Fully canine anti-canine PD-1 antibodies and uses thereof

Abstract

The present disclosure relates to antibodies, binding polypeptides, and scFvs specific for canine programmed death protein 1 (PD-1), nucleic acids encoding them, methods of treatment, and other methods or uses thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 63 / 288,284, filed December 10, 2021, which is incorporated by reference herein in its entirety. [Background technology]

[0002] background The immune checkpoint inhibitors (ICIs), pembrolizumab and nivolumab, have revolutionized the treatment of patients with a variety of cancers, including melanoma, renal cell carcinoma, and non-small cell lung cancer. Such ICI therapy works by blocking receptors that negatively regulate the function of cytotoxic T cells. In effect, ICIs act to block signaling pathways that would otherwise suppress T cell function. Although immune checkpoint receptors normally play a key role in preventing immune responses from becoming toxic, many cancers exploit these negative signals to blunt T cell immune responses against them, often by expressing ligands for immune checkpoint receptors expressed on cytotoxic T cells.

[0003] Immunocompetent dogs develop spontaneous tumors that exhibit distinctive features such as chromosomal abnormalities, molecular subtypes, immune signatures, tumor heterogeneity, metastatic behavior, and response to chemotherapy. However, there are currently no commercially available fully canine anti-canine PD-1 (cPD-1) antibodies in the veterinary setting to realize the potential clinical benefits of checkpoint inhibition in the treatment of canine cancer patients.

[0004] There is a need in the art for the development of non-immunogenic canine antibodies against immune checkpoint molecules that can be used to treat veterinary cancers in canine subjects through blockade of PD-1 on tumor-specific T cells. Summary of the Invention

[0005] overview In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising: a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO:1, 27, or 43; a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO:2, 28, or 44; a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO:3, 29, or 45; a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO:4, 30, or 46; a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO:5, 31, or 47; and a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO:6, 32, or 48.

[0006] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:7, 9, 33, or 49; and a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:11, 35, or 51.

[0007] In some embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 19, 21, 23, 39, or 55, and an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 25, 41, or 57.

[0008] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:7, 9, 33, or 49; and a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:11, 35, or 51.

[0009] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7, 9, 33, or 49; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:11, 35, or 51.

[0010] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 19, 21, 23, 39, or 55, and an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 25, 41, or 57.

[0011] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO:19, 21, 23, 39, or 55, and an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO:25, 41, or 57.

[0012] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO:1, a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO:2, a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO:3, a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO:4, a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO:5, and a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO:6.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:7 or 9; and a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:11.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:19, 21, or 23, and an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:25.

[0015] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:7 or 9; and a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:11.

[0016] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:11.

[0017] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:11.

[0018] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:19, 21, or 23, and an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:25.

[0019] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO:19, and an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO:25.

[0020] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO:21, and an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO:25.

[0021] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO:23, and an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO:25.

[0022] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO:27, a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO:28, a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO:29, a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO:30, a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO:31, and a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO:32.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:33; and a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:35.

[0024] In some embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:39, and an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:41.

[0025] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:33; and a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:35.

[0026] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:33; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:35.

[0027] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:39, and an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:41.

[0028] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO:39, and an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO:41.

[0029] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO:43, a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO:44, a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO:45, a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO:46, a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO:47, and a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO:48.

[0030] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:49; and a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:51.

[0031] In some embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:55, and an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:57.

[0032] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:49; and a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:51.

[0033] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:49; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:51.

[0034] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:55, and an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:57.

[0035] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), the antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO:55, and an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO:57.

[0036] In some embodiments, the antibody or antigen-binding fragment thereof is or is derived from one of an IgG, IgM, IgE, IgA, or IgD molecule. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a full length antibody, a Fab, a single chain variable fragment (scFv), sc(Fv)2, dsFv, Fab, Fab', (Fab')2, and a diabody. In some embodiments, the antibody is a full length antibody. In some embodiments, the antibody is a canine antibody or a caninized antibody. In some embodiments, the antibody is a canine antibody. In some embodiments, the antibody binding fragment is an scFv. In some embodiments, the specific binding of the antibody or antigen-binding fragment thereof to cPD-1 interferes with the interaction of cPD-1 with a ligand of cPD-1 (e.g., canine programmed death-ligand 1).

[0037] In one aspect, disclosed herein is a canine antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1).

[0038] In one aspect, disclosed herein are canine antibodies or antigen-binding fragments thereof that specifically bind to canine programmed death protein 1 (cPD-1), including the antibodies or antigen-binding fragments thereof described herein. In some embodiments, the specific binding of the antibody or antigen-binding fragment thereof to cPD-1 interferes with the interaction of cPD-1 with a ligand of cPD-1 (e.g., canine programmed death-ligand 1).

[0039] In one aspect, the present invention provides a heavy chain variable region comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 27, and 43, HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 28, and 44, and HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 29, and 45; and a light chain variable region comprising light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 30, and 46, LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 31, and 47, and LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: Disclosed herein is a single chain variable fragment (scFv) that specifically binds to canine programmed death protein 1 (cPD-1), comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of NO:6, 32, and 48, wherein the heavy chain variable region and the light chain variable region are connected by a linker.

[0040] In one aspect, disclosed herein is a single chain variable fragment (scFv) that specifically binds canine programmed death protein 1 (cPD-1), comprising: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7, 9, 33, or 49; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:11, 35, or 51, wherein the heavy chain variable region and the light chain variable region are connected by a linker.

[0041] In one aspect, disclosed herein is a single chain variable fragment (scFv) that specifically binds to canine programmed death protein 1 (cPD-1), comprising an amino acid sequence as set forth in SEQ ID NO:13, 15, 17, 37, or 53.

[0042] In some embodiments, the specific binding of the antibody or antigen-binding fragment thereof to cPD-1 disrupts the interaction of cPD-1 with a ligand of cPD-1 (e.g., canine programmed death-ligand 1).

[0043] In one aspect, disclosed herein is a bispecific molecule comprising an antibody or antigen-binding fragment thereof described herein, a canine antibody described herein, or an scFv described herein linked to a molecule having a second binding specificity.

[0044] In one aspect, disclosed herein is an immunoconjugate comprising an antibody or antigen-binding fragment thereof described herein, a canine antibody described herein, or an scFv described herein, or a bispecific molecule described herein, linked to a therapeutic agent.

[0045] In one aspect, disclosed herein is an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof described herein, a canine antibody described herein, or an scFv described herein.

[0046] In one aspect, disclosed herein is an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the polynucleotide sequence set forth in SEQ ID NO:8, 10, 34, or 50; and a light chain variable region encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the polynucleotide sequence set forth in SEQ ID NO:12, 36, or 52.

[0047] In some embodiments, the heavy chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence set forth in SEQ ID NO:8, 10, 34, or 50. In some embodiments, the light chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence set forth in SEQ ID NO:12, 36, or 52.

[0048] In one aspect, disclosed herein is an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof comprising: a heavy chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:8; and a light chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:12.

[0049] In one aspect, disclosed herein is an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof comprising: a heavy chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:10; and a light chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:12.

[0050] In one aspect, disclosed herein is an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof comprising: a heavy chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:34; and a light chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:36.

[0051] In one aspect, disclosed herein is an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof comprising: a heavy chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:50; and a light chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:52.

[0052] In some embodiments, the antibody or antigen-binding fragment thereof is an IgG, IgM, IgE, IgA, or IgD molecule, or is derived from one of these. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, a Fab, a single-chain variable fragment (scFv), a sc(Fv)2, a dsFv, a Fab, a Fab', a (Fab')2, and a diabody. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a canine antibody or a caninized antibody. In some embodiments, the antibody is a canine antibody.

[0053] In one aspect, disclosed herein is an isolated nucleic acid encoding a single chain variable fragment (scFv) comprising: a heavy chain variable region comprising the nucleotide sequence set forth in SEQ ID NO:8, 10, 34, or 50; and a light chain variable region comprising the nucleotide sequence set forth in SEQ ID NO:12, 36, or 52, wherein the heavy chain variable region and the light chain variable region are connected by a linker.

[0054] In one aspect, disclosed herein is an isolated nucleic acid encoding a single chain variable fragment (scFv) comprising a polynucleotide sequence as set forth in SEQ ID NO:14, 16, 18, 38, or 54.

[0055] In some embodiments, the antibody or antigen-binding fragment thereof, or scFv specifically binds to canine programmed death protein 1 (cPD-1). In some embodiments, the specific binding of the antibody or antigen-binding fragment thereof, or scFv to cPD-1 disrupts the interaction of cPD-1 with a ligand of cPD-1 (e.g., canine programmed death-ligand 1).

[0056] In one aspect, the vector disclosed herein comprises the isolated nucleic acid described herein.In some embodiments, the vector is an expression vector.In some embodiments, the vector is selected from the group consisting of DNA vector, RNA vector, plasmid, lentivirus vector, adenovirus vector, adeno-associated virus vector and retrovirus vector.

[0057] In one aspect, disclosed herein is a host cell comprising the isolated nucleic acid described herein or the vector described herein.In some aspects, the host cell is of eukaryotic or prokaryotic origin.In some aspects, the host cell is of mammalian origin.In some aspects, the host cell is of bacterial origin.In some aspects, the host cell is a Chinese hamster ovary cell.

[0058] In one aspect, disclosed herein is a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described herein, a canine antibody as described herein, or an scFv as described herein, a bispecific molecule as described herein, or an immunoconjugate as described herein, and a pharma- ceutical acceptable excipient, carrier, or diluent.In some embodiments, the pharmaceutical composition is formulated for intravenous administration.In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration.In some embodiments, the pharmaceutical composition is formulated for intratumoral administration.

[0059] In one aspect, disclosed herein is a kit comprising an antibody or antigen-binding fragment thereof described herein, a canine antibody described herein, or an scFv described herein, a bispecific molecule described herein, an immunoconjugate described herein, or a pharmaceutical composition described herein and instructions for use.

[0060] In one aspect, disclosed herein is a method for increasing T cell proliferation and reducing T cell exhaustion in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment thereof described herein, a canine antibody described herein, or an scFv described herein, a bispecific molecule described herein, an immunoconjugate described herein, or a pharmaceutical composition described herein.

[0061] In one aspect, disclosed herein is a method for increasing IFNγ production in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment thereof described herein, a canine antibody described herein, or an scFv described herein, a bispecific molecule described herein, an immunoconjugate described herein, or a pharmaceutical composition described herein.

[0062] In one aspect, disclosed herein is a method for inhibiting the canine programmed death protein 1 (cPD-1) signaling pathway in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment thereof described herein, a canine antibody described herein, or an scFv described herein, a bispecific molecule described herein, an immunoconjugate described herein, or a pharmaceutical composition described herein.

[0063] In some embodiments, the subject has cancer, sepsis or septic shock, or a chronic infection (eg, a viral infection).

[0064] In one aspect, disclosed herein is a method for treating a disease or condition in a subject in need thereof, comprising administering an antibody or antigen-binding fragment thereof as described herein, a canine antibody as described herein, or an scFv as described herein, a bispecific molecule as described herein, an immunoconjugate as described herein, or a pharmaceutical composition as described herein. In some embodiments, the disease or condition comprises cancer, sepsis or septic shock, or a chronic infection (e.g., viral infection). In some embodiments, the method further comprises administering one or more additional therapies (e.g., chemotherapy) or interventions.

[0065] In some embodiments, the cancer is associated with the canine programmed death protein 1 (cPD-1) signaling pathway. In some embodiments, a ligand for cPD-1 (e.g., canine programmed death-ligand 1) is expressed on the subject's cancer cells. In some embodiments, cPD-1 is expressed on the subject's immune cells. In some embodiments, the immune cells comprise T cells (e.g., activated T cells), B cells, natural killer (NK) cells, regulatory T cells, macrophages, or dendritic cells (DCs). In some instances, the immune cells comprise activated T cells. In some instances, the immune cells comprise tumor infiltrating lymphocytes. In some embodiments, the cancer is selected from the group consisting of melanoma (e.g., metastatic malignant melanoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), head and neck cancer (e.g., squamous cell carcinoma of the head and neck), cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma (e.g., B cell lymphoma), adrenal gland cancer, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytic tumor, bone cancer, brain and spinal cord cancer, metastatic brain tumor, carotid body tumor, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, fibroplastic bone imperfecta, fibroblastoma ... ossium), fibrous dysplasia of bone, gallbladder or bile duct cancer, gestational trophoblastic disease, germ cell tumors, hematologic malignancies, hepatocellular carcinoma, pancreatic islet cell tumors, Kaposi's sarcoma, kidney cancer, lipoma / benign lipomatous tumors, liposarcoma / malignant lipomatous tumors, medulloblastoma, meningioma, Merkel cell carcinoma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndromes, neuroblastoma, neuroendocrine tumors, papillary thyroid cancer, parathyroid tumors, childhood cancer, peripheral nerve sheath tumors, pheochromocytoma, pituitary tumors, prostate cancer, posterior uveal melanoma, rare hematological disorders, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid metastatic cancer, uterine cancer, or any combination thereof. In some embodiments, the cancer comprises cervical cancer, lung cancer, liver cancer, ovarian cancer, skin cancer including melanoma and squamous cell carcinoma, colon cancer, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, pancreatic cancer, head cancer, and neck cancer.

[0066] In some embodiments, the antibody or antigen-binding fragment thereof, canine antibody, scFv, bispecific molecule, or immunoconjugate specifically binds to cPD-1. In some embodiments, the binding of the antibody or antigen-binding fragment thereof, canine antibody, scFv, bispecific molecule, or immunoconjugate to cPD-1 disrupts the interaction of cPD-1 with a ligand of cPD-1 (e.g., canine programmed death-ligand 1). In some embodiments, the subject is a dog.

[0067] In one aspect, disclosed herein is a method of making a pharmaceutical composition described herein, the method comprising mixing an antibody or antigen-binding fragment thereof described herein, a canine antibody described herein, or an scFv described herein, a bispecific molecule described herein, or an immunoconjugate described herein with a pharma- ceutically acceptable excipient, carrier, or diluent.

[0068] In one aspect, disclosed herein is a method of making a composition for treating cancer, sepsis or septic shock, or a chronic infection (e.g., a viral infection), comprising mixing an antibody or antigen-binding fragment thereof described herein, a canine antibody described herein, or an scFv described herein, a bispecific molecule described herein, or an immunoconjugate described herein with a pharma- ceutically acceptable excipient, carrier, or diluent. In some embodiments, the cancer is selected from the group consisting of melanoma (e.g., metastatic malignant melanoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), head and neck cancer (e.g., squamous cell carcinoma of the head and neck), cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma (e.g., B cell lymphoma), adrenal cancer, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytic tumor, bone cancer, brain and spinal cancer, metastatic brain tumor, carotid body tumor, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, fibroplastic bone imperfection, fibrous dysplasia of bone, gallbladder cancer or bile duct cancer, pregnancy-associated cancer, or gestational age-related cancer. trophoblastic disease, germ cell tumors, hematologic malignancies, hepatocellular carcinoma, islet cell tumors, Kaposi's sarcoma, kidney cancer, lipoma / benign lipomatous tumors, liposarcoma / malignant lipomatous tumors, medulloblastoma, meningioma, Merkel cell carcinoma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndromes, neuroblastoma, neuroendocrine tumors, papillary thyroid cancer, parathyroid tumors, childhood cancer, peripheral nerve sheath tumors, pheochromocytoma, pituitary tumors, prostate cancer, posterior uveal melanoma, rare hematological disorders, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid metastatic cancer, uterine cancer, or any combination thereof. In some embodiments, the cancer comprises cervical cancer, lung cancer, liver cancer, ovarian cancer, skin cancer including melanoma and squamous cell carcinoma, colon cancer, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, pancreatic cancer, head cancer, and neck cancer.

[0069] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof described herein, a canine antibody described herein, or an scFv described herein, a bispecific molecule described herein, or an immunoconjugate described herein, or a pharmaceutical composition described herein, for use as a medicament.

[0070] In one aspect, disclosed herein is an antibody or antigen-binding fragment thereof described herein, a canine antibody described herein, or an scFv described herein, a bispecific molecule described herein, or an immunoconjugate described herein, or a pharmaceutical composition described herein for use as a medicament for treating cancer, sepsis or septic shock, or a chronic infection (e.g., a viral infection).

[0071] In one aspect, disclosed herein is the use of an antibody or antigen-binding fragment thereof described herein, a canine antibody described herein, or an scFv described herein, a bispecific molecule described herein, or an immunoconjugate described herein, or a pharmaceutical composition described herein for the manufacture of a medicament.

[0072] In one aspect, disclosed herein is the use of an antibody or antigen-binding fragment thereof described herein, a canine antibody described herein, or an scFv described herein, a bispecific molecule described herein, or an immunoconjugate described herein, or a pharmaceutical composition described herein, for the manufacture of a medicament for treating cancer, sepsis or septic shock, or a chronic infection (e.g., a viral infection).

[0073] In one aspect, disclosed herein is a method for producing the antibody or antigen-binding fragment thereof described herein, the dog antibody described herein, or the scFv described herein, the bispecific molecule described herein, comprising culturing the host cell described herein.In some embodiments, the method further comprises incubating the host cell described herein in cell culture medium under conditions sufficient to allow expression and secretion of the antibody or antigen-binding fragment thereof described herein, the dog antibody described herein, or the scFv described herein.

