Treatment antibodies

Abstract

The present disclosure provides isolated canine antibodies or antigen-binding portions thereof that bind to an epitope in the extracellular domain of canine PD-1, compositions comprising same, and methods of their use.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to UK Patent Application No. 2300904.6, filed January 20, 2023, and UK Patent Application No. 2310818.6, filed July 14, 2023, the contents of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy, created on January 17, 2024, is named P44613WO1.xml and is 299.008 bytes in size.

[0003] Overview The present invention relates to antibodies that modulate the PD-1 signaling pathway for the treatment of inflammatory diseases in companion animals.

[0004] The human programming death receptor 1 (PD-1) protein is encoded by the PDCD1 gene and expressed as a 32-kDa 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 an inhibitory member of the extended CD28 / CTLA-4 family of T cell regulatory factors. Other members of this family include CD28, CTLA-4, ICOS, and BTLA. PD-1 exists as a monomer lacking the unpaired cysteine ​​residue characteristic of other CD28 family members (Zhang 2004 Immunity 20:337-47). Its cytoplasmic domain contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based switch motifs (ITSMs) that are phosphorylated during signal transduction (Riley 2009 Immunol Rev 229(1):114-25).

[0005] PD-1 is expressed on B cells, T cells, and monocytes (Agata 1996). In PDCD1- / - mice that develop autoimmune diseases, the role of PD-1 in maintaining immune self-tolerance has been demonstrated (Nishimura 1999 Immunity 11:141-51, Nishimura 2001 Science 291(5502):319-22). Thus, the PD-1 pathway regulates antigen responses and balances autoimmunity and tolerance.

[0006] 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, as well as non-hematopoietic cell lineages (reviewed in Francisco 2010 Immunol Rev 236:219-42). 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 can be upregulated by inflammatory cytokines.

[0007] PD-1 binds to its corresponding ligand, PD-L1, and attenuates T cell responses. Antibodies that bind to PD-1 and block PD-L1 binding are used in human cancer therapy to enhance tumor-specific CD8+ T cell immunity and tumor cell clearance by the immune system. PD-1 is known as an immunoinhibitory protein that negatively regulates TCR signaling. The interaction between PD-1 and PD-L1 can function as an immune checkpoint, resulting in, for example, a reduction in tumor-infiltrating lymphocytes, reduced T cell receptor-mediated proliferation, and / or immune evasion by cancer cells. Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or PD-L2, and the effect is additive when PD-1 interactions with both PD-L1 and PD-L2 are blocked.

[0008] Upon activation and costimulation by antigen-presenting cells (APCs), T cell expression of PD-1 is induced. PD-1 engagement with its ligand on the APC crosslinks PD-1 and clusters it 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 Src-homology-2 domain-containing tyrosine phosphatases (SHP1 / 2), which attenuate various components of T cell receptor (TCR) signaling. T cell activation is suppressed, resulting in reduced cytokine responses, proliferation, and cytolytic activity. This downregulation of T cell function prevents overstimulation and helps tolerize cells to weakly immunogenic self-antigens.

[0009] The PD-1 pathway can be exploited in cancer or infection, allowing tumors or viruses to evade effective immune recognition and causing T cells to exhibit an "exhausted" phenotype. PD-L1 has also been shown to be expressed in many tumor types, including urothelial, ovarian, breast, cervical, colon, pancreatic, gastric, melanoma, glioblastoma, and non-small cell lung cancer (reviewed in 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 unresponsive through PD-1 signaling and therefore are unable to eliminate their targets. T regulatory cells (Tregs) have also been shown to express high levels of PD-1, further suppressing antitumor responses (Lowther 2016 JCI Insight 1(5):85935).

[0010] Disruption of PD-1:PD-L1 interaction enhances T cell activity. Anti-PD-1 monoclonal antibodies have been shown to block the interaction between PD-1 and its ligand (Wang 2014 Cancer Immunol Res 2(9):846-56). T cell function can be enhanced in vitro by PD-1 blockade, 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 in vitro PD-1 blockade using the antibody OPDIVO (nivolumab) and can be resistant to Treg suppression (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), and renal cell carcinoma (RCC). It has shown improved overall survival compared to chemotherapy in NSCLC patients. Another PD-1 blocking antibody, KEYTRUDA® (pembrolizumab), has shown responses in human NSCLC patients refractory to CTLA-4 blockade. Both OPDIVO® and KEYTRUDA® functionally block the interaction of human PD-1 with its ligands.

[0011] Cross-linking a combination of anti-PD-1 and anti-CD3 antibodies on a membrane can induce human PD-1 signaling (Bennett 2003 J Immunol 170:711-18, Keir 2005 J Immunol 175:7372-7379). This function can be detrimental during anti-tumor responses because it suppresses T cell activity. When suppression of T cell responses is desired, agonistic anti-PD-1 antibodies or antibodies with effector function can be used to treat immune-related diseases such as rheumatoid arthritis.

[0012] Antibodies that recognize canine PD-1 are described in WO2015 / 091910, WO2015 / 091911, and WO2015 / 091914. Mice were immunized with canine PD-1 to generate monoclonal antibodies from which CDR sequences were extracted and placed into a canine immunoglobulin scaffold in a process called "caninization."

[0013] Antibody caninization is an artificial process that combines a mouse CDR repertoire with a canine immunoglobulin framework. This process assumes that the mouse CDR sequences will function relatively well in canine sequences when introduced in vivo. That is, it assumes that the antibody specificity of the CDR regions will be maintained for binding when these regions are removed from the naturally occurring antibody molecule. Furthermore, the use of these chimeric antibodies assumes that the presence of mouse CDR sequences in the canine molecule will not otherwise provoke any anti-mouse antibody response in the dog into which the chimeric antibody is introduced. The immunogenicity of antibody therapeutics can result in diminished efficacy over time. This is particularly relevant for chronic diseases that require repeated dosing.

[0014] 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 were evaluated in vitro and used in a pilot study to determine their safety profile and clinical efficacy in spontaneously occurring canine cancers, such as advanced oral melanoma. These antibodies are rat monoclonal antibodies with the heavy and light chain variable regions of 4F12-E6 fused to the constant region of canine IgG type A [see also WO2016 / 006241].

[0015] A successful anti-canine PD1 antibody must have some degree of efficacy in the target population, in that the antibody aids in the elimination and / or reduction of the size of a specific type of tumor. While capable of causing a reduction in tumor size, the antibody must also have an acceptable safety profile. The safety profile involves a balance between the antibody's effectiveness in tumor reduction and the severity of any off-target effects. In addition to the safety profile, the antibody's pharmacokinetic (pK) profile must also allow the antibody to remain in the body long enough to be active. Even if the above criteria are met, the antibody must be able to be manufactured on a large scale at an economically viable cost.

[0016] To the inventors' knowledge, no anti-canine PD1 antibody has been published that has high efficacy in the target population and an acceptable safety profile with any sufficient and robust statistical power. In addition, to date, there are no published pK profiles of fully caninized anti-canine PD1 antibodies in the literature. In addition, there are no published caninized anti-canine PD1 antibodies that can be produced economically on a large scale.

[0017] WO2018 / 189520 and WO2020 / 074874 describe transgenic mouse models for the expression of canine antibody repertoires. Advantageously, this approach allows for the utilization of highly evolved in vivo mechanisms, such as germline hypermutation, to generate high-affinity antibodies with optimal biophysical properties. In response to antigen challenge, canine mice produce high-affinity antibodies with canine-like CDRH3 lengths that exhibit broad epitope coverage. In vivo maturation can yield antibodies with favorable and desirable biophysical properties, such as higher stability and lower aggregation tendency.

[0018] There is a need for improved treatments as well as drugs that treat the underlying cause of disease rather than symptoms, particularly treatments that are safe, have a long duration of action, and are effective in covering a wider range of patients, especially non-responders. Summary of the Invention

[0019] The present invention relates to a method for identifying one of the following epitopes of the extracellular domain of canine PD-1: i) an epitope including T76, Q83, E84, L128, P129, and P130 in SEQ ID NO: 2 ii) An isolated canine antibody or antigen-binding portion thereof that binds to an epitope comprising D58, D61, N74, T76, and Y127 in SEQ ID NO: 2, The invention relates to an isolated canine antibody or antigen-binding portion thereof, wherein the epitope is determined using epitope mapping techniques.

[0020] The present invention also provides an isolated canine antibody, or antigen-binding portion thereof, that binds to the extracellular domain of canine PD-1, wherein the antibody: 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; 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; 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 CDR1 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. a LC CDR3 sequence comprising or consisting of a CDR2 sequence and 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; 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 CDR3 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. a CDR1 sequence and an LC 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;an LC CDR3 sequence comprising or consisting of a CDR2 sequence and 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 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: 209, or an amino acid sequence having at least 70%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 209. 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; an 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 an 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 an HC 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. a CDR1 sequence and an HC 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; a CDR2 sequence and an HC 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;a CDR3 sequence 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 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 an 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; The present invention relates to an isolated canine antibody or antigen-binding portion thereof, comprising:

[0021] The present invention also provides an isolated canine antibody, or antigen-binding portion thereof, that binds to canine PD-1, wherein the antibody: a) 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; 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; 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, 9, 10, 11, 12, 13, or 14 amino acid differences compared to SEQ ID NO: 20; and an LC CDR1 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. a CDR2 sequence and an 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 b) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 207, or an amino acid sequence having one or two amino acid differences compared to SEQ ID NO: 207; an HC CDR2 sequence comprising or consisting of SEQ ID NO: 208, or an amino acid sequence having one, two, three, four, or five amino acid differences compared to SEQ ID NO: 208; an HC CDR3 sequence comprising or consisting of SEQ ID NO: 209, or an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen amino acid differences compared to SEQ ID NO: 209; and an LC CDR1 sequence comprising or consisting of SEQ ID NO: 210, or an amino acid sequence having one, two, or three amino acid differences compared to SEQ ID NO: 210; and an LC CDR2 sequence comprising or consisting of SEQ ID NO: 211, or an amino acid sequence having one or two amino acid differences compared to SEQ ID NO: 211; and an LC CDR2 sequence comprising or consisting of SEQ ID NO: 212, or an amino acid sequence having one, two, three, or four amino acid differences compared to SEQ ID NO: 212. CDR3 sequence, or c) an 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; an 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; an 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 an 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 an LC CDR1 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. a CDR2 sequence and an 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; The present invention relates to an isolated canine antibody or antigen-binding portion thereof, comprising:

[0022] The present invention also provides an antibody or antigen-binding portion thereof, the antibody or antigen-binding portion thereof comprising: i) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 17, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 18, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 19, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 20, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 21, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 22; ii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 27, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 28, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 29, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 30, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 31, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 32. iii) an HC CDR1 sequence comprising or consisting of SEQ ID NO:7, an HC CDR2 sequence comprising or consisting of SEQ ID NO:8, an HC CDR3 sequence comprising or consisting of SEQ ID NO:9, an LC CDR1 sequence comprising or consisting of SEQ ID NO:10, an LC CDR2 sequence comprising or consisting of SEQ ID NO:11, and an LC CDR3 sequence comprising or consisting of SEQ ID NO:12; iv) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 37, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 38, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 39, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 40, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 41, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 42. v) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 47, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 48, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 49, an LC CDR1 sequence comprising SEQ ID NO: 50, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 51, and an 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) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 87, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 88, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 89, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 90, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 91, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 92; x) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 97, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 98, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 99, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 100, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 101, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 102; xi) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 107, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 108, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 109, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 110, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 111, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 112; xii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 117, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 118, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 119, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 120, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 12, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 122; xiii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 127, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 128, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 129, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 130, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 131, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 132; xiv) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 137, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 138, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 139, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 140, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 141, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 142; xv) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 147, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 148, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 149, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 150, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 151, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 152; xvi) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 157, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 158, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 159, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 160, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 161, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 162; xvii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 167, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 168, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 169, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 170, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 171, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 172; xviii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 177, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 178, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 179, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 180, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 181, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 182; xix) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 187, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 188, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 189, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 190, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 191, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 192; xx) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 197, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 198, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 199, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 200, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 201, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 202; xxi) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 207, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 208, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 209, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 210, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 211, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 212; xxii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 217, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 218, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 219, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 220, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 221, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 222; xxiii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 227, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 228, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 229, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 230, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 231, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 232; xxiv) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 237, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 238, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 239, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 240, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 241, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 242; xxv) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 247, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 248, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 249, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 250, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 251, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 252; xxvi) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 257, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 258, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 259, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 260, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 261, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 262; xxvii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 267, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 268, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 269, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 270, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 271, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 272; xxviii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 277, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 278, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 279, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 280, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 281, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 282; or xxix) An antibody or antigen-binding portion thereof having an HC CDR1 sequence comprising or consisting of SEQ ID NO: 287, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 288, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 289, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 290, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 291, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 292.

[0023] 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 thereto, 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.

[0024] The present invention also provides an antibody or antigen-binding portion thereof, the antibody or antigen-binding portion thereof comprising: 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 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 thereto, 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 thereto; 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 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 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 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 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 thereto, 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 thereto; 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 thereto, 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 thereto; or 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 thereto, 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 thereto; or

[0025] 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 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 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 thereto, 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 thereto; 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 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 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 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 l) 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 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 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 thereto, 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 thereto; 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 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 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 thereto, 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 thereto; or

[0026] 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 thereto, 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 thereto; 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 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 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 thereto, 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 thereto; 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 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 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 thereto, 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 thereto; 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 thereto, 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 thereto; 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 thereto, 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 thereto; or

[0027] 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 thereto, 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 thereto; 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 thereto, 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 thereto; 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 thereto, 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 thereto; 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 thereto, 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 thereto; 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 thereto, 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 thereto; 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 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 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 thereto, 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 thereto; The present invention relates to an antibody or antigen-binding portion thereof, having the following structure:

[0028] The present invention also relates to pharmaceutical compositions comprising the antibodies or antigen-binding portions thereof described herein.

[0029] The present invention also relates to an antibody or antigen-binding portion described herein, or a pharmaceutical composition described herein, for use in the treatment of a disease.

[0030] 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 described herein, or a pharmaceutical composition described herein.

[0031] In one embodiment, the disease is cancer or a tumor.

[0032] The present invention also relates to nucleic acid sequences encoding the antibodies or antigen-binding portions thereof described herein.

[0033] The present invention also relates to vectors comprising the nucleic acid sequences described herein.

[0034] The present invention also relates to host cells comprising the nucleic acid sequences described herein, or the vectors described herein.

[0035] The present invention also relates to kits comprising an antibody or antigen-binding portion thereof described herein, or a pharmaceutical composition described herein.

[0036] The present invention also relates to a method for producing a canine antibody that binds to PD-1, comprising culturing the isolated host cells described herein and recovering the antibody.

[0037] The present invention also provides a method for producing a canine antibody that binds to PD-1, 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 a library of antibodies from the mice; c) isolating antibodies from the library.

[0038] The present invention also relates to a method for detecting PD-1 protein or the extracellular domain of PD-1 protein in a biological sample from a canine subject, comprising contacting the biological sample with an antibody, or antigen-binding portion thereof, described herein, wherein the antibody, or antigen-binding portion thereof, is linked to a detectable label.

[0039] The present invention also relates to a method of inhibiting tumor growth or metastasis, comprising contacting tumor cells with an effective amount of an antibody or antigen-binding portion thereof described herein, or a pharmaceutical composition described herein.

[0040] The present invention also relates to a method of killing tumor cells that express PD-1, comprising contacting the cells with an antibody described herein or a pharmaceutical composition described herein, such that killing of the PD-1-expressing cells occurs.

[0041] The present invention also relates to a binding agent comprising an antibody or antigen-binding portion thereof 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.

[0042] The present invention also relates to immunoconjugates comprising an antibody or antigen-binding portion thereof described herein, or a binding agent described herein.

[0043] The present invention also relates to a method of modulating an immune response, comprising administering an antibody or fragment thereof described herein, or a pharmaceutical composition described herein, or a binding agent described herein, or an immunoconjugate described herein.

[0044] The present invention also relates to combination therapies comprising an antibody or fragment thereof described herein, or a pharmaceutical composition described herein, or a binding agent described herein, or an immunoconjugate described herein, and an additional therapeutic moiety.

