence comprising SEQ ID NO: 84, or a sequence having at least 40%, 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 86, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0367] j) an HC variable region sequence comprising SEQ ID NO: 94, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0368] k) an HC variable region sequence comprising SEQ ID NO: 104, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0369] 1) an HC variable region sequence comprising SEQ ID NO: 114, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or

[0370] m) an HC variable region sequence comprising SEQ ID NO: 124, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0371] n) an HC variable region sequence comprising SEQ ID NO: 134, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 136, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0372] o) a HC variable region sequence comprising SEQ ID NO: 144, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0373] p) an HC variable region sequence comprising SEQ ID NO: 154, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 156, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0374] q) an HC variable region sequence comprising SEQ ID NO: 164, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 176, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0375] r) an HC variable region sequence comprising SEQ ID NO: 174, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0376] s) an HC variable region sequence comprising SEQ ID NO: 184, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 186, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0377] t) an HC variable region sequence comprising SEQ ID NO: 194, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or

[0378] u) an HC variable region sequence comprising SEQ ID NO: 204, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 206, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0379] v) an HC variable region sequence comprising SEQ ID NO: 214, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 216, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0380] w) an HC variable region sequence comprising SEQ ID NO: 224, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 226, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0381] x) an HC variable region sequence comprising SEQ ID NO: 234, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 236, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or

[0382] y) an HC variable region sequence comprising SEQ ID NO: 244, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 246, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0383] z) an HC variable region sequence comprising SEQ ID NO: 254, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 256, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0384] aa) an HC variable region sequence comprising SEQ ID NO: 264, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 266, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0385] bb) an HC variable region sequence comprising SEQ ID NO: 274, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 276, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or

[0386] cc) an HC variable region sequence comprising SEQ ID NO: 284, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto, and an LC variable region sequence comprising SEQ ID NO: 286, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto, or

[0387] dd) an HC variable region sequence comprising SEQ ID NO: 24, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof.

[0388] 8. The antibody or antigen binding portion thereof according to the preceding clause, wherein the antigen binding portion thereof is a scFv, Fv, heavy chain or single domain antibody.

[0389] 9. The isolated canine antibody, or antigen-binding portion thereof, of the preceding clause that binds to canine PD-1, wherein the antibody, or antigen-binding portion thereof, competes with PD-L1 and / or PD-L2.

[0390] 10. The antibody or antigen-binding portion thereof according to the preceding clause, wherein the antibody or antigen-binding portion thereof blocks the interaction of PD-1 with PD-L1 and / or PD-L2 and / or prevents a cellular response associated with the interaction of PD-1 with PD-L1 and / or PD-L2.

[0391] 11. The antibody, or antigen-binding portion thereof, of the preceding clause, wherein the antibody, or antigen-binding portion thereof, is conjugated to a therapeutic moiety.

[0392] 12. The antibody, or antigen-binding portion thereof, of clause 11, wherein the therapeutic moiety is a second antibody, or antigen-binding portion thereof.

[0393] 13. The antibody, or antigen-binding portion thereof, of clause 12, wherein the second antibody, or antigen-binding portion thereof, binds to a different target.

[0394] 14. The antibody, or antigen-binding portion thereof, of the preceding clause, wherein the antibody, or antigen-binding portion thereof, is conjugated to an additional moiety selected from the group consisting of a half-life extending moiety, a label, a cytotoxin, a liposome, a nanoparticle, or a radioisotope.

[0395] 15. The antibody or antigen-binding portion thereof according to clause 14, wherein the half-life extending moiety is selected from: an albumin binding moiety, a transferrin binding moiety, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human serum albumin, a fragment of human serum albumin, an albumin binding peptide or a single domain antibody that binds to human serum albumin.

[0396] 16. A pharmaceutical composition comprising the antibody, or antigen-binding portion thereof, according to the preceding clause.

[0397] 17. The antibody or antigen-binding portion thereof according to any one of clauses 1 to 15, or the pharmaceutical composition according to clause 16, for use in treating a disease.

[0398] 18. A method of treating a disease in a canine subject in need thereof, the method comprising administering an effective amount of the antibody, or antigen-binding portion thereof, according to any one of clauses 1 to 15, or the pharmaceutical composition according to clause 16.

[0399] 19. The antibody or antigen-binding portion thereof, or pharmaceutical composition according to clause 17, or the method according to clause 18, wherein the disease is cancer or a tumor.

[0400] 20. The antibody, or antigen-binding portion thereof, or pharmaceutical composition according to clause 17 or 19, or the method according to clause 18 or 19, further comprising separately administering to the subject another therapeutic agent.

[0401] 21. The antibody or antigen-binding portion thereof, or pharmaceutical composition or method according to clause 20, wherein the therapeutic agent is a cytotoxic or radiotoxic agent, an immunosuppressant, an immunomodulator, or an antibody or antibody fragment thereof.

[0402] 22. The antibody or antigen-binding portion thereof, or pharmaceutical composition or method according to clause 21, wherein the immunomodulator is a cytokine or chemokine.

[0403] 23. A nucleic acid sequence encoding the antibody, or antibody antigen-binding portion thereof, according to any one of clauses 1 to 15.

[0404] 24. A nucleic acid sequence according to clause 23, comprising a sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 5, or SEQ ID NO: 13 and SEQ ID NO: 15, or SEQ ID NO: 23 and SEQ ID NO: 25, or SEQ ID NO: 33 and SEQ ID NO: 35, or SEQ ID NO: 43 and SEQ ID NO: 45, or SEQ ID NO: 53 and SEQ ID NO: 55, or SEQ ID NO: 63 and SEQ ID NO: 65, or SEQ ID NO: 73 and SEQ ID NO: 75, or SEQ ID NO: 83 and SEQ ID NO: 85, or SEQ ID NO: 93 and SEQ ID NO: 95, or SEQ ID NO: 103 and SEQ ID NO: 105, or SEQ ID NO: 113 and SEQ ID NO: 115, or SEQ ID NO: 123 and SEQ ID NO: 125, or SEQ ID NO: 134 and SEQ ID NO: 136. SEQ ID NO: 133 and SEQ ID NO: 135, or SEQ ID NO: 143 and SEQ ID NO: 145, or SEQ ID NO: 153 and SEQ ID NO: 155, or SEQ ID NO: 163 and SEQ ID NO: 165, or SEQ ID NO: 173 and SEQ ID NO: 175, or SEQ ID NO: 183 and SEQ ID NO: 185, or SEQ ID NO: 193 and SEQ ID NO: 195, or SEQ ID NO: 203 and SEQ ID NO: 205, or SEQ ID NO: 213 and SEQ ID NO: 215, or SEQ ID NO: 223 and SEQ ID NO: 225, or SEQ ID NO: 233 and SEQ ID NO: 235, or SEQ ID NO: 243 and SEQ ID NO: 245, or SEQ ID NO: 253 and SEQ ID NO: 255, or SEQ ID NO: 264 and SEQ ID NO: 265 NO: 263 and SEQ ID NO: 265, or SEQ ID NO: 273 and SEQ ID NO: 275, or SEQ ID NO: 283 and SEQ ID NO: 285, or a sequence with at least 75% sequence identity to any of the foregoing sequences.

[0405] 25. A vector comprising the nucleic acid sequence according to any one of clauses 23 to 24.

[0406] 26. A host cell comprising the nucleic acid sequence according to any one of clauses 23 to 24 or the vector according to clause 25.

[0407] 27. A kit comprising the antibody, or antigen-binding portion thereof, according to any one of clauses 1 to 15 or the pharmaceutical composition according to clause 16.

[0408] 28. The kit according to clause 27, further comprising a reagent for detecting the antibody or antigen-binding portion thereof.

[0409] 29. A method for preparing a canine antibody that binds to PD-1, the method comprising culturing the isolated host cell according to clause 26 and recovering the antibody.

[0410] 30. A method for preparing a canine antibody that binds to PD-1, the method comprising the steps of:

[0411] a) immunizing a transgenic mouse expressing a nucleic acid construct comprising a canine heavy chain V gene and a canine light chain V gene with a PD-1 antigen,

[0412] b) generating an antibody library from said mouse, and

[0413] c) isolating antibodies from said library.

[0414] 31. A method for detecting a PD-1 protein or an extracellular domain of a PD-1 protein in a biological sample from a canine subject, the method comprising contacting the biological sample with the antibody, or antigen-binding portion thereof, according to any one of clauses 1 to 15, wherein the antibody, or antigen-binding portion thereof, is linked to a detectable label.

[0415] 32. The method according to clause 31, wherein the biological sample is a biopsy sample, a tissue sample, a blood sample, a serum sample, a plasma sample or a lymph fluid sample.

[0416] 33. A method of inhibiting tumor growth or metastasis, the method comprising contacting tumor cells with an effective amount of the antibody or antigen-binding portion thereof according to any one of clauses 1 to 15, or the pharmaceutical composition according to clause 16.

[0417] 34. A method of killing a tumor cell expressing PD-1, the method comprising contacting the cell with the antibody according to any one of clauses 1 to 15 or the pharmaceutical composition according to clause 16, such that killing of the cell expressing PD-1 occurs.

[0418] 35. The method according to clause 34, wherein the tumor cells are canine tumor cells.