[0074] INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0075] The following detailed description of the preferred embodiments of the present disclosure will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present disclosure, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0076] [Figure 1-1]Enrichment of the canine scFv phage display library after panning against the canine PD-1 antigen. Recombinant canine PD-1 was adsorbed to microtiter plates overnight at 4°C. Wells were washed and blocked with 2% milk in PBS (MPBS) for 1 h at 37°C. The initial unpanned library (P0) and libraries of phages obtained after each round of selection (P1-P4) were added to the coated plates and incubated for 1 h at 37°C. Plates were washed with PBS supplemented with 0.1% Tween (PBST) and bound phages were detected with a 1:5000 dilution of HRP-conjugated anti-M13 mAb in MPBS. Bound anti-M13 mAb was detected with ABTS substrate. OD was read at 405 nm after 30 min using a Molecular Devices SpectraMax 340 spectrophotometer. [Figure 1-2] See description of Figure 1-1. [Figure 2-1] Figures 2A-2B show the binding of randomly selected scFv-expressing phage clones from panning rounds 3 and 4 to canine PD-1. Polyclonal phage from the fourth round of panning against cPD-1 was used as a positive control in wells coated with cPD-1 and a negative control in wells coated with human (h)CD19 (Figure 2A). Polyclonal phage from the third round of panning against hCD19 in an unrelated study was used as a positive control in wells coated with hCD19 and a negative control for wells coated with cPD-1 (Figure 2A). In Figure 2B, polyclonal phage from the fourth round of panning against canine (c)IL13Rα2 in an unrelated study similarly served as a negative and positive control with wells coated with cPD-1 and cIL13Rα2, respectively. [Figure 2-2] See description of Figure 2-1. [Diagram 3]Figure 1 shows the ability of unique soluble scFv clones to bind cPD-1 by ELISA. 19 unique soluble scFvs were tested for binding to cPD-1 by ELISA. Increasing amounts of biotinylated canine PD-1 were captured on a streptavidin (SA)-coated ELISA plate. 0.25μg / ml of soluble hemagglutinin (HA)-tagged scFvs were added to the wells and bound scFvs were detected using an AP-conjugated anti-HA antibody. An irrelevant soluble HA-tagged scFv against a MERS (Middle East Respiratory Syndrome) antigen from a different project was used as a negative control. [Figure 4] Figure 1 shows a PD-1:PD-L1 inhibition assay. ELISA plates were coated with streptavidin. Soluble biotinylated canine PD-1 was incubated with increasing concentrations of canine PD-L1-Fc, either alone or in the presence of selected soluble anti-cPD-1 scFvs or irrelevant soluble scFvs against MERS. The pre-incubated mixtures were added to the ELISA plate and binding of PD-L1 to PD-1 was detected using an anti-Fc-AP conjugate. Boxed and numbered clones inhibited PD-1:PD-L1 interaction. [Figure 5-1] Figure 2 shows binding of soluble canine anti-PD-1 scFv to cell surface expressed PD-1. The scaffold human erythrocytic leukemia cell line K562 was genetically edited to ablate FcγRII (CD32) (KTδ32). Retroviral transduction of edited cells to express cPD-1 was performed and PD-1 positive target cells were selected in puromycin. Soluble purified HA-tagged scFv was incubated with target cells and bound scFv was detected using an anti-HA antibody. Soluble scFv against MERS and clone 3-7, which showed low affinity binding to cPD-1 by ELISA, were used as negative controls. [Figure 5-2] See description of Figure 5-1. [Figure 6]Figure 1 shows the assessment of full canine anti-PD-1 IgG4 binding to cell surface expressed PD-1 by flow cytometry. KTδ32 cells were retrovirally transduced to express cPD-1 and target positive cells were selected with puromycin. Full canine IgG4-HA tagged clones against canine PD-1 or an irrelevant MERS antibody were used to stain KTδ32 and KTδ32.cPD-1. Bound IgG4 molecules were detected using an anti-HA antibody. [Figure 7] We show that P3C6 antibody variants bind to cPD-1. P3C6 was selected for further development and testing based on functional analysis and cell surface binding. Mutations were introduced into the framework regions to increase the yield / developability of P3C6. Full-length IgG was generated and the variants were tested for their ability to bind soluble cPD-1 by ELISA. P3C6mut3.1 and P3C6mut3.2 have 4 and 5 amino acid substitutions, respectively, from the parent P3C6 clone, with only one amino acid difference between the variant sequences. P4B1 and P4-14 were used as positive controls. [Figure 8] Figure 2 shows that P3C6 antibody variants bind to cPD-1. KTδ32 cells were retrovirally transduced to express cPD-1 and target-positive cells were selected with puromycin. To confirm binding to cell surface expressed PD-1, cells were labeled with two P3C6 variants (mut3.1 and mut3.2), or P4B1. Bound antibody was detected with an anti-HA antibody. An irrelevant anti-MERS antibody was used as a negative control. Plots are gated on viable 7AAD- cells. [Figure 9] 9A-9B show the yield of P3C6 antibody mutants from transient transfection of 293T cells. [Figure 10-1]Figure 1 shows that P3C6 mutant antibodies enhance the proliferative capacity of canine T cells. Canine PBMCs were labeled with cell trace violet (CTV) and stimulated with 2.5 μg / ml concanavalin A in the presence of P4B1, P3C6 (mut3.1 and mut3.2) full length IgG, or an irrelevant MERS control antibody. Cells were labeled with anti-CD5 antibody and analyzed by flow cytometry at 96 hours. Plots are gated on viable CD5+ cells. Data from one dog are shown. Cells show increased proliferative capacity and predicted fold expansion when stimulated in the presence of anti-PD-1 IgG compared to MERS antibody. [Figure 10-2] See description of Figure 10-1. [Figure 11] Figure 2 shows that P3C6 IgG4 variants promote the production of IFN-γ from mitogen-activated canine T cells. Canine PBMCs were stimulated with 2.5 mg / ml concanavalin A in the presence of P4B1, P3C6 (mut3.1 and mut3.2) IgG4, or an irrelevant MERS antibody. Supernatants were harvested after 96 hours and assayed by ELISA for the presence of IFN-γ. Data from two dogs are shown. Both P3C6 variants show increased IFN-γ production when compared to the anti-MERS antibody and a second anti-PD-1 clone (P4B1). [Figure 12] Figure 1 shows that clone A6 binds to cell surface cPD-1. To confirm binding to cell surface expressed cPD-1, KTδ32 and KTδ32.cPD-1 cells were labeled with two P3C6 mutants (mut3.1 and mut3.2), or clone A6 reformatted as IgG4. Bound antibodies were detected using an anti-HA antibody. An antibody against an unrelated MERS virus antigen was used as a negative control. Plots are gated on viable 7AAD- cells. Where indicated, clones were pre-incubated with soluble cPD-1 to block antigen binding sites. Pre-blocking resulted in abolishment of clone A6 binding and reduced cell surface labeling of clones P3C6 mut3.1 and mut3.2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0077] Detailed Description Canine antibody phage display libraries can be used to identify antigen-specific single chain variable fragment (scFv) fusion proteins that specifically bind to canine programmed death protein 1 (cPD-1). These canine PD-1-specific scFvs can then be converted into full-length antibodies containing constant regions of canine origin, making them suitable for in vivo use in canine subjects. Full canine antibodies have a lower risk of inducing an immune response in canine subjects than mouse, chimeric, or caninized antibodies. In some cases, full canine anti-PD-1 can be non-immunogenic and effective in checkpoint inhibition in the treatment of canine cancer patients. Incorporating such full canine antibody therapy against canine PD-1 into the veterinary armamentarium can improve outcomes for pet dogs with malignant tumors.

[0078] Furthermore, this disclosure can shed light on questions in human immuno-oncology. The use of full canine anti-PD-1 in immune-competent canine cancer patients with spontaneous tumors that share similar features to their human counterparts can produce results that inform human clinical trial design. The development of full canine anti-PD-1 provides an important comparative tool for translational research in human immuno-oncology, allowing certain combination therapies, including checkpoint blockade, to be evaluated.

[0079] The present disclosure relates to an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (PD-1), comprising: (a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO:1, 27, or 43; (b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO:2, 28, or 44; (c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO:3, 29, or 45; (d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO:4, 30, or 46; (e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO:5, 31, or 47; and (f) a light chain complementarity determining region 3 (HCDR4) comprising the amino acid sequence set forth in SEQ ID NO:6, 32, or 48. The present invention provides an antibody or antigen-binding fragment thereof, comprising a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in NO:6, 32, or 48.

[0080] In some aspects, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (PD-1), the antibody or antigen-binding fragment thereof comprising: (a) a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:7, 9, 33, or 49; and (b) a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:11, 35, or 51.

[0081] In some aspects, disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (PD-1), the antibody or antigen-binding fragment thereof comprising: (a) an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 19, 21, 23, 39, or 55; and (b) an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 25, 41, or 57.

[0082] In some aspects, provided herein is a canine antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1).

[0083] In some aspects, the antibody is selected from the group consisting of: (a) a heavy chain variable region comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 27, and 43, HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 28, and 44, and HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 29, and 45; and (b) a light chain variable region comprising light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 30, and 46, LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 31, and 47, and LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: Provided herein is a single chain variable fragment (scFv) that specifically binds to canine programmed death protein 1 (cPD-1), comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of NO:6, 32, and 48, wherein the heavy chain variable region and the light chain variable region are connected by a linker.

[0084] In some aspects, provided herein is a single chain variable fragment (scFv) comprising: (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7, 9, 33, or 49; and (b) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:11, 35, or 51, wherein the heavy chain variable region and the light chain variable region are connected by a linker.

[0085] In some aspects, provided herein is a single chain variable fragment (scFv) that specifically binds canine programmed death protein 1 (cPD-1), comprising an amino acid sequence as set forth in SEQ ID NO:13, 15, 17, 37, or 53.

[0086] Also provided are bispecific molecules, immunoconjugates, nucleic acids, vectors, host cells, kits, compositions for the canine PD-1-specific antibodies and antibody binding fragments or scFvs described herein, and methods of making and using the same for the treatment of cancer in a subject (e.g., a canine subject).

[0087] definition In the detailed description, certain specific details are given to provide a thorough understanding of the various embodiments. However, one skilled in the art will understand that the provided embodiments can be practiced without these details. Unless the context otherwise requires, throughout this specification and the following claims, the word "comprise" and its conjugations, such as "comprises" and "comprising," should be interpreted in an open, inclusive sense, i.e., "including but not limited to." As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the term "or" is generally used in the sense including "and / or," unless the context clearly dictates otherwise. In addition, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0088] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0089] As used herein, the term "about" refers to an amount that is 10% or less closer to the amount stated.As used herein, the term "individual", "patient" or "subject" refers to an individual who has been diagnosed with, is suspected to be suffering from, or is at risk of developing at least one disease that the compositions and methods described are useful for treating.In certain embodiments, the individual is a mammal.In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak.In certain embodiments, the individual is a dog or canine.

[0090] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin from a natural source or a recombinant source, or can be an immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetrameric immunoglobulin molecule that contains two heavy chain polypeptides and two light chain polypeptides. Each polypeptide chain contains three complementarity determining regions (CDRs) that bind to an antigen, defining the antigen specificity of the antibody.

[0091] As used herein, the terms "antibody" and "antibodies" can also include polypeptides or polypeptide complexes derived from full-length antibodies. These polypeptide complexes can be naturally occurring or can be constructed from single chain antibodies or antibody fragments and retain antigen-specific binding ability. The antibodies of the present disclosure can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab')2, as well as single chain antibodies, scFv, canine antibodies, canine antibodies, humanized antibodies, and human antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).Many techniques known in the art can be used to prepare suitable antibodies, e.g., recombinant antibodies, monoclonal antibodies, or polyclonal antibodies (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). Genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be produced from hybridomas or plasma cells. Random combination of heavy and light chain gene products produces a large pool of antibodies with various antigen specificities (see, for example, Kuby, Immunology (3rd ed. 1997)). Techniques for producing single chain or recombinant antibodies (U.S. Patent Nos. 4,946,778, 4,816,567) can be adapted to produce the antibodies of the present disclosure.Also, transgenic mice, or other organisms such as other mammals, can be used to express humanized or human antibodies, and canine or canine antibodies (see, e.g., U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995). Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to a selected antigen (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)). Antibodies can also be made bispecific, i.e., capable of recognizing two different antigens (see, e.g., WO 93 / 08829; Traunecker et al., EMBO J. 10:3655-3659 (1991); and Suresh et al., Methods in Enzymology 121:210 (1986)). The antibodies can also be heteroconjugates, such as two covalently linked antibodies, or immunotoxins (see, for example, U.S. Pat. No. 4,676,980, WO 91 / 00360; WO 92 / 200373; and EP 03089).

[0092] As used herein, a molecule, peptide, polypeptide, antibody, or antibody fragment may be referred to as "bispecific" or "dual specific", including grammatical equivalents. Bispecific molecules have the ability to specifically bind to at least two structurally distinct targets. Specific binding can be achieved by binding two distinct binding moieties that are structurally distinct at the molecular level, including, but not limited to, distinct, non-identical amino acid sequences; or by binding with high affinity (e.g., about 1×10 -6 The specific binding of a single binding moiety to two structurally distinct targets with a KD of less than 1000 ng / mol / L can be the result of a single binding moiety capable of specifically binding to two structurally distinct targets. A molecule, peptide, polypeptide, antibody, or antibody fragment referred to as "multispecific" refers to a molecule that has the ability to specifically bind to at least three structurally distinct targets. A "bispecific antibody", including grammatical equivalents, refers to a bispecific molecule that preserves at least one fragment of an antibody capable of specifically binding to a target, such as a variable region, heavy or light chain, or one or more complementarity determining regions from an antibody molecule. A "multispecific antibody", including grammatical equivalents, refers to a multispecific molecule that preserves at least one fragment of an antibody capable of specifically binding to a target, such as a variable region, heavy or light chain, or one or more complementarity determining regions from an antibody molecule.

[0093] The "linker" herein is also referred to as a "linker sequence", "spacer", "tethering sequence", or grammatical equivalents thereof. The "linker" referred to herein connects two separate molecules that themselves have target binding, catalytic activity, or are naturally expressed and assembled as separate polypeptides or contain separate domains of the same polypeptide. For example, two separate binding moieties or heavy / light chain pairs. A number of strategies can be used to covalently link molecules to each other. The linkers described herein can be utilized to link the light chain variable region and the heavy chain variable region in an scFv molecule; or can be used to tether an scFv or other antigen-binding fragment to the N-terminus or C-terminus of an antibody heavy chain; or to the N-terminus or C-terminus of a light chain to create a bispecific or multispecific binding molecule. These include, but are not limited to, polypeptide linkages between the N-terminus and C-terminus of a protein or protein domain, linkages via disulfide bonds, and linkages via chemical cross-linking reagents. In one aspect of this embodiment, the linker is a peptide bond made by recombinant techniques or peptide synthesis. The linker peptide can primarily comprise the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should be of a length that is reasonable to link two molecules such that they assume the correct conformation relative to each other to retain the desired activity. In one embodiment, the linker is about 1-50 amino acids in length, or about 1-30 amino acids in length. In one embodiment, a linker of 1-20 amino acids in length can be used. Useful linkers include, for example, glycine-serine polymers (where n is an integer of at least 1), including (GS)n, (GSGGS)n (SEQ ID NO:65), (GGGGS)n (SEQ ID NO:67), and (GGGS)n (SEQ ID NO:66), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.Exemplary linkers for linking antibody fragments or single chain variable fragments can include AAEPKSS (SEQ ID NO:78), AAEPKSSDKTHTCPPCP (SEQ ID NO:79), GGGG (SEQ ID NO:80), or GGGGDKTHTCPPCP (SEQ ID NO:81). Alternatively, a variety of non-proteinaceous polymers can find use as linkers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.

[0094] The terms "complementarity determining region" and "CDR", which are synonymous with "hypervariable region" or "HVR", are known in the art to refer to non-contiguous sequences of amino acids in antibody variable regions that confer antigen specificity and / or binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3), and each light chain variable region has three CDRs (CDR-L1, CDR-L2, CDR-L3). "Framework region" and "FR" are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, each full-length heavy chain variable region has four FRs (FR-H1, FR-H2, FR-H3, and FR-H4), and each full-length light chain variable region has four FRs (FR-L1, FR-L2, FR-L3, and FR-L4). The precise amino acid sequence boundaries of a given CDR or FR can be determined by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745. ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 Jan;27(1):55-77 (the "IMGT" numbering scheme); Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun 8;309(3):657-70 (the "Aho" numbering scheme); and Whitelegg NR and Rees AR, "WAM: an improved algorithm for modelling antibodies on the WEB," Protein Eng. 2000 Dec;13(12):819-24 (the "AbM" numbering scheme). In certain embodiments, the CDRs of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof.

[0095] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, Kabat scheme is based on structural alignment, while Chothia scheme is based on structural information. The numbering of both Kabat and Chothia schemes is based on the length of the most common antibody region sequences, and insertions are addressed by insertion letters, e.g. "30a", and deletions are found in some antibodies. The two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. In certain embodiments, the CDRs of the antibodies described herein can be defined by the IMGT method.

[0096] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a natural antibody (V H and V L ) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three CDRs (see, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). H Domain or V L A V domain may be sufficient to confer antigen-binding specificity. Furthermore, an antibody that binds to a particular antigen may have a V domain derived from the antibody that binds to the antigen. H Domain or V L The domains are then each bound to a complementary V L Domain or V H Libraries of domains can be used to screen and isolated (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0097] The specific binding or binding of the antibody molecule described herein refers to the binding mediated by one or more CDR portions of the antibody.Not all CDRs are required for specific binding.Specific binding can be demonstrated, for example, by ELISA against a particular described target or antigen, which shows a significant increase in binding compared to isotype control antibody.

[0098] "Epitope" refers to the binding determinant of the antibody or fragment described herein that is minimally required for specific binding of the antibody or fragment to the target antigen. When the target antigen is a polypeptide, the epitope may be a continuous or discontinuous epitope. A continuous epitope is formed by one region of the target antigen, whereas a discontinuous epitope may be formed from two or more separate regions. A discontinuous epitope can be formed, for example, when the target antigen adopts a tertiary structure that brings together two amino acid sequences to form a three-dimensional structure to which the antibody binds. When the target antigen is a polypeptide, the epitope will generally be multiple amino acids linked into a polypeptide chain. A continuous epitope can include 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids. An epitope can include a consecutive polymer of amino acids, but not every amino acid of the polymer can be contacted by an amino acid residue of the antibody. Such non-contacting amino acids may be important for the structure and linkage of contacting amino acids, and therefore still constitute part of the epitope.Those skilled in the art can determine whether any given antibody binds to the epitope of a reference antibody, for example, by cross-blocking experiments with the reference antibody.In certain aspects, antibodies that bind to the same epitope of the described antibody are described herein.In certain aspects, antibodies that are competitively blocked by the described antibodies are described herein.In certain aspects, antibodies that compete for binding with the described antibodies are described herein.