[0045] The invention is further described in the following non-limiting figures. [Brief explanation of the drawings]

[0046] [Figure 1] Figure 1 shows the fragments of 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 mouse IgG2a, containing the genetic annotation hinge-constant heavy2-constant heavy3-constant heavy secretory (H-CH2-CH3-CHS), and "6His" refers to the hexahistidine tag. [Figure 2] Figure 1 shows the binding intensity of anti-PD-1 antibodies to HEK293 plasma membrane-embedded full-length canine PD-1 protein as measured by flow cytometry. Cells stably transfected with canine PD-1 were incubated with anti-canine PD-1 mAb, followed by detection with an anti-canine IgG FITC secondary antibody. [Figure 3A] Figure 1 shows AlexaFluor 647-labeled PD-L1ECD-mFc binding to PD-1-expressing HEK293 cells previously incubated with 2 μg / mL of anti-PD-1 antibody in a flow cytometry-based assay. [Figure 3B] Figure 1 shows AlexaFluor 647-labeled PD-L2ECD-mFc binding to PD-1-expressing HEK293 cells previously incubated with 2 μg / mL of anti-PD-1 antibody in a flow cytometry-based assay. [Figure 4A] 1 is a table showing SPR-based affinity and kinetics of anti-PD-1 antibodies performed as multi-cycle kinetic experiments in either monovalent (PD-1 analyte) or bivalent (mAb analyte) formats. [Figure 4B]

[0033] Figure 1 shows a table showing the apparent affinities of anti-PD-1 antibodies using a flow cytometry-based affinity assay. Apparent affinities were derived by fitting the data to an "agonist vs. response" four-parameter logistic curve in Prism 8.3.0 (GraphPad) and then reporting the concentration at which the signal is 50% maximal. [Figure 5A] Table showing the percentage of mAb2 binding when mAb1 was pre-bound to saturating conditions using the "parallel" SPR-based method. Values ​​below 15% indicate complete blocking of the pair of mAbs, while values ​​above 75% indicate negligible blocking of mAb2 binding by mAb1. Darkest boxes are -5% to 15%. [Figure 5B] A correlation matrix of the binding profile of each mAb relative to the profiles of all other mAbs is shown. To construct this table, Pearson product-moment correlation coefficients were calculated between the relative binding values ​​of each mAb pair. Pearson product-moment correlation coefficients ≥ 0.97 were highlighted. [Figure 5C] A visual representation of the resulting epitope bins is shown. [Figure 6] Figure 1 shows the binding of anti-PD-1 antibodies to concanavalin A-activated CD5+ canine T cells isolated from healthy dogs. The PBMCFc receptor was blocked with canine gamma globulin, and anti-PD-1 mAb was detected by flow cytometry using anti-canine IgG FITC. [Figure 7] Figure 1 shows a functional in vitro assay of IFN-γ secretion performed using supernatants collected from PBMCs stimulated with concanavalin A in the presence of 10 μg / mL of anti-PD-1 mAb or 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-gamma ELISA. [Figure 8A] Figure 1 shows PD-1 / PD-L1 receptor blockade in a luciferase reporter assay using 3 μg / mL of anti-PD-1 mAb. [Figure 8B]Receptor blockade of selected anti-PD-1 mAbs in a luciferase reporter assay using concentrations ranging from 666 nM to 0.03 nM is shown. IC50 concentrations are shown in nM. Reporter Jurkat cells express chimeric canine (ectodomain) and human (transmembrane and intracellular domain) PD-1 TCRs specific for known MHC-peptide complexes and transcribe luciferase under the control of an NFAT-regulated promoter. Raji antigen-presenting cells then express MHC-peptide complexes bound by Jurkat TCRs and full-length canine PD-L1. In the absence of inhibition, TCR signaling is suppressed by PD-1 signaling, resulting in low levels of luciferase transcription. High levels of PD-1 receptor blockade result in undiminished TCR signaling and high levels of luciferase transcription. [Figure 9A] Structural representations are shown using canine PD-1 and PD-L1 extracellular domain (PD-1 ECD and PD-L1 ECD, respectively) homology models, highlighting in black the residue positions identified in the PMX-126 binding epitope on PD-1. [Figure 9B] Sequence-based representation of the PD-1 extracellular domain with highlighted secondary structure features and residue positions identified in the PMX-126 binding epitope on PD-1 (gray). [Figure 10A] Structural representations are shown using canine PD-1 and PD-L1 extracellular domain (PD-1 ECD and PD-L1 ECD, respectively) homology models, highlighting in black the residue positions identified in the PMX-127 binding epitope on PD-1. [Figure 10B] Sequence-based representation of the PD-1 extracellular domain with highlighted secondary structure features and residue positions identified in the PMX-127 binding epitope on PD-1 (gray). [Figure 11] The sequence listing for full-length canine PD-1 is shown. The predicted extracellular domain (ECD) is highlighted in gray. [Figure 12A]Flow cytometry and histogram plots demonstrating binding of PMX-127 antibody to PD-1 receptor expressed on the cell surface of T cells from lymphoma-bearing dogs (n=2) are shown, pre-gated on live single CD5+ T cells. [Figure 12B] Flow cytometry and histogram plots (n=2) demonstrating binding of the PMX-127 antibody to PD-1 receptors expressed on the cell surface of T cells from dogs with transitional cell carcinoma. Pre-gated on live single CD5+ T cells. [Figure 13] Enhanced 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 of anti-PD-1 mAb or Ritux-def2 isotype control. After 96 hours, supernatants were collected and the amount of canine IFN-γ was measured in triplicate using a canine IFN-gamma ELISA. [Figure 14A] Pharmacokinetic analysis showing linear antibody concentration-time curves of canine anti-PD-1 antibody in healthy beagle dogs following a single intravenous administration of low and high doses (mean ± SD, n=3) of PMX-126. The presence of antibody in plasma was assessed by direct ELISA using purified canine PD-1 Fc-tag protein. [Figure 14B] Pharmacokinetic analysis showing the antibody concentration-time curves of canine anti-PD-1 antibody in healthy beagle dogs after a single intravenous administration of low and high doses of PMX-127 (mean ± SD, n=3). The presence of antibody in plasma was assessed by direct ELISA using purified canine PD-1 Fc-tag protein. [Figure 14C] Table showing the results of non-compartmental analysis of PMX-126 or PMX-127 antibodies demonstrating a similar half-life of 5 days. VD(L): volume of distribution. Cmax: maximum observed plasma concentration. AUC: area under the plasma concentration versus time curve from time 0 to 28 days. [Figure 15]Summary table showing potential adverse events following a single intravenous administration of canine PD-1 antibodies PMX-126 and PMX-127 to laboratory beagle dogs. Changes may be mild, transient, and not specific to antibody treatment. [Figure 16A] 1 shows an SPR-based assay graph demonstrating the lack of cross-reactivity of anti-canine mAbs PMX-126 and PMX-127 to recombinant human PD-1 protein at a concentration of 900 nM. [Figure 16B] The ELISA assay graph shows that PMX-126 and PMX-127 do not cross-react with human PD-1 protein up to a concentration of 100 μg / mL. The anti-human PD-1 mAb pembrolizumab was included as a control to demonstrate that the recombinant human PD-1 protein is active. [Figure 17] FIG. 10 is a receptor occupancy graph demonstrating that the 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 presented as mean±SD. [Figure 18A] Volcano plot showing differentially expressed genes at day 7 after treatment with the canine anti-PD-1 antibody PMX-126 compared to pretreatment in experimental dogs. Highlighted genes met the significance criteria of a log2 fold change >1.5 and a p-value <0.05. Horizontal lines on the plot indicate statistical significance. Thus, differentially expressed (DE) genes with high statistical significance are shown from bottom to top. Highly DE genes are located at the extreme horizontal positions of the plot; therefore, the more to the right, the greater the gene expression at day 7 compared to pretreatment. [Figure 18B]Volcano plot showing differentially expressed genes at day 7 after treatment with the canine anti-PD-1 antibody PMX-127 compared to pretreatment in experimental dogs. Highlighted genes met the significance criteria of a log2 fold change >1.5 and a p-value <0.05. Horizontal lines on the plot indicate statistical significance. Thus, differentially expressed (DE) genes with high statistical significance are shown from bottom to top. Highly DE genes are located at the extreme horizontal positions of the plot; therefore, the more to the right, the greater the gene expression at day 7 compared to pretreatment. DETAILED DESCRIPTION OF THE INVENTION

[0047] Table 1. Examples of amino acid residues and conservative amino acid substitutions Table 2. SEQ ID NOs for the amino acid and nucleotide sequences of each anti-PD-1 antibody included herein.

[0048] Detailed Description Embodiments of the present invention will now be further described. In the following sections, different embodiments are described. Each aspect so defined can be combined with any other aspect or aspects, unless expressly indicated to the contrary.

[0049] Generally, the nomenclature used in connection with cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, and the techniques described herein are well known and commonly used in the art.Unless otherwise indicated, the methods and techniques of the present disclosure are generally carried out according to the conventional methods well known in the art, and are carried out as described in various general and more specific references cited and discussed throughout this specification.For example, see Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., 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 ed., Vols 1 and 2, Ontermann and Dubel, eds., Springer-Verlag, Heidelberg (2010).

[0050] Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, 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.

[0051] The present inventors have developed fully canine antibodies that specifically bind to canine PD-1. These antibodies were generated in transgenic rodents expressing canine V, D, and J genes. Therefore, the antibodies are less likely to be immunogenic upon administration to canine subjects than caninized or chimeric antibodies. Furthermore, because these antibodies can be used directly without further modifications to their variable regions, there is no risk of reducing affinity or otherwise compromising the antibody. Other techniques risk introducing development or efficacy liabilities through the ex vivo combination of canine-derived antibody sequences with antibody sequences from another species, typically a rodent. Therefore, preferably, the present invention relates to an isolated canine antibody, or antigen-binding portion thereof, that binds to canine PD-1.

[0052] 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, as well as isolated nucleic acids, isolated recombinant expression vectors, and isolated host cells for making such binding proteins. Methods of using the antibodies disclosed herein to detect canine PD-1 and methods of treating disease are also provided. In another aspect, the present invention provides binding molecules, including antibodies or antigen-binding portions thereof described herein, 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. The properties of the antibodies and antigen-binding portions thereof of the present invention can be utilized in the therapeutic methods and uses, and pharmaceutical formulations described herein.

[0053] In one embodiment of the present invention, an isolated canine antibody or antigen-binding fragment 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 that has 1, 2, 3, 4, or 5 amino acid differences compared to SEQ ID NO: 17; an 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; an 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 an 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 an LC CDR1 sequence comprising or consisting of SEQ ID NO: 21, or an amino acid sequence that has 1, 2, 3, 4, or 5 amino acid differences compared to SEQ ID NO: 21. a LC CDR3 sequence comprising or consisting of a CDR2 sequence and 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; 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, 10, 11, 12, 13, 14, 15, or 16 amino acid differences compared to SEQ ID NO:209; and a LC CDR3 sequence comprising or consisting of SEQ ID NO:210, or an amino acid sequence having 1, 2, or 3 amino acid differences compared to SEQ ID NO:210. a CDR1 sequence and a LC comprising or consisting of SEQ ID NO: 211, or an amino acid sequence having one or two amino acid differences compared to SEQ ID NO: 211;a LC CDR3 sequence comprising or consisting of a CDR2 sequence and 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; 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 CDR3 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 an 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 an 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.

[0054] In one embodiment of the invention, the amino acid differences are selected from conservative amino acid substitutions and / or non-conservative amino acid substitutions.

[0055] 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, the antibody or antigen-binding fragment thereof comprising: i) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 17, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 18, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 19, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 20, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 21, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 22; ii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 47, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 48, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 49, an LC CDR1 sequence comprising SEQ ID NO: 50, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 51, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 52; iii) an HC CDR1 sequence comprising SEQ ID NO: 57, an HC CDR2 sequence comprising SEQ ID NO: 58, an HC CDR3 sequence comprising SEQ ID NO: 59, an LC CDR2 sequence comprising SEQ ID NO: 60 iv) an HC CDR1 sequence comprising or consisting of SEQ ID NO:37, an HC CDR2 sequence comprising or consisting of SEQ ID NO:38, an HC CDR3 sequence comprising or consisting of SEQ ID NO:39, an LC CDR1 sequence comprising or consisting of SEQ ID NO:40, an LC CDR2 sequence comprising or consisting of SEQ ID NO:41, and an LC CDR3 sequence comprising or consisting of SEQ ID NO:42; v) an HC CDR1 sequence comprising or consisting of SEQ ID NO:87, an HC CDR2 sequence comprising or consisting of SEQ ID NO:88, an HC CDR3 sequence comprising or consisting of SEQ ID NO:89, an LC CDR1 sequence comprising or consisting of SEQ ID NO:90, an LC CDR2 sequence comprising or consisting of SEQ ID NO:91, and an LC CDR3 sequence comprising or consisting of SEQ ID NO:92; vi) an HC CDR1 sequence comprising or consisting of SEQ ID NO:137, an HC CDR2 sequence comprising or consisting of SEQ ID NO:138, an HC CDR2 sequence comprising or consisting of SEQ ID NO:139vii) an LC CDR1 sequence comprising or consisting of SEQ ID NO: 157, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 158, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 159, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 160, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 161, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 162;

[0056] viii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 177, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 178, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 179, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 180, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 181, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 182; ix) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 187, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 188, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 189, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 190, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 191, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 153; x) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 207, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 208, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 209, an LC CDR3 sequence comprising or consisting of SEQ ID NO: 210 1) 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; 2) 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; 3) 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: 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; HC comprising or consisting of CDR1 sequence SEQ ID NO: 238a HC CDR2 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;

[0057] xiv) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 247, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 248, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 249, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 250, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 251, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 252; xv) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 257, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 258, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 259, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 260, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 261, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 262; xvi) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 267, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 268, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 269, an LC CDR3 sequence comprising or consisting of SEQ ID NO: 270 xvii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 117, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 118, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 119, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 120, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 121, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 122; xviii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 127, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 128, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 129, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 130, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 131, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 132; xviiii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 167 HC comprising or consisting of CDR1 sequence SEQ ID NO: 168xx) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 197, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 198, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 199, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 200, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 201, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 202; xxi) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 97, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 98, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 99, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 100, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 101, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 102. CDR3 sequence, xxii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 277, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 278, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 279, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 280, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 281, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 282;

[0058] xxiii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 287, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 288, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 289, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 290, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 291, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 292; xxiv) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 107, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 108, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 109, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 110, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 111, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 112; xxv) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 147, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 148, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 149 151, and LC CDR3 sequence comprising or consisting of SEQ ID NO: 152; xxvi) HC CDR1 sequence comprising or consisting of SEQ ID NO: 7, HC CDR2 sequence comprising or consisting of SEQ ID NO: 8, HC CDR3 sequence comprising or consisting of SEQ ID NO: 9, LC CDR1 sequence comprising or consisting of SEQ ID NO: 10, LC CDR2 sequence comprising or consisting of SEQ ID NO: 11, and LC CDR3 sequence comprising or consisting of SEQ ID NO: 12; xxvii) HC CDR1 sequence comprising or consisting of SEQ ID NO: 27, HC CDR2 sequence comprising or consisting of SEQ ID NO: 28, HC CDR3 sequence comprising or consisting of SEQ ID NO: 29, LC CDR1 sequence comprising or consisting of SEQ ID NO: 30, LC CDR2 sequence comprising or consisting of SEQ ID NO: 31, and LC CDR3 sequence comprising or consisting of SEQ ID NO: 32; xxviii) HC CDR1 sequence comprising or consisting of SEQ ID NO: 67 HC comprising or consisting of CDR1 sequence SEQ ID NO: 68xxix) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 77, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 78, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 79, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 80, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 81, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 82.

[0059] 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 to the CDRs as set forth in i) to xxviii) above.

[0060] In one 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 thereto, 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.

[0061] In another related embodiment of the invention, 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) SEQ ID NO: 44, or a sequence having at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto. % sequence identity thereto, or c) 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 thereto, and an LC 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 an LC 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, or or d) an LC 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 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 thereto; or e) 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 thereto. f) 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 thereto, 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 thereto;or 90% sequence identity thereto; or g) an LC variable region sequence comprising SEQ ID NO: 154, 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: 156, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or h) 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 thereto, 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 thereto;

[0062] or i) 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 thereto, 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 thereto; or j) 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 thereto; and and SEQ ID NO: 206, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or k) 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 thereto, 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 thereto; or l) a sequence m) 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 thereto, 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 thereto; or m) 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 thereto, and an LC variable region sequence comprising SEQ ID NO: 23 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or n) 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 thereto; 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 thereto; or o) SEQ ID NO: 254,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: 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 sequence 266, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or q) an 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; and an 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;

[0063] or r) 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 thereto, and an 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) an HC variable region sequence comprising SEQ ID NO: 164, or a sequence having at least 45%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto, and a LC variable region sequence comprising SEQ ID NO: 127, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto. or t) an LC 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: 196 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or u) a sequence or v) 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 thereto, and an 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) 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 thereto, and SEQ ID NO: 276. or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or w) 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 x) 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 thereto; and z) an 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 thereto; or z) 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 z) 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;

[0064] or aa) 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 thereto, 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 thereto; or bb) an 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. or cc) 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 thereto, and an 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; The present invention provides an antibody or antigen-binding portion thereof that binds to canine PD-1, having the formula:

[0065] The antibody or antigen-binding portion thereof according to the present invention binds to one of the following epitopes of the extracellular domain of canine PD-1: i) T76, Q83, E84, L128, P129, P130, or ii) capable of binding to D58, D61, N74, T76, Y127; The epitope was determined using epitope mapping techniques. 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 shows the residue numbering of canine PD-1.

[0066] The antibody or antigen-binding portion thereof according to the present invention binds to one of the following epitopes of the extracellular domain of canine PD-1: i) T76, Q83, E84, L128, P129, and P130 in SEQ ID NO: 2 ii) capable of binding to D58, D61, N74, T76, and Y127 in SEQ ID NO: 2; The epitope is determined using epitope mapping techniques.

[0067] Epitope mapping techniques that can be used to determine whether an antibody binds to a particular epitope include site-directed mutagenesis epitope mapping, shotgun mutagenesis epitope mapping, oligopeptide scanning (e.g., pepscan analysis), hydrogen-deuterium exchange, optionally combined with mass spectrometry, cross-linking mass spectrometry, cross-linking mass spectrometry, X-ray crystallography, electron microscopy, NMR analysis, in silico methods using, for example, computer-generated structure prediction, or a combination of the aforementioned techniques. In one 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 technique described in Example 17 herein.

