Canine monoclonal antibodies against canine cytotoxic T lymphocyte-associated protein 4 (CTLA-4)

Abstract

The present invention relates to canine antibodies, binding polypeptides, and scFvs specific for canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is entitled to priority under 35 USC §119(e) to U.S. Provisional Application No. 63 / 177,692, filed April 21, 2021, which is incorporated by reference herein in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under HHSN261201800042C awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] 2. Background of the Invention Advances in the understanding of the interactions between the immune system and cancer have led to the successful development of immunotherapies against tumors in human patients. These new immune-based treatments have met with great success in the clinic and are increasingly becoming frontline treatments. Indeed, human cancer immunotherapy is now recognized as one of the mainstays of treatment alongside surgery, radiation therapy, and chemotherapy. The field of veterinary cancer immunotherapy has also progressed rapidly over the past decade, although the availability of animal-specific therapeutics, and a full understanding of their specific effects, remain a challenge.

[0004] Checkpoint blockade therapies are a promising immunotherapy strategy that work by inhibiting receptors that negatively regulate T cells. In effect, these therapies remove the natural "brakes," called immune checkpoints, that suppress T cell function. Although these negative receptors normally play a key role in preventing immune responses from becoming toxic, many cancers exploit this negative signaling to blunt T cell-based immune responses directed against them. One of the key immune checkpoint receptors that regulates T cells is cytotoxic T lymphocyte protein 4 or CTLA-4. CTLA-4 is normally expressed on activated T cells and delivers a negative signal to T cells upon binding with its ligands, B7-1 (CD80) and B7-2 (CD86), which are normally expressed on antigen-presenting cells. In this way, CTLA-4 acts like a governor to the engine, preventing overactivation of the immune system. CTLA-4-blocking antibodies such as ipilimumab were the first checkpoint inhibitor therapies to receive FDA approval for use in human patients and have proven useful in treating immunogenic cancers such as melanoma.

[0005] Thus, there is a need in the art for the development of canine antibodies that can be used to treat veterinary cancers in canine subjects through blocking CTLA-4 on tumor-specific T cells. The present invention addresses and fulfills this need. Summary of the Invention

[0006] As described herein, the present invention relates to canine antibodies, binding polypeptides, and scFvs specific for canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Also included are methods and compositions for treating disease, particularly cancer, in canine patients comprising the anti-CTLA-4 antibodies, binding polypeptides, and scFvs of the present invention.

[0007] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof, comprising an antigen-binding domain that specifically binds to an epitope of canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), The antigen-binding domain comprises: i. 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:6, 25, 40, or 74; and ii. 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:8, 27, or 42; Includes; the heavy chain variable region comprises three heavy chain complementarity determining regions (HCDRs), HCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 3, 22, and 37); and The light chain variable region comprises three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 4, 23, and 38), LCDR2 comprises an amino acid sequence selected from the group consisting of (VDG, GNY, and GNS), and LCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 5, 24, and 39); Antibodies or antigen-binding fragments thereof are provided.

[0008] In certain embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, a Fab, and a single-chain variable fragment (scFv).

[0009] In certain aspects, the antibody or antigen-binding fragment thereof is a full-length antibody.

[0010] In certain embodiments, the antibody is a canine antibody.

[0011] In a particular embodiment, the antibody or antigen-binding fragment thereof of claim 1 , wherein the antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 6, 25, 40, or 74.

[0012] In certain embodiments, the antigen binding domain consists of a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:6, 25, 40, or 74.

[0013] In a specific aspect, the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antigen-binding domain comprises a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:8, 27, or 42.

[0014] In a specific aspect, the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antigen-binding domain consists of a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8, 27, or 42.