[0099] The term "antibody fragment" refers to a polypeptide comprising or derived from a portion of an intact antibody, including the antigen-binding determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, single domain antibodies, such as camelid antibodies, composed of either the VL domain or the VH domain that exhibit sufficient affinity for the target (Riechmann, 1999, Journal of Immunological Methods 231:25-38), and multispecific antibodies formed from antibody fragments. Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, e.g., a fragment comprising a non-naturally occurring arrangement, e.g., having two or more antibody regions or chains linked by a synthetic linker, e.g., a polypeptide linker, and / or not generated by enzymatic digestion of a naturally occurring intact antibody. In some aspects, the antibody fragment is an scFv.

[0100] Fab or Fab' fragments contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' or Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab' fragments are generated by cleavage of disulfide bonds at the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those skilled in the art. Fab and F(ab')2 fragments lack the Fragment Crystallizable (Fc) region of intact antibodies, are cleared more rapidly from the circulation of animals, and can have less nonspecific tissue binding than intact antibodies. An "Fv" fragment is the smallest fragment of an antibody that contains a complete target recognition and binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VHVL dimer. In some cases, six CDRs confer target binding specificity to the antibody. However, in some cases, even a single variable domain (or half of an Fv that contains only three CDRs specific for a target) can have the ability to recognize and bind to a target. Single domain antibodies (sdAbs) / single chain fragments are composed of a single VH or VL domain that exhibits sufficient affinity for an antigen. Antibody fragments also include canine or caninized antibodies, or portions of canine or caninized antibodies. scFv (single chain Fv) refers to an antibody binding fragment that contains the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. Generally, scFv polypeptides further contain a polypeptide linker between the VH and VL domains, which allows the scFv to form a structure that is favorable for target binding.

[0101] The term "diabody" refers to small antibody fragments prepared by constructing scFv fragments with a short linker (about 5-10 residues) between the VH and VL domains such that interchain but not intrachain pairing of the variable domains is achieved, resulting in a bivalent fragment, i.e., a fragment with two antigen binding sites. Bispecific diabodies are heterodimers of two "crossover" scFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.

[0102] The term "linear antibody" generally refers to an antibody that comprises a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions with complementary light chain polypeptides. Linear antibodies can be bispecific or monospecific.

[0103] "Antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0104] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation. Kappa light chain and lambda light chain refer to the two major antibody light chain isotypes.

[0105] The term "synthetic antibody" as used herein means an antibody made using recombinant DNA techniques, such as an antibody expressed by a bacteriophage as described herein. The term should also be taken to mean an antibody that has been made by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein, or an amino acid sequence that specifies the antibody, and which DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques that are available and well known in the art.

[0106] The term "antigen" or "Ag" as used herein is defined as a molecule that elicits an immune response. This immune response can involve either antibody production or activation of specific immunocompetent cells, or both. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA that contains a nucleotide sequence or a partial nucleotide sequence that encodes a protein that elicits an immune response, as the term is used herein, therefore encodes an "antigen." Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be made, synthesized, or derived from a biological sample. Such biological samples can include, but are not limited to, a tissue sample, a tumor sample, a cell, or a biological fluid.

[0107] The term "anti-tumor effect" as used herein refers to a biological effect that can be manifested by a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, or an amelioration of various physiological symptoms associated with a cancerous condition. "Anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present disclosure in preventing the development of tumors in the first place.

[0108] As used herein, the term "autologous" is intended to refer to any material derived from the same individual that is subsequently reintroduced into the individual.

[0109] "Allogeneic" refers to a graft derived from a different animal of the same species.

[0110] "Xenogeneic" refers to a graft derived from an animal of a different species.

[0111] The term "cancer" as used herein is defined as a disease characterized by rapid and uncontrolled proliferation of abnormal cells.Cancer cells can spread locally or through bloodstream and lymphatic system to other parts of the body.Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain tumor, lymphoma, leukemia, lung cancer, etc.In a particular embodiment, the cancer is medullary thyroid cancer.

[0112] As used herein, the term "conservative sequence modification" is intended to refer to an amino acid modification that does not significantly affect or change the binding properties of the antibody containing that amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody of the present disclosure 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 that has a similar side chain. Families of amino acid residues that have similar side chains are 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 in the CDR regions of the antibodies of the present disclosure can be replaced with other amino acid residues from the same side chain family, and the modified antibodies can be tested for their ability to bind to GFRα4 using the functional assays described herein.

[0113] "Costimulatory ligand," as that term is used herein, includes molecules expressed by antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.) that specifically bind to a cognate costimulatory molecule on a T cell, thereby providing signals that mediate T cell responses, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by binding of the TCR / CD3 complex to a peptide-loaded MHC molecule. Costimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to the Toll ligand receptor, and ligands that specifically bind B7-H3. Costimulatory ligands also include antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40L, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83, among others.

[0114] "Costimulatory molecule" refers to a cell surface molecule expressed by T cells that specifically binds to a costimulatory ligand expressed by an antigen presenting cell (APC), thereby providing a "secondary signal" that, in combination with a "primary signal" delivered through the interaction of MHC / HLA antigens with the T cell receptor (TCR), results in optimal T cell activation, including, but not limited to, cytokine production and proliferation. Costimulatory molecules include, but are not limited to, ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40L, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0115] The term "dysregulated," when used in the context of levels of expression or activity of PD-1 or its ligands PD-L1 and PD-L2, refers to a level of expression or activity that differs from the level of expression or activity of PD-1 or its ligands in an otherwise identical healthy animal, organism, tissue, cell, or component thereof. The term "dysregulated" also refers to altered regulation of the levels of PD-1, PD-L1 expression and activity compared to regulation in an otherwise identical healthy animal, organism, tissue, cell, or component thereof.

[0116] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene in a cell or other biological system produces the protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0117] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNAs can contain introns.

[0118] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result. Such result may include, but is not limited to, inhibition of viral infection, as determined by any means suitable in the art.

[0119] As used herein, "endogenous" refers to any material that originates or is produced within an organism, cell, tissue, or system.

[0120] As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue, or system.

[0121] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0122] "Expression vector" refers to a vector that contains a recombinant polynucleotide, which contains an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses that incorporate recombinant polynucleotides (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0123] "Homologous" as used herein refers to the identity of subunit sequences between two polymer molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. If a subunit position in both of the two molecules is occupied by the same monomeric subunit; for example, if a position in each of the two DNA molecules is occupied by adenine, they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in the two sequences (e.g., 5 positions in a polymer 10 subunits in length) are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matching or homologous, the two sequences are 90% homologous.

[0124] "Humanized" or "caninized" and "chimeric" forms of non-human or non-canine (e.g., murine) antibodies are immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human or non-canine immunoglobulin. For the most part, humanized, caninized, and chimeric antibodies are human or canine immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human or non-canine species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some cases, residues from the Fv framework region (FR) of the human or canine immunoglobulin are replaced by corresponding non-human or non-canine residues. Furthermore, humanized, caninized, and chimeric antibodies can include residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, humanized, caninized, and chimeric antibodies will contain substantially all of at least one, and typically two, variable domains, where all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are of a human or canine immunoglobulin sequence. Caninized and chimeric antibodies will also optimally contain at least a portion of an immunoglobulin constant region (Fc), typically that of a canine immunoglobulin.

[0125] "Fully canine" or "canine antibody" refers to an immunoglobulin, such as an antibody, having an amino acid sequence that corresponds to that of an antibody produced by a canine or canine cell, including a canine antibody library, or a non-canine source utilizing a canine antibody repertoire or other canine antibody coding sequence. The term excludes caninized forms of non-canine antibodies that contain a non-canine antigen-binding region, such as those in which all or substantially all CDRs are non-canine. In some cases, a fully canine antibody does not contain a portion of an antibody sequence from a non-canine species. Canine antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact canine antibodies or intact antibodies with canine variable regions in response to antigen challenge. Such animals typically contain all or a portion of a canine immunoglobulin locus that replaces an endogenous immunoglobulin locus or is extrachromosomally present or randomly integrated into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin locus is generally inactivated. Canine antibodies can also be derived from canine antibody libraries, including phage display and cell-free libraries, that contain antibody coding sequences derived from the canine repertoire.

[0126] As used herein, "instruction material" includes publications, recordings, drawings, or any other medium of expression that can be used to communicate the usefulness of the disclosed compositions and methods. The instruction material of the disclosed kits can be, for example, affixed to a container containing the disclosed nucleic acids, peptides, and / or compositions, or shipped together with a container containing the disclosed nucleic acids, peptides, and / or compositions. Alternatively, the instruction material can be shipped separately from the container with the intention that the instruction material and the compound will be used jointly by the recipient.

[0127] "Identity" as used herein refers to percent (%) sequence identity to a reference polypeptide sequence, and is the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences to achieve the maximum percent sequence identity, introducing gaps if necessary, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of known ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, % amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is the property of Genentech, Inc., and the source code has been submitted with user documentation to the US Copyright Office, Washington DC, 20559, where it is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0128] In the situation where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (or alternatively, it can be said that a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0129] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0130] In the context of the present disclosure, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0131] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that a nucleotide sequence encoding a protein may contain introns in some versions.

[0132] The term "operably linked" refers to the functional link between a regulatory sequence and a heterologous nucleic acid sequence, resulting in the expression of the latter.For example, a first nucleic acid sequence is functionally linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.For example, a promoter is functionally linked to a coding sequence when the promoter affects the transcription or expression of the coding sequence.Generally, the DNA sequences that are functionally linked are contiguous and, when necessary to link two protein coding regions, are in the same reading frame.

[0133] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0134] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acid is a polymer of nucleotides. Thus, nucleic acid and polynucleotide as used herein are interchangeable. Those skilled in the art have the general knowledge that nucleic acid is a polynucleotide, which can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR™, and the like, and by synthetic means.

[0135] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can make up a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers, and longer chains, of which there are many types, and which are commonly referred to in the art as proteins. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0136] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell or introduced synthetic machinery required to initiate the specific transcription of a polynucleotide sequence.

[0137] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0138] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes a gene product to be produced in a cell under most or all physiological conditions of the cell.

[0139] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

[0140] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes a gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0141] "Signal transduction pathway" refers to the biochemical relationship between various signal transduction molecules that play a role in transmitting a signal from one part of a cell to another part of a cell. The phrase "cell surface receptor" includes molecules and complexes of molecules that can receive a signal and transmit the signal across the plasma membrane of a cell. One example of a "cell surface receptor" is human GFRα4.

[0142] "Single-chain antibody" refers to the antibody formed by recombinant DNA technology, in which the heavy and light fragments of immunoglobulin chains are linked together using engineered amino acid spans to reproduce the Fv region of antibody as a single polypeptide.Various methods for producing single-chain antibody are known, including those described in U.S. Patent No. 4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041.

[0143] The term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response can be elicited. As used herein, a "subject" or "patient" can be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals. Preferably, the subject is a canine.

[0144] As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell that is separated from other cell types that it is normally associated with in its naturally occurring state. In some cases, a population of substantially purified cells refers to a homogenous population of cells. In other cases, the term simply refers to a cell that is separated from the cells that it naturally associates with in its natural state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.

[0145] As used herein, the term "therapeutic" refers to treatment and / or prophylaxis. A therapeutic effect is achieved by suppression, amelioration, or eradication of a disease state.

[0146] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0147] As used herein, the phrases "under transcriptional control" or "operably linked" mean that the promoter is in the correct location and orientation relative to the polynucleotide so as to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0148] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

[0149] As used herein, the term "specifically binds" refers to an antibody or ligand that recognizes and binds to a cognate binding partner protein present in a sample (e.g., a stimulatory molecule and / or a costimulatory molecule present on a T cell) but does not substantially recognize or bind to other molecules in the sample.

[0150] The term "stimulation" refers to a primary response induced by binding of a stimulatory molecule (e.g., the TCR / CD3 complex) with its cognate ligand, thereby mediating a signaling event, such as, but not limited to, signaling through the TCR / CD3 complex. Stimulation can mediate changes in expression of certain molecules, such as downregulation of TGF-β, and / or remodeling of cytoskeletal structure.

[0151] "Stimulatory molecule," as that term is used herein, means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell and / or a tumor cell.

[0152] As used herein, "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.) or tumor cell, can specifically bind to a cognate binding partner (herein referred to as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies, among others.

[0153] Ranges: Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an invariant limitation on the scope of the disclosure. Thus, the description of a range should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0154] Binding polypeptides, antibodies, and scFvs In aspects, disclosed herein are antibodies or antigen-binding fragments thereof (or "binding polypeptides" used interchangeably) that are characterized by certain functional features or properties of the antibodies or antigen-binding fragments thereof. For example, the binding polypeptides and antibodies specifically bind to canine programmed death protein 1 ("cPD-1" or "canine PD-1" used interchangeably). The binding polypeptides and antibodies of the present disclosure can bind to canine PD-1 with high affinity. The binding polypeptides and antibodies of the present disclosure can specifically recognize canine PD-1 protein naturally expressed on a cell. In some cases, the binding polypeptides and antibodies of the present disclosure do not cross-react with other surface molecules on the cell. The binding polypeptides and antibodies of the present disclosure can specifically bind to canine PD-1 protein naturally expressed on a cell such that such binding prevents canine PD-1 from binding to its natural ligand, such as canine programmed death-ligand 1 ("cPD-L1" or "canine PD-L1"). In some cases, the cell is a tumor cell (e.g., melanoma, non-small cell lung cancer, and hepatocellular carcinoma). In some cases, the cell is an immune cell. In some cases, the immune cell comprises a T cell (e.g., an activated T cell), a B cell, a natural killer (NK) cell, a regulatory T cell, a macrophage, or a dendritic cell (DC). In some cases, the immune cell comprises an activated T cell. In some cases, the immune cell comprises a tumor-infiltrating lymphocyte. In some cases, cPD-L1 is overexpressed on a tumor cell. In some cases, the antibody or antigen-binding fragment thereof can bind to cPD-1 and inhibit the cPD-1 signaling pathway.

[0155] In certain aspects, the disclosure provides an antibody or antigen-binding domain thereof that specifically binds to canine programmed death protein 1 (PD-1). In some cases, the antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region 1 (HCDR1). In some cases, the antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region 2 (HCDR2). In some cases, the antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region 3 (HCDR3). In some cases, the antibody or antigen-binding fragment thereof comprises a light chain complementarity determining region 1 (LCDR1). In some cases, the antibody or antigen-binding fragment thereof comprises a light chain complementarity determining region 2 (LCDR2). In some cases, the antibody or antigen-binding fragment thereof comprises a light chain complementarity determining region 3 (LCDR3). In some cases, the antibody or antigen-binding fragment thereof comprises an antigen-binding domain that specifically binds to canine programmed death protein 1 (PD-1). In certain embodiments, the antigen binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), and a light chain variable region comprising three light chain complementarity determining regions (LCDRs).

[0156] In certain aspects, the disclosure provides an isolated binding polypeptide comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, 27, or 43. Also provided is an isolated binding polypeptide comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, 28, or 44. Also provided is an isolated binding polypeptide comprising an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, 29, or 45. Also provided is an isolated binding polypeptide comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, 30, or 46. Also provided is an isolated binding polypeptide comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, 31, or 47. Also provided is an isolated binding polypeptide comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:6, 32, or 48.

[0157] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO:1, 27, or 43, a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO:2, 28, or 44, a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO:3, 29, or 45, a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO:4, 30, or 46, a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO:5, 31, or 47, and a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO:6, 32, or 48. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1). In some cases, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:7, 9, 33, or 49. In some cases, the antibody or antigen-binding fragment comprises a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:11, 35, or 51. In some cases, the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:19, 21, 23, 39, or 55. In some cases, the antibody or antigen-binding fragment comprises an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:25, 41, or 57.

[0158] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:7, 9, 33, or 49, and a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:11, 35, or 51. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1).

[0159] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7, 9, 33, or 49; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:11, 35, or 51. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1).

[0160] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 19, 21, 23, 39, or 55, and an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 25, 41, or 57. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1).

[0161] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19, 21, 23, 39, or 55, and an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 25, 41, or 57. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1).

[0162] In certain aspects, the disclosure provides an isolated binding polypeptide comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the isolated binding polypeptide is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (PD-1). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:7 or 9. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 19, 21, or 23. In some embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 25.

[0163] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:7 or 9; and a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:11. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1).

[0164] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:11. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1).

[0165] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:11. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1).

[0166] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 19, 21, or 23, and an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 25. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1).

[0167] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19, and an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 25. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1).

[0168] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 21, and an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 25. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1).

[0169] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23, and an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 25. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1).

[0170] In certain aspects, the present disclosure provides an isolated binding polypeptide comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:27, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:28, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:29, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:30, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:31, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:32. In some embodiments, the isolated binding polypeptide is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (PD-1). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:33. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 39. In some embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 41.

[0171] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 33; and a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 35. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1).

[0172] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 33; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 35. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1).

[0173] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 39, and an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 41. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1).

[0174] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 39, and an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 41. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1).

[0175] In certain aspects, the present disclosure provides an isolated binding polypeptide comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:43, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:44, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:45, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:46, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:47, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:48. In some embodiments, the isolated binding polypeptide is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (PD-1). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:49. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:51. In some embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:55. In some embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:57.

[0176] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 49; and a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 51. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1).

[0177] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 49; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 51. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1).

[0178] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 55, and an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 57. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1).

[0179] In certain aspects, the disclosure provides an antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 55, and an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 57. In some cases, the antibody or antigen-binding fragment thereof specifically binds to canine programmed death protein 1 (PD-1).