[0419] 36. A binding agent comprising the antibody, or antigen binding portion thereof, of any one of clauses 1 to 15, wherein the antibody, or antigen binding portion thereof, is linked to a second antibody, or antigen binding portion thereof, that binds to a second target.

[0420] 37. The binding agent of clause 36, wherein the second target binding agent is selected from any one of the list comprising LAG-3, OX40, OX40L, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand, CD3, CD8, CD28, CD4 or ICAM-1.

[0421] 38. An immunoconjugate comprising the antibody, or antigen-binding portion thereof, according to any one of clauses 1 to 15, or the binding agent according to clause 36 or 37.

[0422] 39. A method of modulating an immune response, the method comprising administering the antibody or fragment thereof according to any one of clauses 1 to 15, or the pharmaceutical composition according to clause 16, or the binding agent according to any one of clauses 36 to 37, or the immunoconjugate according to any one of clauses 38 to 39.

[0423] 40. A combination therapy comprising: an antibody or fragment thereof according to any one of clauses 1 to 15; or a pharmaceutical composition according to clause 16; or a binding agent according to any one of clauses 36 to 37; or an immunoconjugate according to any one of clauses 38 to 39, and an additional therapeutic moiety.

[0424] 41. The combination therapy of clause 40, wherein the additional therapeutic moiety is an antibody, optionally an antibody that binds an immuno-oncology target; or a chemotherapeutic agent.

[0425] Table 2. Sequence IDs for the amino acid and nucleotide sequences of each anti-PD-1 antibody included in the specification.

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450] The present invention will be further illustrated in the following non-limiting examples.

[0451] Examples

[0452] Example 1: Canine PD-1, PD-L1, and PD-L2 Sequences

[0453] After verification of sequence homology with well-annotated human and mouse orthologs, the canine PD-1 (ENSCAFG00000013184), PD-L1 (ENSCAFG00000002120), and PD-L2 (ENSCAFG00000002121) coding sequences were synthesized (and codon-optimized where appropriate) based on sequences predicted from genes found in the canine reference genome assembly (CanFam3.1), which was found in the Ensembl genome browser. The coding sequences of canine PD-1, PD-L1, and PD-L2 were confirmed by RNA sequencing according to standard methods.

[0454] Example 2: Expression of canine PD-1 in HEK cells and MEF cells

[0455] Human embryonic kidney (HEK) 293 cells were grown as monolayers in 90 mm round tissue culture plates in DMEM / F12 (Life Technologies, CA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma Aldrich) at 37°C in a humidified atmosphere containing 5% CO2. HEK293 cells were transfected with PD-1, PD-L1, or PD-L2 using Lipofectamine LTX and Plus reagent (Life Technologies, CA, USA) according to the manufacturer's recommendations. Stable integration of the plasmid expression cassette was ensured by using PiggyBac transposon elements in the expression vectors and by adding 1% w / w of the transposase-encoding plasmid along with the expression plasmid. Stably transfected cells were selected 48 hours after transfection with 3 µg / mL puromycin for a total of 6 days.

[0456] Mouse embryonic fibroblasts (MEFs) were grown as monolayers on 90 mm round tissue culture plates in DMEM high glucose medium (Life Technologies) supplemented with 10% FBS and 1X MEM non-essential amino acids (Gibco) containing L-glutamine at 37°C in a humidified atmosphere with 5% CO2. Cells were transfected with PD-1 using Lipofectamine LTX Plus reagent (Life Technologies, CA, USA) according to the manufacturer's recommendations. Stable integration of the plasmid expression cassette was ensured by using PiggyBac transposon elements in the expression vector and by adding 1% w / w of the transposase-encoding plasmid along with the expression plasmid. Stably transfected cells were selected 48 hours after transfection with 3 µg / mL puromycin for a total of 6 days.

[0457] Example 3: Expression of recombinant soluble canine PD-1, PD-L1, and PD-L2 proteins

[0458] Chinese hamster ovary (CHO) cells grown in F17 medium (Gibco) supplemented with 4 mM L-glutamine at 37°C in a humidified atmosphere with 8% CO2 were stably transfected with expression plasmids encoding PD-1ECD-mFc, PD-1ECD-6His, PD-L1ECD-mFc, and PD-L2ECD-mFc (ECD = extracellular domain). PD-1ECD, PD-L1ECD, and PD-L2ECD refer to the predicted extracellular domains of PD-1, PD-L1, and PD-L2, respectively. The "-mFc" suffix refers to the Fc portion of murine IgG2a, which contains the gene annotation: hinge-constant heavy 2-constant heavy 3-constant heavy secretory (H-CH2-CH3-CHS), and "6His" refers to a hexahistidine tag ( Figure 1 ).

[0459] Stable integration of the plasmid expression cassette was ensured by using the PiggyBac transposon element in the expression vector and by adding 1% w / w of the transposase-encoding plasmid along with the expression plasmid. 3E+06 cells were transfected with 2 µg of the expression plasmid encoding the appropriate protein and 20 ng of the transposase-expressing plasmid. Briefly, DNA was mixed with polyethyleneimine (PEI, 25 kDa linear, PolySciences) at a 1:3 (w / w) DNA:PEI ratio in 50 µL of Opti-MEM, incubated at room temperature for 15 minutes, and then added dropwise to the cell culture.

[0460] After 24 h, cells were selected with 25 μg / mL puromycin for a total of 8 days. 6100 mL of culture medium was inoculated and incubated at 32°C, 8% CO2, and shaking at 150 rpm. On days 1, 4, 7, and 10, 4% HyClone Cell Boost 7a Supplement, 0.4% HyClone Cell Boost 7b Supplement, and 1% glucose were added to the culture medium. Culture supernatants were collected on day 12. Supernatants were collected by centrifugation at 1000 g for 15 minutes and then subjected to a 0.2 µm filtration step. Proteins were purified from the supernatants by affinity chromatography using an AKTA FPLC system (Cytiva) using either standard Protein A affinity chromatography (for Fc-tagged proteins) or Ni-NTA IMAC (for 6His-tagged proteins). In all cases, proteins were immediately buffer-exchanged into PBS and concentrated using 10 kDa MWCO Amicon spin filter devices. After four rounds of buffer exchange, proteins were filtered through 0.2 µm and quantified by absorbance at 280 nm.

[0461] Example 4: Immunity

[0462] Ky9 TM Mice are immunized, which are transgenic mouse platforms capable of producing antibodies with canine variable domains, for example, essentially as described in WO2018 / 189520 and WO2020 / 074874. Compared to wild-type mice, transgenic mice are modified in such a way that immunoglobulin heavy chain (IGH) and light chain (IGL) variable region (V) genes, IGH D region genes, and IGH J region genes from dogs are inserted into the mice, which enables the mice to produce antibody heavy chains containing variable antibody regions and constant regions expressed from canine DNA.

[0463] The constant region can be a rodent immunoglobulin (IG) constant region, thereby producing a chimeric heavy chain carrying a canine variable region and a murine constant region. Information about such a chimeric antibody chain variable region or nucleic acid comprising the variable region can be used to generate fully canine antibodies, for example for therapeutic use in dogs.

[0464] Mice were immunized using a prime-boost regimen by intraperitoneal (IP) administration of 2.5E+06 MEF cells stably expressing PD-1 in 50% PBS and 50% Sigma Adjuvant system adjuvant (Sigma-Aldrich). Spleens and mesenteric lymph nodes were harvested 10 days after the final boost. Alternatively, mice were immunized three to four times with a recombinant canine PD-1 extracellular domain protein (PD-1ECD-mFc) at two or three-week intervals. The protein immunogen was delivered simultaneously at two sites: intraperitoneally and subcutaneously in 50% PBS and 50% Sigma Adjuvant system adjuvant (Sigma-Aldrich). Spleens and mesenteric lymph nodes were harvested 4 days after the final boost.

[0465] Determination of Serum Titers: Mice were bled before immunization and 10 days after the first boost. Blood was collected using microvette 200 Z-gel tubes (Starstedt AG & Co. KG) and serum was separated. Serum titers of PD-1-specific IgG were assessed by measuring the binding of serum dilutions to HEK293 cells expressing PD-1. Immune sera were serially diluted in FACS buffer and incubated with 1×105 HEK293 cells stably expressing PD-1 for 1 hour at 4°C, followed by washing in 200 µL of FACS buffer. Specific IgG was detected using a BB700-conjugated monoclonal antibody (BD OptiBuild™, Beckman Dickinson) targeting mouse isotypes IgG1, IgG2a, and IgG2b at a dilution of 1:400 for 1 hour at 4°C, followed by washing in 200 µL of FACS buffer. HEK293 cells were analyzed by flow cytometry using a Beckton-Coulter Cytoflex. Data were analyzed using FlowJo version 10.6.1. Background thresholds were set using a 1:100 dilution of preimmune serum.

[0466] Example 5: Isolation of Antibody-Producing Cells and Identification of Antibody Sequences of Candidate Molecules

[0467] Tissue Isolation: Spleens and mesenteric lymph nodes were harvested from immunized mice. Splenocytes were prepared by mincing the spleen into fragments and forcing them through a 45 µm cell strainer (Falcon) while rinsing on ice with RPMI-1640 (Lonza, Basel, CH) + 10% FBS. A similar procedure was used for lymph node-derived lymphocytes. Cells were pelleted at 300 g for 5 minutes and then suspended in PBS + 2% FBS + 1 mM EDTA (if used directly for flow cytometry) or frozen at -150°C in 90% FBS + 10% DMSO for later use.