[0015] In another aspect, the present invention provides a method for producing a composition comprising: i. a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:6; and ii. A light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8. The present invention includes an isolated antibody or antigen-binding fragment thereof comprising:

[0016] In another aspect, the present invention provides a method for producing a composition comprising: i. a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 3, 22, and 37); and ii. a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 4, 23, and 38), LCDR2 comprises an amino acid sequence selected from the group consisting of (VDG, GNY, and GNS), and LCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 5, 24, and 39). Including, the heavy chain variable region and the light chain variable region are separated by a linker; It comprises a single-chain variable fragment (scFv) that contains an antigen-binding domain that specifically binds to an epitope of canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

[0017] In another aspect, the present invention provides a method for producing a composition comprising: i. a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:6, 25, 40, or 74; and ii. a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8, 27, or 42; Including, the heavy chain variable region and the light chain variable region are separated by a linker; Includes single chain variable fragments (scFv).

[0018] In another aspect, the invention includes a single chain variable fragment (scFv) comprising the amino acid sequence set forth in SEQ ID NO:29, 44, or 76.

[0019] In another aspect, the invention includes a single chain variable fragment (scFv) consisting of the amino acid sequence set forth in SEQ ID NO:29, 44, or 76.

[0020] In another aspect, the present invention provides a full-length antibody comprising an antigen-binding domain that specifically binds to an epitope of canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), comprising: The antigen-binding domain comprises: i. a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 3, 22, and 37); and ii. a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 4, 23, and 38), LCDR2 comprises an amino acid sequence selected from the group consisting of (VDG, GNY, and GNS), and LCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 5, 24, and 39). The antibody may be a full-length antibody, including

[0021] In another aspect, the present invention provides a method for producing a composition comprising: i. a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:6, 25, 40, or 74; and ii. a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8, 27, or 42; The antibody may be a full-length antibody, including

[0022] In another aspect, the invention includes a full-length antibody comprising a heavy chain amino acid sequence set forth in SEQ ID NO:10, 12, 14, 16, 31, 46, 71, 72, or 73, and a light chain amino acid sequence set forth in SEQ ID NO:18, 33, or 48.

[0023] In another aspect, the present invention includes a full-length antibody consisting of a heavy chain amino acid sequence set forth in SEQ ID NO:10, 12, 14, 16, 31, 46, 71, 72, or 73, and a light chain amino acid sequence set forth in SEQ ID NO:18, 33, or 48.

[0024] In another aspect, the invention includes an isolated nucleic acid encoding any of the preceding claimed scFv or full-length antibodies.

[0025] In another aspect, the present invention provides an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof comprising an antigen-binding domain that specifically binds to an epitope of canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), comprising: The antigen-binding domain comprises: i. 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 SEQ ID NO:7, 26, 41, or 75; and ii. 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 amino acid sequence of the light chain variable region set forth in SEQ ID NO:9, 28, or 43; Includes; the heavy chain variable region comprises three heavy chain complementarity determining regions (HCDRs), HCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 3, 22, and 37); and The light chain variable region comprises three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 4, 23, and 38), LCDR2 comprises an amino acid sequence selected from the group consisting of (VDG, GNY, and GNS), and LCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 5, 24, and 39); Includes isolated nucleic acids.

[0026] In certain embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, a Fab, and a single-chain variable fragment (scFv).

[0027] In certain embodiments, the antibody is a full-length antibody.

[0028] In certain embodiments, the antibody is a canine antibody.

[0029] In certain embodiments, the heavy chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence set forth in SEQ ID NO:7, 26, 41, or 75.

[0030] 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:7, 26, 41, or 75.

[0031] In certain embodiments, the light chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence set forth in SEQ ID NO:9, 28, or 43.

[0032] 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:9, 28, or 43.

[0033] In another aspect, the present invention provides a method for producing a composition comprising: i. a heavy chain variable region encoded by a nucleic acid sequence comprising a polynucleotide sequence set forth in SEQ ID NO:7, 26, 41, or 75; and ii. a light chain variable region encoded by a nucleic acid sequence comprising a polynucleotide sequence set forth in SEQ ID NO:9, 28, or 43; The present invention also includes an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof comprising:

[0034] In another aspect, the present invention provides a method for producing a composition comprising: i. a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 3, 22, and 37); and ii. a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 4, 23, and 38), LCDR2 comprises an amino acid sequence selected from the group consisting of (VDG, GNY, and GNS), and LCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 5, 24, and 39). The present invention also includes an isolated nucleic acid encoding a single chain variable fragment (scFv) comprising:

[0035] In another aspect, the present invention provides a method for producing a composition comprising: i. a heavy chain variable region comprising the nucleotide sequence set forth in SEQ ID NO:7, 26, 41, or 75; and ii. a light chain variable region comprising the nucleotide sequence set forth in SEQ ID NO:9, 28, or 43. Including, the heavy chain variable region and the light chain variable region are separated by a linker; The present invention includes an isolated nucleic acid encoding a single chain variable fragment (scFv).