[0068] 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 in the extracellular domain of canine PD-1: i) T76, Q83, E84, L128, P129, and P130 in SEQ ID NO: 2 ii) binds to D58, D61, N74, T76, and Y127 in SEQ ID NO: 2; epitopes are determined using epitope mapping techniques; 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; 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; 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. a LC CDR3 sequence comprising or consisting of a CDR2 sequence and 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; 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 CDR3 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. a CDR1 sequence and an LC 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;an LC CDR3 sequence comprising or consisting of a CDR2 sequence and 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 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: 209, or an amino acid sequence having at least 70%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 209. 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; an 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 an 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 an HC 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. a CDR1 sequence and an HC 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; a CDR2 sequence and an HC 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;a CDR3 sequence 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 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 an 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; Includes:

[0069] 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 in the extracellular domain of canine PD-1: i) T76, Q83, E84, L128, P129, and P130 in SEQ ID NO: 2 ii) binds to D58, D61, N74, T76, and Y127 in SEQ ID NO: 2; epitopes are determined using epitope mapping techniques; 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; 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; 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, 9, 10, 11, 12, 13, or 14 amino acid differences compared to SEQ ID NO: 20; and an LC CDR1 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. a LC CDR3 sequence comprising or consisting of a CDR2 sequence and 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; 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, 10, 11, 12, 13, 14, 15, or 16 amino acid differences compared to SEQ ID NO:209; and a LC CDR3 sequence comprising or consisting of SEQ ID NO:210, or an amino acid sequence having 1, 2, or 3 amino acid differences compared to SEQ ID NO:210. a CDR1 sequence and a LC comprising or consisting of SEQ ID NO: 211, or an amino acid sequence having one or two amino acid differences compared to SEQ ID NO: 211;a LC CDR3 sequence comprising or consisting of a CDR2 sequence and 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; 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 CDR3 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. a CDR1 sequence and an 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 an 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; Includes:

[0070] In another related embodiment of the 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 in the extracellular domain of canine PD-1: i) T76, Q83, E84, L128, P129, and P130 in SEQ ID NO: 2 ii) binds to D58, D61, N74, T76, and Y127 in SEQ ID NO: 2; epitopes are determined using epitope mapping techniques; The antibody or antigen-binding fragment thereof is selected from the group consisting of: i) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 17, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 18, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 19, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 20, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 21, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 22; ii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 47, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 48, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 49, an LC CDR1 sequence comprising SEQ ID NO: 50, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 51, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 52; iii) an HC CDR1 sequence comprising SEQ ID NO: 57, an HC CDR2 sequence comprising SEQ ID NO: 58, an HC CDR3 sequence comprising SEQ ID NO: 59, an LC CDR1 sequence comprising SEQ ID NO: 60, an LC CDR2 sequence comprising SEQ ID NO: 61, and an LC CDR3 sequence comprising SEQ ID NO: 62. CDR3 sequences, iv) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 37, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 38, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 39, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 40, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 41, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 42, v) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 87, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 88, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 89, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 90, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 91, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 92, vi) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 137, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 138, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 139, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 141, CDR1 sequence, LC comprising or consisting of SEQ ID NO: 142vii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 157, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 158, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 159, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 160, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 161, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 162;

[0071] viii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 177, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 178, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 179, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 180, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 181, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 182; ix) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 187, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 188, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 189, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 190, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 191, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 153; x) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 207, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 208, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 209, an LC CDR3 sequence comprising or consisting of SEQ ID NO: 210 1) 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; 2) 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; 3) 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: 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; HC comprising or consisting of CDR1 sequence SEQ ID NO: 238xiv) 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; xv) 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 CDR1 sequence comprising or consisting of SEQ ID NO: 261 xvi) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 267, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 268, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 269, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 270, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 271, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 271;

[0072] xvii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 117, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 118, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 119, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 120, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 121, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 122; xviii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 127, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 128, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 129, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 130, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 131, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 132; xviiii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 167, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 168, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 169 xx) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 197, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 198, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 199, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 200, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 201, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 202; xxi) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 97, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 98, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 99, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 100, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 101, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 102. CDR3 sequence, xxii) HC comprising or consisting of SEQ ID NO: 277 CDR1 sequence, HC comprising or consisting of SEQ ID NO: 278xxiii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 287, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 288, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 289, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 290, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 291, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 292; xxiv) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 107, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 108, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 109, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 110, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 111 xxv) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 147, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 148, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 149, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 150, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 151, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 152;

[0073] xxvi) an HC CDR1 sequence comprising or consisting of SEQ ID NO:7, an HC CDR2 sequence comprising or consisting of SEQ ID NO:8, an HC CDR3 sequence comprising or consisting of SEQ ID NO:9, an LC CDR1 sequence comprising or consisting of SEQ ID NO:10, an LC CDR2 sequence comprising or consisting of SEQ ID NO:11, and an LC CDR3 sequence comprising or consisting of SEQ ID NO:12; xxvii) an HC CDR1 sequence comprising or consisting of SEQ ID NO:27, an HC CDR2 sequence comprising or consisting of SEQ ID NO:28, an HC CDR3 sequence comprising or consisting of SEQ ID NO:29, an LC CDR1 sequence comprising or consisting of SEQ ID NO:30, an LC CDR2 sequence comprising or consisting of SEQ ID NO:31, and an LC CDR3 sequence comprising or consisting of SEQ ID NO:32; xxviii) an HC CDR1 sequence comprising or consisting of SEQ ID NO:67, an HC CDR2 sequence comprising or consisting of SEQ ID NO:68, an HC CDR3 sequence comprising or consisting of SEQ ID NO:69, an LC CDR1 sequence comprising or consisting of SEQ ID NO:70, an LC CDR3 sequence comprising or consisting of SEQ ID NO:71; xxix) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 77, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 78, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 79, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 80, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 81, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 82.

[0074] In one embodiment of the present invention, an antibody or antigen-binding portion thereof that binds to one of the following epitopes of the extracellular domain of canine PD-1: i) T76, Q83, E84, L128, P129, and P130 in SEQ ID NO: 2 ii) D58, D61, N74, T76, Y127 in SEQ ID NO: 2; Epitopes are determined using epitope mapping techniques and include 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 thereto, 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, for example, an LC variable region sequence comprising SEQ ID NO: 6.

[0075] In another related embodiment of the invention, an 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 including T76, Q83, E84, L128, P129, and P130 in SEQ ID NO: 2 ii) an epitope comprising D58, D61, N74, T76, and Y127 in SEQ ID NO: 2; Epitopes were determined using epitope mapping techniques and identified as: 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 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 thereto; or b) an LC 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. or c) 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 thereto, and an LC 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 an LC 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 an LC 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, or d) an LC 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 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 thereto; or e) 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 90% sequence identity thereto; or g) an LC variable region sequence comprising SEQ ID NO: 154, 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: 156, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or h) SEQ ID NO: 174, or an HC variable region sequence comprising 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: 176 or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or i) SEQ ID NO: 184 or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto. or j) an HC variable region sequence comprising a sequence having SEQ ID NO: 204 or a sequence having at least 40%, 45%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto, 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 thereto; k) an LC 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, 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 thereto;

[0076] or l) 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 thereto, 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 thereto; or m) 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 thereto; and and SEQ ID NO: 236, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or n) 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 thereto; 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 thereto; or o) a sequence or p) 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 thereto, 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 thereto; or p) 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 thereto, and SEQ ID NO: 26 6, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or q) an 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; and an 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) 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 thereto, and a LC variable region sequence comprising SEQ ID NO: 165, or a sequence having at least 45%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto, and a 166, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; 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 thereto, and an 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 thereto;

[0077] or u) 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 thereto, and an 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) 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 thereto; and or x) 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 x) 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; or y) an 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 an 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) 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 thereto, and an LC variable region sequence comprising SEQ ID NO: 145, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or z) an LC 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 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 thereto; or aa) SEQ ID NO: 24,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: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 a LC variable region sequence comprising SEQ ID NO:65, or a sequence having at least 45%, 50%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto. 66, or a sequence having at least 35%, 40%, 70%, 75%, 80%, 85%, or 90% sequence identity thereto; or cc) 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 thereto; and an 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; The present invention provides a method for manufacturing a semiconductor device, comprising:

[0078] In one embodiment, the antibody, or antigen-binding portion thereof, binds to an epitope in the extracellular domain of canine PD-1 extracellular domain comprising D58, D61, N74, T76, Y127 set forth in SEQ ID NO: 2, and 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 CDR3 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, and an 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 thereof 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 thereto, 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 thereto.

[0079] In one embodiment, the antibody, or antigen-binding portion thereof, binds to an epitope in the extracellular domain of canine PD-1 extracellular domain comprising D58, D61, N74, T76, Y127 set forth in SEQ ID NO: 2, and the antibody comprises 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, an 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 an 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 an HC CDR4 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 an 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 an 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.

[0080] In one embodiment, the antibody or antigen-binding portion thereof binds to an epitope in the extracellular domain of canine PD-1 extracellular domain comprising T76, Q83, E84, L128, P129, P130 set forth in SEQ ID NO:2, and the antibody or antigen-binding portion thereof comprises 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 an 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 an 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 an 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 an 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 an 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 thereof 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 thereto, 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 thereto.

[0081] In one embodiment, the antibody or antigen-binding portion thereof binds to an epitope in the extracellular domain of canine PD-1 extracellular domain comprising T76, Q83, E84, L128, P129, P130 set forth in SEQ ID NO: 2, and the antibody or antigen-binding portion thereof comprises an HC CDR1 sequence comprising or consisting of SEQ ID NO: 27, or an amino acid sequence which has 1, 2, 3, 4, or 5 amino acid differences compared to SEQ ID NO: 27, and an HC CDR2 sequence comprising or consisting of SEQ ID NO: 28, or an amino acid sequence which has 1, 2, 3, 4, 5, or 6 amino acid differences compared to SEQ ID NO: 28, and an HC CDR3 sequence comprising or consisting of SEQ ID NO: 29, or an amino acid sequence which has 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid differences compared to SEQ ID NO: 29, and an HC CDR4 sequence comprising or consisting of SEQ ID NO: 30, or an amino acid sequence which has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acid differences compared to SEQ ID NO: 30. and an 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 differences compared to SEQ ID NO:31, and an 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 an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid differences compared to SEQ ID NO:32.

[0082] The term PD-1 refers to a protein also known as CD279 (cluster of differentiation 279), which is a protein on the surface of T cells and B cells. The antibody and its antigen-binding portion 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 specified, the term PD-1 as used herein refers to canine PD-1 or the T cell and B cell antigen PD-1. In humans and dogs, PD-1 is encoded by the PDCD1 gene.

[0083] Unless otherwise specified, the term PD-1 as used herein 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.

[0084] The terms "PD-1 binding molecule / protein / polypeptide / agent / moiety," "PD-1 antigen-binding molecule / protein / polypeptide / agent / moiety," "anti-PD-1 antibody," and "anti-PD-1 antibody or antigen-binding portion thereof" all refer to a molecule capable of specifically binding to canine PD-1 antigen. Binding can be demonstrated by standard methods, for example, by reference to a negative control test using an antibody of irrelevant specificity. The term "PD-1 binding molecule / agent" includes PD-1 binding proteins.

[0085] Antibodies or antigen-binding portions thereof, including multispecific, e.g., bispecific or trispecific, binding agents described herein that "bind" or "are capable of binding" to an antigen of interest, i.e., canine PD-1, are antibodies or antigen-binding portions thereof that bind to the antigen with sufficient affinity such that they are useful as therapeutic agents in targeting cells or tissues that express the antigen PD-1 described herein.

[0086] Binding molecules of the invention, including the antibodies and multivalent or multispecific binding agents described herein, specifically bind to canine PD-1. In other words, binding to the PD-1 antigen is measurably different from nonspecific interactions. As shown in the Examples, antibodies of the invention do not cross-react with mouse PD-1. Furthermore, as shown in the Examples, antibodies of the invention do not cross-react with human PD-1. Preferably, antibodies of the invention bind to canine PD-1.

[0087] As used herein, the terms "specific binding" or "specifically binds" or "specific for" a particular polypeptide or epitope on a particular polypeptide target means, for example, 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 -13 In one embodiment, the term "specific binding" as used herein refers to a molecule having a KD of at least about 10 M or less for a 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 M, or lower. In one embodiment, the term "specific binding" refers to at least single- or double-digit nanomolar or picomolar binding. In one embodiment, the term "specific binding" refers to binding in which a molecule binds to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes. In one embodiment, the KD of a binding molecule of the invention for a target is determined using monovalent and / or bivalent binding assays, such as the monovalent and bivalent binding assays described in Example 9. In one embodiment, the term "specific binding" as used herein refers to a binding of at least about 10 to a target as determined by a bivalent binding assay. -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 -12M, alternatively at least about 10 -13 A binding molecule can be represented by a molecule having a KD of about 10 M or less as determined by a bivalent binding assay. -8 M~10 -13 M, 10 -9 M~10 -13 M, 10 -10 M~10 -13 M, 10 -11 M~10 -13 M, 10 -12 M~10 -13 In one embodiment, the binding molecule may have a KD for the target of about 10 as determined by a bivalent binding assay. -10 M~10 -13 M. In one embodiment, the term "specific binding," as used herein, refers to a binding affinity for a target of at least about 10, as determined by monovalent binding assays. -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 A binding molecule can be represented by a molecule having a KD of about 10 M or less as determined by a monovalent binding assay. -8 M~10 -13 M, 10 -9 M~10 -13 M, 10 -10 M~10 -13 M, 10 -11 M~10 -13 M, 10 -12 M~10 -13 In one embodiment, the binding molecule may have a KD for the target of about 10 as determined by a monovalent binding assay. -8 M~10 -11 It may have a KD of the target of M.

[0088] The term "antibody," as used herein, refers broadly to any immunoglobulin (Ig) molecule, or antigen-binding portion thereof, composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof that retains the essential epitope-binding characteristics of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art.

[0089] 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 is composed of 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 is composed of one domain, CL.

[0090] The heavy and light chain variable regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each heavy and light chain variable region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0091] Immunoglobulin molecules generally can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgAI, and IgA2), or subclass.

[0092] In dogs, there are four IgG heavy chains, designated A, B, C, and D. These heavy chains represent four different subclasses of canine IgG, designated IgG-A, IgG-B, IgG-C, and IgG-D. The DNA and amino acid sequences of these four heavy chains were first identified by Tang et al. (Vet. Immunol. Immunopathol. 80:259-270 (2001)). The amino acid and DNA sequences for these heavy chains are also available from the GenBank database (IgGA: accession number AAL35301.1, IgGB: accession number AAL35302.1, IgGC: accession number AAL35303.1, IgGD: accession number AAL35304.1). Canine antibodies also contain two types of light chains, kappa and lambda (GenBank Accession No. kappa light chain amino acid sequence ABY57289.1, GenBank Accession No. ABY55569.1). The antibodies herein may have lambda or kappa light chains. In one embodiment, the light chain is a lambda light chain.

[0093] The term "CDR" refers to the complementarity determining region in antibody variable sequence. In each of the variable regions of heavy and light chains, there are three CDRs, designated CDR1, CDR2, and CDR3. The term "CDR set" refers to a group of three CDRs that occur in a single variable region that can bind to antigen. The exact boundaries of these CDRs can be defined differently according to different systems known in the art.

[0094] The Kabat complementarity-determining regions (CDRs) are based on sequence variability and are 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, USDapartment of Health and Human Services, NIH Publication No. 91-3242). Chothia instead refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The Kabat numbering system is generally used to refer to residues within the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain). 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 specified, the IMGT numbering scheme is used herein.

[0095] In another embodiment, the invention relates to an isolated monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region that is the product of or derived from the canine IGHV3-5VH gene, the canine IGHV3-19VH gene, or the canine IGHV4-1VH gene, wherein the antibody specifically binds to canine PD-1. In another embodiment, the invention relates to an isolated monoclonal antibody, or antigen-binding portion thereof, comprising a light chain variable region that is the product of or derived from the canine IGKV2-9VK gene, the canine IGKV2-5VK gene, the canine IGLV3-3VL gene, or the canine IGLV3-11VL gene, wherein the antibody specifically binds to canine PD-1.

[0096] Antibodies against PD-1 according to the present invention may be canine, humanized, chimeric, feline, or caninized antibodies.

[0097] Chimeric antibodies are recombinant proteins that contain variable domains comprising 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.

[0098] As used herein, the term "caninized antibody" refers to a form of recombinant antibody containing sequences from both canine and non-canine (e.g., murine) antibodies. Generally, caninized antibodies contain substantially all of at least one or more, typically two, variable domains, with all or substantially all of the hypervariable loops corresponding 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 framework) from canine immunoglobulin sequences. Caninized antibodies may contain both three heavy chain CDRs and three light chain CDRS from a mouse or human antibody, together with a canine framework or a modified canine framework. The modified canine framework may contain one or more amino acid changes that can further optimize the effectiveness of the caninized antibody, for example, to increase binding to its target. For example, the non-canine sequence of the hypervariable loop may be further compared to the canine sequence, and as many residues as possible may be changed to resemble the authentic canine sequence.

[0099] As used herein, the term "felineized antibody" refers to a form of recombinant antibody containing sequences from both feline and non-feline (e.g., canine) antibodies. Generally, felineized antibodies contain substantially all of at least one or more, typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-felineized immunoglobulin, and all or substantially all of the framework (FR) regions (and typically all or substantially all of the remaining framework) from a felineized immunoglobulin sequence. A felineized antibody may contain both three heavy chain CDRs and three light chain CDRS from a mouse or human antibody, along with a feline or modified feline framework. The modified feline framework may contain one or more amino acid changes that can further optimize the efficacy of the felineized antibody, for example, to increase binding to its target. For example, the non-feline sequence of a hypervariable loop may be further compared to the feline sequence, and as many residues as possible may be changed to resemble the authentic feline sequence.

[0100] A "speciesized" antibody (e.g., humanized, caninized, chimeric, feline) is one that has been engineered to resemble an antibody of the target species. In one embodiment, a "speciesized" antibody is more than about 80%, 85%, or 90% similar to an antibody of the target species.

[0101] In contrast, the complete canine antibodies of the present invention have canine variable regions and do not contain complete or partial CDRs or FRs from other species. Advantageously, the complete canine antibodies described herein are obtained from transgenic mice containing canine immunoglobulin sequences. Antibodies produced in these immunized mice are developed through in vivo B cell signaling and development to allow natural affinity maturation, including in vivo V(D)J recombination, in vivo junctional diversification, in vivo pairing of heavy and light chains, and in vivo hypermutation. Complete canine antibodies produced in this manner generate antibodies with optimal properties for potential development, minimizing lengthy lead optimization before large-scale production. Advantageously, such complete canine antibodies present the lowest possible risk of immunogenicity when introduced into patient animals, thereby facilitating repeated dosing regimes. Considering that ex vivo mAb engineering runs the risk of introducing developmental liability, immunogenicity, and reduced affinity (as outlined above), the fully canine antibodies of the present invention are therefore most likely to be an effective therapy in a clinical context.

[0102] In one embodiment, the antibody or antibody fragment is a canine antibody or antibody fragment, hi one embodiment, the antibody is a fully canine antibody.

[0103] 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 that cross-reacts with canine PD-1 is felinized. The antibody or antibody fragment that cross-reacts with feline PD-1 may have any of the characteristics described herein. In one embodiment, the antibody or antibody fragment cross-reacting 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, 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, 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 HC CDR3 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 an 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 an 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.

[0104] The term "antigen-binding site" refers to the portion of an antibody or antibody fragment that comprises the area that specifically binds to an antigen. An antigen-binding site may be provided by one or more antibody variable domains. Preferably, the antigen-binding site is contained within the relevant VH and VL of an antibody or antibody fragment.

[0105] As used herein, the terms antigen-binding portion or antigen-binding fragment or portion or fragment of an antibody are used interchangeably. In one embodiment of the present invention, there is provided an antibody or antigen-binding portion thereof that binds to canine PD-1, wherein the antigen-binding portion thereof is an scFv, Fv, heavy chain, or single domain antibody, e.g., a VH single domain antibody.

[0106] 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. Thus, recombinant functional antibody fragments, such as Fab (fragment antibody), scFv (single-chain variable fragment), and single-domain antibodies (dAb), have been used to develop therapeutic agents as alternatives to mAb-based therapeutic agents.

[0107] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-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 results in six hypervariable loops (three loops each from the H and L chains) that contribute 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 containing only three HVRs specific for an antigen) has the ability to recognize and bind to an antigen, albeit with lower affinity than the entire binding site. A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing the VH and VL antibody domains connected in a single polypeptide chain.