[0468] Cell Sorting: Prior to antigen-specific cell sorting, T / erythroid cells were depleted using biotinylated anti-CD90.2 and anti-ter119 antibodies and then bound to streptavidin fast balls according to the manufacturer's instructions (StemCell Technologies UK). Bulk cell sorting was then performed using a BD FACSAria Fusion flow cytometer. Class-switched B cells were identified using the markers CD19, IgM, and IgD, while plasmablasts and plasma cell populations within spleen and lymph node (SP+LN) cells were labeled using CD138 (Syndecan-1) + CD267 (TACI). Within the class-switched B cell population, cells expressing a BCR specific for canine PD-1 were detected using an amine-labeled AlexaFluo647-conjugated PD-1 ECD-mFC fusion protein and sorted by FACS. Markers for identification of unwanted cell populations and dead cells (CD8a; CD4; fixable viability dye) were included in all staining panels to exclude these cells.

[0469] Sorted cells were prepared for antibody repertoire analysis using the 10X Genomics Chromium Single Cell Immunoassay System and V(D)J Kit (10X Genomics) according to the manufacturer's instructions. The nucleotide sequences of the expressed antibodies were determined by Illumina sequencing.

[0470] The variable immunoglobulin region comprises the VDJ region of the immunoglobulin nucleotide sequence of the heavy gene and the VJ region of the immunoglobulin nucleotide sequence of Igκ and Igλ. Within a clonal family, there are subfamilies that have shared mutations within their V(D)J segments that occur during immunoglobulin gene recombination and somatic hypermutation. Different clonal families that exhibit unique V(D)J segment usage typically exhibit different binding properties. During recombination and hypermutation, cells with higher affinity for antibodies to the antigen are selected, and if low-affinity clones from the same lineage have neutralizing function, the affinity typically increases with further mutation; for example, WO2015 / 040401 Figure 6 A family of clusters is shown.

[0471] A clonal family is typically defined by the related immunoglobulin heavy chain and / or light chain V(D)J sequences of two or more clonal cells. Related immunoglobulin heavy chain V(D)J sequences can be identified by their shared usage of V(D)J gene segments. An example of analyzing antibody sequences from sorted Ag-specific single B cells is shown in Figure 5 of WO2015 / 040401, which shows antibody sequences arranged and clustered by heavy chain V gene family usage to generate the displayed phylogenetic tree. From phylogenetic trees such as these, candidate clones are selected.

[0472] Example 6: Recombinant expression of monoclonal antibodies

[0473] The heavy and light chain variable region sequences of the selected candidate clones were synthesized and cloned into an expression vector containing sequences encoding an effector-deficient canine IgGB constant heavy chain region (see WO2023012496) and a wild-type canine λ constant 5 light chain region or a wild-type canine κ constant light chain region. The expression vectors encoding the heavy and light chains were co-transfected into CHO cells for stable expression. For antibody production, 3×10 6 Each selected CHO cell was seeded in 3 mL of culture medium and incubated at 32°C, 8% CO2, and shaking at 200 rpm. On days 1, 4, 7, and 10, 4% HyClone Cell Boost 7a supplement, 0.4% HyClone Cell Boost 7b supplement, and 1% glucose were added to the culture medium. Culture supernatants were collected on day 12, and IgG concentrations were determined by measuring protein A binding using surface plasmon resonance (Biacore 8K, Stovam).

[0474] Example 7: Flow cytometry cell binding assay

[0475] Because PD-1 is a transmembrane protein, the most conformationally correct protein is embedded in the plasma membrane. Flow cytometric measurement of binding to cells stably transfected with canine PD-1 allows for the measurement of membrane-embedded protein binding. 1E+05 HEK293 cells stably transfected with canine PD-1 were incubated in CHO supernatant containing 2 µg / mL IgG for one hour at 4°C. The cells were washed twice in 200 µL of FACS buffer and then incubated with a 1:300 dilution of goat anti-canine H+L-FITC-conjugated secondary antibody (Abcam) at 4°C for 30 minutes in the dark. The cells were then washed once in 200 µL of FACS buffer, resuspended in 200 µL of FACS buffer, and acquired on a BD Accuri C6 Plus flow cytometer. Figure 2 The results shown indicate that mAbs PMX125-144 all bind to canine full-length PD-1 in a transmembrane format, with binding comparable to that of the benchmark anti-canine PD-1 binding mAbs Givimab, 3B7-D, and 1B9 Def2. Givimab is a canine PD-1 binding IgGB kappa antibody, the full-length heavy and light chain amino acid sequences of which are described in: WHO Drug Information, Vol. 30, No. 4, 2016 ( https: / / www.antibodysociety.org / wordpress / wp-content / uploads / 2017 / 07 / INN-2017-Reference-26.pdf 3B7-D9 is a PD-1 binding IgGD κ antibody whose variable regions are described in US2017 / 0158764A1 (variable heavy SEQ ID NO 2, variable light SEQ ID NO 3). Here, its light chain variable region is grafted onto a wild-type canine κ constant (also known as IGKC) sequence, and its heavy chain variable region is grafted onto a wild-type canine IgGD constant (also known as IGHG4). 1B9 Def2 is an IgGB Def2 κ antibody whose variable regions are described in WO2020 / 103885A1 (variable heavy SEQ ID NO 50, variable light SEQ ID NO 52). Here, its light chain variable region is grafted onto a wild-type canine κ constant sequence (also known as IGKC), and its heavy chain variable region is grafted onto PetMedix's "Def2" modified IgGB constant Fc domain, which is described in WO2023012496.

[0476] Example 8: PD-L1 / PD-L2 Blockade Assay Using Flow Cytometry

[0477] Blocking the interaction between PD-1 and its ligands PD-L1 and PD-L2 is the primary mechanism of action of checkpoint inhibitors targeting this axis. Blocking this interaction in vitro is presumably a prerequisite for in vivo function.

[0478] Flow cytometry-based ligand blocking assay

[0479] 1E+05 HEK293 cells stably transfected with canine PD-1 were incubated in CHO supernatant containing 2 µg / mL recombinant IgG for one hour at 4°C. The cells were then washed twice with 200 µL FACS buffer and incubated with 1 µg / mL phycoerythrin-labeled PD-L1ECD-mFc or PDL2ECD-mFc for one hour at 4°C. The cells were then washed once in 200 µL FACS buffer, resuspended in 200 µL FACS buffer, and acquired on a Beckman-Coulter CytoFlex flow cytometer. Figure 3A and Figure 3B The data presented show that mAbs PMX125-PMX144 all effectively blocked the binding of PD-L1 to PD-1, and a subset also blocked the binding of PD-L2 to PD-1, with blocking effects superior to those of the control antibody 1B9 Def2. A subset of PD-L1 blockers also blocked the binding of PD-L2 to PD-1, but only PMX126, PMX128, PMX129, PMX133, and PMX142 blocked the binding of PD-L2 to PD-1 better than 1B9 Def2.

[0480] Example 9: Affinity determination – SPR and flow cytometry

[0481] SPR-based affinity assay

[0482] Monomeric affinity determination based on SPR was performed in a multi-cycle kinetics format. Approximately 100 RU of purified monoclonal antibody was captured on the Fc2 of a Series S Protein A chip (Sytovan). Following capture, monomeric PD-1 ECD-6His protein was injected at 30 µL / min in two-fold dilutions (from 100 nM to 0.13 nM), with an association time of 150 s and a dissociation time of 700 s. Bivalent affinity determination based on SPR was performed in a multi-cycle kinetics format. Approximately 170 RU of purified PD-1 ECD-mFc protein was captured on the Fc2 of a Series S CM5 chip (Sytovan). Following capture, monoclonal antibody was injected at 30 µL / min in two-fold dilutions (from 11.1 nM to 0.04 nM), with an association time of 180 s and a dissociation time of 1000 s. At the end of each cycle, the chip surface was regenerated with a 120-s pulse of 10 mM glycine, pH 1.9, at 30 µL / min. Data were analyzed using the Biacore Insight software package (Sytofran) by fitting to a global 1:1 binding model and performing kinetic analysis. Ks values ​​for the analyzed mAbs were in the single-digit (or double-digit for PMX-152) nanomolar range when assayed in monovalent mode and in the single- or double-digit picomolar range when assayed in bivalent mode. Figure 4A ).

[0483] Apparent affinity determination based on flow cytometry

[0484] 5E+04 HEK cells stably expressing PD-1 were incubated for 2 hours at 4°C in 250 µL FACS buffer containing purified mAbs diluted at 3-fold concentrations (ranging from 300 nM to 10 pM). The cells were then washed twice in 200 µL FACS buffer and incubated in a 1:300 dilution of goat anti-dog H+L-FITC-conjugated secondary antibody (Abcam) at 4°C in the dark for 30 minutes. The cells were then washed once in 200 µL FACS buffer and resuspended in 200 µL FACS buffer and acquired on a BD Accuri C6 Plus flow cytometer. The apparent affinity was then calculated by fitting the data to a "[agonist] vs. response" 4-parameter logistic curve in Prism 8.3.0 (GraphPad) and reporting the concentration at which the signal reached 50% of the maximum value. For all mAbs analyzed, the resulting EC values ​​were measured. 50 The values ​​(apparent affinities) were all in the single-digit nanomolar range ( Figure 4B ).