[0180] Permissible variations in complementarity determining region (CDR) sequences will be known to those of skill in the art. For example, in some embodiments, an isolated binding polypeptide comprises a complementarity determining region (HCDR or LCDR) that comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 27, 28, 29, 30, 31, 32, 43, 44, 45, 46, 47, or 48. In some embodiments, the isolated binding polypeptide comprises a complementarity determining region (HCDR or LCDR) comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs:1, 2, 3, 4, 5, or 6. In some embodiments, the isolated binding polypeptide comprises a complementarity determining region (HCDR or LCDR) comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs:27, 28, 29, 30, 31, or 32.In some embodiments, the isolated binding polypeptide comprises a complementarity determining region (HCDR or LCDR) comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs:43, 44, 45, 46, 47, or 48. In some embodiments, the isolated binding polypeptide is an antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (PD-1).

[0181] In some embodiments, the isolated binding polypeptide binds to programmed death protein 1 (PD-1), e.g., canine PD-1. In some embodiments, the binding polypeptide comprises an antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG molecule, an IgM molecule, an IgE molecule, an IgA molecule, or an IgD molecule. In some embodiments, the antibody or antigen-binding fragment thereof is derived from an IgG molecule, an IgM molecule, an IgE molecule, an IgA molecule, or an IgD molecule. In some embodiments, the antigen-binding fragment is selected from the group consisting of a full-length antibody, a Fab, a single-chain variable fragment (scFv), a single-domain antibody, a sc(Fv)2, a dsFv, a Fab, a Fab', a (Fab')2, and a diabody. In further embodiments, the antibody is a full-length antibody. In still further embodiments, the antibody or antigen-binding fragment is a canine antibody or a caninized antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a canine antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a canine antibody. In some embodiments, the antibody or antigen-binding fragment is a canine IgG4 antibody. In some embodiments, the antibody or antigen-binding fragment is an scFv. In some embodiments, the antibody or antigen-binding fragment is a canine scFv or caninized scFv. In some embodiments, the antibody or antigen-binding fragment is a canine scFv. In some embodiments, the specific binding of the antibody or antigen-binding fragment to cPD-1 interferes with the interaction of cPD-1 with a ligand of cPD-1 (e.g., canine programmed death-ligand 1). In some embodiments, the specific binding of the antibody or antigen-binding fragment to cPD-1 inhibits the interaction of cPD-1 with a ligand of cPD-1 (e.g., canine programmed death-ligand 1). In some embodiments, the specific binding of the antibody or antigen-binding fragment to cPD-1 inhibits a signaling pathway activated by the interaction of cPD-1 with a ligand of cPD-1 (e.g., canine programmed death-ligand 1). In some embodiments, the specific binding of the antibody or antigen-binding fragment thereof to cPD-1 inhibits the cPD-1 signaling pathway.

[0182] In certain embodiments, a binding polypeptide comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:7, 9, 33, or 49. In certain embodiments, a binding polypeptide comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:7, 9, 33, or 49. In certain embodiments, a binding polypeptide comprises a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:7, 9, 33, or 49.

[0183] In certain embodiments, a binding polypeptide comprises a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:11, 35, or 51. In certain embodiments, a binding polypeptide comprises a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:11, 35, or 51. In certain embodiments, a binding polypeptide comprises a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:11, 35, or 51.

[0184] Also provided is an isolated binding polypeptide comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7 or 9, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:11.

[0185] Also provided is an isolated binding polypeptide comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:33, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:35.

[0186] Also provided is an isolated binding polypeptide comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:49, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:51.

[0187] In certain embodiments, the present disclosure includes antibodies that bind to the same epitope on canine PD-1 as the antibodies of the present disclosure (i.e., antibodies that have the ability to cross-compete with any of the antibodies of the present disclosure for binding to canine PD-1). In preferred embodiments, the reference antibody for cross-competition studies can be one of the antibodies described herein (e.g., P3C6mut3.1, P3C6mut3.2, P4B1, or A6 in Table 1 and Example 1). For example, Biacore analysis, ELISA assays, or flow cytometry can be used to demonstrate cross-competition with the antibodies of the present disclosure. The ability of the test antibody to inhibit, for example, P3C6mut3.1 or P3C6mut3.2 binding to cPD-1 demonstrates that the test antibody can compete with P3C6mut3.1 or P3C6mut3.2 for binding to cPD-1, and thus is considered to bind to the same epitope of PD-1 as P3C6mut3.1 or P3C6mut3.2.

[0188] The antibodies of the present disclosure can be prepared by using an antibody or any fragment thereof having one or more of the VH and / or VL sequences disclosed herein as starting material to engineer an engineered antibody, which can have altered properties compared to the starting antibody. The antibody can be engineered by modifying one or more amino acids in one or both variable regions (i.e., VH and / or VL), for example, in one or more CDR regions and / or in one or more framework regions. Additionally or alternatively, the antibody can be engineered by modifying residues in the constant region, for example, to change the effector function of the antibody.

[0189] Also provided is a canine antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1). The canine antibody or antigen-binding fragment thereof can be generated from a canine antibody phage display library. A process for generating a canine antibody or antigen-binding fragment thereof that specifically binds to cPD-1 is provided in Example 1. In some cases, such a canine antibody comprises any of the antigen-binding fragments or binding peptides disclosed herein. In some cases, the specific binding of the antibody or antigen-binding fragment thereof to cPD-1 interferes with the interaction of cPD-1 with a ligand of cPD-1 (e.g., canine programmed death-ligand 1).

[0190] Also provided are single chain variable fragments (scFv) that specifically bind to canine programmed death protein 1 (PD-1). Optionally, the scFv comprises an antigen-binding domain. Optionally, the specific binding of the antibody or antigen-binding fragment thereof to cPD-1 disrupts the interaction of cPD-1 with a ligand of cPD-1 (e.g., canine programmed death-ligand 1).

[0191] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins (e.g., mouse, dog, or human) covalently linked to form a VH:VL heterodimer. The heavy (VH) and light (VL) chains are either directly linked or linked by a peptide-encoding linker that connects the N-terminus of the VH to the C-terminus of the VL or the C-terminus of the VH to the N-terminus of the VL. In some embodiments, the antigen-binding domain (e.g., the PD-1 binding domain) comprises an scFv with the following configuration from N-terminus to C-terminus: VH-linker-VL. In some embodiments, the antigen-binding domain comprises an scFv with the following configuration from N-terminus to C-terminus: VL-linker-VH. One of skill in the art would be able to select an appropriate configuration for use in the present disclosure.

[0192] The linker is usually rich in glycine for flexibility and serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO 2014 / 087010, the contents of which are incorporated herein by reference in their entirety. Various linker sequences are known in the art, including, but not limited to, glycine serine (GS) linkers, e.g., (GS) n , (GSGGS) n (SEQ ID NO:65), (GGGS) n (SEQ ID NO:66), and (GGGGS) n (SEQ ID NO:67), where n represents an integer of at least 1. Exemplary linker sequences include, but are not limited to, TIFF2025503400000001.tif27156 and the like. One of skill in the art would be able to select an appropriate linker sequence for use in the present disclosure. In one embodiment, the scFv of the present disclosure comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL are represented by the nucleic acid sequence An amino acid sequence that can be encoded by TIFF2025503400000002.tif11128 TIFF2025503400000003.tif4128, where the arginine residue R is present as a result of including a nucleotide sequence for the restriction endonuclease Xba I. It will be appreciated by those skilled in the art that the presence of the restriction sites in the linker, together with those flanking the scFv construct, is useful for performing heavy / light chain "swapping" experiments for antibody optimization, if desired.

[0193] Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be expressed from nucleic acids containing sequences encoding VH and VL, as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. Patent Application Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity have been described (see, e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40)). Agonistic scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0194] In certain embodiments, the antigen-binding domain of the scFv comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) and a light chain variable region comprising three light chain complementarity determining regions (LCDRs). HCDR1 comprises the amino acid sequence (SEQ ID NO: 1, 27, or 43), and / or HCDR2 comprises the amino acid sequence (SEQ ID NO: 2, 28, or 44), and / or HCDR3 comprises the amino acid sequence (SEQ ID NO: 3, 29, or 45), and / or LCDR1 comprises the amino acid sequence (SEQ ID NO: 4, 30, or 46), and / or LCDR2 comprises the amino acid sequence (SEQ ID NO: 5, 31, or 47), and / or LCDR3 comprises the amino acid sequence (SEQ ID NO: 6, 32, or 48). The heavy chain variable region and the light chain variable region are connected by a linker.

[0195] Also provided is a single chain variable fragment (scFv) comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7, 9, 33, or 49, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:11, 35, or 51. The heavy chain variable region and the light chain variable region are connected by a linker.

[0196] In another aspect, there is provided a single chain variable fragment (scFv) comprising the amino acid sequence shown in SEQ ID NO: 13, 15, 17, 37, or 53. In another aspect, there is provided a single chain variable fragment (scFv) consisting of the amino acid sequence shown in SEQ ID NO: 13, 15, 17, 37, or 53.

[0197] Acceptable variations in scFv sequences will be known to those of skill in the art. For example, in some embodiments, the scFv comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs:13, 15, 17, 37, or 53.

[0198] In another aspect, a full length antibody is provided, comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19, 21, 23, 39, or 55, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 25, 41, or 57. In another aspect, a full length antibody is provided, consisting of a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19, 21, 23, 39, or 55, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 25, 41, or 57.

[0199] Acceptable variations in full-length antibody sequences will be known to those of skill in the art. For example, in some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs: 19, 21, 23, 39, or 55, and and a light chain comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NO:25, 41, or 57.

[0200] In certain embodiments, the full-length heavy or light chain of an antibody or antigen-binding fragment thereof of the present disclosure further comprises a leader sequence. In certain embodiments, the leader sequence comprises the amino acid sequence set forth in SEQ ID NO:60. In certain embodiments, the leader sequence is encoded by the nucleotide sequence set forth in SEQ ID NO:59.

[0201] In certain embodiments, the full-length heavy chain of an antibody or antigen-binding fragment thereof of the present disclosure further comprises a linker and an HA tag or a hemagglutinin tag at the carboxy terminus of the polypeptide. In certain embodiments, the heavy chain linker and HA tag comprise the amino acid sequence set forth in SEQ ID NO:62. In certain embodiments, the heavy chain linker and HA tag are encoded by the nucleotide sequence set forth in SEQ ID NO:61.

[0202] In certain embodiments, the scFv of the disclosure further comprises an IgG fragment and a 6xHIS or histidine-hemagglutinin tag at the carboxy terminus of the polypeptide. In certain embodiments, the scFv IgG fragment-6xHIS-hemagglutinin tag comprises the amino acid sequence set forth in SEQ ID NO:64. In certain embodiments, the scFv IgG fragment-6xHIS-hemagglutinin tag is encoded by the nucleic acid sequence set forth in SEQ ID NO:63.

[0203] Table 1: Sequences used in this disclosure TIFF2025503400000004.tif38163TIFF2025503400000005.tif227163TIFF2025503400000006.ti f226163TIFF2025503400000007.tif226163TIFF2025503400000008.tif226163TIFF20255034000 00009.tif227163TIFF2025503400000010.tif226163TIFF2025503400000011.tif226163TIFF202 5503400000012.tif228163TIFF2025503400000013.tif226163TIFF2025503400000014.tif153164

[0204] PD-1 blockade In some aspects, the binding polypeptides, antibodies, or antigen-binding fragments thereof described in the present disclosure can bind to canine PD-1 and thus block the interaction of canine PD-1 with its natural ligand and any downstream signaling pathways associated with such interaction. In some cases, the antibodies or antigen-binding fragments described herein can bind to a region on canine PD-1 that is the same region recognized by the natural ligand of canine PD-1. In some cases, the antibodies or antigen-binding fragments described herein can bind to a region on canine PD-1 that is a different region than the natural ligand of canine PD-1. In some cases, the binding of the antibodies or antigen-binding fragments described herein to canine PD-1 can interfere with the region recognized by the natural ligand of canine PD-1. In some cases, the binding of the antibodies or antigen-binding fragments described herein to canine PD-1 can prevent the interaction of canine PD-1 with its natural ligand, e.g., canine PD-L1. In some cases, binding of the antibodies or antigen-binding fragments described herein to canine PD-1 can inhibit the canine PD-1 signaling pathway, which can be activated by a natural ligand of canine PD-1, such as PD-L1.

[0205] Programmed death protein 1 (PD-1 or PD1), also known as "programmed cell death protein 1" and CD279, is a member of the immunoglobulin superfamily of transmembrane receptors, including both stimulatory and regulatory receptors. The intracellular domain of PD-1 contains two phosphorylation sites located in immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which deliver signals that oppose T cell activation via the T cell receptor (TCR). PD-1 is not expressed on naive resting CD4+ and CD8+ T cells, but is strongly upregulated on the surface of activated T cells. The ligands for PD-1 are PD-L1 and PD-L2, which are part of the B7 family of receptors, and both deliver negative signals to T cells. Expression of both PD-1 ligands can be constitutive or induced in various cell types. PD-L1 is generally expressed on antigen-presenting cells such as macrophages and dendritic cells in response to signals such as lipopolysaccharide (LPS), IFNγ, and GM-CSF, but can also be found on peripheral non-lymphoid cells, including microvascular endothelial cells and organ tissues such as the heart and lung. In particular, IFNγ signaling results in the expression of PD-L1, which contributes to limiting inflammation. PD-L2 is primarily expressed by antigen-presenting cells, but can also be found on a wide variety of tissues, including the gastrointestinal tract, skeletal muscle, and pancreas.

[0206] Both PD-L1 and PD-L2 can be strongly expressed by many cancers as a mechanism to evade immune recognition and killing, effectively blunting T cell responses against them. Indeed, high expression of PD-1 ligands often correlates with poor prognosis and more aggressive disease. Similarly, IFNγ signaling in the tumor microenvironment, for example, caused by the presence of antitumor cytotoxic CD8+ T cells, can stimulate expression of PD-L1 by many types of tumor cells. Tumor-associated expression of PD-1 on tumor-infiltrating T cells and PD-L1 on tumor cells has been identified in numerous primary cancers, including, but not limited to, cervical cancer, lung cancer, liver cancer, ovarian cancer, cancer of the skin, including melanoma and squamous cell carcinoma, colon cancer, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, pancreatic cancer, and head and neck cancer, among others.

[0207] Blocking and inhibiting PD-1 signaling pathway can be used as a treatment strategy for chronic infection, and suppressing PD-1 can reverse or prevent T cell exhaustion and maintain cytotoxic function. Chronic infections include viral infections. Such infections are often characterized by exhaustion or dysfunction of CD8+ T cells, which contributes to the subject's inability to resolve the infection. Blocking PD-1 in animal models of chronic infection (e.g., dogs or pet dogs) can prevent T exhaustion, maintain both the number and function of responding T cells, and improve pathogen clearance and subject survival.

[0208] The fully canine anti-PD-1 disclosed herein can be used to inhibit the PD-1 signaling pathway and serves as an important comparative tool for anti-tumor therapy in dogs and translational research in human immuno-oncology.

[0209] Nucleic acids and expression vectors The present disclosure provides an isolated nucleic acid encoding a polypeptide. The nucleic acid of the present disclosure can include a polynucleotide sequence encoding any one of the binding polypeptides, scFvs, antibodies, or any fragments thereof disclosed herein.

[0210] One aspect of the disclosure includes an isolated nucleic acid encoding a binding polypeptide comprising an antigen-binding domain that specifically binds to canine programmed death protein 1 (PD-1).

[0211] In certain embodiments, the nucleic acid comprises an antigen-binding domain comprising a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein HCDR1 comprises the amino acid sequence (SEQ ID NO:1, 27, or 43), and / or HCDR2 comprises the amino acid sequence (SEQ ID NO:2, 28, or 44), and / or HCDR3 comprises the amino acid sequence (SEQ ID NO:3, 29, or 45), and / or LCDR1 comprises the amino acid sequence (SEQ ID NO:4, 30, or 46), and / or LCDR2 comprises the amino acid sequence (SEQ ID NO:5, 31, or 47), and / or LCDR3 comprises the amino acid sequence (SEQ ID NO:6, 32, or 48).

[0212] In certain embodiments, the binding polypeptide comprises an antibody or an antigen-binding fragment thereof. In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, single chain variable fragment (scFv), single domain antibody, sc(Fv)2, dsFv, Fab, Fab', (Fab')2, and diabody. In certain embodiments, the antibody is a full-length antibody. In certain embodiments, the antibody or antigen-binding fragment is a canine antibody or caninized antibody or a fragment thereof.

[0213] In certain embodiments, the heavy chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to SEQ ID NO:8, 10, 34, or 50.

[0214] In certain embodiments, the heavy chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence set forth in SEQ ID NO:8, 10, 34, or 50.

[0215] In certain embodiments, the heavy chain variable region is encoded by a nucleic acid consisting of the polynucleotide sequence set forth in SEQ ID NO:8, 10, 34, or 50.

[0216] In certain embodiments, the light chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence of the light chain variable region set forth in SEQ ID NO:12, 36, or 52. In certain embodiments, the light chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence set forth in SEQ ID NO:12, 36, or 52. In certain embodiments, the light chain variable region is encoded by a nucleic acid consisting of the polynucleotide sequence set forth in SEQ ID NO:12, 36, or 52.

[0217] Also provided is an isolated nucleic acid encoding a binding polypeptide comprising a heavy chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:8 or 10, and a light chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:12.

[0218] Also provided is an isolated nucleic acid encoding a binding polypeptide comprising a heavy chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:34, and a light chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:36.

[0219] Also provided is an isolated nucleic acid encoding a binding polypeptide comprising a heavy chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:50, and a light chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:52.

[0220] Also provided is an isolated nucleic acid encoding a single chain variable fragment (scFv) comprising a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein HCDR1 comprises the amino acid sequence (SEQ ID NO: 1, 27, and 43), and / or HCDR2 comprises the amino acid sequence (SEQ ID NO: 2, 28, or 44), and / or HCDR3 comprises the amino acid sequence (SEQ ID NO: 3, 29, and 45), and / or LCDR1 comprises the amino acid sequence (SEQ ID NO: 4, 30, or 46), and / or LCDR2 comprises the amino acid sequence (SEQ ID NO: 5, 31, or 47), and / or LCDR3 comprises the amino acid sequence (SEQ ID NO: 6, 32, or 48).