[0036] In another aspect, the invention includes an isolated nucleic acid encoding a single chain variable fragment (scFv) comprising a polynucleotide sequence set forth in SEQ ID NO:30, 45, or 77.

[0037] In another aspect, the invention includes an isolated nucleic acid encoding a single chain variable fragment (scFv) consisting of the polynucleotide sequence set forth in SEQ ID NO:30, 45, or 77.

[0038] In another aspect, the present invention includes a vector comprising the isolated nucleic acid of any one of claims 19 to 32.

[0039] In certain embodiments, the vector is an expression vector.

[0040] In certain embodiments, the vector 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.

[0041] In another aspect, the invention includes a host cell comprising the vector of any one of the above aspects or any aspect or embodiment disclosed herein.

[0042] In certain aspects, the host cell is of eukaryotic or prokaryotic origin.

[0043] In certain embodiments, the host cell is of mammalian origin.

[0044] In certain embodiments, the host cell is of bacterial origin.

[0045] In another aspect, the present invention includes a method for producing an antibody or antigen-binding fragment thereof that binds to canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), comprising culturing a host cell of any one of the above aspects or any aspect or embodiment disclosed herein.

[0046] In another aspect, the invention includes a pharmaceutical composition comprising a full-length antibody or scFv of any one of the above aspects or any aspect or embodiment disclosed herein and a pharma- ceutically acceptable carrier.

[0047] In another aspect, the present invention includes a method for treating cancer in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment thereof of any one of the above aspects or any aspect or embodiment disclosed herein.

[0048] In certain aspects, the cancer is associated with cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

[0049] In certain embodiments, CTLA-4 is expressed on cancer-associated cells.

[0050] In certain embodiments, the cancer-associated cells are T lymphocytes.

[0051] In certain embodiments, the antibody or antigen-binding fragment thereof specifically binds to canine CTLA-4.

[0052] In certain embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, a Fab, and a single-chain variable fragment (scFv).

[0053] In certain aspects, the antibody or antigen-binding fragment thereof is a full-length antibody.

[0054] In certain embodiments, the antibody is a caninized antibody.

[0055] In certain embodiments, the subject is a dog. [Brief description of the drawings]