[0108] The scFv fragment (approximately 25 kDa) consists of two variable domains, VH and VL. Naturally, the VH and VL domains tend to associate and dissociate non-covalently through hydrophobic interactions. However, stable fragments can be engineered by linking the domains with a hydrophilic flexible linker to create a single-chain Fv (scFv).

[0109] The smallest antigen-binding fragment is a single variable fragment, i.e., a variable heavy (VH) or variable light (VL) chain domain. The VH and VL domains are each capable of binding to an antigen. Binding to the respective light / heavy chain partners, or indeed the presence of other portions of the complete antibody, is not required for target binding. Antigen-binding entities of antibodies reduced in size to one single domain (corresponding to a VH or VL domain) are commonly referred to as "single-domain antibodies" or "immunoglobulin single variable domains." Thus, single-domain antibodies (approximately 12-15 kDa) have either a VH or a VL domain.

[0110] In one aspect, the invention relates to an isolated single domain antibody, isolated variable single domain, or 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 and PD-L1 and / or PD-L2.

[0111] 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, 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. Accordingly, canine heavy chain single variable domain antibodies are within the scope of the present invention.

[0112] 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, which does not comprise a VL domain.

[0113] The term "isolated" single domain antibody refers to a single domain antibody that is substantially free from other single domain antibodies, antibodies or antibody fragments having different antigen specificities. Furthermore, an isolated single domain antibody may be substantially free of other cellular material and / or chemicals.

[0114] In one embodiment of the present invention, an isolated canine antibody, or antigen-binding portion thereof, is provided that binds to PD-1, wherein the antibody, or antigen-binding portion thereof, competes with PD-L1 and / or PD-L2.

[0115] In related embodiments of the 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 cellular responses associated with the interaction of PD-1 with PD-L1 and / or PD-L2.

[0116] As used herein, a "blocking antibody or antibodies" or a "neutralizing antibody or antibodies" refers to an antibody whose binding to PD-1 results in inhibition of at least one biological activity of PD-1. For example, an antibody of the invention may prevent or block PD-1 binding to PD-L1 and / or PD-L2. In one embodiment, an antibody of the invention blocks PD-1 binding to PD-L1. In one embodiment, an antibody of the invention blocks PD-1 binding to PD-L2.

[0117] Each single VH domain antibody comprises three CDRs and four FRs arranged from amino-terminus to carboxy-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 (VH) domain having the following formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0118] Modifications to the C- or N-terminal VH framework sequences may be made to antibodies of the invention to improve their properties, for example, the VH domain may contain a C-terminal extension or an N-terminal deletion.

[0119] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation, carbohydrate addition) that may be present in minor amounts. Such monoclonal antibodies may be derived from a single B cell or plasma cell. Monoclonal antibodies are highly specific and are directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma cultures, uncontaminated by other immunoglobulins.

[0120] The term "antigen-binding site" refers to the portion of an antibody or antibody fragment that comprises the area that specifically binds to an antigen. An antigen-binding site can be provided by one or more antibody variable domains. An antigen-binding site is typically contained within the associated VH and VL of an antibody or antibody fragment.

[0121] The term "epitope" or "antigenic determinant" refers to a site on the surface of an antigen to which an immunoglobulin, antibody, or antibody fragment specifically binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. This term specifically includes linear and conformational epitopes. Epitopes within a protein antigen can be formed from both contiguous amino acids (usually linear epitopes) or non-contiguous amino acids juxtaposed by tertiary folding of the protein (usually conformational epitopes). Epitopes formed from contiguous amino acids are typically, but not always, retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain 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 the epitope bound by a given antibody or antibody fragment (i.e., epitope mapping) are well known in the art and include, for example, immunoblot and immunoprecipitation assays, in which overlapping or adjacent peptides are tested for reactivity with a given antibody or antibody fragment.

[0122] An antibody binds to "essentially the same epitope" as a reference antibody when the two antibodies recognize the same or sterically overlapping epitopes. The most widely used and rapid method for determining whether two epitopes bind to the same or sterically overlapping epitopes is a competitive assay, which can be configured in different formats using either labeled antigen or labeled antibody. The epitope may or may not be a three-dimensional surface feature of the antigen. In some embodiments, the antibody may bind to, for example, a monomer / single subunit and block the formation of an active form. Preferably, the antibody binds to the extracellular domain of PD-1.

[0123] Proteolytic digestion of antibodies releases different fragments called Fv (Fragment variable), Fab (Fragment antigen binding), and Fc (Fragment crystallization). The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The constant domains of the Fc fragment are responsible for mediating the effector functions of the antibody.

[0124] The present invention extends to antigen-binding portions or antigen-binding fragments of antibodies. The terms "binding portion" and "fragment" are used interchangeably herein. Antibody fragments are portions of antibodies, such as F(ab')2, Fab, Fv, scFv, heavy chain, light chain, variable heavy chain (VH), variable light chain (VL) domains, etc. Functional fragments of full-length antibodies retain the target specificity of the full-length antibody. Thus, recombinant functional antibody fragments, such as Fab (fragment, antibody), scFv (single-chain variable fragment), and single-domain antibodies (dAb), have been used to develop therapeutic agents as alternatives to mAb-based therapeutics.

[0125] The present invention also extends to antibody mimetics comprising the sequences described herein.

[0126] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-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 results in six hypervariable loops (three loops each from the H and L chains) that contribute 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 containing only three HVRs specific for an antigen) has the ability to recognize and bind to an antigen, albeit with lower affinity than the entire binding site.

[0127] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment (scFv fragment) (approximately 25 kDa) consisting of two variable domains, VH and VL, connected to a single polypeptide chain. Naturally, the VH and VL domains tend to associate and dissociate non-covalently via hydrophobic interactions. However, stable fragments can be engineered by linking the domains with a hydrophilic, flexible linker to create a single-chain Fv (scFv).

[0128] The smallest antigen-binding fragment is a single variable fragment, i.e., a variable heavy (VH) or variable light (VL) chain domain. The VH and VL domains are each capable of binding to an antigen. Binding to the respective light / heavy chain partners, or indeed the presence of other portions of an intact antibody, is not required for target binding. Antigen-binding entities of antibodies reduced in size to one single domain (corresponding to a VH or VL domain) are generally referred to as "single-domain antibodies" or "immunoglobulin single variable domains." Thus, single-domain antibodies (approximately 12-15 kDa) have either a VH or VL domain, i.e., they lack the other portions of an intact antibody. The term "dAb," for "domain antibody," generally refers to a single immunoglobulin variable domain (VH, VHH, or VL) polypeptide that specifically binds to an antigen.

[0129] The term "isolated" refers to a moiety that is isolated 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. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0130] 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 of the reference polypeptide to be compared, after aligning the sequences and, in some embodiments, after introducing gaps as necessary to achieve the maximum percentage homology, without considering any conservative substitutions as part of sequence identity.Therefore, the homology percentage between two amino acid sequences is equal to the identity percentage between the two sequences.N-terminal or C-terminal extensions, tags, or insertions should not be interpreted as reducing identity or homology.Methods and computer programs for alignment are well known.The identity percentage between two amino acid sequences can be determined using well-known mathematical algorithms.

[0131] "Amino acid" as used herein refers to one of the 20 naturally occurring amino acids or any non-natural analogues that can be present at a specific defined position. Amino acid encompasses both naturally occurring amino acids and synthetic amino acids. However, in most cases, when proteins are recombinantly produced, only naturally occurring amino acids are used.

[0132] As used herein, "substitution of an amino acid residue" with another amino acid residue in the amino acid sequence of a heterodimeric protein or polypeptide (e.g., an antibody) described herein is equivalent to "replacing an amino acid residue" with another amino acid residue and indicates that a particular amino acid residue at a particular 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 done using standard techniques available to those of skill in the art, such as recombinant DNA technology. Amino acids are altered compared to the native (wild-type / germline) sequence as found in nature in wild-type (wt), but can be generated in IgG molecules that contain other changes compared to the native sequence. "Wild-type" or "WT" or "native" herein refers to 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.

[0133] An antibody or antigen-binding portion thereof according to the invention has one or more of the following properties: a) binds to canine PD-1; b) blocking or reducing the functional interaction of PD-1 with PD-L1 and / or PD-L2; c) shows PD-1 blockade as demonstrated in the examples; d) increasing the secretion of IFN-γ from canine PBMCs, as shown in the Examples; e) binds to canine PD-1 on activated canine T cells, as shown in the Examples; f) capable of binding to cells expressing canine PD-1; g) provides good stability as shown in the examples; h) does not cross-react with human PD-1; i) has a half-life of 1 to 20 days; j) capable of increasing or upregulating T cell activation, and / or k) one or more genes involved in T cell proliferation, activation and / or differentiation may be upregulated, such as IFGGC1, CD3D, ICOS, CXCL10, CD80, CCR5, IL-7 and / or INF-gamma, and / or GZMB, PRF1, CD28, CXCR3, CTLA-4, CD8A, CD8B, TBX21, GATA3, IL2RA, CD40LG and / or IRF1.

[0134] These properties can be measured by methods known in the art, such as those disclosed in the Examples.

[0135] For example, blocking the functional interaction of PD-1 with PD-L1 and / or PD-L2 means that the antibody reduces or abolishes the binding of PD-1 to PD-L1 and / or PD-L2, thereby abolishing or reducing signaling through these pathways.

[0136] The antibodies or antigen-binding portions thereof according to the invention have a preferred half-life. In some embodiments, the antibody or antigen-binding portion thereof exhibits 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, or 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.

[0137] Increased or upregulated T cell activation can be assessed by comparing the level of T cell activation with a reference value. The reference value can be the level of T cell activation from a subject before treatment with the antibody of the present invention. The reference value can be the level of T cell activation from a healthy subject. The reference value can be the level of T cell activation from a subject with a disease. The subject with a disease may or may not be receiving therapy.

[0138] The activation of T cells can be measured using any suitable method known in the art. Examples of suitable methods for determining T cell activation are provided in Example 12 and Example 20 herein. In one embodiment, an increase in the secretion of IFN-γ can indicate an increase in T cell activation. In one embodiment, T cell activation can be determined by the upregulation of specific genes related to T cell activation, for example, the upregulation of one or more of IFGGC1, CD3D, ICOS, CXCL10, CD80, CCR5, IL-7, and / or INF-gamma.

[0139] The upregulation of one or more genes selected from IFGGC1, CD3D, ICOS, CXCL10, CD80, CCR5, IL-7, and / or INF-gamma can be assessed by comparing the level, e.g., expression level, of the gene with a reference value. The reference value can be the level of one or more of the genes from a subject before treatment with an 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 be receiving therapy.

[0140] The term "IFGGC1" refers to the gene encoding the 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 costimulator receptor. The term "CXCL10" refers to the gene encoding the C-X-C motif chemokine ligand 10. The term "CD80" refers to the gene encoding the cluster of differentiation 80. The term "CCR5" refers to the gene encoding the C-C chemokine receptor type 5.

[0141] 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, VL CDR amino acid sequences, VH amino acid sequences, and VL amino acid sequences of the PMX molecules shown in Table 2 are particularly within the scope of the present invention.

[0142] 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.

[0143] Variants of the above antibodies and antigen-binding portions are also within the scope of the present invention.

[0144] A variant of an antibody or antigen-binding portion thereof (e.g., VH or VL) described herein has 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 modifications are conservative sequence modifications.

[0145] 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 an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into an antibody of the present invention, or its antigen-binding portion, by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody of the invention can be replaced with other amino acid residues from the same side chain family, and the modified antibodies can be tested for retained function (i.e., PD-1 binding) using the functional assays described herein.

[0146] Therefore, these amino acid changes can typically be made without altering the biological activity, function, or other desired properties of the polypeptide, such as its affinity for an antigen or its specificity. Generally, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. Furthermore, substitutions of amino acids with similar structures or functions are unlikely to destroy the biological activity of the polypeptide. Abbreviations for the amino acid residues comprising the polypeptides and peptides described herein, as well as conservative substitutions for these amino acid residues, are set forth in Table 1 below. Table 1. Examples of amino acid residues and conservative amino acid substitutions [Table 1]

[0147] In some embodiments, the present invention provides an antibody or antigen-binding portion thereof that is a variant of the antibody or antigen-binding portion thereof compared to an amino acid sequence selected from any one of SEQ ID NOs: 3 to 292, which comprises one or more sequence modifications and has improved one or more properties, such as binding affinity, specificity, thermal stability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility, compared to an unmodified antibody or fragment thereof. Modifications of modified effector function activity are described, for example, in WO2023 / 012496 and WO2021 / 165417.

[0148] In some embodiments, the invention provides an antibody or antigen-binding portion thereof that is a variant of the 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, comprising one or more sequence modifications and having improved half-life compared to an unmodified antibody or antigen-binding portion thereof. Suitable sequence modifications for improving half-life are known in the art and are described, for example, in WO2018 / 073185, WO2020 / 082048, WO2020 / 116560, WO2020 / 142625, WO2021 / 212081, WO2021 / 212081, and WO2022 / 067233. When an antibody or antigen-binding portion thereof comprises a half-life extending sequence modification, the half-life of the molecule may be extended to 1-20 days, 1-19 days, 1-18 days, 1-17 days, 1-16 days, 1-15 days, 1-14 days, 1-13 days, 1-12 days, 1-11 days, 1-10 days, 2-10 days, 3-10 days, 4-10 days, 5-10 days, 1-9 days, 1- It may exhibit a half-life of 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, or 10 to 15 days.

[0149] Suitable methods for measuring properties that may indicate that an antibody can be successfully developed on a large scale include first 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, and aggregation and thermal stability (Tm) studies).

[0150] In one embodiment of the 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, e.g., a cytotoxic radionuclide, a chemical toxin, or a protein toxin. In a further related embodiment of the 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 a related embodiment of the invention, the second antibody or antigen-binding portion thereof binds to a different target. In further related embodiments, the different targets may 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.

[0151] In another embodiment of the invention, there is provided an antibody, or antigen-binding portion thereof, that binds to PD-1, wherein the antibody, or antigen-binding portion thereof, is conjugated to a further moiety selected from a half-life extending moiety, a label, a cytotoxin, a liposome, a nanoparticle, or a radioisotope. In related embodiments of the invention, 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. It will be apparent to 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 invention. When the antibody or antigen-binding portion thereof comprises a half-life extending moiety, the half-life of the molecule may be 1-20 days, 1-19 days, 1-18 days, 1-17 days, 1-16 days, 1-15 days, 1-14 days, 1-13 days, 1-12 days, 1-11 days, 1-10 days, 2-10 days, 3-10 days, 4-10 days, 5-10 days, 1-9 days, 1-8 days, 1-9 days, 1-10 days, 1-11 days, 1-10 days, 2-10 days, 3-10 days, 4-10 days, 5-10 days, 1 ... The antibody or antigen-binding portion thereof may exhibit a half-life of 10 to 15 days, e.g., 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, or 10 to 15 days. When the antibody or antigen-binding portion thereof comprises a half-life extending moiety, the half-life of the molecule may exhibit a half-life in the range of 10 to 15 days, e.g., 10, 11, 12, 13, 14, or 15 days.

[0152]

[0013] In another aspect of the present invention, there is provided a pharmaceutical composition comprising an antibody or antigen-binding portion thereof according to any preceding embodiment. The pharmaceutical composition may be used to treat a disease. In one embodiment, the disease is cancer or a tumor.

[0153] Pharmaceutical compositions according to the invention may comprise an antibody or antigen-binding portion thereof described herein and, optionally, a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical composition" refers to a composition for veterinary use, that is, a composition used to treat companion animals, i.e., a veterinary composition. In a preferred embodiment, the animal being treated is a dog.

[0154] Pharmaceutical compositions may optionally include 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.

[0155] Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical, or subcutaneous administration. Preferably, the compositions are administered parenterally.

[0156] The pharmaceutically acceptable carrier or vehicle may be particulate, such that the composition is in tablet or powder form, for example. The term "carrier" refers to a diluent, adjuvant, or excipient with which the drug-antibody conjugate of the present invention is administered. Such pharmaceutical carriers may be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Carriers may be saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary substances, stabilizers, thickeners, lubricants, and coloring agents may be used. In one embodiment, when administered to animals, the antigen-binding domain, or antibody or composition of the present invention and the pharmaceutically acceptable carrier are sterile. Water is a preferred carrier when the drug-antibody conjugate of the present invention is administered intravenously. Saline solutions and aqueous dextrose and glycerol 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, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0157] The pharmaceutical composition of the present invention can be in the form of liquid, for example, solution, emulsion or suspension.Liquid can be useful for injection, infusion (for example, IV infusion) or subcutaneous delivery.When intended for oral administration, composition is preferably in the form of solid or liquid, and semi-solid, semi-liquid, suspension and gel form are included in the form that is considered as solid or liquid in this specification.

[0158] As a solid composition for oral administration, the composition can be formulated in the form of powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions typically contain one or more inert diluents. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose, or dextrin; disintegrating agents such as alginic acid, sodium alginate, or corn starch; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavors such as peppermint, methyl salicylate, or orange flavor, and coloring agents. When the composition is in the form of a capsule (e.g., a gelatin capsule), it can contain, in addition to the above-mentioned materials, a liquid carrier such as polyethylene glycol, cyclodextrin, or fatty oil.

[0159] Composition can be in liquid form, for example, elixir, syrup, solution, emulsion or suspension.This liquid can be useful for oral administration or injection delivery.When intended for oral administration, composition can contain one or more of sweetener, preservative, dye / colorant and flavor enhancer.In the composition for injection administration, it can also contain one or more of surfactant, preservative, wetting agent, dispersant, suspending agent, buffer, stabilizer and isotonic agent.

[0160] The compositions may be in the form of one or more dosage units. In certain embodiments, it may be desirable to administer the compositions locally to the area in need of treatment or by intravenous injection or infusion.

[0161] In another aspect of the invention, a method of 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, of any preceding embodiment of the invention. The method can be used to treat the disease. In one embodiment, the disease is cancer or a tumor.

[0162] 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.

[0163] In another aspect, the invention relates to the use of an antibody, or antigen-binding portion thereof, described herein in the manufacture of a medicament for the treatment of cancer or tumors.