[0485] Example 10: Epitope Partitioning

[0486] Epitope partitioning was performed using a tandem SPR-based approach. Approximately 200 RU of PD-1 ECD-mFc was amine-coupled to Fc2 on a CM5 chip at pH 5.0 according to standard manufacturer's recommendations (Sytofran). The tandem assay consisted of a 300-second injection of 200 nM mAb 1 onto the chip in "dual" mode, followed immediately by a 300-second injection of 200 nM mAb 2 at a flow rate of 10 µL / min. The low ligand density, along with the high mAb 1 concentration (200 nM) and long injection time (300 s), ensured ligand saturation. A strong signal following mAb 2 injection indicated a noncompetitive binding mode for the mAb pair, while a low or no signal following mAb 2 injection indicated a competitive binding mode. After cycling, the chip surface was regenerated with a 120-second pulse of 10 mM glycine HCl, pH 1.9, at 30 µL / min. The running buffer used was HBS-EP+ (Synova), and data were collected on a Biacore 8K instrument (Synova). Givitumab (WHO Drug Information, Vol. 30, No. 4, 2016), 3B7-D9 (US2017 / 0158764A1), and 1B9 Def2 (WP2020 / 103885A1) are three mAbs that bind to canine PD-1. Figure 5A The fraction of mAb2 bound when mAb1 was pre-bound to saturating conditions is shown. Values ​​below 15% indicate complete blocking of a pair of mAbs, while values ​​above 75% indicate negligible blocking of mAb 2 binding by mAb 1. Analyzing competition between mAbs, in some cases, allows for differentiation of binding epitopes of mAbs that cross-compete in a one-to-one manner. Correlating the relative binding profile of each mAb in the matrix (Relative Binding Value column) with the relative binding profiles of all other mAbs can highlight subtle binding epitope similarities between mAbs when the threshold is appropriately set ( Figure 5B These results indicate that the epitopes bound by PMX126 and PMX151, while overlapping with those bound by the previously characterized mAbs 1B9Def2, Givitar, and 3B7-D9, can be shown to be distinct; however, using this limited panel of mAbs, the binding epitopes of PMX125 and PM127 could not be distinguished from that of 1B9Def2 ( Figure 5B and Figure 5C ).

[0487] Example 11: Binding to activated canine T cells

[0488] Canine PBMCs isolated from healthy dogs were plated in 96-well round-bottom plates at a density of 1 × 10 5Cells were cultured at a concentration of 100 cells / well in complete RPMI medium (RPMI-1640 + 10% FBS + 1 U / mL penicillin + 10 µg / mL streptomycin). Cells were stimulated with 1 µg / mL concanavalin A (ConA) for 96 hours to upregulate PD-1 expression, and then harvested for flow cytometric binding analysis. To assess PD-1 antibody binding on activated T cells, PD-1 antibodies were directly conjugated to AlexaFluor® 647. Cells were resuspended in FACS buffer and incubated with commercially available canine PE-conjugated CD5 (clone YKIX322.3, BioRad) to identify T cells, followed by incubation with AlexaFluor® 647-conjugated PD-1 antibody at a final concentration of 5 µg / mL for 30 minutes at 4°C to detect PD-1. Dead cells were labeled and excluded using the fixable viability dye eFluor 780 (eBioscience). Figure 6 The results shown indicate that all mAbs tested bound to PD-1 on ConA-activated canine T cells.

[0489] Because cancer samples are thought to have higher levels of PD-1 / PD-L1, the ability of PD-1 antibodies to bind to PD-1 on T cells from canine cancer cells was also evaluated. Samples from canine cancer cells were provided by the Department of Oncology at the Queen's Animal Hospital, Royal Veterinary College (RVC), United Kingdom. For this analysis, peripheral blood leukocytes were isolated from canine blood after erythrocyte lysis (Ebsen). Cells were stained with anti-canine PE-conjugated CD5 (clone YKIX322.3, Bio-Rad) and Alexa647-conjugated PMX127 antibodies as described above and analyzed by flow cytometry. To assess PD-1 expression on T cells, lymphocytes were first gated according to forward scatter and side scatter properties, and then CD5 + The population was gated. For analysis of PD-1 + Gate of cells based on CD5 + Binding of the population to an isotype control antibody. Figure 12A and Figure 12B The results shown confirm that PD-1 antibodies detect canine CD5 + The ability of T cells to express PD-1.

[0490] Example 12: Functional in vitro assay IFN-γ secretion assay

[0491] Blood from healthy beagle dogs was collected in EDTA tubes. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood after density centrifugation using Ficoll-Paque PLUS 1.077 g / mL (Sterofam) in Leucosep tubes (Greiner Bio-One). The isolated PBMCs were washed twice, resuspended in complete RPMI-1640 medium (Lonza), and cultured at 2.5 × 10 5 Cells were plated in triplicate into 96-well round-bottom plates at a concentration of 10 cells / well. Cells were stimulated with 1 μg / ml concanavalin A (ConA) and incubated for 96 hours. Antibodies were added at the time of stimulation at a final concentration of 10 μg / mL. To measure IFN-γ secretion, supernatants were collected after 96 hours of incubation, and IFN-γ levels were quantified by sandwich ELISA using the Canine IFN-γ ELISA BASIC kit (Mabtech AB) according to the manufacturer's recommended protocol. To evaluate T cell proliferation, after 96 hours of conA stimulation, cells were pelleted at 400 g for 5 minutes, resuspended in PBS + 3% FBS + 2 mM EDTA (FACS buffer), and stained with anti-canine CD5-RPE (Bio-Rad), fixable viability dye eFluor 780 (Invitrogen), and anti-mouse / rat Ki-67 eFluor 450 (Invitrogen) for 30 minutes at 4°C. Cells were pelleted and washed twice in FACS buffer and acquired on a Beckman-Coulter Cytoflex flow cytometer. Lymphocytes were first gated using FSC and SSC profiling, and viable cells, CD5 + Cells and Ki-67 + The data were analyzed using FlowJo version 10.6.1. Figure 7 The results shown demonstrate that several mAbs were able to significantly increase IFN-γ secretion in canine PBMCs, an indicator of increased T cell activation. PMX126, PM127, PM131, PMX151, and PM152 were able to increase T cell activation to levels above those observed for Givilumab and 1B9 Def2.

[0492] The effect of PD-1 blockade on INF-γ production in blood samples from cancer-bearing dogs (obtained from RVC, as described above) was evaluated. Canine leukocytes isolated from cancer-bearing dogs were stimulated with 1 mg / ml of ConA in the presence of PMX126 or PMX127, and the cultures were incubated for 96 hours. Controls included cells without added antibody and an irrelevant IgG isotype-matched antibody. After 96 hours of incubation, supernatants were collected and the concentration of IFN-γ in the supernatants was measured by ELISA (R&D Systems, USA). Increased IFN-γ production was observed with both PMX126 and PMX127 antibodies compared to controls ( Figure 13 ).

[0493] Example 13: Canine Cell-Based PD-1 Signaling Reporter Gene Assay

[0494] To evaluate the immunological activity of candidate antibodies, we tested their ability to disrupt PD-1 / PD-L1 inhibitory signaling in a bioluminescent reporter PD-1 / PD-L1 cell-based assay adapted from a commercially available cell line (Invitrogen, France). In this assay, NFAT-luc reporter Jurkat T cells were genetically engineered to express chimeric canine (extracellular domain) / human (transmembrane and intracellular domains) PD-1 instead of full-length human PD-1, and Raji antigen-presenting B cells were engineered to overexpress full-length canine PD-L1. Raji-Null cells, which do not express PD-L1, were used as a positive control for bioluminescent signaling. Briefly, 1 × 10 cells / well were added to a total volume of 200 µL. 5 Chimeric PD-1 NFAT-luc Jurkat cells and 5×10 4 Canine PD-L1 Raji-APC cells were co-cultured in the presence of 3 µg / mL of anti-canine PD-1 antibody candidates or control antibodies. The co-cultures were incubated overnight at 37°C, and bioluminescence was measured by adding the luciferase detection reagent QUANTI-Luc Gold (Invitrogen, France) according to the manufacturer's instructions. Figure 8A The results shown in Figure 3 and Figure 4 demonstrate that all mAbs tested have the ability to inhibit PD-1 signaling by interfering with the canine PD-1 / PD-L1 interaction. In this assay, PMX125, PMX126, and PMX127 resulted in significantly greater signaling blockade compared to Givitumab and 1B9 Def2.

[0495] Example 14: In vivo evaluation: MC-38 tumor therapy in a caninized mouse model

[0496] Transgenic C57BL / 6 mice carrying chimeric PD-1 were created by methods known to those skilled in the art and named C57BL / 6 cPD-1 / cPD-1 , this chimeric PD-1 carries the murine transmembrane and intracellular domains (encoding residues 170VIG-WPL288) and the canine extracellular domain (encoding residues 27SPD-QGL169) consisting of exon 2 and part of exon 3. A PD-L1-positive murine colorectal cancer cell line, called MC-38, is commonly used to evaluate human checkpoint inhibitors. This cell line can be stably transfected using the PiggyBac transposase system to constitutively express the canine PD-L1 ortholog (MC-38 cPD -1+ ). In transgenic C57BL / 6 cPD-1 / cPD-1 In mice, MC-38 cPD-1+ The cell line was able to grow at the same rate as MC-38. cPD-1+ Three days after cells were subcutaneously seeded in a 50% v / v formulation of PBS and Matrigel hESC (Corning), or when tumors reached 150 mm 3 Tumor size was monitored by measuring the tumor in two dimensions and calculating the volume using the following formula: tumor volume = ((width) 2 × length) / 2.