[0221] Also provided is an isolated nucleic acid encoding a single chain variable fragment (scFv) comprising a heavy chain variable region comprising the nucleotide sequence set forth in SEQ ID NO:8 or 10; and / or a light chain variable region comprising the nucleotide sequence set forth in SEQ ID NO:12. The heavy chain variable region and the light chain variable region are connected by a linker.

[0222] Also provided is an isolated nucleic acid encoding a single chain variable fragment (scFv) comprising the polynucleotide sequence set forth in SEQ ID NO: 14, 16, 18, 38, or 54. Also provided is an isolated nucleic acid encoding a single chain variable fragment (scFv) consisting of the polynucleotide sequence set forth in SEQ ID NO: 14, 16, 18, 38, or 54.

[0223] Also provided is an isolated nucleic acid encoding a full-length antibody comprising a heavy chain polynucleotide sequence set forth in SEQ ID NO:20, 22, 40, or 56, and a light chain polynucleotide sequence set forth in SEQ ID NO:26, 42, or 58. Also provided is an isolated nucleic acid encoding a full-length antibody consisting of a heavy chain polynucleotide sequence set forth in SEQ ID NO:20, 22, 40, or 56, and a light chain polynucleotide sequence set forth in SEQ ID NO:26, 42, or 58.

[0224] Acceptable variations in nucleic acid sequences will be known to one of skill in the art. For example, in some embodiments, a nucleic acid encoding a full-length heavy chain comprises a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the nucleotide sequences set forth in SEQ ID NOs:20, 22, 40, or 56, and a nucleic acid encoding a full-length light chain comprises a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the nucleotide sequences set forth in SEQ ID NOs:20, 22, 40, or 56. The present invention includes a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the nucleotide sequences set forth in SEQ ID NO:26, 42, or 58.

[0225] In certain embodiments, the nucleic acid of the present disclosure comprises a first polynucleotide sequence and a second polynucleotide sequence. The first polynucleotide sequence and the second polynucleotide sequence can be connected by a linker. For example, in certain embodiments, the heavy chain variable region and the light chain variable region of the scFv are connected by a linker. In certain embodiments, the nucleic acid comprises, from 5' to 3', the first polynucleotide sequence, the linker, and the second polynucleotide sequence. In certain embodiments, the nucleic acid comprises, from 5' to 3', the second polynucleotide sequence, the linker, and the first polynucleotide sequence.

[0226] Another aspect of the present disclosure provides a vector, comprising any one of the isolated nucleic acids disclosed herein.In certain embodiments, the vector is selected from the group consisting of DNA vector, RNA vector, plasmid, lentivirus vector, adenovirus vector, adeno-associated virus vector and retrovirus vector.In certain embodiments, the vector is an expression vector.

[0227] Also provided is a host cell comprising any of the vectors or nucleic acids disclosed herein. The host cell can be of eukaryotic, prokaryotic, mammalian, or bacterial origin. Non-limiting examples of cells that can be used to express the antigen-binding fragments of anti-cPD-1 antibodies or scFvs disclosed herein include human embryonic kidney (HEK) cell lines (e.g., HEK293), Chinese hamster ovary (CHO) cell lines, baby hamster kidney (BHK) cell lines, COS cell lines, Madin Darby canine kidney (MDCK) cell lines, and HeLa cell lines. In some cases, the host cell is a Chinese hamster ovary cell.

[0228] Also provided herein are methods of producing an antibody, binding polypeptide, or scFv that binds canine PD-1, the methods comprising culturing a host cell. In some embodiments, the methods further comprise incubating the host cell in cell culture medium under conditions sufficient to allow expression and secretion of an anti-cPD-1 antibody or antigen-binding fragment thereof, or scFv, as described herein.

[0229] In some aspects, the nucleic acids of the disclosure can be operably linked to transcriptional control elements, such as promoters and enhancers. Suitable promoter and enhancer elements are known to those of skill in the art.

[0230] In certain embodiments, the nucleic acid is operably linked to a promoter. In certain embodiments, the promoter is a phosphoglycerate kinase-1 (PGK) promoter.

[0231] For expression in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P, and trc. For expression in eukaryotic cells, suitable promoters include, but are not limited to, light and / or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoter; promoters present in long terminal repeats from retroviruses; mouse metallothionein-I promoter; and tissue-specific promoters known in various technical fields. Suitable reversible promoters, including reversible inducible promoters, are known in the art. Such reversible promoters can be isolated and derived from many organisms, for example, eukaryotes and prokaryotes. For example, the modification of reversible promoters from a first organism for use in a second organism, such as a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, is well known in the art.Such reversible promoters, and systems based on such reversible promoters but also including additional regulatory proteins, include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator protein (A1cR), etc.), tetracycline-regulated promoters (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g., metallothionein promoter system, etc.), pathogenesis-associated regulated promoters (e.g., salicylic acid-regulated promoters, ethylene-regulated promoters, benzothiadiazole-regulated promoters, etc.), temperature-regulated promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulated promoters, synthetic inducible promoters, etc.

[0232] For expression in yeast cells, suitable promoters are constitutive promoters such as the ADH1 promoter, the PGK1 promoter, the ENO promoter, the PYK1 promoter, etc.; or regulatable promoters such as the GAL1 promoter, the GAL10 promoter, the ADH2 promoter, the PHOS promoter, the CUP1 promoter, the GALT promoter, the MET25 promoter, the MET3 promoter, the CYC1 promoter, the HIS3 promoter, the ADH1 promoter, the PGK promoter, the GAPDH promoter, the ADC1 promoter, the TRP1 promoter, the URA3 promoter, the LEU2 promoter, the ENO promoter, the TP1 promoter, and the AOX1 (e.g., for use in Pichia). The selection of the appropriate vector and promoter is well within the level of one skilled in the art. Promoters suitable for use in prokaryotic host cells include, but are not limited to, the bacteriophage T7 RNA polymerase promoter; the trp promoter; the lac operon promoter; hybrid promoters, such as the lac / tac hybrid promoter, the tac / trc hybrid promoter, the trp / lac promoter, the T7 / lac promoter; the trc promoter; the tac promoter, and the like; the araBAD promoter; in vivo regulated promoters, such as the ssaG promoter or related promoters (see, for example, U.S. Patent Application Publication No. 20040131637), the pagC promoter (Pulkkinen and Miller, J. Bacteriol. (1991) 173(1): 86-93; Alpuche-Aranda et al., Proc. Natl. Acad. Sci. USA (1992) 89(21): 10079-83), the nirB promoter (Harborne et al. Mol. Micro. (1992) 6:2805-2813) and the like (e.g., Dunstan et al., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine (2004) 22:3243-3255; and Chatfield et al., Biotechnol.(1992) 10:888-892); sigma 70 promoters, e.g., consensus sigma 70 promoters (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); stationary phase promoters, e.g., dps promoters, spv promoters, and the like; promoters from pathogenicity island SPI-2 (see, e.g., WO96 / 17951); actA promoters (see, e.g., Shetron-Rama et al., Infect. Immun. (2002) 70:1087-1096); rpsM promoters (see, e.g., Valdivia and Falkow Mol. Microbiol. (1996). 22:367); tet promoters (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds), Topics in Molecular and Structural Biology, Protein--Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); SP6 promoter (see, e.g., Melton et al., Nucl. Acids Res.(1984) 12:7035). Strong promoters suitable for use in prokaryotes such as Escherichia coli include, but are not limited to, Trc, Tac, T5, T7, and PLambda. Non-limiting examples of operators for use in bacterial host cells include, but are not limited to, the lactose promoter operator (the LacI repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator), the tryptophan promoter operator (when complexed with tryptophan, the TrpR repressor protein has a conformation that binds to the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and the tac promoter operator (see, e.g., deBoer et al., Proc. Natl. Acad. Sci. USA (1983) 80:21-25). .

[0233] Other examples of suitable promoters include the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive high levels of expression of any polynucleotide sequence operably linked to it. Other constitutive promoter sequences can also be used, including but not limited to the Simian Virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, EF-1 alpha promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present disclosure. The use of inducible promoters provides a molecular switch that can turn on expression of the operably linked polynucleotide sequence when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0234] In some embodiments, the locus or construct or transgene containing a suitable promoter is irreversibly switched through the induction of an inducible system. Suitable systems for the induction of irreversible switching are well known in the art, for example, induction of irreversible switching can use Cre-lox mediated recombination (see, for example, Fuhrmann-Benzakein, et al., Proc. Natl. Acad. Sci. USA (2000) 28:e99, the present disclosure of which is incorporated herein by reference). Any suitable combination of recombinases, endonucleases, ligases, recombination sites, etc., known in the art, can be used to create irreversibly switchable promoters. The methods, mechanisms, and requirements for performing site-specific recombination described elsewhere herein find use in the creation of irreversibly switched promoters and are well known in the art. See, e.g., Grindley et al. Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.), the present disclosures of which are incorporated herein by reference.

[0235] The nucleic acids of the present disclosure can be present in an expression vector and / or a cloning vector. Expression vectors can include a selection marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like. Numerous suitable vectors and promoters are known to those of skill in the art, and many are commercially available for making recombinant constructs of interest. The following vectors are provided by way of example and should not be construed as limiting in any way: Bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0236] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker that is functional in the expression host can be present. Suitable expression vectors include viral vectors (e.g., vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984, and WO 95 / 00655); adeno-associated virus (e.g., Ali et al., Hum. Gene Ther. (1998) 9: 81-86; Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4: 683 690; Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci.USA (1993) 90: 10613-10617); SV40; herpes simplex virus; viral vectors based on human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319-23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus).

[0237] Additional expression vectors suitable for use include, but are not limited to, lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpes virus vectors, engineered hybrid virus vectors, transposon-mediated vectors, etc. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY, and in other virology and molecular biology manuals. Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

[0238] Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0239] In some aspects, an expression vector (e.g., lentiviral vector) can be used to introduce a nucleic acid into a host cell. Thus, an expression vector (e.g., lentiviral vector) of the present disclosure can include a nucleic acid encoding a polypeptide. In some aspects, an expression vector (e.g., lentiviral vector) will include additional elements that aid in the functional expression of a polypeptide encoded therein. In some aspects, an expression vector that includes a nucleic acid encoding a polypeptide further includes a mammalian promoter. In one aspect, the vector further includes an elongation factor-1-alpha promoter (EF-1α promoter). The use of the EF-1α promoter can increase the efficiency of expression of downstream transgenes. A physiological promoter (e.g., EF-1α promoter) can be less likely to induce integration-mediated genotoxicity and can suppress the ability of a retroviral vector to transform stem cells. Other physiological promoters suitable for use in vectors (e.g., lentiviral vectors) are known to those of skill in the art and can be incorporated into the vectors of the present disclosure. In some aspects, a vector (e.g., lentiviral vector) further includes non-essential cis-acting sequences that can improve titer and gene expression. One non-limiting example of a non-essential cis-acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS), which is important for efficient reverse transcription and nuclear import. Other non-essential cis-acting sequences are known to those skilled in the art and can be incorporated into the vectors (e.g., lentiviral vectors) of the present disclosure. In some embodiments, the vector further comprises a post-transcriptional regulatory element. The post-transcriptional regulatory element can improve the translation of RNA, improve the expression of the transgene, and stabilize the RNA transcript. One example of a post-transcriptional regulatory element is the Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE). Thus, in some embodiments, the vector for the present disclosure further comprises a WPRE sequence. A variety of post-transcriptional regulatory elements are known to those skilled in the art and can be incorporated into the vectors (e.g., lentiviral vectors) of the present disclosure.The vectors of the present disclosure can further include additional elements, such as a rev response element (RRE) for RNA transport, a packaging sequence, and 5' and 3' long terminal repeats (LTRs). The term "long terminal repeat" or "LTR" refers to a domain of base pairs located at the end of a retroviral DNA, including the U3, R, and U5 regions. LTRs generally provide functions required for retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and for viral replication. In one embodiment, the vectors of the present disclosure (e.g., lentiviral vectors) include a 3'U3 deleted LTR. Thus, the vectors of the present disclosure (e.g., lentiviral vectors) can include any combination of elements described herein to enhance the efficiency of functional expression of the transgene. For example, the vectors of the present disclosure (e.g., lentiviral vectors) can include a WPRE sequence, a cPPT sequence, an RRE sequence, a 5'LTR, a 3'U3 deleted LTR' in addition to a nucleic acid encoding a CAR.

[0240] The vector of the present disclosure can be a self-inactivating vector. As used herein, the term "self-inactivating vector" refers to a vector in which the 3'LTR enhancer promoter region (U3 region) is modified (e.g., by deletion or substitution). Self-inactivating vectors can prevent viral transcription beyond the first round of viral replication. Thus, self-inactivating vectors can infect and then be integrated into the host genome (e.g., mammalian genome) only once, and cannot spread further. Thus, self-inactivating vectors can greatly reduce the risk of producing replication-competent viruses.

[0241] In some embodiments, the nucleic acid of the present disclosure can be RNA, for example, in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those skilled in the art; any known method can be used to synthesize RNA comprising a sequence encoding a polypeptide of the present disclosure. Methods for introducing RNA into a host cell are known in the art. See, for example, Zhao et al. Cancer Res. (2010) 15: 9053. The introduction of RNA comprising a nucleotide sequence encoding a polypeptide of the present disclosure into a host cell can be performed in vitro, ex vivo, or in vivo. For example, host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) can be electroporated with RNA comprising a nucleotide sequence encoding a polypeptide of the present disclosure in vitro or ex vivo.

[0242] To evaluate the expression of a polypeptide or a part thereof, the expression vector introduced into the cell can also contain either a selection marker gene or a reporter gene, or both, so as to facilitate the identification and selection of expressing cells from a population of cells that are to be transfected or infected through a viral vector. In some embodiments, the selection marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selection marker and the reporter gene can be adjacent to the appropriate regulatory sequence to allow expression in the host cell. Useful selection markers include, but are not limited to, antibiotic resistance genes.

[0243] Reporter genes are used to identify cells that may be transfected and to evaluate the functionality of regulatory sequences.Generally, reporter genes are genes that are not present in or expressed by recipient organisms or tissues, and code for a polypeptide whose expression is manifested by some easily detectable property, for example, enzymatic activity.The expression of reporter genes is evaluated at a suitable time after DNA is introduced into recipient cells.Suitable reporter genes can include, but are not limited to, genes that code for luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).

[0244] In some aspects, the nucleic acids of the disclosure provide for the production of a polypeptide described herein, e.g., in a host cell. In some aspects, the nucleic acids of the disclosure provide for the amplification of nucleic acids encoding the polypeptides.

[0245] Immunoconjugates The present disclosure also provides immunoconjugates comprising any of the anti-cPD-1 antibodies or antigen-binding fragments, or scFvs disclosed herein. In some embodiments, the immunoconjugate comprises a disclosed antibody or antigen-binding fragment, or scFv, linked to an agent. In some embodiments, the immunoconjugate comprises a bispecific molecule disclosed herein, linked to an agent. The agent can be a therapeutic agent. The agent can be a diagnostic agent.

[0246] For diagnostic agents, suitable agents may include a detectable label. Non-limiting examples of detectable labels include radioisotopes for whole body imaging, as well as radioisotopes, enzymes, fluorescent labels, and other suitable antibody tags for sample testing. Detectable labels that can be linked to any of the anti-cPD-1 antibodies described herein are presented with particulate labels including metal sols such as colloidal gold, e.g., N2S2, N3S, or N4 type peptide chelators. 125 I or 99m They can be any of a variety of types currently used in the field of in vitro diagnostics, including isotopes such as Tc, chromophores including fluorescent, luminescent, phosphorescent, etc., as well as enzyme labels that convert a given substrate into a detectable marker, and polynucleotide tags that become evident after amplification, such as by the polymerase chain reaction. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, and the like. For example, the label can be the enzyme alkaline phosphatase, which is detected by measuring the presence or formation of chemiluminescence following conversion of 1,2 dioxetane substrates such as adamantyl methoxyphosphoryloxyphenyl dioxetane (AMPPD), 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.1 3,7}decan}-4-yl)phenyl phosphate disodium (CSPD), and CDP and CDP-STAR®, or other luminescent substrates well known to those of skill in the art, e.g., suitable lanthanide chelates such as terbium(III) and europium(III). The detection means will be determined by the label selected. Appearance of the label or its reaction products can be achieved using the naked eye, if the label is particulate and accumulates at appropriate levels, or using instruments such as spectrophotometers, luminometers, fluorometers, and the like, all in accordance with standard practice.

[0247] In some embodiments, the conjugation method results in substantially (or almost) non-immunogenic bonds, such as peptide bonds (i.e., amide bonds), sulfide bonds, (steric hindrance), disulfide bonds, hydrazone bonds, and ether bonds.These bonds are almost non-immunogenic and show reasonable stability in serum; see, for example, Senter, PD, Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO 2009 / 059278; WO 95 / 17886.

[0248] Depending on the biochemical properties of the agent and the anti-cPD-1 antibody or scFv, different conjugation strategies can be used. If the agent is naturally occurring or recombinant of 50-500 amino acids, standard procedures describing the chemistry for the synthesis of protein conjugates are available in textbooks and can be easily followed by one of skill in the art (see, e.g., Hackenberger, CPR, and Schwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In some embodiments, reaction of a maleimide agent with a cysteine ​​residue in the antibody or agent is used. This is a coupling chemistry that is particularly suitable, for example, when Fab or Fab' fragments of an antibody are used. Alternatively, in some embodiments, coupling to the C-terminus of the antibody or agent is performed. C-terminal modification of proteins, e.g., Fab fragments, can be performed as described (Sunbul, M. and Yin, J., Org. Biomol. Chem. 7(2009) 3361-3371).