[0164] Cancers that may be treated by an antibody or antigen-binding portion thereof, pharmaceutical composition, and / or method according to the invention are selected from bone cancer, pancreatic cancer, skin cancer, head or 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, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, kidney cancer, soft tissue sarcoma, urethral cancer, bladder cancer, renal cancer, lung cancer, non-small cell lung cancer, thymic cancer, urothelial cortical cancer, prostate cancer, mesocortical cancer, paracortical cancer, lymphoma, such as, for example, Hodgkin's cancer, non-Hodgkin's disease, gastric cancer, and multiple myeloma. Canine cancers that may be targeted by an antibody or antigen-binding portion thereof, pharmaceutical composition, and / or method according to the invention are selected from breast cancer (cancer of the tissues lining the internal organs), osteosarcoma (bone cancer), melanoma (skin cancer), and hemangiosarcoma (cancer of the walls of blood vessels).

[0165] In further related embodiments of the present invention, the method of the previous embodiment is provided, wherein the antibody or antigen-binding portion thereof, pharmaceutical composition, or embodiments thereof further comprises separately administering another therapeutic agent to the subject. In related embodiments, the other therapeutic agent is a radiotoxic agent, an immunosuppressant, an immunomodulatory agent, or an antibody or antibody fragment thereof. In related embodiments, the immunomodulatory agent is a cytokine, a chemokine, or an anti-cancer therapy. The therapeutic agent is a compound or molecule useful in treating a disease. Examples of therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptotic agents, anti-angiogenic agents, boron compounds, photoactive agents or dyes, and radioisotopes. The antibody molecule includes a complete antibody or a fragment thereof (e.g., Fab, F(ab'), Fv, single-chain Fv fragment (scFv), or a single-domain antibody, e.g., a VH domain, or an antibody mimetic protein). Anti-cancer therapy may include therapeutic agents or radiation therapy, and may include gene therapy, viral therapy, RNA therapy, bone marrow transplantation, nanotherapy, targeted anti-cancer therapy, or oncolytic drugs. Examples of other therapeutic agents include other checkpoint inhibitors, anti-tumor agents, immunogenic agents, attenuated cancer cells, tumor antigens, tumor-derived antigens, or antigen-presenting cells such as dendritic cells pulsed with nucleic acids, immunostimulatory cytokines (e.g., IL-2, IFNα2, GM-CSF), targeted small molecules and biomolecules (e.g., agents that bind to tumor-specific antigens, including signal transduction pathway components, e.g., tyrosine kinase modulators and receptor tyrosine kinase inhibitors, and EGFR antagonists), anti-inflammatory agents, cytotoxic agents, radiotoxic agents, or immunosuppressants, as well as cells transfected with genes encoding immunostimulatory cytokines (e.g., GM-CSF), and chemotherapy. In one embodiment, antibodies are used in combination with surgery.

[0166] In one embodiment, an antibody or pharmaceutical composition of the invention is administered in combination with an immunomodulatory agent, a checkpoint modulator, an agent involved in T cell activation, a tumor microenvironment modifier (TME), or a tumor-specific target. For example, an immunomodulatory agent can be an inhibitor of an immune checkpoint molecule chosen from one or more inhibitors of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, CEACAM, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFRβ, 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 immunomodulatory agent can be an activator of a costimulatory molecule selected from one or more agonists of 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.

[0167] In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody selected from Nivolumab®, Pembrolizumab®, Pidilizumab®, or Gilvetmab®.

[0168] In one embodiment of the present invention, the composition is administered simultaneously 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 compound of the present invention, preferably at least 1 hour, 5 hours, 12 hours, 1 day, 1 week, 1 month, more preferably several months (for example, up to 3 months) before or after the administration of the compound or composition of the present invention.

[0169] The antibodies or antigen-binding portions or pharmaceutical compositions herein can be administered at the same time as or at different times, e.g., simultaneously, separately, or sequentially, other therapies or therapeutic compounds or therapies. In one embodiment, the other therapy can be any additional PD-1, PD-L1, or PD-L2 therapy.

[0170] The amount of therapeutic agent that is effective / active in treating a particular disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can be optionally used to help identify the optimal dosage range. The exact dose to be used in the composition also depends on the route of administration and the severity of the disorder or disease, and should be determined according to the judgment of the physician and the individual circumstances of each patient. Factors such as age, weight, sex, diet, administration time, excretion rate, host condition, drug combination, reaction sensitivity, and disease severity should be taken into consideration.

[0171] Typically, this amount is at least about 0.01% of the antibody, antigen-binding domain, or fragment thereof of the present invention by weight of the composition. When intended for oral administration, this amount can vary from about 0.1% to about 80% by weight of the composition. Preferred oral compositions may contain about 4% to about 50% of the antibody, or fragment thereof, of the present invention by weight of the composition.

[0172] In one embodiment, the antibody or antigen-binding portion thereof is administered 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, or 0.5 to 40 mg / kg. , 0.6-40 mg / kg, 0.2-30 mg / kg, 0.3-30 mg / kg, 0.4-30 mg / kg, 0.5-30 mg / kg, 0.6-30 mg / kg, 0.2-20 mg / kg, 0.3-20 mg / kg, 0.4-20 mg / kg, 0.5-20 mg / kg, 0.6-20 mg / kg, 0.2-10 mg / kg, 0.3-10 mg / kg, 0.4-10 mg / kg, 0.5-10 mg / kg, or 0.6-10 mg / kg. 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.

[0173] 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.

[0174] For administration by injection, such as intravenous or subcutaneous injection, the composition may typically comprise about 0.01 mg / kg to about 250 mg / kg of the subject's body weight, e.g., 0.1 mg / kg to about 250 mg / kg, e.g., about 0.1 mg / kg to 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 doses of less than 0.1 mg / kg are 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. Dosing schedules can vary, for example, from once weekly to once every 2, 3, or 4 weeks, or 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 3 to 4 weeks, for example, 0.5 mg / ml or 2.5 mg / ml every 3 to 4 weeks. In some embodiments, the dose is selected to provide long-term depletion of PD-1-positive cells, allowing for a 3 to 4 week interval between doses. Multiple doses, preferably up to about 6 or more repeated doses, may be administered.

[0175] In one embodiment, after treatment, the subject has no disease progression for 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 no disease progression for 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.

[0176] In one embodiment, the number of days alive, disease free or disease progression free is at least 2 months, or at least 3 months, or at least 4 months, such as at least 5 months, for example at least 6 months.

[0177] In one embodiment, the number of days alive, disease-free, or disease progression-free is at least 9 months, 200 days, 300 days, or 3 years or more. In one embodiment, it is at least 1 year, 2 years, 3 years, or more. The invention provides a method of treating or preventing a PD-1 mediated disease or disorder 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 invention.

[0178] As used herein, "treat," "treating," or "treatment" means inhibiting or alleviating a disease or disorder. For example, treatment can include postponing the onset of symptoms associated with a disease or disorder and / or reducing the severity of such symptoms that would or are expected to occur with the disease. These terms include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, these terms indicate that beneficial results are achieved in at least some treated mammals, e.g., canine patients. Many medical treatments are effective in some, but not all, patients who receive treatment. For example, in the treatment of B-cell lymphoma, improvement in symptoms can be assessed by measuring lymph nodes after treatment and observing a reduction in lymph node size as an indicator of successful treatment.

[0179] The term "subject" or "patient" refers to a dog who is the object of treatment, observation, or experiment. For the avoidance of doubt, human treatment is excluded.

[0180] In another aspect, the invention provides a nucleic acid sequence encoding an antibody, or antibody antigen-binding portion thereof, according to any preceding embodiment of the invention. Such DNA sequences may be 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 or SEQ ID NOs: 163 and 165, or SEQ ID NOs: 173 and 175, or SEQ ID NOs: 183 and 185, or 193 and 195, or 203 and 205, or SEQ ID NOs: 213 and 215, or SEQ ID NOs: 223 and 225, or SEQ ID NOs: 233 and 235, or SEQ ID NOs: 243 and 245, or SEQ ID NOs: 253 and 255, or SEQ ID NOs: 263 and 265, or SEQ ID NOs: 273 and 275, or SEQ ID NOs: 283 and 285, or a sequence having at least 75%, 80%, 90% or 95% sequence identity thereto.

[0181] In another aspect, the present invention provides a vector comprising the above-described nucleic acid sequence.

[0182] In another aspect, the invention provides a host cell comprising a nucleic acid according to any preceding embodiment, or a vector as described above.

[0183] In another aspect, the invention provides a kit comprising an antibody or antigen-binding portion thereof according to any preceding embodiment, or a pharmaceutical composition according to the preceding embodiment, hi related embodiments, the kit further comprises reagents for detecting the antibody or antigen-binding portion thereof.

[0184] In another aspect, the present invention provides kits for detecting PD-1 for diagnosis, prognosis, or disease monitoring, comprising an antibody or antigen-binding portion thereof of the present invention. Such kits may contain other components, packaging, instructions, or materials to aid in the detection of PD-1 protein. The kits may include a labeled antibody or antigen-binding portion thereof of the present invention described above and one or more compounds for detecting the label.

[0185] In another aspect, the invention provides an antibody, or antigen-binding portion thereof, of the invention packaged in lyophilized form or packaged in an aqueous medium.

[0186] In another aspect, the invention provides a method for producing a canine antibody that binds to PD-1, the method comprising culturing an isolated host cell of the invention and recovering the antibody.

[0187] In a further aspect, the invention provides a method for making a canine antibody that binds to PD-1, 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 a library of antibodies from the mice; and c) isolating antibodies from the library.

[0188] In another further embodiment, the present invention provides a method for detecting PD-1 protein or the extracellular domain of PD-1 protein in a biological sample from a canine subject, 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, tissue, blood, serum, plasma, or lymph sample. The method can be performed in vivo, in vitro, or ex vivo.

[0189] In another aspect, the invention provides a method of inhibiting tumor growth or metastasis, comprising contacting tumor cells with an effective amount of an antibody or antigen-binding portion thereof according to the invention, or a pharmaceutical composition according to the invention.

[0190] In a still further aspect, the invention provides a method of killing a tumor cell that expresses PD-1, the method comprising contacting the cell with an antibody or antigen-binding portion thereof of the invention, or a pharmaceutical composition according to the invention, such that killing of the PD-1-expressing cell occurs.

[0191] In one embodiment, the tumor cells are canine tumor cells.

[0192] In a further 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 still further embodiments, 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.

[0193] The present invention also relates to the following non-limiting provisions:

[0194] 1. One of the following epitopes of the extracellular domain of canine PD-1: i) an epitope including T76, Q83, E84, L128, P129, and P130 in SEQ ID NO: 2 ii) An isolated canine antibody or antigen-binding portion thereof that binds to an epitope comprising D58, D61, N74, T76, and Y127 in SEQ ID NO: 2, An isolated canine antibody or antigen-binding portion thereof, the epitope of which is determined using epitope mapping techniques.

[0195] 2. The antibody is 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 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 CDR1 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. a CDR2 sequence and an 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; an 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; an 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 an 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 an LC CDR1 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. a CDR2 sequence and an LC CDR3 sequence comprising or consisting of an amino acid sequence 2 having at least 70%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO:3, or 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 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. a CDR1 sequence and an 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 an 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; 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; 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 CDR1 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. The antibody or antigen-binding portion thereof of clause 1, comprising a CDR2 sequence and an 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.

[0196] 3. The antibody is a) 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; 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; 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, 9, 10, 11, 12, 13, or 14 amino acid differences compared to SEQ ID NO: 20; and an LC CDR1 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. a CDR2 sequence and an 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 b) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 207, or an amino acid sequence having one or two amino acid differences compared to SEQ ID NO: 207; an HC CDR2 sequence comprising or consisting of SEQ ID NO: 208, or an amino acid sequence having one, two, three, four, or five amino acid differences compared to SEQ ID NO: 208; an HC CDR3 sequence comprising or consisting of SEQ ID NO: 209, or an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen amino acid differences compared to SEQ ID NO: 209; and an LC CDR1 sequence comprising or consisting of SEQ ID NO: 210, or an amino acid sequence having one, two, or three amino acid differences compared to SEQ ID NO: 210; and an LC CDR2 sequence comprising or consisting of SEQ ID NO: 211, or an amino acid sequence having one or two amino acid differences compared to SEQ ID NO: 211; and an LC CDR2 sequence comprising or consisting of SEQ ID NO: 212, or an amino acid sequence having one, two, three, or four amino acid differences compared to SEQ ID NO: 212. CDR3 sequence, or c) an 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; an 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; an 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 an 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 an LC CDR1 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. 3. The antibody or antigen-binding portion thereof of clause 1 or 2, comprising a CDR2 sequence 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.

[0197] 4. An isolated canine antibody, or antigen-binding portion thereof, that binds to canine PD-1, wherein the antibody: a) 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; 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; 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, 9, 10, 11, 12, 13, or 14 amino acid differences compared to SEQ ID NO: 20; and an LC CDR1 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. a CDR2 sequence and an 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 b) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 207, or an amino acid sequence having one or two amino acid differences compared to SEQ ID NO: 207; an HC CDR2 sequence comprising or consisting of SEQ ID NO: 208, or an amino acid sequence having one, two, three, four, or five amino acid differences compared to SEQ ID NO: 208; an HC CDR3 sequence comprising or consisting of SEQ ID NO: 209, or an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen amino acid differences compared to SEQ ID NO: 209; and an LC CDR1 sequence comprising or consisting of SEQ ID NO: 210, or an amino acid sequence having one, two, or three amino acid differences compared to SEQ ID NO: 210; and an LC CDR2 sequence comprising or consisting of SEQ ID NO: 211, or an amino acid sequence having one or two amino acid differences compared to SEQ ID NO: 211; and an LC CDR2 sequence comprising or consisting of SEQ ID NO: 212, or an amino acid sequence having one, two, three, or four amino acid differences compared to SEQ ID NO: 212. CDR3 sequence, or c) an 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; an 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; an 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 an 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 an LC CDR1 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. a CDR2 sequence and an 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; 1. An isolated canine antibody or antigen-binding portion thereof, comprising:

[0198] 5. The antibody or antigen-binding portion thereof, i) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 17, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 18, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 19, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 20, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 21, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 22; ii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 27, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 28, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 29, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 30, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 31, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 32. iii) an HC CDR1 sequence comprising or consisting of SEQ ID NO:7, an HC CDR2 sequence comprising or consisting of SEQ ID NO:8, an HC CDR3 sequence comprising or consisting of SEQ ID NO:9, an LC CDR1 sequence comprising or consisting of SEQ ID NO:10, an LC CDR2 sequence comprising or consisting of SEQ ID NO:11, and an LC CDR3 sequence comprising or consisting of SEQ ID NO:12; iv) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 37, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 38, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 39, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 40, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 41, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 42. v) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 47, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 48, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 49, an LC CDR1 sequence comprising SEQ ID NO: 50, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 51, and an 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) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 87, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 88, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 89, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 90, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 91, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 92; x) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 97, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 98, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 99, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 100, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 101, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 102; xi) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 107, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 108, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 109, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 110, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 111, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 112; xii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 117, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 118, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 119, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 120, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 12, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 122; xiii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 127, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 128, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 129, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 130, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 131, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 132;

[0199] xiv) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 137, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 138, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 139, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 140, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 141, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 142; xv) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 147, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 148, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 149, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 150, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 151, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 152; xvi) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 157, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 158, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 159, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 160, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 161, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 162; xvii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 167, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 168, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 169, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 170, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 171, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 172; xviii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 177, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 178, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 179, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 180, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 181, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 182; xix) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 187, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 188, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 189, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 190, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 191, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 192; xx) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 197, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 198, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 199, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 200, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 201, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 202; xxi) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 207, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 208, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 209, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 210, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 211, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 212; xxii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 217, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 218, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 219, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 220, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 221, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 222; xxiii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 227, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 228, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 229, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 230, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 231, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 232;

[0200] xxiv) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 237, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 238, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 239, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 240, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 241, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 242; xxv) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 247, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 248, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 249, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 250, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 251, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 252; xxvi) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 257, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 258, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 259, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 260, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 261, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 262; xxvii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 267, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 268, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 269, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 270, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 271, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 272; xxviii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 277, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 278, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 279, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 280, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 281, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 282; or xxix) An antibody or antigen-binding portion thereof according to any one of the preceding clauses, having an HC CDR1 sequence comprising or consisting of SEQ ID NO: 287, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 288, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 289, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 290, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 291, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 292.

[0201] 6. The antibody or antigen-binding portion thereof of the preceding clause, 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 thereto, 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.

[0202] 7. The antibody or antigen-binding portion thereof, 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 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 thereto, 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 thereto; 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 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 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 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 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 thereto, 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 thereto; 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 thereto, 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 thereto; or 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 thereto, 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 thereto; 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 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 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 thereto, 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 thereto; 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 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 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 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

[0203] l) 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 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 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 thereto, 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 thereto; 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 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 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 thereto, 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 thereto; 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 thereto, 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 thereto; 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 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 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 thereto, 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 thereto; 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 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 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 thereto, 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 thereto; 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 thereto, 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 thereto; or

[0204] 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 thereto, 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 thereto; 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 thereto, 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 thereto; 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 thereto, 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 thereto; 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 thereto, 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 thereto; or

[0205] 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 thereto, 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 thereto; 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 thereto, 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 thereto; 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 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 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 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; 10. The antibody or antigen-binding portion thereof of the preceding clause, having:

[0206] 8. The antibody or antigen-binding portion thereof according to the preceding clause, wherein the antigen-binding portion thereof is an scFv, Fv, heavy chain or single domain antibody.

[0207] 9. The isolated canine antibody, or antigen-binding portion thereof, of the preceding clause, which binds to canine PD-1, wherein the antibody, or antigen-binding portion thereof, competes with PD-L1 and / or PD-L2.

[0208] 10. The antibody, or antigen-binding portion thereof, of 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.

[0209] 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.

[0210] 12. The antibody or antigen-binding portion thereof of clause 11, wherein the therapeutic moiety is a second antibody or antigen-binding portion thereof.

[0211] 13. The antibody or antigen-binding portion thereof of clause 12, wherein said second antibody or antigen-binding portion thereof binds to a different target.

[0212] 14. The antibody, or antigen-binding portion thereof, of the preceding clause, wherein the antibody, or antigen-binding portion thereof, is conjugated to a further moiety selected from a half-life extending moiety, a label, a cytotoxin, a liposome, a nanoparticle, or a radioisotope.

[0213] 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.

[0214] 16. A pharmaceutical composition comprising an antibody or antigen-binding portion thereof according to the preceding clause.