[0497] Example 15: Epitope Mapping

[0498] In order to determine the precise binding site of the monoclonal antibody to the protein, including interacting residues, epitope mapping can be performed. Several methods are available, including X-ray crystallography, cross-linking mass spectrometry, hydrogen-deuterium exchange mass spectrometry (HDX-MS), or alanine scanning mutagenesis, all of which are known to those skilled in the art. Alanine scanning mutagenesis consists of sequentially mutating residues to alanine, measuring the strength of the interaction of the variants compared to the wild-type protein, and inferring the target residues that contribute significantly to binding. Alanine was chosen because its side chain is small, consisting of only one methyl group, and its standard Ramachandran plot characteristics make it unlikely to significantly affect the local alpha carbon structure around the mutation.

[0499] Example 16: In Vivo Canine Study

[0500] The lead candidate antibody was administered to healthy beagle dogs at 3 or 0.6 mg / kg body weight via intravenous infusion. Pharmacokinetics (pK) was monitored by direct ELISA using the target of interest. The pK was expected to be linear with an elimination half-life of approximately 8-15 days.

[0501] Canine PD-1 antibody plasma concentrations were measured using ELISA. To perform this assay, ELISA 96-well plates (Thermofisher) were coated overnight at 4°C with 5 µg / mL of in-house produced canine PD-1 Fc tag protein. After washing and blocking steps, serial dilutions of canine plasma (12-point dilutions starting from a 250 µg / mL concentration) and goat anti-canine IgG HRP-conjugated secondary antibody (Bethyl labs) were added. Canine PD-1 plasma concentrations were determined using SuperSignalELISA Pico chemiluminescent substrate (Thermo Fisher Scientific). Quantification of bound PMX126 and PMX127 antibodies in plasma was determined by measuring optical density absorbance at 450 nm on a CLARIOstar microplate reader. Plasma concentrations of PMX126 and PMX127 antibodies were calculated from the standard curve using a single-phase decay model in GraphPad Prism software. A linear pK and a half-life of 5 days were observed. ( Figure 14A , B, C)

[0502] Safety and tolerability are other key readouts. Veterinarians closely monitored for immune-related adverse events (irAEs) and performed complete blood counts and liver enzyme activity. In multiple-dose studies, anti-drug antibody (ADA) assays were performed using standard methods, including acid-dissociation or ion-dissociation ELISAs, as previously described. 1,2 ) Measure the presence of ADA and closely monitor anti-drug immunogenicity.

[0503] In an exploratory single-dose target animal safety and pK study, animals were monitored for adverse events during their lifetime. Animal clinical observations, body weight monitoring, hematology, and serum biochemistry analysis were performed on all animals before treatment and once a week throughout the study period. Following investigational therapy in dogs and cats, adverse events were classified using the Veterinary Cooperative Oncology Group (VCOG)-Common Terminology Criteria for Adverse Events (VCOG-CTCAE v2). Single intravenous administration of PMX126 and PMX127 to healthy beagle dogs at doses of 0.6 and 3 mg / kg was well tolerated with no effect on body weight or clinical observations. Clinical pathology analysis showed only mild and transient changes, which were likely unrelated to treatment ( Figure 15 ).

[0504] Example 17: Alanine scanning mutagenesis

[0505] Alanine scanning experiments were performed by creating a panel of canine PD-1 mutant plasmids containing a single codon substitution encoding the amino acid alanine at each position in the predicted extracellular domain (L25-L166). Each mutant plasmid and the wild-type plasmid were transfected into HEK293 cells using PiggyBac transposase technology combined with transgene-specific drug selection to generate 134 stable cell lines. All cell lines were continuously cultured under selective pressure. To control for variable expression levels of PD-1 mutants in the 134 cell lines, PD-1-binding mAbs that failed to block PD-L1 binding were identified as PD-1 expression controls (IM84-187 and IM84-223). The inability of IM84-187 and IM84-223 to interfere with the binding of PMX-126 and PMX-127 to PD-1 was fully validated using SPR real-time competition assays and flow cytometry endpoint competition assays. Using standard amine coupling chemistry, the test mAbs PMX-126 and PMX-127, as well as the control mAbs IM84-187 and IM84-223, were directly labeled with AlexaFluor-647 and AlexaFluor-555, respectively. In each experiment, each mutant stable cell line was co-stained with one test mAb (PMX-126 or PMX-127) and one control mAb (IM84-187 or IM187-223) at concentrations of 0.9 µg / mL (test mAb) and 4.1 µg / mL (control mAb). Binding to each stable cell line was measured by flow cytometry. Cell lines expressing a lower proportion of the test mAb binding compared to the control mAb were inferred to be involved in test mAb binding. This was determined by a non-diagonal downward shift in the 2D scatter plots for AlexaFluor-647 (vertical axis) and AlexaFluor-555 (horizontal axis). The shift in proportional signal intensity between the AlexaFluor-647 signal and the AlexaFluor-555 signal was attributed to the different amounts of membrane-bound PD-1 on the cell surface of different stable cell lines; this could be due to factors such as the copy number or location of transgene integration or the expression level of the mutant PD-1 protein. Using this method, several residue positions that interact with PMX-126 (D58, D61, N74, T76, and Y127) and PMX-127 (T76, Q83, E84, L128, P129, and P130) were identified and displayed on the schematic structure of the PD-1 / PD-L1 extracellular domain ( Figure 9 A, B and Figure 10 A, B).

[0506] Example 18: Cross-reactivity with human PD-1

[0507] The cross-reactivity of the anti-canine mAbs PMX-126 and PMX-127 with the human PD-1 protein was tested by SPR and ELISA. In the SPR-based assay, approximately 300 RU of the antibodies PMX-126 and PMX-127 were captured on flow cell 2 of a Series S Sensor Chip Protein A (Sytovan), and recombinant canine PD-1-ECD-6His protein (produced in-house) or recombinant human PD-1-ED-6His protein (Bio-Techne, product number BP164049-1mg) was injected at a concentration of 900 nM and a flow rate of 30 µL / min for 180 seconds. The assay was also performed using a capture analog of pembrolizumab (Biosynth AG) to ensure that the human PD-1 ECD-6His protein was properly folded and active. Neither PMX-126 nor PMX-127 cross-reacted with the human PD-1ECD-6His protein, and pembrolizumab bound only to the human PD-1ECD-6His protein ( Figure 16A ELISA assays were performed as follows: Maxisorp plates (Nunc) were coated with 100 µL of recombinant canine PD-1-ECD-6His protein or 100 µL of recombinant human PD-1-ECD-6His protein (both at a concentration of 3 µg / mL) overnight at 4°C. The plates were then washed twice with PBS-Tween 20, blocked with 2% albumin fraction V in PBS for 1 hour, washed again with PBS-Tween 20, and incubated with 100 µL of a 12-point, three-fold dilution series of unlabeled mAb (top concentration: 100 µg / mL) for 1 hour at room temperature. Antibody binding was detected using a 1:8000 dilution of anti-canine IgG-HRP conjugate (for pembrolizumab, anti-human IgG-HRP conjugate was used), and the ELISA was developed with 100 µL of TMB substrate solution (Thermo Fisher Scientific). The reaction was stopped with 50 µL of 1 M H2SO4, and the plates were read at 450 nm. Neither PMX-126 nor PMX-127 cross-reacted with human PD-1ECD-6His protein, even at concentrations as high as 100µg / mL ( Figure 16B ).

[0508] Example 19: Receptor Occupancy

[0509] Receptor occupancy (RO) of PD-1 antibodies on T cells was determined using a flow cytometric assay to identify unoccupied PD-1 receptors in PBMC samples from healthy beagle dogs treated with PMX-126, PMX-127, or a control antibody. After isolating PBMCs using a previously described method, PBMC samples from dogs treated with PMX-126 were incubated with PE-conjugated mouse anti-canine CD5 antibodies (Bio-Rad) and Alexa 647-conjugated PMX-126 or anti-canine IgG isotype control at 4°C for 30 minutes in the dark. PBMC samples from dogs treated with PMX127 were incubated with PE-conjugated mouse anti-canine CD5 antibodies and Alexa 647-conjugated PMX-127 or anti-canine IgG isotype control. Samples were centrifuged at 400 × g for 3 minutes at 4°C and washed twice in PBS. Binding of PMX-126 or PMX-127 antibodies to PD-1 molecules on T cells was detected by flow cytometry ( Figure 17 ).