[0249] In general, site-specific reactions and covalent couplings are based on converting natural amino acids into amino acids with reactivity orthogonal to that of other functional groups present. For example, specific cysteines in rare sequence contexts can be enzymatically converted to aldehydes (Frese, MA, and Dierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible to obtain the desired amino acid modifications by taking advantage of the specific enzymatic reactivity of certain enzymes with natural amino acids in a given sequence context (see, for example, Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier, A. et al. Chem. Biol. 15 (2008) 128-136; protease-catalyzed C-N bond formation is used by Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403). Site-specific reactions and covalent couplings can also be achieved by selective reaction of the terminal amino acid with an appropriate modifying reagent.

[0250] The reactivity of N-terminal cysteines with benzonitrile (see Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used to achieve site-specific covalent coupling.

[0251] Native chemical ligation can also rely on C-terminal cysteine ​​residues (Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96). US6437095 B1 describes a conjugation method based on the faster reaction of cysteines within a stretch of negatively charged amino acids with cysteines located in a stretch of positively charged amino acids.

[0252] The agent can also be a synthetic peptide or peptidomimetic.When the polypeptide is chemically synthesized, amino acids with orthogonal chemical reactivity can be incorporated during such synthesis (see, for example, de Graaf, AJ et al., Bioconjug. Chem. 20 (2009) 1281-1295).Conjugation of such peptides to linkers is standard chemistry, since a wide variety of orthogonal functional groups are of concern and can be introduced into synthetic peptides.

[0253] To obtain a single labeled polypeptide, the 1:1 stoichiometric conjugate can be separated from other conjugation by-products by chromatography. This procedure can be facilitated by using dye-labeled binding pair members and charged linkers. By using this type of labeled, strongly negatively charged binding pair member, the single conjugate polypeptide is easily separated from unlabeled polypeptides and polypeptides carrying more than one linker, since the difference in charge and molecular weight can be used for separation. Fluorescent dyes can be useful for purifying the complex from unbound components, such as labeled monovalent conjugates.

[0254] In some embodiments, the agent attached to the anti-cPD-1 antibody is selected from the group consisting of a binding moiety, a labeling moiety, and a biologically active moiety.

[0255] The anti-cPD-1 antibodies described herein can also be conjugated to a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In an ADC, the antibody and therapeutic agent are preferably conjugated via a cleavable linker, such as a peptidyl linker, a disulfide linker, or a hydrazone linker. In some embodiments, the linker is a peptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID NO:78), Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. ADCs can be prepared as described in U.S. Patent Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publication Nos. WO 02 / 096910; WO 07 / 038658; WO 07 / 051081; WO 07 / 059404; WO 08 / 083312; and WO 08 / 103693; U.S. Patent Application Publication Nos. 20060024317; 20060004081; and 20060247295.

[0256] The anti-cPD-1 antibodies or scFv described herein can also be used to detect canine PD-1, e.g., cPD-1 in a tissue or tissue sample. The antibodies can be used, for example, in an ELISA assay or flow cytometry. In some embodiments, the anti-cPD-1 antibody is contacted with cells, e.g., cells in a tissue, for a suitable amount of time for specific binding to occur, and then a reagent is added, e.g., an antibody that detects the anti-cPD-1 antibody. Exemplary assays are provided in the Examples. The anti-cPD-1 antibody can be a complete canine antibody. The anti-cPD-1 antibody can be a caninized or chimeric antibody. An exemplary method for detecting cPD-1 in a sample (cell or tissue sample) includes (i) contacting the sample with an anti-cPD-1 antibody for a time sufficient to allow specific binding of the anti-cPD-1 antibody to the cPD-1 in the sample, and (2) contacting the sample with a detection reagent, e.g., an antibody that specifically binds to the anti-cPD-1 antibody, such as the Fc region of the anti-cPD-1 antibody, thereby detecting cPD-1 bound by the anti-cPD-1 antibody. A wash step can be included after incubation with the antibody and / or detection reagent. The anti-cPD-1 antibody for use in these methods does not need to be linked to a label or detection agent, as a separate detection agent can be used.

[0257] bispecific molecules The anti-cPD-1 antibodies, antigen-binding fragments thereof, or scFvs described herein can be used to form bispecific molecules. The anti-cPD-1 antibodies, or antigen-binding portions thereof, can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or a ligand for a receptor), to create a bispecific molecule that binds to at least two different binding sites or target molecules.

[0258] For example, an anti-cPD-1 antibody can be linked to an antibody or scFv that specifically binds to any protein that can be used as a potential target for combination treatment. The antibodies described herein can in fact be derived or linked to more than one other functional molecule to create multispecific molecules that bind to more than two different binding sites and / or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create the bispecific molecules described herein, the antibodies described herein can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or other methods) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, such that a bispecific molecule results.

[0259] Provided herein are bispecific molecules comprising at least one first binding specificity for canine PD-1 and a second binding specificity for a second target epitope. In some embodiments described herein where the bispecific molecule is multispecific, the molecule can further comprise a third binding specificity.

[0260] In some embodiments, the bispecific molecules described herein comprise as binding specificities at least one antibody or antibody fragment thereof, including, for example, Fab, Fab', F(ab')2, Fv, or single chain Fv (scFv). The antibody can also be a light or heavy chain dimer, or any minimal fragment thereof, such as an Fv or single chain construct.

[0261] In some embodiments, canine monoclonal antibodies are used in the bispecific molecules, hi other embodiments, other antibodies can be used in the bispecific molecules described herein, including murine, chimeric, and caninized monoclonal antibodies.

[0262] The bispecific molecules described herein can be prepared by conjugating the constituent binding specificities using methods known in the art.For example, each binding specificity of the bispecific molecule can be made separately and then conjugated to each other.When the binding specificities are proteins or peptides, various coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linkers include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Fiu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229:81-83, and Glennie et al. (1987) J. Immunol. 139: 2367-2375. Some conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

[0263] When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In some embodiments, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

[0264] Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAh x mAh, mAh x Fab, mAh x (scFv)2, Fab x F(ab')2, or ligand x Fab fusion protein. The bispecific antibody can include an antibody that includes an scFv at the C-terminus of each heavy chain. The bispecific molecules described herein can be single chain molecules that include one single chain antibody and a binding determinant, or single chain bispecific molecules that include two binding determinants. The bispecific molecule can include at least two single chain molecules. Methods for preparing bispecific molecules are described, for example, in U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030; U.S. Patent No. 4,881,175; U.S. Patent No. 5,132,405; U.S. Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. Patent No. 5,013,653; U.S. Patent No. 5,258,498; and U.S. Patent No. 5,482,858.

[0265] Binding of a bispecific molecule to its specific target can be confirmed using art-recognized methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays generally detects the presence of a protein-antibody complex of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

[0266] Treatment Method In some aspects, the present disclosure provides a method for inhibiting the canine programmed death protein 1 (cPD-1) signaling pathway in a subject in need thereof, comprising administering to the subject an antibody, binding polypeptide, and scFv described herein, or a bispecific molecule, immunoconjugate, or composition comprising any one of these. In some cases, the subject has cancer, sepsis or septic shock, or a chronic infection (e.g., viral infection). The composition can include a pharmaceutical composition and can further include a pharma- ceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition can be administered to the subject (e.g., a dog or canine).

[0267] In some aspects, the present disclosure provides a method for increasing T cell proliferation and reducing T cell exhaustion in a subject in need thereof, comprising administering to the subject an antibody, binding polypeptide, and scFv described herein, or a bispecific molecule, immunoconjugate, or composition comprising any one of them. In some cases, the subject has cancer, sepsis or septic shock, or a chronic infection (e.g., viral infection). The composition can comprise a pharmaceutical composition and can further comprise a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition can be administered to the subject (e.g., dog or canine). In some cases, T cell proliferation in the canine subject is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% compared to a comparable canine subject that was not given the antibodies, binding polypeptides, and scFvs described herein, or a bispecific molecule, immunoconjugate, or composition comprising any one of these described herein. In some cases, T cell exhaustion in the canine subject is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% compared to a comparable canine subject that was not given the antibodies, binding polypeptides, and scFvs described herein, or a bispecific molecule, immunoconjugate, or composition comprising any one of these described herein. In some embodiments, the T cells are effector T cells. In some embodiments, the T cells are CD5+ T cells.

[0268] In some aspects, the present disclosure provides a method for increasing IFNγ production in a subject in need thereof, comprising administering to the subject an antibody, binding polypeptide, and scFv, or a bispecific molecule, immunoconjugate, or composition comprising any one of the antibodies, binding polypeptides, and scFvs described herein. In some cases, the subject has cancer, sepsis or septic shock, or a chronic infection (e.g., viral infection). The composition can include a pharmaceutical composition and can further include a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition can be administered to the subject (e.g., dog or canine). In some cases, IFNγ production in the canine subject is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% compared to a comparable canine subject that did not receive the antibodies, binding polypeptides, and scFv described herein, or a bispecific molecule, immunoconjugate, or composition comprising any one of these described herein. In some embodiments, the T cells are CD5+ T cells.

[0269] In one aspect, the present disclosure provides a method for treating a disease or condition in a subject in need thereof, comprising administering to the subject an antibody, binding polypeptide, and scFv, or a bispecific molecule, immunoconjugate, or composition comprising any one of the antibodies, binding polypeptides, and scFvs described herein. The disease or condition includes cancer, sepsis or septic shock, or chronic infection (e.g., viral infection). The composition can include a pharmaceutical composition and can further include a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition can be administered to the subject (e.g., dog or canine).

[0270] In certain aspects, described herein are antibodies, binding polypeptides, and scFvs described herein, or bispecific molecules, immunoconjugates, or compositions comprising any one of these, for use as a medicament.

[0271] In certain aspects, described herein are antibodies, binding polypeptides, and scFvs described herein, or bispecific molecules, immunoconjugates, or compositions comprising any one of these, for use as a medicament for treating cancer, sepsis or septic shock, or chronic infection.

[0272] In certain aspects, described herein is the use of an antibody, binding polypeptide, and scFv described herein, or a bispecific molecule, immunoconjugate, or composition comprising any one of these, for the manufacture of a medicament.

[0273] In certain aspects, described herein is the use of an antibody, binding polypeptide, and scFv described herein, or a bispecific molecule, immunoconjugate, or composition comprising any one of these, for the manufacture of a medicament for treating cancer, sepsis or septic shock, or a chronic infection (e.g., a viral infection).

[0274] Treatment refers to a method that aims to improve or restore the condition being treated. With respect to cancer, treatment includes, but is not limited to, reducing tumor volume, reducing tumor volume growth, increasing progression-free survival, or overall life expectancy. In certain embodiments, treatment will affect the remission of the cancer being treated. In certain embodiments, treatment includes use as a prophylactic or maintenance dose intended to prevent the recurrence or progression of previously treated cancer or tumor. It is understood by those skilled in the art that not all individuals respond equally to the treatment administered, or do not respond at all, but these individuals are nevertheless considered to be treated.

[0275] The method includes administering to the subject an isolated binding polypeptide comprising a heavy chain variable region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7, 9, 33, or 49, and a light chain variable region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:11, 35, or 51.

[0276] In certain embodiments, the cancer is associated with the programmed death protein 1 (PD-1) signaling pathway. In certain embodiments, PD-1 is expressed on immune cells of the subject. In certain embodiments, the immune cells are T lymphocytes. In certain embodiments, the immune cells are tumor-infiltrating lymphocytes. In certain embodiments, a ligand of PD-1, such as PD-L1 or PD-L2, is expressed on cancer cells of the subject. In certain embodiments, canine programmed death-ligand 1 is overexpressed on cancer cells of the subject.

[0277] In certain embodiments, the binding polypeptide specifically binds to programmed death protein 1 (PD-1). In certain embodiments, the binding polypeptide comprises an antibody or an antigen-binding fragment thereof. In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, single chain variable fragment (scFv), single domain antibody, sc(Fv)2, dsFv, Fab, Fab', (Fab')2, and diabody. In certain embodiments, the antibody is a full-length antibody. In certain embodiments, the antibody or antigen-binding fragment is a canine antibody or an antigen-binding fragment thereof.

[0278] In certain embodiments, the method further comprises administering one or more additional therapies or interventions. Such additional therapies or interventions are not limited, and can include any therapeutic agent or small molecule drug that is useful for treating a subject in need thereof. In some embodiments, the additional therapies or interventions are administered with the antibody or its antigen-binding fragment, or scFv, or bispecific molecule or immunoconjugate comprising thereof, as combination therapy. Non-limiting examples of additional therapies or interventions include chemotherapy (e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine), radiation therapy, immunotherapy, and other targeted therapy.

[0279] In certain embodiments, the cancer or tumor is a solid cancer or tumor. In certain embodiments, the cancer or tumor is a blood cancer or tumor. In certain embodiments, the cancer or tumor includes breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testis, and liver tumors. In certain embodiments, tumors that can be treated with the antibodies of the present disclosure include adenoma, adenocarcinoma, angiosarcoma, astrocytoma, epithelial carcinoma, embryonal tumor, glioblastoma, glioma, hemangioendothelioma, hemangiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and / or teratoma. In certain aspects, the tumor / cancer is selected from the group consisting of acral lentigo melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytic tumor, Bartholin's adenocarcinoma, basal cell carcinoma, bronchial adenocarcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma, chondrosarcoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymoma, Swing's sarcoma, sarcoma), focal nodular hyperplasia, gastronoma, germ cell tumors, glioblastoma, glucagonoma, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatocellular carcinoma, insulinite, intraepithelial neoplasia, intraepithelial squamous cell neoplasia, invasive squamous cell carcinoma, large cell carcinoma, liposarcoma, lung cancer, lymphoblastic leukemia, lymphocytic leukemia, leiomyosarcoma, melanoma, The tumor is selected from the group consisting of malignant melanoma, malignant mesothelial tumor, nerve sheath tumor, medulloblastoma, medulloepithelioma, mesothelioma, mucoepidermoid carcinoma, myeloid leukemia, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, ovarian cancer, papillary serous adenocarcinoma, pituitary tumor, plasmacytoma, pseudosarcoma, prostate cancer, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, squamous cell carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous cell carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vaginal / vulvar carcinoma, VIPpoma, and Wilms' tumor.In certain embodiments, the tumors / cancers treated with one or more antibodies of the present disclosure include brain tumors, head and neck cancer, colorectal cancer, acute myeloid leukemia, pre-B cell acute lymphoblastic leukemia, bladder cancer, astrocytoma, preferably grade II, III, or IV astrocytoma, glioblastoma, glioblastoma multiforme, small cell carcinoma, and non-small cell carcinoma, preferably non-small cell lung cancer, lung adenocarcinoma, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer, prostate adenocarcinoma, and breast cancer, preferably ductal carcinoma, and / or breast cancer. In certain embodiments, the cancers treated with one or more antibodies of the present disclosure include glioblastoma. In certain embodiments, the cancers treated with one or more antibodies of the present disclosure include pancreatic cancer. In certain embodiments, the cancers treated with one or more antibodies of the present disclosure include ovarian cancer. In certain embodiments, the cancers treated with one or more antibodies of the present disclosure include lung cancer. In certain embodiments, the cancer treated with one or more antibodies of the present disclosure comprises prostate cancer.In certain embodiments, the cancer treated with one or more antibodies of the present disclosure comprises colon cancer.In certain embodiments, the cancer treated comprises glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer.In certain embodiments, the cancer is resistant to other treatments.In certain embodiments, the cancer treated is recurrent.

[0280] In some embodiments, the cancer is selected from the group consisting of melanoma (e.g., metastatic malignant melanoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), head and neck cancer (e.g., squamous cell carcinoma of the head and neck), cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma (e.g., B cell lymphoma), adrenal cancer, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytic tumor, bone cancer, brain and spinal cancer, metastatic brain tumor, carotid body tumor, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, fibroplastic bone imperfection, fibrous dysplasia of bone, gallbladder cancer or bile duct cancer, pregnancy-associated cancer, or gestational age-related cancer. trophoblastic disease, germ cell tumors, hematologic malignancies, hepatocellular carcinoma, islet cell tumors, Kaposi's sarcoma, kidney cancer, lipoma / benign lipomatous tumors, liposarcoma / malignant lipomatous tumors, medulloblastoma, meningioma, Merkel cell carcinoma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndromes, neuroblastoma, neuroendocrine tumors, papillary thyroid cancer, parathyroid tumors, childhood cancer, peripheral nerve sheath tumors, pheochromocytoma, pituitary tumors, prostate cancer, posterior uveal melanoma, rare hematological disorders, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid metastatic cancer, uterine cancer, or any combination thereof.

[0281] In some cases, the cancer includes cervical cancer, lung cancer, liver cancer, ovarian cancer, cancer of the skin including melanoma and squamous cell carcinoma, colon cancer, bladder cancer, breast cancer, kidney cancer, esophageal cancer, stomach cancer, pancreatic cancer, head cancer, and neck cancer.

[0282] The compositions of the present disclosure can be administered at dosages and routes and times determined in appropriate preclinical and clinical experiments and tests. The compositions can be administered multiple times at dosages within these ranges. The administration of the compositions can be combined with other methods useful for treating the desired disease or condition, as determined by those skilled in the art. In certain embodiments, the antibody can be administered to a subject in need thereof by any route suitable for administration of an antibody-containing pharmaceutical composition, such as, for example, subcutaneous, intraperitoneal, intravenous, intramuscular, intratumoral, or intracerebral. In certain embodiments, the antibody is administered intravenously. In certain embodiments, the antibody is administered subcutaneously. In certain embodiments, the antibody is administered intratumorally. In certain embodiments, the antibody is administered at a suitable dosing schedule, such as, for example, weekly, twice weekly, monthly, twice monthly, once every two weeks, once every three weeks, or once a month. In certain embodiments, the antibody is administered once every three weeks. The antibody can be administered in any therapeutically effective amount. In certain embodiments, the therapeutically acceptable amount is about 0.1 mg / kg to about 50 mg / kg. In certain embodiments, the therapeutically acceptable amount is about 1 mg / kg to about 40 mg / kg. In certain embodiments, the therapeutically acceptable amount is about 1 mg / kg to about 20 mg / kg. In certain embodiments, the therapeutically acceptable amount is about 1 mg / kg to about 10 mg / kg. In certain embodiments, the therapeutically acceptable amount is about 5 mg / kg to about 30 mg / kg. In certain embodiments, the therapeutically acceptable amount is about 5 mg / kg to about 20 mg / kg. A therapeutically effective amount includes an amount sufficient to ameliorate one or more symptoms associated with the disease or affliction being treated.