[0215] 17. An antibody or antigen-binding portion thereof according to any one of clauses 1 to 15, or a pharmaceutical composition according to clause 16, for use in the treatment of a disease.

[0216] 18. 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 according to any one of clauses 1 to 15, or a pharmaceutical composition according to clause 16.

[0217] 19. The antibody or antigen-binding portion thereof or pharmaceutical composition of clause 17, or the method of clause 18, wherein the disease is cancer or tumor.

[0218] 20. The antibody or antigen-binding portion thereof or pharmaceutical composition of clause 17 or 19, or the method of clause 18 or 19, further comprising separately administering to the subject another therapeutic agent.

[0219] 21. The antibody or antigen-binding portion thereof or pharmaceutical composition or method of clause 20, wherein the therapeutic agent is a cytotoxic or radiotoxic agent, an immunosuppressant, an immunomodulatory agent, or an antibody or antibody fragment thereof.

[0220] 22. The antibody or antigen-binding portion thereof or pharmaceutical composition or method of clause 21, wherein the immunomodulatory agent is a cytokine or chemokine.

[0221] 23. A nucleic acid sequence encoding the antibody or antigen-binding portion thereof of any one of clauses 1 to 15.

[0222] 24. 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: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: 24. The nucleic acid sequence of clause 23, comprising a sequence selected from 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: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 having at least 75% sequence identity to any one of the aforesaid sequences.

[0223] 25. A vector comprising a nucleic acid sequence according to clause 23 or 24.

[0224] 26. A host cell comprising a nucleic acid sequence according to clause 23 or 24, or a vector according to clause 25.

[0225] 27. A kit comprising an antibody or antigen-binding portion thereof according to any one of clauses 1 to 15, or a pharmaceutical composition according to clause 16.

[0226] 28. The kit of clause 27, further comprising reagents for detection of the antibody or antigen-binding portion thereof.

[0227] 29. A method for producing a canine antibody that binds to PD-1, comprising culturing the isolated host cell of clause 26 and recovering the antibody.

[0228] 30. A method for producing a canine antibody that binds to PD-1, comprising: 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 a library of antibodies from the mice; c) isolating antibodies from the library.

[0229] 31. A method for detecting PD-1 protein or the extracellular domain of 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 according to any one of clauses 1 to 15, wherein the antibody or antigen-binding portion thereof is linked to a detectable label.

[0230] 32. The method of clause 31, wherein the biological sample is a biopsy, tissue, blood, serum, plasma, or lymph sample.

[0231] 33. A method for inhibiting tumor growth or metastasis, comprising contacting tumor cells with an effective amount of an antibody or antigen-binding portion thereof according to any one of clauses 1 to 15, or a pharmaceutical composition according to clause 16.

[0232] 34. A method of killing tumor cells that express PD-1, comprising contacting the cells with an antibody according to any one of clauses 1 to 15, or a pharmaceutical composition according to clause 16, so as to cause killing of the cells that express PD-1.

[0233] 35. The method of clause 34, wherein the tumor cells are canine tumor cells.

[0234] 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.

[0235] 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.

[0236] 38. An immunoconjugate comprising an antibody or antigen-binding portion thereof according to any one of clauses 1 to 15, or a binding agent according to clause 36 or 37.

[0237] 39. A method of modulating an immune response, comprising administering 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.

[0238] 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 a further therapeutic moiety. 41. The combination therapy according to clause 40, wherein the further therapeutic moiety is an antibody (optionally an antibody that binds to an immuno-oncological target), or a chemotherapeutic agent.

[0239] Table 2. SEQ ID NOs for the amino acid and nucleotide sequences of each anti-PD-1 antibody included herein [Table 2-1] JPEG2026508096000004.jpg124169 [Table 2-2] JPEG2026508096000006.jpg154169 [Table 2-3] JPEG2026508096000008.jpg118169 [Table 2-4] JPEG2026508096000010.jpg124169 [Table 2-5] JPEG2026508096000012.jpg149169 [Table 2-6] JPEG2026508096000014.jpg118169 [Table 2-7] JPEG2026508096000016.jpg124169 Table 2-8 JPEG2026508096000018.jpg154169 Table 2-9 JPEG2026508096000020.jpg113169 Table 2-10 JPEG2026508096000022.jpg102169 Table 2-11 JPEG2026508096000024.jpg96169 Table 2-12 Table 2-13 JPEG2026508096000027.jpg102169 Table 2-14 JPEG2026508096000029.jpg112169 Table 2-15 Table 2-16 JPEG2026508096000032.jpg107169 Table 2-17 JPEG2026508096000034.jpg91169 Table 2-18 Table 2-19 JPEG2026508096000037.jpg150169 Table 2-20 JPEG2026508096000039.jpg112169 Table 2-21 Table 2-22 JPEG2026508096000042.jpg107169

[0240] The present invention is further illustrated in the following non-limiting examples. [Example]

[0241] Example 1 Canine PD-1, PD-L1 and PD-L2 sequences Canine PD-1 (ENSCAFG00000013184), PD-L1 (ENSCAFG00000002120), and PD-L2 (ENSCAFG00000002121) coding sequences were synthesized (codon-optimized, where appropriate) according to the predicted sequences from the genes found in the canine reference genome assembly (CanFam3.1) found in the Ensemble Genome Browser after verifying sequence homology with well-annotated human and mouse orthologs. The coding sequences of canine PD-1, PD-L1, and PD-L2 were confirmed by RNA sequencing according to standard approaches.

[0242] Example 2 Expression of canine PD-1 in HEK and MEF cells Human embryonic kidney (HEK) 293 cells were grown on 90 mm round tissue culture plates as monolayers in DMEM / F12 (Life Technologies, California, 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 either PD-1, PD-L1, or PD-L2 using Lipofectamine LTX with Plus reagent (Life Technologies, California, 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 including a 1% w / w transposase-encoding plasmid along with the expression plasmid. Stably transfected cells were selected with 3 μg / mL puromycin for 48 hours post-transfection, for a total of 6 days.

[0243] Mouse embryonic fibroblasts (MEFs) were grown as monolayers in DMEM high glucose (Life Technologies) with L-glutamine supplemented with 10% FBS and 1x MEM non-essential amino acids (Gibco) on 90 mm round tissue culture plates at 37°C in a humidified atmosphere and 5% CO2. Cells were transfected with PD-1 using Lipofectamine LTX with Plus reagent (Life Technologies, California, USA) according to the manufacturer's recommendations. Stable integration of the plasmid expression cassette was ensured by using a PiggyBac transposon element in the expression vector and including a 1% w / w transposase-encoding plasmid along with the expression plasmid. Stably transfected cells were selected with 3 μg / mL puromycin for 48 hours post-transfection, for a total of 6 days.

[0244] Example 3 Expression of recombinant soluble canine PD-1, PD-L1 and PD-L2 proteins Chinese hamster ovary (CHO) cells grown in F17 medium (Gibco) supplemented with 4 mM L-glutamine were stably transfected with expression plasmids encoding PD-1ECD-mFc, PD-1ECD-6His, PD-L1ECD-mFc, and PD-L2ECD-mFc (ECD = extracellular domain) at 37 °C and 8% CO in a humidified environment. 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 mouse IgG2a, containing the genetic annotation hinge-constant heavy2-constant heavy3-constant heavy secretory (H-CH2-CH3-CHS), and "6His" refers to the hexahistidine tag (Figure 1).

[0245] Stable integration of the expression cassette of the plasmid was ensured by using PiggyBac transposon elements in the expression vector and including a 1% w / w transposase-encoding plasmid along with the expression plasmid. 3E+06 cells were transfected with 2 μg of expression plasmid encoding the appropriate protein and 20 ng of transducing expression plasmid. Briefly, DNA was mixed with polyethyleneimine (PEI, 25 kDa linear, PolySciences) at a 1:3 ratio (w / w) of DNA:PEI in 50 μL of Opti-MEM, incubated at room temperature for 15 minutes, and then added dropwise to the cell culture.

[0246] After 24 hours, cells were selected with 25 μg / mL puromycin for a total of 8 days. After selection, cells were cultured at 3 × 10 6 Cells were seeded in 100 mL of culture medium at 100°C and incubated at 32°C, 8% CO2 with shaking at 150 rpm. 4% HyClone Cell Boost 7a supplement + 0.4% HyClone Cell Boost 7b supplement + 1% glucose was added to the medium on days 1, 4, 7, and 10. Culture supernatants were collected on day 12. Supernatants were harvested by centrifugation at 1000 g for 15 min, followed by a 0.2 μm filtration step. Proteins were purified from the supernatants by affinity chromatography using standard protein A affinity chromatography (for Fc-tagged proteins) or by a Ni-NTA IMAC approach (for 6His-tagged proteins) using an AKTA FPLC system (Cytiva). In all cases, proteins were immediately buffer-exchanged into PBS and concentrated using a 10 kDa MWCO Amicon spin filter device. After four rounds of buffer exchange, proteins were 0.2 μm filtered and quantified by absorbance at 280 nm.

[0247] Example 4 Immunization: For example, Ky9™ mice, a transgenic mouse platform capable of generating antibodies with canine variable domains, were immunized substantially as described in WO2018 / 189520 and WO2020 / 074874. The transgenic mice have been modified compared to wild-type mice by the insertion of immunoglobulin heavy (IGH) and light chain (IGL) variable (V) region genes, IGH D region genes, and IGH J region genes from a dog into the mouse, allowing for the production of antibody heavy chains comprising variable antibody regions derived from the expression of canine DNA in the mouse in combination with constant regions.

[0248] The constant region can be a rodent immunoglobulin (IG) constant region, resulting in the production of a chimeric heavy chain having a canine variable region and a mouse constant region. Information about the variable region of such a chimeric antibody chain, or nucleic acid containing the variable region of such a chimeric antibody chain, can be used to generate complete canine antibodies, for example, for therapeutic use in dogs.

[0249] Mice were immunized with a prime-boost regimen by intraperitoneal (IP) administration of 2.5E+06 MEF cells stably expressing PD-1, formulated in 50% PBS and 50% Sigma Adjuvant System adjuvant (Sigma Aldrich). Ten days after the final boost, spleens and mesenteric lymph nodes were harvested. Alternatively, mice were immunized three or four times with recombinant canine PD-1 extracellular domain protein (PD-1ECD-mFc) at two-week or three-week intervals. The protein immunogen was delivered simultaneously at two sites: intraperitoneally and subcutaneously, formulated in 50% PBS and 50% Sigma Adjuvant System adjuvant (Sigma Aldrich). Four days after the final boost, spleens and mesenteric lymph nodes were harvested.

[0250] Serum titer determination: Mice were bled before immunization and 10 days after the first boost. Blood was collected and serum separated using Microvette 200Z gel tubes (Starstedt AG & Co. KG). Serum titers of PD-1-specific IgG were assessed by measuring the binding of serum dilutions to PD-1-expressing HEK293 cells. Immune serum was serially diluted in FACS buffer and incubated with 1 × 10 HEK293 cells stably expressing PD-1 for 1 hour at 4°C, followed by washing with 200 μL of FACS buffer. Specific IgG was detected with a trio of BB700-conjugated monoclonal antibodies against mouse isotypes IgG1, IgG2a, and IgG2b (BD OptiBuild™, Becton Dickinson) at a 1:400 dilution for 1 hour at 4°C, followed by washing with 200 μL of FACS buffer. The geometric mean fluorescence intensity of HEK293 cells was calculated by flow cytometry using a Beckton-Coulter Cytoflex. Data were analyzed with FlowJo version 10.6.1. A 1:100 dilution of pre-immune serum was used to set the background threshold.

[0251] Example 5 Isolation of antibody-producing cells and identification of antibody sequences of candidate molecules Tissue isolation: Spleens and mesenteric lymph nodes were harvested from immunized mice. Splenocytes were prepared by cutting spleens into fragments and forcing them through a 45 μm cell strainer (Falcon) while rinsing with RPMI-1640 (Lonza, Basel, CH) + 10% FBS on ice. A similar process was used for lymphocytes from lymph nodes. Cells were pelleted at 300 g for 5 min and then resuspended in PBS + 2% FBS + 1 mM EDTA for direct use in flow sorting or frozen at -150°C in 90% FBS + 10% DMSO for later use.

[0252] Cell sorting: Prior to antigen-specific cell sorting, T / erythroid cells were depleted using biotinylated anti-CD90.2 and anti-ter119 antibodies, followed by binding to streptavidin rapid flow spheres according to the manufacturer's instructions (StemCell Technologies UK). Bulk cell sorting was then performed using a BD FACSAria Fusion flow sorter. The markers CD19, IgM, and IgD were used to identify class-switched B cells, and CD138 (Syndecan-1) + CD267 (TACI) were used to mark plasmablast and plasma cell populations within spleen + lymph node cells (SP + LN). 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-1ECD-mFC fusion protein and sorted by FACS. Markers to identify unwanted cell populations and dead cells (CD8a, CD4, fixable viability dyes) were included in all staining panels to exclude these cells.

[0253] Sorted cells were prepared for antibody profiling using the 10XGenomics Chromium Single Cell Immune Profiling System and V(D)J kit (10XGenomics) according to the manufacturer's instructions. The nucleotide sequences of expressed antibodies were determined by Illumina sequencing.

[0254] Variable immunoglobulin regions include the VDJ regions of the immunoglobulin nucleotide sequences for heavy genes and the VJ regions of the immunoglobulin nucleotide sequences for Igκ and Igλ. Within a clonal family, there are subfamilies with shared mutations within their V(D)J segments that arise during immunoglobulin gene recombination and somatic hypermutation. Different clonal families that exhibit unique V(D)J segment usage typically exhibit different binding characteristics. During recombination and hypermutation, cells with antibodies with higher affinity for antigen are selected, while low-affinity clones from the same lineage retain neutralizing function. Affinity typically increases with further mutations; for example, clustered families are shown in Figure 6 of WO2015 / 040401.

[0255] A clonal family is generally defined by the related immunoglobulin heavy and 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 use of V(D)J gene segments. An example of analysis of antibody sequences from sorted Ag-specific single B cells is shown in Figure 5 of WO2015 / 040401, which shows antibody sequences aligned by heavy chain V gene family usage and clustered to generate a displayed phylogenetic tree. Candidate clones were selected from these phylogenetic trees.

[0256] Example 6 Recombinant expression of monoclonal antibodies 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 region (see WO2023 / 012496) and a wild-type canine lambda constant 5 light region or a wild-type canine kappa constant light region. The expression vectors encoding the heavy and light chains were co-transfected into CHO cells to obtain stable expression. For antibody production, 3 x 10 6Selected CHO cells were seeded in 3 mL of culture medium and incubated at 32°C, 8% CO2 with shaking at 200 rpm. 4% HyClone Cell Boost 7a supplement + 0.4% HyClone Cell Boost 7b supplement + 1% glucose was added to the medium on days 1, 4, 7, and 10. Culture supernatants were collected on day 12, and IgG concentrations were determined by assaying against protein A using surface plasmon resonance (Biacore 8K, Cytiva).

[0257] Example 7 Flow cytometry cell binding assay Because PD-1 is a transmembrane protein, most conformationally correct proteins are embedded in the plasma membrane. Binding to plasma membrane-embedded proteins can be measured by flow cytometry by measuring binding to cells stably transfected with canine PD-1. 1E+05 HEK293 cells stably transfected with canine PD-1 were incubated in CHO supernatant containing 2 μg / mL IgG for 1 hour at 4°C. Cells were washed twice with 200 μL of FACS buffer and then incubated with a 1:300 dilution of goat anti-canine H+L-FITC conjugated secondary antibody (Abcam) for 30 minutes at 4°C in the dark. Cells were then washed once with 200 μL of FACS buffer, resuspended in 200 μL of FACS buffer, and acquired on a BD Accuri C6 Plus flow cytometer. The results shown in Figure 2 indicate that all mAbs PMX125-144 bind to canine full-length PD-1 in a transmembrane form, to a degree comparable to that of the benchmark anti-canine PD-1 binding mAbs Gilvetmab, 3B7-D, and 1B9 Def2. Gilvetmab is a canine PD-1 binding IgGB kappa antibody whose full-length heavy and light chain amino acid sequences are listed 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 kappa antibody with variable regions listed in US2017 / 0158764A1 (variable heavy SEQ ID NO: 2, variable light SEQ ID NO: 3). wherein the light chain variable region is grafted onto the wild-type canine kappa constant (also known as IGKC) sequence and the heavy chain variable region is grafted onto the wild-type IgGD canine constant (also known as IGHG4). 1B9 Def2 is an IgGB Def2 kappa antibody with variable regions described in WO2020 / 103885A1 (variable heavy SEQ ID NO: 50, variable light SEQ ID NO: 52).Here, the light chain variable region is grafted onto the wild-type canine kappa constant sequence (also known as IGKC) and the heavy chain variable sequence is grafted onto PetMedix's "Def2" modified IgGB constant Fc domain described in WO2023 / 012496.

[0258] Example 8 PD-L1 / PD-L2 Blocking Assay Using Flow Cytometry Blockade of the interaction between PD-1 and its ligands PD-L1 and PD-L2 is the primary mechanism of action of checkpoint inhibitors that target this axis, and in vitro blockade of this interaction is postulated to be a prerequisite for in vivo function.

[0259] Flow cytometry-based ligand blocking assay 1E+05 HEK293 cells stably transfected with canine PD-1 were incubated in CHO supernatant containing 2 μg / mL recombinant IgG at 4°C for 1 hour. The cells were then washed twice with 200 μL of FACS buffer before being incubated with 1 μg / mL phycoerythrin-labeled PD-L1ECD-mFc or PDL2ECD-mFc at 4°C for 1 hour. The cells were then washed once with 200 μL of FACS buffer, resuspended in 200 μL of FACS buffer, and acquired on a Beckman-Coulter CytoFlex flow cytometer. The data shown in Figure 3A and Figure 3B demonstrate that mAbs PMX125-PMX144 all potently blocked PD-L1 binding to PD-1, and a subset also blocked PD-L2 binding to PD-1 more potently than the control antibody 1B9 Def2. A subset of PD-L1 blockers also block PD-L2 binding to PD-1, but only PMX126, PMX128, PMX129, PMX133, and PMX142 are more potent than 1B9 Def2.