[0510] Example 20: Differential expression in genes associated with T cell function

[0511] Transcriptome analysis was performed to evaluate changes in gene expression profiles in PBMC samples from healthy beagle dogs following treatment with the PMX-126 and PMX-127 antibodies. RNA was isolated from PBMCs using the Quick-DNA / RNA Miniprep Plus Kit (Zymo Research) according to the manufacturer's protocol and quantified using a Nanodrop spectrophotometer and Bioanalyser. Samples were processed at the nCounter Core Facility at University College London (UCL), and genes involved in T cell activation and function were detected using the nCounter Canine IO Panel (NanoString Technologies). Data were analyzed using Rosalind software provided by NanoString Technologies. Genes selected for significant differential expression were based on the following criteria: a log2 fold change of less than or greater than 1.5, and p < 0.05, comparing day 07 samples to pretreatment samples. Data for specific genes were visualized using volcano plots, heat maps, and histograms. Between-group comparisons were performed using pre-treatment samples and day 07 samples from each antibody group (PMX126, PMX127, and isotype-matched controls). Transcriptome analysis showed an increase in T cell activation signatures following treatment with PMX-126 and PMX-127 ( Figure 18A and Figure 18B In particular, upregulation of IFGGC1, CD3D, ICOS, CXCL10, CD80, CCR5, IL-7, and INF-γ was observed.

[0512] References:

[0513] 1.Patton, A., Mullenix, MC, Swanson, SJ and Koren, E., 2005. An aciddissociation bridging ELISA for detection of antibodies directed against therapeutic proteins in the presence of antigen. Journal of immunologicalmethods, 304(1-2), pp. 189-195.

[0514] 2. Jordan, G., Pöhler, A., Guilhot, F., Zaspel, M. and Staack, RF, 2020. High ionic strength dissociation assay (HISDA) for high drug tolerantimmunogenicity testing. Bioanalysis, 12(12), pp. 857-866.

[0515] PD1 sequence

[0516] Wild-type canine PD-1 nucleotide sequence (SEQ ID NO: 1):

[0517] ATGGGGAGCCGGCGGGGGCCCTGGCCGCTCGTCTGGGCCGTGCTGCAGCTGGGCTGGTGGCCAGGATGGCTCCTAGACTCCCCTGACAGGCCCTGGAGCCCGCTCACCTTCTCCCCGGCGCAGCTCACGGTGCAGGAGGGAGAGAACGCCACGTTCACCTGCAGCCTGGCCGACATCCCCGACAGCTTCGTGCTCAACTGGTACCGCCTGAGCCCCCGCAACCAGACGGACAAGCTGGCCGCCTTCCAGGAGGACCGCATCGAGCCGGGCCGGGACAGGCGCTTCCGCGTCACGCGGCTGCCCAACGGGCGGGACTTCCACATGAGCATCGTCGCTGCGCGCCTCAACGACAGCGGCATCTACCTGTGCGGGGCCATCTACCTGCCCCCCAACACACAGATCAACGAGAGTCCCCGCGCAGAGCTCTCCGTGACGGAGAGAACCCTGGAGCCCCCCACACAGAGCCCCAGCCCCCCACCCAGACTCAGCGGCCAGTTGCAGGGGCTGGTCATCGGCGTCACGAGCGTGCTGGTGGGTGTCCTGCTACTGCTGCTGCTGACCTGGGTCCTGGCCGCTGTCTTCCCCAGGGCCACCCGAGGTGCCTGTGTGTGCGGGAGCGAGGACGAGCCTCTGAAGGAGGGCCCCGATGCAGCGCCCGTCTTCACCCTGGACTACGGGGAGCTGGACTTCCAGTGGCGAGAGAAGACGCCGGAGCCCCCGGCGCCCTGTGCCCCGGAGCAGACCGAGTATGCCACCATCGTCTTCCCGGGCAGGCCGGCGTCCCCGGGCCGCAGGGCCTCGGCCAGCAGCCTGCAGGGAGCCCAGCCTCCGAGCCCCGAGGACGGACCCGGCCTGTGGCCCCTC

[0518] Wild-type canine PD-1 amino acid sequence (SEQ ID NO:2):

[0519] MGSRRGPWPLVWAVLQLGWWPGWLLDSPDRPWSPLTFSPAQLTVQEGENATFTCSLADIPDSFVLNWYRLSPRNQTDKLAAFQEDRIEPGRDRRFRVTRLPNGRDFHMSIVAARLNDSGIYLCGAIYLPPNTQINESPRAELSV TERTLEPPTQSPSPPPRLSGQLQGLVIGVTSVLVGVLLLLLLTWVLAAVFPRATRGACVCGSEDEPLKEGPDAAPVFTLDYGELDFQWREKTPEPPAPCAPEQTEYATIVFPGRPASPGRRASASSLQGAQPPSPEDGPGLWPL

[0520] Wild-type canine PD-1 extracellular domain amino acid sequence (SEQ ID NO: 302):

[0521] LDSPDRPWSPLTFSPAQLTVQEGENATFTCSLADIPDSFVLNWYRLSPRNQTDKLAAFQEDRIEPGRDRRFRVTRLPNGRDFHMSIVAARLNDSGIYLCGAIYLPPNTQINESPRAELSVTERT

[0522] PET002 control antibody:

[0523] Givitumab heavy chain (SEQ ID NO: 296)

[0524] EVQLVQSGGDLVKPGGSVRLSCVASGFNIKNTYMHWVRQAPGKGLQWIGRIAPANVDTKYAPKFQGKATISADTAKNTAYMQLNSLRAEDTAVYYCVLIYYDYDGDIDVWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK

[0525] Givinostat monoclonal antibody light chain (SEQ ID NO: 297)

[0526] DIVMTQTPLSLSVSLGEPASISCHASQNINVWLSWYRQKPGQIPQLLIYKASHLHTGVPDRFSGSGSGTDFTLRISRVEADDAGVYYCQQGQSWPLTFGQGTKVEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDSKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

[0527] 3B7-D9 heavy chain (SEQ ID NO: 298)

[0528] EVQLVETGGGLVQPGRSLKLSCVASGFTFNNYWMSWTRQAPGKGLEWVASITNSGVSTYYPDSVKGRFTISRDNAQNTLYLQMNSLRSEDTATYFCTSALNWGYWYFDFWGPGTMVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSTVTVPSSRWPSETFTCNVVHPASNTKVDKPVPKESTCKCISPCPVPESLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPGK

[0529] 3B7-D9 light chain (SEQ ID NO: 299)

[0530] DIVMTQTPSSQAVSAGEKVTMSCKSSQSLLYSENKKNYLAWYQRKPGQSPKLLIYWASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLAVYYCQQYYNFPLTFGSGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

[0531] 1B9 Def2 heavy chain (SEQ ID NO: 300)

[0532] EVQLVQSGAEVKKPGASVKVSCKASGYTFTSFWMNWVRLAPGAGLEWIGRVDPYDSETHYNQKFKDRAILTVDTSTSTAYMELSSLRAGDIAVYYCATQFGFSWLAYWGQGTLVTVSAASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPESLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLGREDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNHIGLPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQKSLSHSPGK

[0533] 1B9 Def2 light chain (SEQ ID NO: 301)

[0534] EIVMTQSPGSLAGSAGESVSINCKSSQSLLYSSNQKNYLAWYQQKPGESPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINNLQAEDVGVYYCQQYYSNPYTFGQGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDSKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

Claims

1. An isolated canine antibody, or antigen-binding portion thereof, that binds to one of the following epitopes of the extracellular domain of canine PD-1: i) an epitope comprising T76, Q83, E84, L128, P129, P130 according to SEQ ID NO: 2 ii) an epitope comprising D58, D61, N74, T76, Y127 according to SEQ ID NO: 2 wherein the epitope is determined using epitope mapping technology.

2. The antibody, or antigen-binding portion thereof, of claim 1, wherein the antibody comprises: a) a HC CDR1 sequence comprising or consisting of: : SEQ ID NO: 17 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 17; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 18 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 18; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 19 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 19; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 20 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 20; and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 21 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 21 has at least 70%, 80%, 85%, 90% or 95% sequence identity; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 22 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 22; or b) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 27 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 27; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 28 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 28; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 29 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 29; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 30 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 30; and a CDR2 sequence comprising or consisting of SEQ ID NO: 31 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 31; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 32 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 32; or c) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 207 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 207; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 208 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 208; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 209 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 209; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 210 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 210; and a CDR2 sequence comprising or consisting of SEQ ID NO: 211, or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 211; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 212, or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 212; or d) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 97 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 97; and an HC CDR2 sequence comprising or consisting of SEQ ID NO: 98 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 98; and an HC CDR3 sequence comprising or consisting of SEQ ID NO: 99 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 99; and an LC CDR1 sequence comprising or consisting of SEQ ID NO: 100 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 100; and an LC a CDR2 sequence comprising or consisting of SEQ ID NO: 101 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 101; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 102 or an amino acid sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:

102.

3. The antibody, or antigen-binding portion thereof, of claim 1 or 2, wherein the antibody comprises: a) a HC CDR1 sequence comprising or consisting of: : SEQ ID NO: 17 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences compared to SEQ ID NO: 17; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 18 or an amino acid sequence having 1, 2, 3, 4, 5 or 6 amino acid differences compared to SEQ ID NO: 18; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 19 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid differences compared to SEQ ID NO: 19; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 20 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid differences compared to SEQ ID NO: 20; and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 21 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences compared to SEQ ID NO: 21; and a LCCDR3 sequence comprising or consisting of SEQ ID NO: 22 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences compared to SEQ ID NO: 22; or b) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 207 or an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 207; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 208 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences from SEQ ID NO: 208; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 209 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acid differences from SEQ ID NO: 209; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 210 or an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 210; and a CDR2 sequence comprising or consisting of SEQ ID NO: 211 or an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 211; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 212 or an amino acid sequence having 1, 2, 3 or 4 amino acid differences from SEQ ID NO: 212; or c) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 97 or an amino acid sequence that has 1, 2 or 3 amino acid differences compared to SEQ ID NO: 97; and an HC CDR2 sequence comprising or consisting of SEQ ID NO: 98 or an amino acid sequence that has 1, 2, 3 or 4 amino acid differences compared to SEQ ID NO: 98; and an HC CDR3 sequence comprising or consisting of SEQ ID NO: 99 or an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid differences compared to SEQ ID NO: 99; and an LC CDR1 sequence comprising or consisting of SEQ ID NO: 100 or an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid differences compared to SEQ ID NO: and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 101 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid differences compared to SEQ ID NO: 100; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 102 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences compared to SEQ ID NO:

102.