[0056] The following detailed description of the preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0057] [Figure 1]1 is a table displaying phage input and output results from library panning against canine CTLA-4 (cCTLA-4). A comprehensive canine IgM / IgG / lambda / Kappa scFv phage display library containing an estimated 40 billion canine scFv transformants was subjected to four rounds of solid-phase selection ("panning") against biotinylated avitag-cCTLA-4. The number of phage in the input and output (eluate) is reported along with the % of phage bound to the target after each panning round and the enrichment of antigen-specific binders. [Diagram 2] Enrichment of a canine phage display library after panning against canine CTLA-4 antigen. Biotinylated avitag-cCTLA-4 was captured on streptavidin-coated microtiter plates overnight at 4°C. Wells were washed with PBS and blocked with 2% milk in PBS (MPBS) for 1 h at 37°C. The initial library (P0) and polyclonal libraries obtained after each round of panning (P1-P4; 1:1000 dilution in MPBS) were added to the coated plates and incubated for 1 h at 37°C. Plates were washed with PBS supplemented with 0.5% Tween (PBST) and bound phages were detected with HRP-conjugated anti-M13 mAb (GE Healthcare) at 1:5000 dilution in 2% milk. Plates were washed again and bound phages were detected with ABTS. OD was read at 405 nm after 30 minutes using a Molecular Devices Spectra Max 340 spectrophotometer. Plates coated with no antigen, canine CD19, and human avitag-CD3 were used as negative control antigens. Polyclonal phage from the fourth round of panning against canine CD19 from a parallel panning campaign was used as a positive control. [Diagram 3]Screening of canine scFv antibodies from panning rounds 3 and 4 for binding to recombinant canine CTLA-4 is shown. Twelve clones from panning round 3 and 12 clones from panning round 4 against canine CTLA-4 were randomly selected and tested for their ability to bind to cCTLA-4 by scFv phage ELISA. All 24 clones bound to cCTLA-4. Nucleotide sequencing of these 24 scFvs revealed 20 unique antibodies, the majority of which (17) contained lambda light chains. No phage and an unrelated MERS (Middle East Respiratory Syndrome Virus)-specific phage were used as negative controls. Polyclonal phage from the fourth round of panning against canine CTLA-4 was used as a positive control. [Figure 4] Binding of clone P4-8 and additional soluble CTLA-4 scFvs (identified via high throughput screening (HTS) expression assays) to cCTLA-4 using ELISA. Unique scFv clones that bind to cCTLA-4 were identified via HTS assays (A1, B10, D5, and G11) in addition to clone P4-8 (Figure 3). Unique, soluble, HA-tagged, purified scFvs were tested for their ability to bind increasing concentrations of cCTLA-4 by ELISA. 0.25 ug / ml of soluble scFv was added to each well and bound scFv was detected using an AP-conjugated anti-HA antibody. All clones bound to cCTLA-4 with various affinities. A soluble single chain against an unrelated MERS protein was used as a negative control. [Diagram 5]Figures 5A-5B show that CTLA-4-specific soluble scFvs inhibit the binding of cCTLA-4 to its cognate ligands CD80 and CD86. 1.0 and 3.0 pmol of recombinant human CD86-Fc chimera (Figure 5A) or human CD80-Fc chimera (Figure 5B) were bound to ELISA plates overnight. Wells were blocked with 5% milk / PBS 0.05% Tween 20. Biotinylated cCTLA-4 extracellular domain (ECD) was preincubated with each soluble scFv at the indicated molar ratio for 1 h at RT and then added to the plate and incubated for 2 h. After washing, cCTLA-4 bound to CD80 or CD86 was detected using streptavidin-AP conjugate followed by AP colorimetric substrate. Biotinylated cCTLA-4 and non-biotinylated cCTLA-4 in the absence of any antibody were used as positive and negative controls. "na" = no antibody. [Figure 6] 1 is a table showing the expression levels of anti-dog IgG. [Figure 7] ELISA analysis of full-length canine anti-canine CTLA-4 mAb. [Figure 8] Figures 8A-8B show assays of inhibition of CTLA-4 binding to its cognate ligands CD80 (Figure 8A) and CD86 (Figure 8B) as purified full-length monoclonal roe IgG. [Figure 9] 1 is a table showing that replacement of the A1 VH framework region with the B10 VH framework region rescues the generation of the anti-canine CTLA-4 clone A1 (the modified clone is labeled mut2). [Figure 10]Evaluation of anti-CTLA-4 mAb clones binding to membrane-expressed canine CTLA-4. K562 cells lacking FcγRII (CD32) were engineered to express canine CTLA-4 (KT32δ.cCTLA4). KT32δ (top) and KT32δ.cCTLA4 (middle) cells were incubated with three anti-CTLA-4 clones reformatted as canine isotype IgG1, and surface labeling was detected using an anti-HA antibody. To confirm antigen-specific binding, anti-CTLA-4 clones were first incubated with soluble cCTLA-4 protein to block antigen-binding sites and then used for cell surface labeling (bottom). [Figure 11] Figures 11A-11C show that scFv clones reformatted as full canine IgG molecules retain their ability to bind cCTLA-4 and inhibit its interaction with recombinant human and canine CD80 and CD86. Full-length bivalent IgG antibodies were generated from isolated scFvs by cloning the VH and VL chains of selected scFvs into separate heavy and light chain expression plasmids. Adherent 293T cells were transiently co-transfected with light and heavy chain plasmids and mAbs were purified from plate supernatants 3 days later using Protein A affinity chromatography. Figure 11A. mAbs