[0283] In the context of this disclosure, preventive, palliative, symptomatic, and / or curative treatments may represent separate aspects of the disclosure. The anti-cPD-1 antibodies or antigen-binding fragments thereof, or scFvs disclosed herein can be administered parenterally, e.g., intravenously, e.g., intramuscularly, e.g., subcutaneously. Alternatively, the antibodies of the disclosure can be administered via a non-parenteral route, e.g., orally or topically. The antibodies of the present disclosure can be administered prophylactically. The antibodies of the present disclosure can be administered therapeutically (on demand).

[0284] Pharmaceutical compositions, kits, and methods of making the compositions Also provided is a pharmaceutical composition comprising any one of the binding polypeptides, scFv, antibodies or antigen-binding fragments disclosed herein.Among the compositions are pharmaceutical compositions and formulations for administration, such as for treating disease or disorder.Also provided is a therapeutic method for administering the pharmaceutical composition to a subject, for example, a dog.

[0285] Pharmaceutical compositions and formulations generally include one or more optional pharma- ceutically acceptable carriers or excipients. In some embodiments, the composition includes at least one additional therapeutic agent.

[0286] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation will be administered. "Pharmaceutically acceptable carrier" refers to ingredients in a pharmaceutical formulation other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In some aspects, the choice of carrier is determined in part by the particular composition and / or by the method of administration. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain a preservative. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (about 10 residues) soluble oligosaccharides, such as glycerol, glycerol, sorbitol ... hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG).

[0287] A buffering agent is included in the composition in some aspects. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0288] The formulation may comprise an aqueous solution. The formulation or composition may also contain more than one active ingredient useful for a particular indication, disease, or condition being treated with the composition, preferably one with complementary activities in the composition, where the respective activities do not adversely affect each other. Such active ingredients are present as appropriate in combination in amounts that are effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmacologic active agents or drugs, such as chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine. The pharmaceutical composition in some embodiments contains the composition in an amount effective to treat or prevent a disease or condition, such as a therapeutically or prophylactically effective amount. The therapeutic or prophylactic effectiveness in some embodiments is monitored by periodic evaluation of the subject being treated. The desired dosage can be delivered by administration of a single bolus of the composition, by administration of multiple boluses of the composition, or by continuous infusion administration of the composition.

[0289] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the composition is administered parenterally. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the composition is administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. The composition in some embodiments is provided as a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which in some aspects can be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within a suitable viscosity range to provide a longer contact period with a particular tissue. Liquid or viscous compositions can contain a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0290] Sterile injectable solutions can be prepared, for example, by incorporating the composition into a solvent in a mixture with a suitable carrier, diluent, or excipient, such as sterile water, saline, glucose, dextrose, etc. The composition can contain auxiliary substances, such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and / or coloring agents, depending on the desired route of administration and preparation. In some aspects, standard texts can be consulted to prepare suitable preparations.

[0291] Various additives that enhance the stability and sterility of the composition can be added, including antibacterial preservatives, antioxidants, chelating agents, and buffers.Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid.Prolonged absorption of injectable pharmaceutical forms can be achieved by the use of agents that delay absorption, such as aluminum monostearate and gelatin.

[0292] Formulations to be used for in vivo administration are generally sterile. Sterility is readily accomplished, for example, by filtration through sterile filtration membranes.

[0293] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein are incorporated herein by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0294] In certain embodiments, the anti-canine PD-1 antibodies of the present disclosure are included in a pharmaceutical composition comprising one or more pharma- ceutically acceptable excipients, carriers, and diluents. Pharmaceutically acceptable excipients, carriers, and diluents can be included to increase the shelf life, stability, or administrability of the antibody. Such compounds include salts, pH buffers, surfactants, anticoagulants, and preservatives. In certain embodiments, the antibodies of the present disclosure are administered suspended in a sterile solution. In certain embodiments, the solution comprises about 0.9% NaCl. In certain embodiments, the solution comprises about 5.0% dextrose. In certain embodiments, the solution further comprises one or more of the following: a buffer, such as acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); a surfactant, such as polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; a polyol / disaccharide / polysaccharide, such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid, such as glycine or arginine; an antioxidant, such as ascorbic acid, methionine; or a chelating agent, such as EDTA or EGTA.

[0295] In certain embodiments, the antibodies of the present disclosure can be lyophilized, shipped / stored, and reconstituted prior to administration. In certain embodiments, the lyophilized antibody formulation includes a bulking agent, such as mannitol, sorbitol, sucrose, trehalose, dextran 40, or a combination thereof. The lyophilized formulation can be contained in a vial composed of glass or other suitable non-reactive material. The antibody, as formulated, whether reconstituted or not, can be buffered at a certain pH, generally below 7.0. In certain embodiments, the pH can be 4.5-7.0, 4.5-6.5, 4.5-6.0, 4.5-5.5, 4.5-5.0, or 5.0-6.0.

[0296] Also described herein are kits that include one or more of the antibodies described herein in a suitable container, as well as one or more additional components selected from instructions for use; diluents, excipients, carriers, and devices for administration.

[0297] In certain embodiments, described herein is a method for making a composition for treating cancer, sepsis or septic shock, or chronic infection (e.g., viral infection), comprising mixing one or more pharma- ceutically acceptable excipients, carriers, or diluents with an antibody of the present disclosure.In certain embodiments, described herein is a method for preparing a cancer treatment for storage or shipment, comprising lyophilizing one or more antibodies of the present disclosure.

[0298] Although the present disclosure has been described in relation to its specific embodiments, it should be understood by those skilled in the art that various modifications can be made and equivalents can be substituted without departing from the true spirit and scope of the present disclosure. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein can be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications can be made to adapt a particular situation, material, composition of matter, process, process step, or step to the objective, spirit, and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto. Although certain embodiments have been described in detail herein, the same will be more clearly understood by reference to the following examples, which are included for illustrative purposes only and are not intended to be limiting. EXAMPLES

[0299] The present disclosure will be further described in detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present disclosure should in no way be interpreted as being limited to the following examples, but rather as embracing any and all variations that become apparent as a result of the teachings provided herein. Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein can be used in carrying out the present disclosure.

[0300] All publications, patent applications, issued patents, and other documents mentioned herein are incorporated by reference herein as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in the texts incorporated by reference are excluded to the extent they conflict with definitions in this disclosure.

[0301] Without further description, it is believed that one of ordinary skill in the art can, using the foregoing description and the following illustrative examples, make and utilize the disclosed compounds and practice the claimed methods. Accordingly, the following examples specifically point out certain aspects of the present disclosure.

[0302] Example 1: Generation of canine anti-canine PD-1 scFv A canine IgM / IgG / lambda / Kappa scFv phage display library containing an estimated 40 billion individual E. coli transformants was constructed from canine B cell mRNA and the pComb3X phagemid vector using oligonucleotide primers based on published canine immunoglobulin heavy and light chain germline genes as previously described (Andris-Widhopf, J., Steinberger, P., Fuller, R., Rader, C., Barbas, CF Generation of antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences in Barbas, CF, Burton, DR, Scott, JK, Silverman GJ Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001) and Lefranc, MP, et al. IMGT, the international ImMunoGeneTics information system. Nucleic Acids Res 37, D1006-1012 (2009)). Aliquots of the library were subjected to four rounds of solid-phase selection ("panning") against canine PD-1. To ensure that the selected phage contained cPD-1-specific scFv phage particles, polyclonal scFv phage from each round of panning were assessed by scFv phage ELISA using cPD-1 as the target antigen (Figure 1). Substantial enrichment of phage for cPD-1 specific binders was identified in the third round of panning (P3) and increased in the fourth round of panning (P4).

[0303] An initial investigation of the positive hit rate was assessed by testing 16 clones from panning round 3 and 16 clones from panning round 4 for their ability to bind cPD-1 by phage ELISA. 10 / 16 clones from P3 and 15 / 16 clones from P4 bound to cPD-1 with a range of apparent affinities (Figures 2A-2B). 14 unique clones were identified from these 25 binders by nucleotide sequencing and produced as soluble scFv for further characterization (Figure 3, left graph).

[0304] Following this initial survey to look at positivity rates, high-throughput screening of over several hundred additional clones was performed. 176 positive clones were isolated, of which an additional eight unique clones were identified. Five of these eight additional clones were generated as soluble scFv for further characterization. Based on the results of soluble scFv binding to cPD-1 (Figure 3), clones 3-4, 3-8, 3-13, 4-2, 4-9, 4-14, P4B1, P3C6, and P4F3 were selected for further analysis of their ability to inhibit the interaction of PD-1 with PD-L1 (Figure 4).

[0305] Five clones were detected that showed evidence of their ability to inhibit the interaction of PD-1 with PD-L1 (shown in numbered boxes in Figure 4). These clones were then tested for their ability to bind membrane-expressed cPD-1. The human erythroleukemia cell line K562 (KTδ32), edited to remove CD32 (to reduce non-specific binding), was engineered to express cPD-1 using a retroviral vector carrying a puromycin selection cassette (KTδ32.cPD-1). HA-tagged soluble scFvs selected via inhibition assays (Figure 4) were incubated with either KTδ32 or KTδ32.cPD-1 cells, and binding was determined using an anti-HA antibody and flow cytometry (Figure 5).

[0306] Clones P3C6 and P4B1, which inhibited the PD-1:PD-L1 protein interaction and bound to cell surface expressed PD-1, were reformatted as full-length intact canine IgG4 molecules and re-tested by flow cytometry for their ability to bind cell surface PD-1 (Figure 6).

[0307] The yield of P3C6 achieved from transient transfection of 293T cells was low, and chain swapping experiments confirmed that this was related to the VH chain of P3C6 (data not shown). Strategic amino acid replacements were made, and the resulting mutant VH chains were paired with the original P3C6 light chain and evaluated for production and cPD-1 binding ability (Figures 7 and 8). Early attempts to mutate P3C6 also resulted in low yields (Figure 9A). After several attempts, the yields of two specific mutants, P3C6 mut 3.1 and mut 3.2, were significantly improved (Figure 9B).

[0308] To determine whether the P3C6 mutants can increase canine T cell proliferation and IFNγ production, canine PBMCs were labeled with Cell Trace Violet (CTV) and stimulated with the mitogen Concanavalin A in the presence of P3C6 mutant IgG4 molecules. CD5+ T cell proliferation was determined by flow cytometry (FIG. 10) and IFNγ production was measured by ELISA 96 hours after stimulation (FIG. 11).

[0309] Given the relatively small number of unique scFvs identified from the initial screening and subsequent high-throughput screening, a second round of high-throughput screening was performed on the 872 screened clones, and over 200 soluble scFv clones present in the expression extracts were identified as binding to cPD-1 by ELISA. These clones were further evaluated for binding by flow cytometry, and three additional unique scFv clones were identified that bound to cPD-1 on the surface of KTδ32 cells. Only one of these clones from panning round 3, clone A6, was successfully reformatted as full-length canine IgG4. The ability of this full-length clone to specifically bind membrane-bound cPD-1 was evaluated in flow cytometry (Figure 12). Clone A6 specifically bound to KTδ32.cPD-1 cells, and this binding was blocked by pre-incubation of IgG4 molecules with soluble cPD-1.

[0310] Enumerated aspects The following enumerated aspects are provided, the numbering of which should not be construed as designating a level of importance.

[0311] Aspect 1 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO:1, 27, or 43; (b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 2, 28, or 44; (c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 3, 29, or 45; (d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO:4, 30, or 46; (e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO:5, 31, or 47; and (f) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 6, 32, or 48; An antibody or antigen-binding fragment thereof comprising:

[0312] Aspect 2 provides: (a) a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:7, 9, 33, or 49; and (b) a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:11, 35, or 51; 2. The antibody or antigen-binding fragment thereof of embodiment 1, comprising:

[0313] Aspect 3 provides: (a) an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 19, 21, 23, 39, or 55; and (b) an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 25, 41, or 57. 3. The antibody or antigen-binding fragment thereof of embodiment 1 or 2, comprising:

[0314] Aspect 4 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:7, 9, 33, or 49; and (b) a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:11, 35, or 51; An antibody or antigen-binding fragment thereof comprising:

[0315] Aspect 5 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7, 9, 33, or 49; and (b) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:11, 35, or 51. An antibody or antigen-binding fragment thereof comprising:

[0316] Aspect 6 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 19, 21, 23, 39, or 55; and (b) an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 25, 41, or 57. An antibody or antigen-binding fragment thereof comprising:

[0317] Aspect 7 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19, 21, 23, 39, or 55; and (b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 25, 41, or 57; An antibody or antigen-binding fragment thereof comprising:

[0318] Aspect 8 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO:1; (b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO:2; (c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO:3; (d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO:4; (e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO:5; and (f) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO:6; An antibody or antigen-binding fragment thereof comprising:

[0319] Aspect 9 provides: (a) a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:7 or 9; and (b) a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:11; The antibody or antigen-binding fragment thereof of embodiment 8, comprising:

[0320] Aspect ten provides: (a) an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:19, 21, or 23; and (b) an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:25. 10. The antibody or antigen-binding fragment thereof of embodiment 8 or 9, comprising:

[0321] Aspect 11 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:7 or 9; and (b) a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:11; An antibody or antigen-binding fragment thereof comprising:

[0322] Aspect 12 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7; and (b) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:11; An antibody or antigen-binding fragment thereof comprising:

[0323] Aspect 13 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9; and (b) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:11; An antibody or antigen-binding fragment thereof comprising:

[0324] Aspect 14 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:19, 21, or 23; and (b) an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:25. An antibody or antigen-binding fragment thereof comprising:

[0325] Aspect 15 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO:19; and (b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO:25; An antibody or antigen-binding fragment thereof comprising:

[0326] Aspect 16 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO:21; and (b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO:25; An antibody or antigen-binding fragment thereof comprising:

[0327] Aspect 17 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO:23; and (b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO:25; An antibody or antigen-binding fragment thereof comprising:

[0328] Aspect 18 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO:27; (b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO:28; (c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO:29; (d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO:30; (e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO:31; and (f) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO:32; An antibody or antigen-binding fragment thereof comprising:

[0329] Aspect 19 provides: (a) a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:33; and (b) a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:35. 20. The antibody or antigen-binding fragment thereof of embodiment 18, comprising:

[0330] Aspect 20 provides: (a) an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:39; and (b) an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:41. 20. The antibody or antigen-binding fragment thereof of embodiment 18 or 19, comprising:

[0331] Aspect 21 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:33; and (b) a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:35. An antibody or antigen-binding fragment thereof comprising:

[0332] Aspect 22 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:33; and (b) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:35. An antibody or antigen-binding fragment thereof comprising:

[0333] Aspect 23 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:39; and (b) an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:41. An antibody or antigen-binding fragment thereof comprising:

[0334] Aspect 24 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO:39; and (b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO:41; An antibody or antigen-binding fragment thereof comprising:

[0335] Aspect 25 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO:43; (b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO:44; (c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO:45; (d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO:46; (e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO:47; and (f) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO:48; An antibody or antigen-binding fragment thereof comprising:

[0336] Aspect 26 provides: (a) a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:49; and (b) a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:51; 26. The antibody or antigen-binding fragment thereof of embodiment 25, comprising:

[0337] Aspect 27 provides: (a) an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:55; and (b) an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:57; 27. The antibody or antigen-binding fragment thereof of embodiment 25 or 26, comprising:

[0338] Aspect 28 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:49; and (b) a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:51; An antibody or antigen-binding fragment thereof comprising:

[0339] Aspect 29 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:49; and (b) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:51; An antibody or antigen-binding fragment thereof comprising:

[0340] Aspect 30 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) an immunoglobulin heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:55; and (b) an immunoglobulin light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:57; An antibody or antigen-binding fragment thereof comprising:

[0341] Aspect 31 provides: An antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), (a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO:55; and (b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO:57; An antibody or antigen-binding fragment thereof comprising:

[0342] Aspect 32 provides: 32. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 31, which is an IgG molecule, an IgM molecule, an IgE molecule, an IgA molecule, or an IgD molecule, or is derived from one of these.

[0343] Aspect 33 provides: 33. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 32, wherein the antibody or antigen-binding fragment is selected from the group consisting of a full-length antibody, Fab, a single-chain variable fragment (scFv), sc(Fv)2, dsFv, Fab, Fab', (Fab')2, and a diabody.

[0344] Aspect 34 provides: 34. The antibody or antigen-binding fragment thereof according to any one of aspects 1 to 33, wherein the antibody is a full-length antibody.

[0345] Aspect 35 provides: 35. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 34, wherein the antibody is a canine antibody or a caninized antibody.

[0346] Aspect 36 provides: The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 35, wherein the antibody is a canine antibody.

[0347] Aspect 37 provides: 34. The antibody or antigen-binding fragment thereof according to any one of aspects 1 to 33, wherein the antibody-binding fragment is an scFv.

[0348] Aspect 38 provides: 38. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 37, wherein the specific binding of the antibody or antigen-binding fragment thereof to cPD-1 interferes with the interaction of cPD-1 with its ligand (canine programmed death-ligand 1).

[0349] Aspect 39 provides: A canine antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1).

[0350] Aspect 40 provides: A canine antibody or antigen-binding fragment thereof that specifically binds to canine programmed death protein 1 (cPD-1), comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1 to 36.

[0351] Aspect 41 provides: 41. The canine antibody or antigen-binding fragment thereof of embodiment 39 or 40, wherein the specific binding of the antibody or antigen-binding fragment thereof to cPD-1 interferes with the interaction of cPD-1 with a ligand of cPD-1 (e.g., canine programmed death-ligand 1).