4. An isolated canine antibody, or antigen-binding portion thereof, that binds canine PD-1, wherein the antibody comprises: a) a HC CDR1 sequence comprising or consisting of: : SEQ ID NO: 17 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences compared to SEQ ID NO: 17; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 18 or an amino acid sequence having 1, 2, 3, 4, 5 or 6 amino acid differences compared to SEQ ID NO: 18; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 19 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid differences compared to SEQ ID NO: 19; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 20 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid differences compared to SEQ ID NO: 20; and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 21 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences compared to SEQ ID NO: 21; and a LCCDR3 sequence comprising or consisting of SEQ ID NO: 22 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences compared to SEQ ID NO: 22; or b) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 207 or an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 207; and a HC CDR2 sequence comprising or consisting of SEQ ID NO: 208 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences from SEQ ID NO: 208; and a HC CDR3 sequence comprising or consisting of SEQ ID NO: 209 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acid differences from SEQ ID NO: 209; and a LC CDR1 sequence comprising or consisting of SEQ ID NO: 210 or an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NO: 210; and a CDR2 sequence comprising or consisting of SEQ ID NO: 211 or an amino acid sequence having 1 or 2 amino acid differences from SEQ ID NO: 211; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 212 or an amino acid sequence having 1, 2, 3 or 4 amino acid differences from SEQ ID NO: 212; or c) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 97 or an amino acid sequence that has 1, 2 or 3 amino acid differences compared to SEQ ID NO: 97; and an HC CDR2 sequence comprising or consisting of SEQ ID NO: 98 or an amino acid sequence that has 1, 2, 3 or 4 amino acid differences compared to SEQ ID NO: 98; and an HC CDR3 sequence comprising or consisting of SEQ ID NO: 99 or an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid differences compared to SEQ ID NO: 99; and an LC CDR1 sequence comprising or consisting of SEQ ID NO: 100 or an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid differences compared to SEQ ID NO: and a LC CDR2 sequence comprising or consisting of SEQ ID NO: 101 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid differences compared to SEQ ID NO: 100; and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 102 or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences compared to SEQ ID NO:

102.

5. The antibody, or antigen binding portion thereof, of any preceding claim, wherein the antibody, or antigen binding portion thereof, has: i) a HC CDR1 sequence comprising SEQ ID NO: 17 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 18 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 19 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 20 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 21 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 22 or consisting thereof, ii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 27, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 28, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 29, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 30, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 31, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 32, iii) a HC CDR1 sequence comprising SEQ ID NO: 7 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 8 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 9 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 10 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 11 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 12 or consisting thereof, iv) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 37, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 38, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 39, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 40, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 41, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 42, v) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 47, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 48, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 49, a LC CDR1 sequence comprising SEQ ID NO: 50, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 51, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 52, vi) a HC CDR1 sequence comprising SEQ ID NO: 57, a HC CDR2 sequence comprising SEQ ID NO: 58, a HC CDR3 sequence comprising SEQ ID NO: 59, a LC CDR1 sequence comprising SEQ ID NO: 60, a LC CDR2 sequence comprising SEQ ID NO: 61, and a LC CDR3 sequence comprising SEQ ID NO: 62, vii) a HC CDR1 sequence comprising SEQ ID NO: 67, a HC CDR2 sequence comprising SEQ ID NO: 68, a HC CDR3 sequence comprising SEQ ID NO: 69, a LC CDR1 sequence comprising SEQ ID NO: 70, a LC CDR2 sequence comprising SEQ ID NO: 71, and a LC CDR3 sequence comprising SEQ ID NO: 72, viii) a HC CDR1 sequence comprising SEQ ID NO: 77, a HC CDR2 sequence comprising SEQ ID NO: 78, a HC CDR3 sequence comprising SEQ ID NO: 79, a LC CDR1 sequence comprising SEQ ID NO: 80, a LC CDR2 sequence comprising SEQ ID NO: 81, and a LC CDR3 sequence comprising SEQ ID NO: 82, ix) a HC CDR1 sequence comprising SEQ ID NO: 87 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 88 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 89 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 90 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 91 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 92 or consisting thereof, x) a HC CDR1 sequence comprising SEQ ID NO: 97 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 98 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 99 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 100 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 101 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 102 or consisting thereof, xi) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 107, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 108, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 109, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 110, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 111, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 112, xii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 117, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 118, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 119, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 120, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 12, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 122, xiii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 127, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 128, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 129, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 130, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 131, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 132, xiv) a HC CDR1 sequence comprising SEQ ID NO: 137 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 138 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 139 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 140 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 141 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 142 or consisting thereof, xv) a HC CDR1 sequence comprising SEQ ID NO: 147 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 148 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 149 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 150 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 151 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 152 or consisting thereof, xvi) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 157, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 158, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 159, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 160, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 161, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 162, xvii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 167, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 168, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 169, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 170, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 171, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 172, xviii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 177, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 178, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 179, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 180, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 181, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 182, xix) a HC CDR1 sequence comprising SEQ ID NO: 187 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 188 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 189 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 190 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 191 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 192 or consisting thereof, xx) a HC CDR1 sequence comprising SEQ ID NO: 197 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 198 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 199 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 200 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 201 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 202 or consisting thereof, xxi) a HC CDR1 sequence comprising SEQ ID NO: 207 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 208 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 209 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 210 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 211 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 212 or consisting thereof, xxii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 217, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 218, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 219, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 220, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 221, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 222, xxiii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 227, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 228, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 229, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 230, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 231, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 232, xxiv) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 237, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 238, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 239, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 240, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 241, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 242, xxv) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 247, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 248, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 249, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 250, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 251, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 252, xxvi) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 257, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 258, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 259, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 260, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 261, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 262, xxvii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 267, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 268, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 269, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 270, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 271, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 272, xxviii) a HC CDR1 sequence comprising SEQ ID NO: 277 or consisting thereof, a HC CDR2 sequence comprising SEQ ID NO: 278 or consisting thereof, a HC CDR3 sequence comprising SEQ ID NO: 279 or consisting thereof, a LC CDR1 sequence comprising SEQ ID NO: 280 or consisting thereof, a LC CDR2 sequence comprising SEQ ID NO: 281 or consisting thereof, and a LC CDR3 sequence comprising SEQ ID NO: 282 or consisting thereof, or xxix) a HC CDR1 sequence comprising SEQ ID NO: 287 or consisting of thereof, a HC CDR2 sequence comprising SEQ ID NO: 288 or consisting of thereof, a HC CDR3 sequence comprising SEQ ID NO: 289 or consisting of thereof, a LC CDR1 sequence comprising SEQ ID NO: 290 or consisting of thereof, a LC CDR2 sequence comprising SEQ ID NO: 291 or consisting of thereof, and a LC CDR3 sequence comprising SEQ ID NO: 292 or consisting of thereof.

6. The antibody, or antigen binding portion thereof, of the preceding claim, wherein the antibody, or antigen binding portion thereof, comprises a HC variable region sequence comprising SEQ ID NO: 4, or a sequence having at least 45%, 50%, 60%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereof, e.g., a LC variable region sequence comprising SEQ ID NO:

6.

7. The antibody or antigen binding portion thereof according to the preceding claim, wherein the antibody or antigen binding portion thereof has: a) an HC variable region sequence comprising SEQ ID NO: 4, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or b) an HC variable region sequence comprising SEQ ID NO: 14, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 16, or a sequence having at least 35%, 45%, 55%, 65%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or c) an HC variable region sequence comprising SEQ ID NO: 24, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or d) an HC variable region sequence comprising SEQ ID NO: 34, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or e) an HC variable region sequence comprising SEQ ID NO: 44, or a sequence having at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 46, or a sequence having at least 35%, 45%, 55%, 65%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or f) an HC variable region sequence comprising SEQ ID NO: 54, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 56, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or g) a HC variable region sequence comprising SEQ ID NO: 64, or a sequence having at least 45%, 65%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and a LC variable region sequence comprising SEQ ID NO: 36, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or h) an HC variable region sequence comprising SEQ ID NO: 74, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or i) an HC variable region sequence comprising SEQ ID NO: 84, or a sequence having at least 40%, 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 86, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or j) an HC variable region sequence comprising SEQ ID NO: 94, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or k) an HC variable region sequence comprising SEQ ID NO: 104, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or 1) an HC variable region sequence comprising SEQ ID NO: 114, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or m) an HC variable region sequence comprising SEQ ID NO: 124, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or n) an HC variable region sequence comprising SEQ ID NO: 134, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 136, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or o) an HC variable region sequence comprising SEQ ID NO: 144, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or p) an HC variable region sequence comprising SEQ ID NO: 154, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 156, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or q) an HC variable region sequence comprising SEQ ID NO: 164, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 176, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or r) an HC variable region sequence comprising SEQ ID NO: 174, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or s) an HC variable region sequence comprising SEQ ID NO: 184, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 186, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or t) an HC variable region sequence comprising SEQ ID NO: 194, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or u) an HC variable region sequence comprising SEQ ID NO: 204, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 206, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or v) an HC variable region sequence comprising SEQ ID NO: 214, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 216, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or w) an HC variable region sequence comprising SEQ ID NO: 224, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 226, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or x) an HC variable region sequence comprising SEQ ID NO: 234, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 236, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or y) an HC variable region sequence comprising SEQ ID NO: 244, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 246, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or z) an HC variable region sequence comprising SEQ ID NO: 254, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 256, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or aa) an HC variable region sequence comprising SEQ ID NO: 264, or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 266, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or bb) an HC variable region sequence comprising SEQ ID NO: 274, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 276, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof; or cc) an HC variable region sequence comprising SEQ ID NO: 284, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 286, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereof; or dd) an HC variable region sequence comprising SEQ ID NO: 24, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85% or 90% sequence identity thereof, and an LC variable region sequence comprising SEQ ID NO: 26, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85% or 90% sequence identity thereof.