were assessed for binding to increasing concentrations of cCTLA-4 by ELISA. Figure 11B. mAbs were assessed for their ability to inhibit the interaction of cCTLA-4 with recombinant human CD80 (left graph) and rhuCD86 (right graph) at the indicated molar ratios. Similarly, FIG. 11C shows inhibition of the interaction of cCTLA-4 with recombinant canine CD80 (left graph) and CD86 (right graph) at the indicated molar ratios. [Figure 12A] Figures 12A-12C show that CTLA-4 specific clones exhibit high binding affinity to canine CTLA-4. Figure 12A shows CTLA-4 binding to the A1mut2 antibody. [Figure 12B]Figures 12A-12C show that CTLA-4 specific clones exhibit high binding affinity to canine CTLA-4. Figure 12B shows CTLA-4 binding to the B10 antibody. [Figure 12C] Figures 12A-12C show that CTLA-4 specific clones exhibit high binding affinity to canine CTLA-4. Figure 12C shows CTLA-4 binding to the D5 antibody. [Figure 13] FIG. 1 shows the normal function of CTLA-4 upon binding to its cognate ligand on antigen-presenting cells, and strategies for antibody-based blockade of CTLA-4 signaling. [Figure 14] Figure 1 shows binding of unique soluble CTLA-4 scFv to canine CTLA-4 by ELISA. Unique, soluble, HA-tagged, purified scFv from panning rounds 3 (P3) and 4 (P4) were tested for their ability to bind increasing concentrations of cCTLA-4 by ELISA. 0.25 ug / ml of soluble scFv was added to each well and bound scFv was detected with an AP-conjugated anti-HA antibody. All clones bound to cCTLA-4 with various affinities. A soluble single chain against an unrelated MERS protein was used as a negative control. A commercially available polyclonal anti-CTLA-4 antibody was used as a positive control in one assay (top left graph). [Figure 15] Binding of anti-CTLA-4 mAb clones to activated canine T cells. Canine PBMCs were activated with 2.5 ug / ml concanavalin A, harvested at 48 and 72 hours post-activation, and labeled with each HA-tagged anti-CTLA-4 mAb clone as indicated. Bound anti-CTLA-4 antibody was detected using anti-HA antibody. Plots are gated on CD5+, 7AAD- cells. [Figure 16]Binding of A1mut2 mAb to canine regulatory T cells is shown. Canine PBMCs from dogs with T cell lymphoma were first surface labeled with either A1mut2 or an irrelevant MERS mAb, then permeabilized and labeled with anti-FOXP3 mAb (top). In parallel, after permeabilization, cells were relabeled with either anti-MERS antibodies or A1mut2 and Foxp3 to detect intracellular stores of CTLA-4 (bottom). Plots are gated on CD45+, CD5+, CD4+, and FOXP3+ cells. [Figure 17] Figures 17A-17B show the evaluation of A1mut2 binding to feline CTLA-4. PBMCs from two cats with lymphoid malignancies were surface labeled with CD5 and either HA-tagged A1mut2 or an irrelevant MERS mAb (Figure 17A). The labeled cells were then permeabilized and relabeled with MERS or A1mut2 to detect intracellular stores of CTLA-4 (Figure 17B). Anti-HA secondary antibody alone was used as an additional negative control. Plots are gated on CD5+ lymphocytes. [Figure 18] Figures 18A-18B show that A1mut2 expressed as canine isotypes IgGC (canine IgG3) and IgGB (canine IgG2) fixes human complement. Figure 18A. Serial dilutions of HA-tagged A1mut2 reformatted as each of the four different IgG subclasses were bound to SA-captured biotinylated cCTLA-4. Human serum complement or heat-inactivated (HI) human serum complement was added to the plate and then detected with anti-human C1q IgG HRP conjugate and HRP substrate. Figure 18B. Confirmation of the presence of A1mut2 IgG subclass bound to cCTLA-4. Serial dilutions of A1mut2 IgG bound to canine CTLA-4 were detected using AP-conjugated anti-HA IgG and AP substrate. [Figure 19]Figures 19A-19B show that A1mut2 increases the responder frequency and proliferation capacity of canine T cells. Canine PBMCs were labeled with Cell Trace Violet (CTV) and stimulated with 2.5 ug / ml ConA in the presence of either 10 ug / ml A1mut2 or an irrelevant MERS antibody. Cells were harvested at 72 or 96 hours and labeled with anti-CD5 mAb and the viability dye 7-AAD. Cells were captured on a FACS Canto II and analyzed by FlowJo software. Responder frequency (number of cells that underwent at least one division) and proliferation capacity (average number of daughter cells produced per cell) were determined. Figure 19A. Representative histograms from one dog. Figure 19B. Responder frequency and proliferation capacity of nine healthy dogs calculated 96 hours after stimulation. [Figure 20] Figure 2 shows that A1mut2 increases the production of IFN-γ from canine T cells. Canine PBMCs were stimulated with 2.5 ug / ml ConA in the presence of either A1mut2 or irrelevant MERS antibody at either 10 ug / ml or 20 ug / ml. Supernatants were collected at 96 hours and IFN-γ present in the supernatants was measured by ELISA. Data from triplicate experiments in one healthy donor dog are shown. Horizontal bars represent median values. [Figure 21] 1 is a table showing the kinetic rate constants of canine anti-cCTLA-4 binding, dissociation equilibrium constants, and half-lives of antibody / antigen interactions by selected full-length canine mAbs using surface plasmon resonance (SPR). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] Detailed Description definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any method and material similar or equivalent to those described herein can be used in the practice of the present invention for testing, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terms are used.