[0260] Example 9 Affinity assays - SPR and flow cytometry SPR-based affinity assay SPR-based monomeric affinity assays were performed as multi-cycle kinetic experiments. Approximately 100 RU of purified monoclonal antibody was captured onto Fc2 of a Series S Protein A chip (Cytiva). After capture, monomeric PD-1 ECD-6 His protein was injected at 30 μL / min in two-fold dilutions ranging from 100 nM to 0.13 nM with an association time of 150 seconds and a dissociation time of 700 seconds. SPR-based bivalent affinity assays were performed as multi-cycle kinetic experiments. Approximately 170 RU of purified PD-1 ECD-mFc protein was captured onto Fc2 of a Series SCM5 chip (Cytiva). After capture, monoclonal antibody was injected at 30 μL / min in two-fold dilutions ranging from 11.1 nM to 0.04 nM with an association time of 180 seconds and a dissociation time of 1000 seconds. At the end of each cycle, the chip surface was regenerated with a 120-second pulse of 10 mM glycine, pH 1.9, at 30 μL / min. Data were analyzed using the Biacore Insight package (Cytiva) by fitting to a global 1:1 binding model with kinetic analysis. The analyzed mAbs have Ks values ​​in the single-digit nanomolar range (double-digit in the case of PMX-152) when assayed in monovalent mode, and in the single- or double-digit picomolar range when assayed in bivalent mode (Figure 4A).

[0261] Flow cytometry-based apparent affinity assay 5E+04 HEK cells stably expressing PD-1 were incubated for 2 hours at 4°C in 250 μL of FACS buffer containing purified mAb diluted at 3x concentrations ranging from 300 nM to 10 pM. Cells were then washed twice in 200 μL of FACS buffer and incubated in a 1:300 dilution of goat anti-dog HL-FITC conjugated secondary antibody (Abcam) for 30 minutes at 4°C in the dark. 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. Apparent affinity was then derived by fitting the data to a 4-parameter logistic curve for agonist versus response in Prism 8.3.0 (GraphPad) and reporting the concentration at which the signal was 50% maximal. The resulting EC was measured. 50 The values ​​(apparent affinities) were in the single-digit nanomolar range for all mAbs analyzed (Fig. 4B).

[0262] Example 10 Epitope binning Epitope binning was performed using a "parallel" SPR-based approach. Approximately 200 RU of PD-1 ECD-mFc was amine-coupled to Fc2 at pH 5.0 on a CM5 chip according to standard manufacturer recommendations (Cytiva). The parallel assay consisted of injecting mAb1 in "dual" mode onto the chip at 200 nM for 300 s, followed immediately by injection of mAb2 at 200 nM for 300 s at a flow rate of 10 μL / min. Low ligand density, along with a high mAb1 concentration (200 nM) and long injection time (300 s), ensured ligand saturation. A strong signal after mAb2 injection indicates a noncompetitive binding mode in the mAb pair, whereas a signal after mAb2 injection indicates little or no competitive binding mode in the mAb pair. After completion of the cycle, the chip surface was regenerated with a 120-s pulse of 10 mM glycine HCl (pH 1.9) at 30 μL / min. The running buffer used was HBS-EP+ (Cytiva), and data were collected on a Biacore 8K instrument (Cytiva). Gilvetmab (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 shows the percentage of mAb2 binding when mAb1 was pre-bound under saturating conditions. Values ​​below 15% indicate complete blocking of the pair of mAbs, while values ​​above 75% indicate negligible blocking of mAb2 binding by mAb1. Analysis of the competitive relationship between mAbs can sometimes distinguish between the binding epitopes of mAbs that cross-compete one-to-one. Correlating the relative binding profile of each mAb in the matrix (column of relative binding values) with the relative binding profiles of each other mAb can, if appropriately thresholded, highlight non-trivial binding epitope similarities between mAbs (Figure 5B).These results suggest that PMX126 and PMX151 access epitopes that overlap with, but may be shown to be distinct from, the binding epitopes of the previously characterized mAbs 1B9 Def2, Gilvetmab, and 3B7-D9, although the binding epitopes of PMX125 and PM127 cannot be resolved from that of 1B9 Def2 using this limited set of mAbs (Figures 5B and 5C).

[0263] Example 11 Binding to activated canine T cells Canine PBMCs isolated from healthy dogs were plated at 1 × 10 per well in complete RPMI medium (RPMI-1640 + 10% FBS + 1 U / mL penicillin + 10 μg / mL streptomycin). 5 The cells were cultured in 96-well round-bottom plates at a concentration of 1 μg / ml. Cells were stimulated with 1 μg / ml concanavalin A (ConA) for 96 hours to upregulate PD-1 expression before being harvested for flow cytometry binding analysis. To assess PD-1 antibody binding on activated T cells, Alexa Fluor® 647 was used to directly conjugate PD-1 antibodies. Cells were resuspended in FACS buffer and incubated with commercially available canine PE-conjugated CD5 (clone YKIX322.3, BioRad) to identify T cells, and PD-1 was detected using Alexa 647-conjugated PD-1 antibodies at a final concentration of 5 μg / ml for 30 minutes at 4°C. Fixable viability dye eFluor 780 (eBioscience) was used to label and exclude dead cells. The results, shown in Figure 6, demonstrate that all tested mAbs bind to canine PD-1 on ConA-activated canine T cells.

[0264] Because cancer samples are thought to have higher levels of PD-1 / PD-L1, we also evaluated the ability of PD-1 antibodies to bind to PD-1 on T cells from cancer-bearing dogs. Samples from cancer-bearing dogs were provided by the Oncology Service at the Queen Mother Animal Hospital, Royal Veterinary College (RVC). For this analysis, peripheral blood leukocytes were isolated from canine blood following red blood cell lysis (eBioscience). Cells were stained with anti-canine PE-conjugated CD5 (clone YKIX322.3, BioRad) and Alexa647-conjugated PMX127 antibodies as previously described and analyzed by flow cytometry. For evaluation of PD-1 expression by T cells, lymphocytes were first gated by forward and side scatter characteristics, followed by CD5 cleavage. + The group was gated. PD-1 + Gate for cell analysis: CD5 + The results shown in Figures 12A and 12B were based on the binding of an isotype control antibody by the canine CD5 + We demonstrated the ability of PD-1 antibodies to detect PD-1 expression by T cells.

[0265] Example 12 Functional in vitro assay IFN-γ secretion assay Blood from healthy beagle dogs was collected in EDTA tubes. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood after density centrifugation in Leucosep tubes (Greiner Bio-One) with Ficoll-Paque + 1.077 g / mL (Cytiva). Isolated PBMCs were washed twice and resuspended in complete RPMI-1640 medium (Lonza) at 2.5 × 10 per well. 5Cells were plated in triplicate in 96-well round-bottom plates at a concentration of 1 μg / ml. Cells were stimulated with 1 μg / ml concanavalin A (ConA) and incubated for 96 hours. Antibodies were added at a final concentration of 10 μg / ml at the time of stimulation. For IFN-γ secretion measurements, supernatants were collected after 96 hours of incubation, and IFN-γ levels were quantified by sandwich ELISA using the Canine IFN-Gamma ELISA BASIC Kit (Mabtech AB) according to the manufacturer's recommended protocol. For T cell proliferation assessment, 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-dog 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, washed twice in FACS buffer, and acquired on a Beckman-Coulter Cytoflex flow cytometer. FSC and SSC profiling was used to initially gate lymphocytes and identify live, CD5+, and IL-1+ cells in each sample. + , Ki-67 + The percentage of cells was assessed in triplicate. Data were analyzed using FlowJo version 10.6.1. The results, shown in Figure 7, indicate that several mAbs can significantly increase IFN-γ secretion from canine PBMCs, an indicator of increased T cell activation. PMX126, PM127, PM131, PMX151, and PM152 were able to increase T cell activation above the levels observed with Gilvemab and 1B9 Def2.

[0266] The effect of PD-1 blockade on IFN-gamma production in blood samples from cancer-bearing dogs (obtained from the RVC as described above) was evaluated. Canine leukocytes isolated from cancer-bearing dogs were stimulated with 1 mg / ml ConA in the presence of PMX126 or PMX127, and the cultures were incubated for 96 hours. Controls included cells incubated without antibody and an irrelevant IgG isotype-matched antibody. After 96 hours of incubation, the supernatants were collected, and the concentration of IFN-gamma in the supernatants was measured by ELISA (R&D Systems, USA). Increased IFN-gamma production was observed with both PMX126 and PMX127 antibodies compared to the control (Figure 13).

[0267] Example 13 Caninized cell-based assay PD-1 signaling reporter assay To evaluate the immunological activity of antibody candidates, their ability to inhibit PD-1 / PD-L1 inhibitory signaling was tested in a bioluminescent reporter PD-1 / PD-L1 cell-based assay adapted from a commercially available cell line (Invivogen, France). In this assay, NFAT-luc reporter Jurkat T cells were engineered to express chimeric canine (extracellular domain) / human (transmembrane and intracellular domain) 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 5 5 × 10 chimeric PD-1 NFAT-luc Jurkat cells were plated per well in the presence of 3 μg / mL of anti-canine PD-1 antibody candidate or control antibody. 4The cells were co-cultured with canine PD-L1 Raji-APC cells in a total volume of 200 μL. The co-cultures were incubated overnight at 37°C, and bioluminescence measurements were performed by adding the luciferase detection reagent QUANTI-Luc Gold (Invivogen, France) according to the manufacturer's instructions. The results, shown in Figure 8A and B, indicate that all assayed mAbs had the ability to inhibit PD-1 signaling by disrupting canine PD-1 / PD-L1 interaction. In this assay, PMX125, PMX126, and PMX127 resulted in significantly increased signaling blockade compared to both Gilvetmab and 1B9 Def2.

[0268] Example 14 In vivo evaluation: MC-38 tumor therapy in a caninized mouse model Transgenic C57BL / 6 mice carrying chimeric PD-1 with mouse transmembrane and intracellular domains (encoding residues 170VIG-WPL288) and a canine extracellular domain consisting of exon 2 and part of exon 3 (encoding residues 27SPD-QGL169) were generated by methods known to those skilled in the art. cPD-1 / cPD-1 A PD-L1-positive murine colorectal cancer cell line, designated MC-38, is routinely used for the evaluation of human checkpoint inhibitors. This cell line was stably transfected using the PiggyBac transposase system to express the canine PD-L1 orthologue (MC-38). cPD-1+ ) can be constitutively expressed. Transgenic C57BL / 6 cPD-1 / cPD-1 In mice, MC-38 cPD-1+ The cell line can grow at the same rate as MC-38. 2.5E5 MC-38 in 50% v / v formulation of PBS and Matrigel hESC (Corning) cPD-1+ Three days after subcutaneous inoculation of cells or when tumors reached 150 mm 3When tumor size reaches 100%, administer 2.5 mg / kg of anti-canine PD-1 mAb intraperitoneally once a week. Tumor size is monitored by measuring the tumor in two dimensions and calculating the derived volume using the following formula: tumor volume = ((width)) 2 × length) / 2.

[0269] Example 15 Epitope mapping Epitope mapping can be performed to determine the precise binding site of a monoclonal antibody against a protein, including the interacting residues. 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 involves sequentially mutating residues to alanine, measuring the interaction strength of the variants compared to the wild-type protein, and predicting the target residues that significantly contribute to binding. Alanine is chosen because its small side chain, consisting solely of a methyl group, and its standard Ramachandran plot characteristics make it unlikely to significantly affect the local alpha-carbon structure around the mutation.

[0270] Example 16 In vivo dog studies Lead candidate antibodies will be administered by intravenous infusion to healthy beagles at 3 or 0.6 mg / kg body weight. Pharmacokinetics (pK) will be monitored using a direct ELISA assay using the target of interest. A linear pK relationship is expected with an elimination half-life of approximately 8-15 days.

[0271] Plasma concentrations of canine PD-1 antibodies were detected using ELISA. For 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, serially diluted canine plasma (12-point dilutions from a starting concentration of 250 μg / mL) and a goat anti-canine IgG HRP-conjugated secondary antibody (Bethyl Labs) were added. Canine PD-1 plasma concentrations were determined using SuperSignal ELISA Pico Chemiluminescent Substrate (Thermofisher). Quantitation of bound PMX126 and PMX127 antibodies in plasma was determined by measuring the 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 one-phase decay model in GraphPad Prism software. A linear pK and a 5-day half-life were observed. (Fig. 14A, B, C)

[0272] Safety and tolerability are other key readouts. In particular, immune-related adverse events (irAEs) are closely monitored by veterinarians, and complete blood counts and liver enzyme activity tests are performed. In multi-dose studies, anti-drug immunogenicity is closely monitored by measuring the presence of anti-drug antibodies (ADAs) using standard approaches, including the acid dissociation or ion dissociation ELISA-based ADA assays described previously. 1、2 .

[0273] In the exploratory single-dose target animal safety and pK study, animals were monitored for adverse events during the in-life phase. Animal clinical observations, weight monitoring, hematology, and serum biochemistry analyses were performed before treatment and then weekly throughout the study period for all animals. Adverse events were classified using the Veterinary Cooperative Oncology Group (VCOG)-Common Terminology Criteria for Adverse Events (VCOG-CTCAE v2) following investigational treatment in dogs and cats. Single intravenous administration of PMX126 and PMX127 at doses of 0.6 and 3 mg / kg to healthy beagles did not affect body weight or clinical observations and was well tolerated. Clinical pathology analysis showed only mild and transient changes that may not be related to treatment (Figure 15).

[0274] Example 17 Alanine scanning mutagenesis Alanine scanning experiments were performed by generating 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 and wild-type plasmid was separately transfected into HEK293 cells to generate 134 stable cell lines using PiggyBac transposase technology combined with drug selection of the transgene. All cell lines were continuously cultured under selective pressure. To control for variable expression levels of PD-1 mutants across the 134 cell lines, PD-1-binding mAbs that fail to block PD-L1 binding were identified for use as PD-1 expression controls (IM84-187 and IM84-223). The inability of IM84-187 and IM84-223 to block the binding of PMX-126 and PMX-127 to PD-1 was extensively verified by real-time competition assays using SPR and endpoint competition assays using flow cytometry. Test mAbs PMX-126 and PMX-127 and expression control mAbs IM84-187 and IM84-223 were directly labeled with AlexaFluor-647 and AlexaFluor-555, respectively, using standard amine coupling chemistry. In each experiment, one test mAb (either PMX-126 or PMX-127) and one expression control mAb (IM84-187 or IM187-223) were simultaneously used to co-stain each mutant stable cell line at concentrations of 0.9 μg / mL (test mAb) and 4.1 μg / mL (expression control mAb). Binding to each stable cell line was measured by flow cytometry. Cell lines that showed lower proportional binding of the test mAb to the cell line compared to the expression control mAb were assumed to be responsible for test mAb binding. In practice, this was determined by a downward off-diagonal shift on the 2D scatter plots of AlexaFluor-647 (ordinate) and AlexaFluor-555 (abscissa). The proportional signal intensity shifts of both the AlexaFluor-647 and AlexaFluor-555 signals were attributed to different amounts of membrane-bound PD-1 on the cell surface of the different stable cell lines.This may be caused by factors such as the transgene integration copy number or location, or the expression level of the mutant PD-1 protein. Using this method, we identified several residue positions that interact with PMX-126 (D58, D61, N74, T76, and Y127) and PMX-127 (T76, Q83, E84, L128, P129, and P130), which are shown on the structural representation of the PD-1 / PD-L1 extracellular domain (Figure 9A, B and Figure 10A, B).

[0275] Example 18 Cross-reactivity to human PD-1 The cross-reactivity of the anti-canine mAbs PMX-126 and PMX-127 with human PD-1 protein was tested by SPR and ELISA. In the SPR-based assay, approximately 300 RU of antibodies PMX-126 and PMX-127 were captured on flow cell 2 of a Series S SensorChip Protein A (Cytiva), and recombinant canine PD-1-ECD-6His protein (produced in-house) or recombinant human PD-1-ED-6His protein (Bio-Techne, item BP164049-1mg) was injected at a concentration of 900 nM at a flow rate of 30 μL / min for 180 seconds. The assay was also performed by capturing a pembrolizumab analog (Biosynth AG) to confirm that the human PD-1-ECD-6His protein was correctly folded and active. Neither PMX-126 nor PMX-127 cross-reacted with human PD-1ECD-6His protein; pembrolizumab bound only to human PD-1ECD-6His protein (Figure 16A). ELISA assays were performed by coating 100 μL of recombinant canine PD-1-ECD-6His protein or 100 μL of recombinant human PD-1-ECD-6His protein, both at 3 μg / mL, overnight at 4°C. 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, 3-fold dilution series of unlabeled mAb at a highest concentration of 100 μg / mL for 1 hour at room temperature. Antibody binding was detected using an anti-dog IgG-HRP conjugate (anti-human IgG-HRP conjugate for pembrolizumab) diluted 1:8000, and ELISAs were developed with 100 μL of TMB substrate solution (Thermo Fisher). The reaction was stopped with 50 μL of 1 M H2SO4, and plates were read at 450 nm. Neither PMX-126 nor PMX-127 cross-reacted with human PD-1ECD-6His protein, even at concentrations up to 100 μg / mL (Figure 16B).

[0276] Example 19 Receptor occupancy The receptor occupancy (RO) of PD-1 antibodies on T cells was determined using a flow cytometry assay to identify unoccupied PD-1 receptors in PBMC samples from healthy beagles treated with PMX-126, PMX-127, or control antibodies. After isolating PBMCs using the previously described method, PBMC samples from dogs treated with PMX-126 were incubated with PE-conjugated mouse anti-canine CD5 (Biorad) and Alexa647-conjugated PMX-126 or an anti-canine IgG isotype control in the dark at 4°C for 30 minutes. PBMC samples from dogs treated with PMX127 were incubated with PE-conjugated mouse anti-canine CD5 and Alexa647-conjugated PMX-127 or an 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 (FIG. 17).

[0277] Example 20 Differential expression in genes related to T cell function Transcriptome analysis was performed to assess changes in gene expression profiles in PBMC samples from healthy beagles after treatment with 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 Bioanalyzer. Samples were processed at the nCounter Core Facility at University College London (UCL), UK, for detection of genes involved in T cell activation and function using the nCounter Canine IO Panel (NanoString Technologies). Data were analyzed using Rosalind software provided by NanoString. Criteria for significantly differentially expressed genes were selected based on a log2 fold change of less than or equal to 1.5 and a p<0.05 comparing day 7 samples with pretreatment samples. Data were visualized using volcano plots, heat maps, and histograms of specific genes. Group-wise comparisons were performed using pretreatment samples compared with day 7 samples from each antibody group (PMX126, PMX127, and isotype-matched controls). Transcriptome analysis revealed an increase in T cell activation signatures after treatment with PMX-126 and PMX-127 (Figures 18A and 18B). In particular, upregulation of IFGGC1, CD3D, ICOS, CXCL10, CD80, CCR5, IL-7, and INF-gamma was observed.