8. The antibody or antigen binding portion thereof according to the preceding claim, wherein the antigen binding portion thereof is a scFv, Fv, heavy chain or single domain antibody.

9. The isolated canine antibody, or antigen-binding portion thereof, of the preceding claim, which binds to canine PD-1, wherein the antibody, or antigen-binding portion thereof, competes with PD-L1 and / or PD-L2.

10. The antibody or antigen binding portion thereof according to the preceding claim, wherein the antibody or antigen binding portion thereof blocks the interaction of PD-1 with PD-L1 and / or PD-L2 and / or prevents a cellular response associated with the interaction of PD-1 with PD-L1 and / or PD-L2.

11. The antibody, or antigen binding portion thereof, of the preceding claim, wherein the antibody, or antigen binding portion thereof, is conjugated to a therapeutic moiety.

12. The antibody, or antigen-binding portion thereof, of claim 11, wherein the therapeutic moiety is a second antibody, or antigen-binding portion thereof.

13. The antibody, or antigen binding portion thereof, of claim 12, wherein the second antibody, or antigen binding portion thereof, binds to a different target.

14. The antibody, or antigen binding portion thereof, of the preceding claim, wherein the antibody, or antigen binding portion thereof, is conjugated to an additional moiety selected from the group consisting of a half-life extending moiety, a label, a cytotoxin, a liposome, a nanoparticle, or a radioisotope.

15. The antibody or antigen binding portion thereof of claim 14, wherein the half-life extending moiety is selected from the group consisting of: an albumin binding moiety, a transferrin binding moiety, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human serum albumin, a fragment of human serum albumin, an albumin binding peptide, or a single domain antibody that binds to human serum albumin.

16. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof according to the preceding claim.

17. The antibody or antigen-binding portion thereof according to any one of claims 1 to 15 or the pharmaceutical composition according to claim 16 for use in treating a disease.

18. A method of treating a disease in a canine subject in need thereof, the method comprising administering an effective amount of the antibody, or antigen-binding portion thereof, of any one of claims 1 to 15 or the pharmaceutical composition of claim 16.

19. The antibody or antigen-binding portion thereof or pharmaceutical composition of claim 17 or the method of claim 18, wherein the disease is cancer or a tumor.

20. The antibody or antigen-binding portion thereof, or pharmaceutical composition according to claim 17 or 19, or the method according to claim 18 or 19, further comprising separately administering to the subject another therapeutic agent.

21. The antibody or antigen-binding portion thereof, or pharmaceutical composition or method of claim 20, wherein the therapeutic agent is a cytotoxic or radiotoxic agent, an immunosuppressant, an immunomodulator, or an antibody or antibody fragment thereof.

22. The antibody or antigen-binding portion thereof, or pharmaceutical composition or method of claim 21, wherein the immunomodulator is a cytokine or chemokine.

23. A nucleic acid sequence encoding the antibody or antibody antigen-binding portion thereof according to any one of claims 1 to 15.

24. The nucleic acid sequence of claim 23, comprising a sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 5, or SEQ ID NO: 13 and SEQ ID NO: 15, or SEQ ID NO: 23 and SEQ ID NO: 25, or SEQ ID NO: 33 and SEQ ID NO: 35, or SEQ ID NO: 43 and SEQ ID NO: 45, or SEQ ID NO: 53 and SEQ ID NO: 55, or SEQ ID NO: 63 and SEQ ID NO: 65, or SEQ ID NO: 73 and SEQ ID NO: 75, or SEQ ID NO: 83 and SEQ ID NO: 85, or SEQ ID NO: 93 and SEQ ID NO: 95, or SEQ ID NO: 103 and SEQ ID NO: 105, or SEQ ID NO: 113 and SEQ ID NO: 115, or SEQ ID NO: 123 and SEQ ID NO: 125, or SEQ ID NO: 134 and SEQ ID NO:

136. NO: 133 and SEQ ID NO: 135, or SEQ ID NO: 143 and SEQ ID NO: 145, or SEQ ID NO: 153 and SEQ ID NO: 155, or SEQ ID NO: 163 and SEQ ID NO: 165, or SEQ ID NO: 173 and SEQ ID NO: 175, or SEQ ID NO: 183 and SEQ ID NO: 185, or SEQ ID NO: 193 and SEQ ID NO: 195, or SEQ ID NO: 203 and SEQ ID NO: 205, or SEQ ID NO: 213 and SEQ ID NO: 215, or SEQ ID NO: 223 and SEQ ID NO: 225, or SEQ ID NO: 233 and SEQ ID NO: 235, or SEQ ID NO: 243 and SEQ ID NO: 245, or SEQ ID NO: 253 and SEQ ID NO: 255, or SEQ ID NO: 264 and SEQ ID NO: 266 NO: 263 and SEQ ID NO: 265, or SEQ ID NO: 273 and SEQ ID NO: 275, or SEQ ID NO: 283 and SEQ ID NO: 285, or a sequence with at least 75% sequence identity to any of the foregoing sequences.

25. A vector comprising the nucleic acid sequence according to any one of claims 23 to 24.

26. A host cell comprising the nucleic acid sequence according to any one of claims 23 to 24 or the vector according to claim 25.

27. A kit comprising the antibody or antigen-binding portion thereof according to any one of claims 1 to 15 or the pharmaceutical composition according to claim 16.

28. The kit of claim 27, further comprising reagents for detecting the antibody or antigen-binding portion thereof.

29. A method for preparing a canine antibody that binds to PD-1, the method comprising culturing the isolated host cell of claim 26 and recovering the antibody.

30. A method for preparing a canine antibody that binds to PD-1, the method comprising the steps of: a) immunizing a transgenic mouse expressing a nucleic acid construct comprising a canine heavy chain V gene and a canine light chain V gene with a PD-1 antigen, b) generating an antibody library from said mouse, and c) isolating antibodies from said library.

31. A method for detecting a PD-1 protein or an extracellular domain of a PD-1 protein in a biological sample from a canine subject, the method comprising contacting the biological sample with the antibody, or antigen-binding portion thereof, according to any one of claims 1 to 15, wherein the antibody, or antigen-binding portion thereof, is linked to a detectable label.

32. The method of claim 31, wherein the biological sample is a biopsy sample, a tissue sample, a blood sample, a serum sample, a plasma sample, or a lymph fluid sample.

33. A method of inhibiting tumor growth or metastasis, the method comprising contacting tumor cells with an effective amount of the antibody or antigen-binding portion thereof according to any one of claims 1 to 15 or the pharmaceutical composition according to claim 16.

34. A method for killing tumor cells expressing PD-1, the method comprising contacting the cells with the antibody according to any one of claims 1 to 15 or the pharmaceutical composition according to claim 16, such that killing of the cells expressing PD-1 occurs.

35. The method of claim 34, wherein the tumor cells are canine tumor cells.

36. A binding agent comprising the antibody, or antigen binding portion thereof, of any one of claims 1 to 15, wherein the antibody, or antigen binding portion thereof, is linked to a second antibody, or antigen binding portion thereof, that binds to a second target.

37. The binding agent of claim 36, wherein the second target binding agent is selected from any one of the list comprising LAG-3, OX40, OX40L, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand, CD3, CD8, CD28, CD4 or ICAM-1.

38. An immunoconjugate comprising the antibody or antigen binding portion thereof according to any one of claims 1 to 15 or the binding agent according to claim 36 or 37.

39. A method of modulating an immune response comprising administering the antibody or fragment thereof according to any one of claims 1 to 15, or the pharmaceutical composition according to claim 16, or the binding agent according to any one of claims 36 to 37, or the immunoconjugate according to any one of claims 38 to 39.

40. A combination therapy comprising: The antibody or fragment thereof according to any one of claims 1 to 15; or the pharmaceutical composition of claim 16; or the binding agent of any one of claims 36 to 37; or the immunoconjugate of any one of claims 38 to 39, and an additional therapeutic moiety.

41. The combination therapy of claim 40, wherein the additional therapeutic moiety is an antibody, optionally an antibody that binds an immuno-oncology target; or a chemotherapeutic agent.

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