[0059] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

[0060] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0061] As used herein, "about" when referring to a measurable value such as an amount, a temporal duration, and the like, is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are appropriate for carrying out the disclosed methods.

[0062] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin from natural or recombinant sources, or an immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetrameric immunoglobulin molecule that contains two heavy and two light polypeptide chains. Each polypeptide chain contains three complementarity determining regions (CDRs) that bind to antigens, defining the antigen specificity of the antibody.

[0063] As used herein, the terms "antibody" and "antibodies" can also include polypeptides or polypeptide complexes derived from full-length antibodies. These polypeptide complexes may be naturally occurring or constructed from single chain antibodies or antibody fragments and retain antigen-specific binding ability. The antibodies of the present invention 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), caninized 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).

[0064] The term "antibody fragment" refers to a polypeptide that comprises or is derived from a portion of an intact antibody and includes 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 consisting of either the VL or 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 also include human or humanized antibodies or portions of human or humanized antibodies.

[0065] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0066] 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 and lambda light chains refer to the two major antibody light chain isotypes.

[0067] As used herein, the term "synthetic antibody" refers to an antibody produced 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 produced 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.

[0068] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may 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 will be readily apparent that the present invention 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 will be readily apparent that an antigen can be generated, 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.

[0069] As used herein, the term "anti-tumor effect" refers to the biological effect manifested by the reduction of tumor volume, the reduction of tumor cell number, the reduction of metastasis number, the increase of life span, or the improvement of various physiological symptoms associated with cancerous conditions. "Anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention in preventing the development of tumors in the first place.

[0070] 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.

[0071] "Allogeneic" refers to a graft derived from a different animal of the same species.

[0072] "Xenogeneic" refers to a graft derived from an animal of a different species.

[0073] As used herein, the term "cancer" is defined as a disease characterized by rapid and uncontrolled proliferation of abnormal cells.Cancer cells can spread locally or through the 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, kidney cancer, liver cancer, brain tumors, lymphoma, leukemia, lung cancer, etc.In a particular embodiment, the cancer is medullary thyroid cancer.