[0352] Aspect 42 provides: (a) a heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 27, and 43, HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 28, and 44, and HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 29, and 45; and (b) a light chain variable region comprising a light chain complementarity determining region 1 (LCDR1), an LCDR2, and an LCDR3, wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 30, and 46, LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 31, and 47, and LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 32, and 48. Including, The heavy chain variable region and the light chain variable region are connected by a linker. A single-chain variable fragment (scFv) that specifically binds canine programmed death protein 1 (cPD-1).

[0353] Aspect 43 provides: (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7, 9, 33, or 49; and (b) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:11, 35, or 51. Including, The heavy chain variable region and the light chain variable region are connected by a linker. A single-chain variable fragment (scFv) that specifically binds to canine programmed death protein 1 (cPD-1).

[0354] Aspect 44 provides: A single-chain variable fragment (scFv) that specifically binds to canine programmed death protein 1 (cPD-1), comprising the amino acid sequence set forth in SEQ ID NO: 13, 15, 17, 37, or 53.

[0355] Aspect 45 provides: The scFv of any one of aspects 42-44, wherein the specific binding of the antibody or antigen-binding fragment thereof to cPD-1 disrupts the interaction of cPD-1 with its ligand (canine programmed death-ligand 1).

[0356] Aspect 46 provides: A bispecific molecule comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1 to 38, the canine antibody of any one of embodiments 39 to 41, or the scFv of any one of embodiments 42 to 45, linked to a molecule having a second binding specificity.

[0357] Aspect 47 provides: An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 38, the canine antibody according to any one of embodiments 39 to 41, the scFv according to any one of embodiments 42 to 45, or the bispecific molecule of embodiment 46, linked to a therapeutic agent.

[0358] Aspect 48 provides: An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 38, the canine antibody according to any one of embodiments 39 to 41, or the scFv according to any one of embodiments 42 to 45.

[0359] Aspect 49 provides: 1. An isolated nucleic acid encoding an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising: (a) a heavy chain variable region encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to a polynucleotide sequence set forth in SEQ ID NO:8, 10, 34, or 50; and (b) a light chain variable region encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the polynucleotide sequence set forth in SEQ ID NO: 12, 36, or 52. An isolated nucleic acid comprising:

[0360] Embodiment 50 provides: 50. The isolated nucleic acid of embodiment 49, wherein the heavy chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence as set forth in SEQ ID NO:8, 10, 34, or 50.

[0361] Aspect 51 provides: 51. The isolated nucleic acid of embodiment 49 or 50, wherein said light chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence as set forth in SEQ ID NO: 12, 36, or 52.

[0362] Embodiment 52 provides: (a) a heavy chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:8; and (b) a light chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:12. An isolated nucleic acid encoding an antibody or antigen-binding fragment thereof comprising:

[0363] Embodiment 53 provides: (a) a heavy chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:10; and (b) a light chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:12. An isolated nucleic acid encoding an antibody or antigen-binding fragment thereof comprising:

[0364] Embodiment 54 provides: (a) a heavy chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:34; and (b) a light chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:36. An isolated nucleic acid encoding an antibody or antigen-binding fragment thereof comprising:

[0365] Embodiment 55 provides: (a) a heavy chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:50; and (b) a light chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:52. An isolated nucleic acid encoding an antibody or antigen-binding fragment thereof comprising:

[0366] Embodiment 56 provides the following: 56. The isolated nucleic acid of any one of embodiments 49 to 55, wherein the antibody or antigen-binding fragment thereof is an IgG molecule, an IgM molecule, an IgE molecule, an IgA molecule, or an IgD molecule, or is derived from one of these.

[0367] Embodiment 57 provides: 57. The isolated nucleic acid of any one of embodiments 49 to 56, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, Fab, a single chain variable fragment (scFv), sc(Fv)2, dsFv, Fab, Fab', (Fab')2, and a diabody.

[0368] Embodiment 58 provides: 58. The isolated nucleic acid of any one of aspects 49 to 57, wherein the antibody is a full-length antibody.

[0369] Aspect 59 provides: 59. The isolated nucleic acid of any one of aspects 49 to 58, wherein the antibody is a canine antibody or a caninized antibody.

[0370] Embodiment 60 provides: 60. The isolated nucleic acid of any one of aspects 49 to 59, wherein the antibody is a canine antibody.

[0371] Aspect 61 provides: (a) a heavy chain variable region comprising the nucleotide sequence set forth in SEQ ID NO:8, 10, 34, or 50; and (b) a light chain variable region comprising the nucleotide sequence set forth in SEQ ID NO: 12, 36, or 52. Including, The heavy chain variable region and the light chain variable region are connected by a linker. An isolated nucleic acid encoding a single chain variable fragment (scFv).

[0372] Embodiment 62 provides: An isolated nucleic acid encoding a single chain variable fragment (scFv), comprising a polynucleotide sequence as set forth in SEQ ID NO:14, 16, 18, 38, or 54.

[0373] Embodiment 63 provides: 63. The isolated nucleic acid of any one of aspects 49 to 62, wherein the antibody or antigen-binding fragment thereof, or the scFv specifically binds to canine programmed death protein 1 (cPD-1).

[0374] Embodiment 64 provides the following: 57. The isolated nucleic acid of embodiment 56, wherein the specific binding of the antibody or antigen-binding fragment thereof, or the scFv to cPD-1 disrupts the interaction of cPD-1 with its ligand (canine programmed death-ligand 1).

[0375] Embodiment 65 provides: A vector comprising the isolated nucleic acid of any one of embodiments 48 to 64.

[0376] Embodiment 66 provides the following: The vector of embodiment 65, which is an expression vector.

[0377] Aspect 67 provides: 67. The vector of embodiment 65 or 66, which is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and a retroviral vector.

[0378] Embodiment 68 provides the following: A host cell comprising the isolated nucleic acid of any one of embodiments 48 to 64, or the vector of any one of embodiments 65 to 67.

[0379] Aspect 69 provides: 70. The host cell of embodiment 68, which is of eukaryotic or prokaryotic origin.

[0380] Embodiment 70 provides: 70. The host cell of embodiment 68 or 69, which is of mammalian origin.

[0381] Aspect 71 provides: 70. The host cell of embodiment 68 or 69, which is of bacterial origin.

[0382] Aspect 72 provides: 71. The host cell of any one of embodiments 68 to 70, which is a Chinese hamster ovary cell.

[0383] Aspect 73 provides: A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 38, the canine antibody according to any one of embodiments 39 to 41, or the scFv according to any one of embodiments 42 to 45, the bispecific molecule of embodiment 46, or the immunoconjugate of embodiment 47, and a pharma- ceutically acceptable excipient, carrier or diluent.

[0384] Aspect 74 provides: 74. The pharmaceutical composition of embodiment 73, formulated for intravenous administration.

[0385] Aspect 75 provides: 74. The pharmaceutical composition of embodiment 73, formulated for subcutaneous administration.

[0386] Aspect 76 provides: The pharmaceutical composition of embodiment 73, formulated for intratumoral administration.

[0387] Aspect 77 provides: A kit comprising the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 38, the canine antibody according to any one of embodiments 39 to 41, or the scFv according to any one of embodiments 42 to 45, the bispecific molecule of embodiment 46, the immunoconjugate of embodiment 47, or the pharmaceutical composition of embodiment 73, and instructions for use.

[0388] Aspect 78 provides: A method for increasing T cell proliferation and reducing T cell exhaustion in a subject in need thereof, the method comprising the step of administering to said subject the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 38, the canine antibody according to any one of embodiments 39 to 41, or the scFv according to any one of embodiments 42 to 45, the bispecific molecule of embodiment 46, or the immunoconjugate of embodiment 47, or the pharmaceutical composition of embodiment 73.

[0389] Aspect 79 provides: A method for increasing IFNγ production in a subject in need thereof, the method comprising the step of administering to said subject the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 38, the canine antibody according to any one of embodiments 39 to 41, or an scFv according to any one of embodiments 42 to 45, the bispecific molecule of embodiment 46, or the immunoconjugate of embodiment 47, or the pharmaceutical composition of embodiment 73.

[0390] Embodiment 80 provides: A method for inhibiting the canine programmed death protein 1 (cPD-1) signalling pathway in a subject in need thereof, comprising the step of administering to said subject the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 38, the canine antibody according to any one of embodiments 39 to 41, or an scFv according to any one of embodiments 42 to 45, the bispecific molecule of embodiment 46, or the immunoconjugate of embodiment 47, or the pharmaceutical composition of embodiment 73.

[0391] Aspect 81 provides: The method of any one of embodiments 78-80, wherein the subject has cancer, sepsis or septic shock, or a chronic infection.

[0392] Embodiment 82 provides the following: A method for treating a disease or condition in a subject in need thereof, comprising the step of administering to said subject the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 38, the canine antibody according to any one of embodiments 39 to 41, or an scFv according to any one of embodiments 42 to 45, the bispecific molecule of embodiment 46, or the immunoconjugate of embodiment 47, or the pharmaceutical composition of embodiment 73.

[0393] Embodiment 83 provides the following: The method of embodiment 82, wherein the disease or condition comprises cancer, sepsis or septic shock, or a chronic infection.

[0394] Embodiment 84 provides the following: The method of any one of embodiments 78-83, further comprising administering one or more additional therapies or interventions.

[0395] Embodiment 85 provides the following: The method of any one of embodiments 81, 83, or 84, wherein said cancer is associated with the canine programmed death protein 1 (cPD-1) signaling pathway.

[0396] Embodiment 86 provides the following: The method of embodiment 85, wherein a ligand for cPD-1 (canine programmed death-ligand 1) is expressed on the cancer cells of the subject.

[0397] Aspect 87 provides: The method of any one of aspects 80-86, wherein cPD-1 is expressed on immune cells of the subject.

[0398] Aspect 88 provides: The method of embodiment 87, wherein said immune cells comprise T cells, B cells, natural killer (NK) cells, regulatory T cells, macrophages, dendritic cells (DCs), or tumor infiltrating lymphocytes.

[0399] Aspect 89 provides: The cancers include melanoma, prostate cancer, head and neck cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, adrenal gland cancer, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytic tumor, bone cancer, brain and spinal cord cancer, metastatic brain tumor, carotid body tumor, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, fibroplastic bone imperfecta ossium), fibrous dysplasia of bone, gallbladder or bile duct cancer, gestational trophoblastic disease, germ cell tumor, hematologic malignancy, hepatocellular carcinoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, lipoma / benign lipomatous tumor, liposarcoma / malignant lipomatous tumor, medulloblastoma, meningioma, Merkel cell carcinoma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, papillary thyroid cancer, parathyroid tumor, childhood cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare hematological disorder, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymic cancer, thymoma, thyroid metastatic cancer, uterine cancer, or any combination thereof.

[0400] Aspect 90 provides: The method of any one of embodiments 81-88, wherein the cancer comprises cervical cancer, lung cancer, liver cancer, ovarian cancer, skin cancer including melanoma and squamous cell carcinoma, colon cancer, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, pancreatic cancer, head cancer, and neck cancer.

[0401] Aspect 91 provides: 91. The method of any one of aspects 78 to 90, wherein the antibody or antigen-binding fragment thereof, the canine antibody, the scFv, the bispecific molecule, or the immunoconjugate specifically binds to cPD-1.

[0402] Aspect 92 provides: The method of embodiment 91, wherein binding of the antibody or antigen-binding fragment thereof, the canine antibody, the scFv, the bispecific molecule, or the immunoconjugate to cPD-1 disrupts the interaction of cPD-1 with a ligand of cPD-1 (e.g., canine programmed death-ligand 1).

[0403] Aspect 93 provides: The method of any one of aspects 78 to 92, wherein the subject is a dog.

[0404] Aspect 94 provides: A method of producing a pharmaceutical composition according to embodiment 73, comprising the step of mixing an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 38, a canine antibody according to any one of embodiments 39 to 41, or an scFv according to any one of embodiments 42 to 45, a bispecific molecule according to embodiment 46, or an immunoconjugate according to embodiment 47, with a pharma- ceutically acceptable excipient, carrier or diluent.

[0405] Aspect 95 provides: A method for making a composition for treating cancer, sepsis or septic shock, or a chronic infection, the method comprising the step of mixing an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 38, a canine antibody according to any one of embodiments 39 to 41, or an scFv according to any one of embodiments 42 to 45, a bispecific molecule according to embodiment 46, or an immunoconjugate according to embodiment 47, with a pharma- ceutically acceptable excipient, carrier or diluent.

[0406] Aspect 96 provides: The cancer is selected from the group consisting of melanoma (e.g., metastatic malignant melanoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), head and neck cancer (e.g., squamous cell carcinoma of the head and neck), cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma (e.g., B cell lymphoma), adrenal gland cancer, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytic tumor, bone cancer, brain and spinal cord cancer, metastatic brain tumor, carotid body tumor, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, fibroplastic bone imperfection, fibrous dysplasia of bone, gallbladder or bile duct cancer, gestational trophoblastic disease, embryonal cell carcinoma, fibroblast ... 96. The method of embodiment 95, comprising treating or preventing the progression of a pulmonary artery disease, a pulmonary sarcoma ...

[0407] Aspect 97 provides: The method of embodiment 95, wherein the cancer comprises cervical cancer, lung cancer, liver cancer, ovarian cancer, skin cancer including melanoma and squamous cell carcinoma, colon cancer, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, pancreatic cancer, head cancer, and neck cancer.

[0408] Aspect 98 provides the following: An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 38, a canine antibody according to any one of embodiments 39 to 41, or an scFv according to any one of embodiments 42 to 45, a bispecific molecule according to embodiment 46, an immunoconjugate according to embodiment 47, or a pharmaceutical composition according to embodiment 73 for use as a medicament.

[0409] Aspect 99 provides: The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 38, the canine antibody according to any one of embodiments 39 to 41, or the scFv according to any one of embodiments 42 to 45, the bispecific molecule of embodiment 46, the immunoconjugate of embodiment 47, or the pharmaceutical composition of embodiment 73, for use as a medicament for treating cancer, sepsis or septic shock, or a chronic infection.

[0410] The embodiment 100 provides: Use of an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 38, a canine antibody according to any one of embodiments 39 to 41, or an scFv according to any one of embodiments 42 to 45, a bispecific molecule according to embodiment 46, an immunoconjugate according to embodiment 47, or a pharmaceutical composition according to embodiment 73 for the manufacture of a medicament.

[0411] Aspect 101 provides: Use of an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 38, a canine antibody according to any one of embodiments 39 to 41, or an scFv according to any one of embodiments 42 to 45, a bispecific molecule according to embodiment 46, an immunoconjugate according to embodiment 47, or a pharmaceutical composition according to embodiment 73 for the manufacture of a medicament for the treatment of cancer, sepsis or septic shock, or a chronic infection.

[0412] Aspect 102 provides: A method for producing an antibody or antigen-binding fragment thereof according to any one of aspects 1 to 38, a canine antibody according to any one of aspects 39 to 41, or an scFv according to any one of aspects 42 to 45, the method comprising the step of culturing a host cell according to any one of aspects 68 to 72.

[0413] Aspect 103 provides: The method of embodiment 102, further comprising the step of incubating a host cell according to any one of embodiments 68 to 72 in a cell culture medium under conditions sufficient to allow expression and secretion of the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 38, the canine antibody according to any one of embodiments 39 to 41, or the scFv according to any one of embodiments 42 to 45.

[0414] Other Aspects The recitation of a list of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0415] The disclosures of any and all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety.Although the present disclosure has been disclosed in relation to specific embodiments, it is clear that other embodiments and variations of the present disclosure may be devised by those skilled in the art without departing from the true spirit and scope of the present disclosure.The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. (a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 1, 27, or 43; (b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 2, 28, or 44; (c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 3, 29, or 45; (d) a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 4, 30, or 46; (e) a light chain complementarity-determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 5, 31, or 47; and (f) a light chain complementarity-determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 6, 32, or 48; An antibody or antigen-binding fragment thereof comprising:

2. (a) a heavy chain variable region comprising an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:7, 9, 33, or 49; and (b) a light chain variable region comprising an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 11, 35, or 51; The antibody or antigen-binding fragment thereof of claim 1, comprising:

3. (a) an immunoglobulin heavy chain comprising an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 19, 21, 23, 39, or 55; and (b) an immunoglobulin light chain comprising an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 25, 41, or 57; The antibody or antigen-binding fragment thereof of claim 1, comprising: (a) a heavy chain variable region comprising an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:7, 9, 33, or 49; and (b) a light chain variable region comprising an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 11, 35, or 51; An antibody or antigen-binding fragment thereof comprising: (a) an immunoglobulin heavy chain comprising an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 19, 21, 23, 39, or 55; and (b) an immunoglobulin light chain comprising an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 25, 41, or 57; An antibody or antigen-binding fragment thereof comprising:

6. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a canine antibody or a caninized antibody.

7. (a) a heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 27, and 43, HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 28, and 44, and HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 29, and 45; and (b) a light chain variable region comprising a light chain complementarity determining region 1 (LCDR1), an LCDR2, and an LCDR3, wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 30, and 46, LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 31, and 47, and LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 32, and 48. Including, The heavy chain variable region and the light chain variable region are connected by a linker. Single chain variable fragment (scFv).

8. (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7, 9, 33, or 49; and (b) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11, 35, or 51; Including, The heavy chain variable region and the light chain variable region are connected by a linker. Single chain variable fragment (scFv).

9. A bispecific molecule comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 6, or the scFv of claim 7 or 8, linked to a molecule with a second binding specificity.

10. 10. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 6, the scFv of claim 7 or 8, or the bispecific molecule of claim 9, linked to a therapeutic agent.

11. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 6, or the scFv of claim 7 or 8.

12. A vector comprising the isolated nucleic acid of claim 11.

13. 13. A host cell comprising the isolated nucleic acid of claim 11 or the vector of claim 12.

14. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 6, the scFv of claim 7 or 8, the bispecific molecule of claim 9, or the immunoconjugate of claim 10, and a pharmaceutically acceptable excipient, carrier, or diluent.

15. A pharmaceutical composition as described in claim 14 for use in treating a disease or condition in a subject, optionally wherein the disease or condition includes cancer, sepsis or septic shock, or a chronic infection, and optionally wherein the subject is a dog.

Images