[0278] References: 1. Patton, A., Mullenix, MC, Swanson, S Jand Koren, E., 2005. An acid dissociation bridging ELISA for detection of antibodies directed against therapeutic proteins in the presence of antigen. Journal of immunological methods, 304(1-2), pp. 189-195. 2. Jordan, G., Pohler, A., Guilhot, F., Zaspel, M. and Staack, RF, 2020. High ionic strength dissociation assay (HISDA) for high drug tolerant immunogenicity testing. Bioanalysis, 12(12), pp. 857-866.

[0279] PD1 sequence Wild-type canine PD-1 nucleotide sequence (SEQ ID NO: 1): ATGGGGAGCCGGCGGGGGCCCTGGCCGCTCGTCTGGGCCGTGCTGCAGCTGGGCTGGTGGCCAGGATGGCTCCTAGACTCCCCTGACAGGCCCTGGAGCCCGCTCACCTTCTCCCCGGCGCAGCTCACGGTGCAGGAGGGAGAGAACGCCACGTTCACCTGCAGCCTGGCCGACATCCCCGACAGCTTCGTGCTCAACTGGTACCGCCTGAGCCCCCGCAACCAGACGGACAAGCTGGCCGCCTTCCAGGAGGACCGCATCGAGCCGGGCCGGGACAGGCGCTTCCGCGTCACGCGGCTGCCCAACGGGCGGGACTTCCACATGAGCATCGTCGCTGCGCGCCTCAACGACAGCGGCATCTACCTGTGCGGGGCCATCTACCTGCCCCCCAACACACAGATCAACGAGAGTCCCCGCGCAGAGCTCTCCGTGACGGAGAGAACCCTGGAGCCCCCCACACAGAGCCCCAGCCCCCCACCCAGACTCAGCGGCCAGTTGCAGGGGCTGGTCATCGGCGTCACGAGCGTGCTGGTGGGTGTCCTGCTACTGCTGCTGCTGACCTGGGTCCTGGCCGCTGTCTTCCCCAGGGCCACCCGAGGTGCCTGTGTGTGCGGGAGCGAGGACGAGCCTCTGAAGGAGGGCCCCGATGCAGCGCCCGTCTTCACCCTGGACTACGGGGAGCTGGACTTCCAGTGGCGAGAGAAGACGCCGGAGCCCCCGGCGCCCTGTGCCCCGGAGCAGACCGAGTATGCCACCATCGTCTTCCCGGGCAGGCCGGCGTCCCCGGGCCGCAGGGCCTCGGCCAGCAGCCTGCAGGGAGCCCAGCCTCCGAGCCCCGAGGACGGACCCGGCCTGTGGCCCCTC Wild-type canine PD-1 amino acid sequence (SEQ ID NO: 2): MGSRRGPWPLVWAVLQLGWWPGWLLDSPDRPWSPLTFSPAQLTVQEGENATFTCSLADIPDSFVLNWYRLSPRNQTDKLAAFQEDRIEPGRDRRFRVTRLPNGRDFHMSIVAARLNDSGIYLCGAIYLPPNTQINESPRAELSV TERTLEPPTQSPSPPPRLSGQLQGLVIGVTSVLVGVLLLLLLTWVLAAVFPRATRGACVCGSEDEPLKEGPDAAPVFTLDYGELDFQWREKTPEPPAPCAPEQTEYATIVFPGRPASPGRRASASSLQGAQPPSPEDGPGLWPL Wild-type canine PD-1 extracellular domain amino acid sequence (SEQ ID NO: 302): LDSPDRPWSPLTFSPAQLTVQEGENATFTCSLADIPDSFVLNWYRLSPRNQTDKLAAFQEDRIEPGRDRRFRVTRLPNGRDHMSIVAARLNDSGIYLCGAIYLPPNTQINESPRAELSVTERT PET002 control antibody: Gilvetmab heavy chain (SEQ ID NO: 296) EVQLVQSGGDLVKPGGSVRLSCVASGFNIKNTYMHWVRQAPGKGLQWIGRIAPANVDTKYAPKFQGKATISADTAKNTAYMQLNSLRAEDTAVYYCVLIYYDYDGDIDVWGQG TLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVP RPPDCPKCPOPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERT ISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK Gilvetmab light chain (SEQ ID NO: 297) DIVMTQTPLSLSVSLGEPASISCHASQNINVWLSWYRQKPGQIPQLLIYKASHLHTGVPDRFSGSGSGTDFTLRISRVEADDAGVYYCQQGQSWPLTFGQGTKVEIKRN DAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDSKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD 3B7-D9 heavy chain (SEQ ID NO: 298) EVQLVETGGGLVQPGRSLKLSCVASGFTFNNYWMSWTRQAPGKGLEWVASITNSGVSTYYPDSVKGRFTISRDNAQNTLYLQMNSLRSEDTATYFCTSALNWGYWYFDFWGP GTMVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSTVTVPSSRWPSETFTCNVVHPASNTKVDKPVPKESTCK CISCPVPESLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTIS KARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPGK 3B7-D9 light chain (SEQ ID NO: 299) DIVMTQTPSSQAVSAGEKVTMSCKSSQSLLYSENKKNYLAWYQRKPGQSPKLLIYWASTRESGVPDRFIGSGGTDFTLTISSVQAEDLAVYYCQQYYNFPLTFGSGTKLE IKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD 1B9 Def2 heavy chain (SEQ ID NO: 300) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSFWMNWVRLAPGAGLEWIGRVDPYDSETHYNQKFKDRAILTVDTSTSTAYMELSSLRAGDIAVYYCATQFGFSWLAYWGQGTL VTVSAASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPR PPDCPKCPAPESLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLGREDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNHIGLPPSPIERT ISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQKSLSHSPGK 1B9 Def2 light chain (SEQ ID NO: 301) EIVMTQSPGSLAGSLAGSAGESVSINCKSSQSLLYSSNQKNYLAWYQQKPGESPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINNLQAEDVGVYYCQQYYSNPYTFGQGTKLEI KRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDSKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

Claims

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

2. The antibody a) a LC comprising or consisting of an HC CDR1 sequence set forth in 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; an HC CDR2 sequence set forth in 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 set forth in 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 comprising or consisting of an amino acid sequence set forth in 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. an LC CDR1 sequence 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, and an 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) 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; 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 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. a CDR1 sequence and an 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 an 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 LC comprising or consisting of an HC CDR1 sequence set forth in 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; an HC CDR2 sequence set forth in 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 set forth in 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 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. a CDR1 sequence and an 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 an 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) 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; 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 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.

2. The antibody or antigen-binding portion thereof of claim 1, comprising: a LC CDR1 sequence and an 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.

3. The antibody a) 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; 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; 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, 9, 10, 11, 12, 13, or 14 amino acid differences compared to SEQ ID NO: 20; and an LC CDR1 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. a CDR2 sequence and an 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 b) a HC CDR1 sequence comprising or consisting of SEQ ID NO:207, or an amino acid sequence having one or two amino acid differences compared to SEQ ID NO:207; a HC CDR2 sequence comprising or consisting of SEQ ID NO:208, or an amino acid sequence having one, two, three, four, or five amino acid differences compared to SEQ ID NO:208; a HC CDR3 sequence comprising or consisting of SEQ ID NO:209, or an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen amino acid differences compared to SEQ ID NO:209; and a LC CDR1 sequence comprising or consisting of SEQ ID NO:210, or an amino acid sequence having one, two, or three amino acid differences compared to SEQ ID NO:210; and a LC CDR1 sequence comprising or consisting of SEQ ID NO:211, or an amino acid sequence having one or two amino acid differences compared to SEQ ID NO:

211. a CDR2 sequence and an 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 c) an 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; an 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; an 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 an 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 an LC CDR1 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.

3. The antibody or antigen-binding portion thereof of claim 1 or 2, comprising a LC CDR2 sequence and an 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. 1. An isolated canine antibody, or antigen-binding portion thereof, that binds to canine PD-1, said antibody comprising: a) 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; 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; 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, 9, 10, 11, 12, 13, or 14 amino acid differences compared to SEQ ID NO: 20; and an LC CDR1 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. a CDR2 sequence and an 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 b) a HC CDR1 sequence comprising or consisting of SEQ ID NO:207, or an amino acid sequence having one or two amino acid differences compared to SEQ ID NO:207; a HC CDR2 sequence comprising or consisting of SEQ ID NO:208, or an amino acid sequence having one, two, three, four, or five amino acid differences compared to SEQ ID NO:208; a HC CDR3 sequence comprising or consisting of SEQ ID NO:209, or an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen amino acid differences compared to SEQ ID NO:209; and a LC CDR1 sequence comprising or consisting of SEQ ID NO:210, or an amino acid sequence having one, two, or three amino acid differences compared to SEQ ID NO:210; and a LC CDR1 sequence comprising or consisting of SEQ ID NO:211, or an amino acid sequence having one or two amino acid differences compared to SEQ ID NO:

211. a CDR2 sequence and an 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 c) an 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; an 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; an 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 an 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 an LC CDR1 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. LC CDR2 sequence and an 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, i) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 17, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 18, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 19, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 20, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 21, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 22; ii) an HC CDR1 sequence comprising or consisting of SEQ ID NO:27, an HC CDR2 sequence comprising or consisting of SEQ ID NO:28, an HC CDR3 sequence comprising or consisting of SEQ ID NO:29, an LC CDR1 sequence comprising or consisting of SEQ ID NO:30, an LC CDR2 sequence comprising or consisting of SEQ ID NO:31, and an LC CDR3 sequence comprising or consisting of SEQ ID NO:

32. iii) an HC CDR1 sequence comprising or consisting of SEQ ID NO:7, an HC CDR2 sequence comprising or consisting of SEQ ID NO:8, an HC CDR3 sequence comprising or consisting of SEQ ID NO:9, an LC CDR1 sequence comprising or consisting of SEQ ID NO:10, an LC CDR2 sequence comprising or consisting of SEQ ID NO:11, and an LC CDR3 sequence comprising or consisting of SEQ ID NO:12; iv) an HC CDR1 sequence comprising or consisting of SEQ ID NO:37, an HC CDR2 sequence comprising or consisting of SEQ ID NO:38, an HC CDR3 sequence comprising or consisting of SEQ ID NO:39, an LC CDR1 sequence comprising or consisting of SEQ ID NO:40, an LC CDR2 sequence comprising or consisting of SEQ ID NO:41, and an 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 or consisting of SEQ ID NO:87, a HC CDR2 sequence comprising or consisting of SEQ ID NO:88, a HC CDR3 sequence comprising or consisting of SEQ ID NO:89, a LC CDR1 sequence comprising or consisting of SEQ ID NO:90, a LC CDR2 sequence comprising or consisting of SEQ ID NO:91, and a LC CDR3 sequence comprising or consisting of SEQ ID NO:92; x) a HC CDR1 sequence comprising or consisting of SEQ ID NO:97, a HC CDR2 sequence comprising or consisting of SEQ ID NO:98, a HC CDR3 sequence comprising or consisting of SEQ ID NO:99, a LC CDR1 sequence comprising or consisting of SEQ ID NO:100, a LC CDR2 sequence comprising or consisting of SEQ ID NO:101, and a LC CDR3 sequence comprising or consisting of SEQ ID NO:102; 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) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 117, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 118, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 119, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 120, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 12, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 122; xiii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 127, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 128, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 129, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 130, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 131, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 132; xiv) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 137, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 138, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 139, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 140, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 141, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 142; xv) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 147, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 148, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 149, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 150, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 151, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 152; xvi) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 157, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 158, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 159, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 160, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 161, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 162; xvii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 167, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 168, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 169, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 170, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 171, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 172; xviii) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 177, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 178, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 179, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 180, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 181, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 182; xix) 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 or consisting of SEQ ID NO: 192; xx) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 197, 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 or consisting of SEQ ID NO:207, 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; xxii) an HC CDR1 sequence comprising or consisting of SEQ ID NO:217, an HC CDR2 sequence comprising or consisting of SEQ ID NO:218, an HC CDR3 sequence comprising or consisting of SEQ ID NO:219, an LC CDR1 sequence comprising or consisting of SEQ ID NO:220, an LC CDR2 sequence comprising or consisting of SEQ ID NO:221, and an LC CDR3 sequence comprising or consisting of SEQ ID NO:222; xxiii) an HC CDR1 sequence comprising or consisting of SEQ ID NO:227, an HC CDR2 sequence comprising or consisting of SEQ ID NO:228, an HC CDR3 sequence comprising or consisting of SEQ ID NO:229, an LC CDR1 sequence comprising or consisting of SEQ ID NO:230, an LC CDR2 sequence comprising or consisting of SEQ ID NO:231, and an 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) an HC CDR1 sequence comprising or consisting of SEQ ID NO:257, an HC CDR2 sequence comprising or consisting of SEQ ID NO:258, an HC CDR3 sequence comprising or consisting of SEQ ID NO:259, an LC CDR1 sequence comprising or consisting of SEQ ID NO:260, an LC CDR2 sequence comprising or consisting of SEQ ID NO:261, and an LC CDR3 sequence comprising or consisting of SEQ ID NO:262; xxvii) an HC CDR1 sequence comprising or consisting of SEQ ID NO:267, an HC CDR2 sequence comprising or consisting of SEQ ID NO:268, an HC CDR3 sequence comprising or consisting of SEQ ID NO:269, an LC CDR1 sequence comprising or consisting of SEQ ID NO:270, an LC CDR2 sequence comprising or consisting of SEQ ID NO:271, and an LC CDR3 sequence comprising or consisting of SEQ ID NO:272; xxviii) a HC CDR1 sequence comprising or consisting of SEQ ID NO:277, a HC CDR2 sequence comprising or consisting of SEQ ID NO:278, a HC CDR3 sequence comprising or consisting of SEQ ID NO:279, a LC CDR1 sequence comprising or consisting of SEQ ID NO:280, a LC CDR2 sequence comprising or consisting of SEQ ID NO:281, and a LC CDR3 sequence comprising or consisting of SEQ ID NO:282; or xxix) The antibody or antigen-binding portion thereof of any one of claims 1 to 4, having a HC CDR1 sequence comprising or consisting of SEQ ID NO:287, a HC CDR2 sequence comprising or consisting of SEQ ID NO:288, a HC CDR3 sequence comprising or consisting of SEQ ID NO:289, a LC CDR1 sequence comprising or consisting of SEQ ID NO:290, a LC CDR2 sequence comprising or consisting of SEQ ID NO:291, and a LC CDR3 sequence comprising or consisting of SEQ ID NO:

292.

6. 6. The antibody or antigen-binding portion thereof of any one of claims 1 to 5, 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 thereto, 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, for example, an LC variable region sequence comprising SEQ ID NO:

6.

7. the antibody or antigen-binding portion thereof, 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 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 thereto, 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 thereto; 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 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 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 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 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 thereto, 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 thereto; 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 thereto, 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 thereto; or 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 thereto, 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 thereto; 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 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 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 thereto, 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 thereto; 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 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 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 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 l) 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 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 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 thereto, 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 thereto; 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 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 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 thereto, 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 thereto; 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 thereto, 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 thereto; 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 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 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 thereto, 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 thereto; 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 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 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 thereto, 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 thereto; 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 thereto, 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 thereto; 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 thereto, 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 thereto; 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 thereto, 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 thereto; 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 thereto, 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 thereto; 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 thereto, 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 thereto; 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 thereto, 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 thereto; 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 thereto, 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 thereto; 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 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 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 thereto, 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 thereto; The antibody or antigen-binding portion thereof according to any one of claims 1 to 6, having the following structure:

8. The antibody or antigen-binding portion thereof according to any one of claims 1 to 7, wherein the antigen-binding portion thereof is an scFv, Fv, heavy chain or single domain antibody.

9. 9. The isolated canine antibody or antigen-binding portion thereof of any one of claims 1 to 8, wherein the antibody or antigen-binding portion thereof binds to canine PD-1 in competition with PD-L1 and / or PD-L2.

10. The antibody or antigen-binding portion thereof of any one of claims 1 to 9, 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 any one of claims 1 to 10, 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. 14. The antibody or antigen-binding portion thereof of any one of claims 1 to 13, wherein the antibody or antigen-binding portion thereof is conjugated to a further moiety selected from a half-life extending moiety, a label, a cytotoxin, a liposome, a nanoparticle, or a radioisotope.

15. 15. The antibody or antigen-binding portion thereof of claim 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.

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

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

18. A method of treating a disease in a canine subject in need thereof, the method comprising administering an effective amount of an antibody or antigen-binding portion thereof described in any one of claims 1 to 15, or a pharmaceutical composition described in 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. 20. The antibody or antigen-binding portion thereof or pharmaceutical composition of claim 17 or 19, or the method of claim 18 or 19, further comprising separately administering to the subject another therapeutic agent.

21. 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 immunomodulatory agent, or an antibody or antibody fragment thereof.

22. 22. The antibody or antigen-binding portion thereof, or pharmaceutical composition, or method of claim 21, wherein the immunomodulatory agent 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. 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:133 and SEQ ID NO:135, or SEQ ID NO:.

24. The nucleic acid sequence of claim 23, comprising a sequence selected from 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: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 having at least 75% sequence identity to any one of the foregoing sequences.

25. A vector comprising the nucleic acid sequence of claim 23 or 24.

26. A host cell comprising a nucleic acid sequence according to claim 23 or 24, or a vector according to claim 25.

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

28. 28. The kit of claim 27, further comprising a reagent for detection of the antibody or antigen-binding portion thereof.

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

30. 1. A method for producing a canine antibody that binds to PD-1, comprising: 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 a library of antibodies from said mice; c) isolating antibodies from said library.

31. 16. A method for detecting PD-1 protein or an extracellular domain of PD-1 protein in a biological sample from a canine subject, comprising contacting the biological sample with 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 detectable label.

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

33. 17. A method for inhibiting tumor growth or metastasis, comprising contacting tumor cells with 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.

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

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

36. 16. 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. 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 an antibody or antigen-binding portion thereof according to any one of claims 1 to 15, or a binding agent according to claim 36 or 37.

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

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

41. 41. The combination therapy of claim 40, wherein the further therapeutic moiety is an antibody (optionally an antibody that binds to an immuno-oncological target), or a chemotherapeutic agent.

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