[0074] As used herein, the term "conserved sequence modification" is intended to refer to an amino acid modification that does not significantly affect or change the binding properties of the antibody that contains the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody of the present invention 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), non-polar 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 region of the antibody of the present invention can be replaced with other amino acid residues of the same side chain family, and the altered antibodies can be tested for their ability to bind to GFRα4 using the functional assays described herein.

[0075] "Costimulatory ligand," as that term is used herein, includes molecules on 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 engagement of the TCR / CD3 complex with, for example, a peptide-loaded MHC molecule. Costimulatory ligands 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, in particular, costimulatory molecules presented on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, antibodies that specifically bind B7-H3, and ligands that specifically bind CD83.

[0076] "Costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

[0077] The term "dysregulated," when used in the context of levels of CTLA-4 expression or activity, refers to a level of expression or activity that is different from the level of CTLA-4 expression or activity in an otherwise identical healthy animal, organism, tissue, cell, or component thereof. The term "dysregulated" also refers to an altered regulation of the levels of CTLA-4 expression and activity compared to regulation in an otherwise identical healthy animal, organism, tissue, cell, or component thereof.

[0078] "Encode" 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, having 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 when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. 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 used as a template for transcription of the gene or cDNA can be said to encode the protein or other product of the gene or cDNA.

[0079] 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.

[0080] "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, including, but not limited to, inhibition of viral infection as determined by any means suitable in the art.

[0081] As used herein, "endogenous" refers to any material from or produced within an organism, cell, tissue, or system.

[0082] As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue, or system.

[0083] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0084] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes 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).

[0085] As used herein, "homologous" refers to the identity of subunit sequences between two polymer molecules, for example, between two nucleic acid molecules, for example, 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 that is 10 subunits long) 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.

[0086] "Humanized", "caninized", and "chimeric" forms of non-human (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 the non-human immunoglobulin. For the most part, humanized, caninized, and chimeric antibodies are human or canine immunoglobulins (recipient antibody) in which residues from the complementarity determining regions (CDRs) of the recipient are replaced by residues from the CDRs of a non-human or non-canine species (donor antibody) such as mouse, rat, or rabbit that has the desired specificity, affinity, and capacity. In some cases, residues from the Fv framework regions (FRs) of the human or canine immunoglobulin are replaced by corresponding non-human or non-canine residues. Furthermore, humanized, caninized, and chimeric antibodies can contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to refine and optimize antibody performance. In general, humanized, caninized, and chimeric antibodies are considered to contain substantially all of at least one, typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin, and all or substantially all of the FR regions being of a human, caninized, immunoglobulin sequence. Humanized and chimeric antibodies are also considered to optimally contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The World Health Organization (WHO) International Nonproprietary Nomenclature (INN) Expert Group has defined the requirements for a non-human derived antibody to be considered "humanized". In accordance with the guidelines, a comparison of the candidate antibody to the human sequence should be performed by the International Immunogenetics Information System® (IMGT®) DomainGapAlign tool (www.imgt.org). This tool queries the IMGT® database of antibody germline variable region genes, where alignment scores are generated only for germline sequence variable region exons, thus omitting CDR3 and part of the J region from the analysis.For an antibody to be "humanized," in addition to being "closer to human than other species," the top "hit" must be human and have at least 85% identity to the human sequence, or the antibody will be designated as "chimeric." For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0087] "Fully human" refers to an immunoglobulin, eg, an antibody, whose entire molecule is of human origin or consists of an amino acid sequence identical to a human form of an antibody.

[0088] 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 compositions and methods of the invention. The instruction material of the kits of the invention can be, for example, affixed to a container containing the nucleic acids, peptides, and / or compositions of the invention or shipped together with a container containing the 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.

[0089] As used herein, "identity" refers to the identity of subunit sequences between two polymer molecules, particularly between two amino acid molecules, for example, between two polypeptide molecules. If two amino acid sequences have the same residue at the same position; for example, if a position in each of the two polypeptide molecules is occupied by arginine, they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of positions that match or are identical; for example, if half of the positions in the two sequences (e.g., 5 positions in a polymer that is 10 amino acids long) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are identical or matched, the two amino acid sequences are 90% identical.

[0090] "Isolated" means changed 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 may exist in a substantially purified form, or may exist in a non-native environment, such as, for example, a host cell.

[0091] In the present invention, 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.

[0092] 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 can include introns in some versions.

[0093] As used herein, "lentivirus" refers to a genus of the Retroviridae family.Lentivirus is unique among retroviruses in that it can infect non-dividing cells; it can deliver a significant amount of genetic information to the DNA of host cells, making it one of the most efficient methods of gene delivery vectors.HIV, SIV, and FIV are all examples of lentiviruses.Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0094] The term "operably linked" refers to the functional link between a control sequence and a heterologous nucleic acid sequence, which allows the latter to be expressed. 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 adjacent and, where necessary to join two protein coding regions, are in the same reading frame.

[0095] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0096] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide, which can be hydrolyzed into monomeric "nucleotides". The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, those that are synthesized by recombinant means, i.e., conventional cloning techniques and PCR. (商標)These include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including cloning of nucleic acid sequences from recombinant libraries or cell genomes, using nucleic acid sequences such as those described above, as well as by synthetic means.

[0097] 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 may 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 peptides, oligopeptides, and oligomers, and longer chains, of which there are many types, commonly referred to in the art as proteins. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, mutants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide can be a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0098] As used herein, the term "promoter" 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.

[0099] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence that is required for the expression of a gene product that is 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 that are required for the expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0100] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes a cell to produce the gene product under most or all physiological conditions of the cell.

[0101] 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 substantially produced in a cell only if an inducer corresponding to the promoter is present in the cell.

[0102] 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 substantially produced in a cell only if the cell is a cell of the tissue type corresponding to the promoter.

[0103] A "signal transduction pathway" refers to the biochemical relationship between various signal transduction molecules that play a role in the transmission of 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.

[0104] "Single chain antibody" refers to an antibody formed by recombinant DNA technology in which the heavy and light fragments of immunoglobulin chains are linked together using an engineered amino acid span to reproduce the Fv region of the antibody as a single polypeptide.Various methods for producing single chain antibodies 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.

[0105] 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" may be a human or a 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 human.

[0106] 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 normally associates 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 embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0107] As used herein, the term "therapeutic" refers to treatment and / or prophylaxis. The therapeutic effect is achieved by suppression, amelioration, or eradication of the disease state.

[0108] 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 that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0109] As used herein, the phrases "under transcriptional control" or "operably linked" mean that the promoter is in the correct position and orientation relative to the polynucleotide so as to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0110] 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 bound to 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.

[0111] As used herein, the term "specifically binds" refers to an antibody or ligand that recognizes and binds to its cognate binding partner (e.g., a stimulatory and / or costimulatory molecule present on a T cell) protein present in a sample, but does not substantially recognize or bind to other molecules in the sample.

[0112] 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, for example, downregulation of TGF-β, and / or remodeling of cytoskeletal structure.

[0113] "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.

[0114] 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, among others, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0115] Ranges: Throughout this disclosure, various aspects of the invention may 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 inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose not only the individual numerical values ​​within the range, but also all possible subranges. For example, a description of a range such as 1-6 should be considered to specifically disclose not only the individual numbers within the range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6, but also subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc. This applies regardless of the breadth of the range.

[0116] explanation The present invention is based on the discovery that canine antibody phage display library can be used to identify antigen-specific single chain variable fragment (scFv) fusion proteins that specifically bind to canine origin cytotoxic T-lymphocyte protein 4 (CTLA-4).These canine CTLA-4-specific scFvs can then be converted into full-length antibodies with canine constant regions, making them suitable for in vivo use in canine subjects.Methods and compositions comprising the canine CTLA-4-specific scFvs, antibodies, or antigen-binding fragments of the present invention for the treatment of cancer are also provided.

[0117] Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or CD152 is a member of the immunoglobulin superfamily of transmembrane receptors that is highly homologous to the T-cell costimulatory molecule CD28. CTLA-4 is a member of the immunoglobulin superfamily of transmembrane receptors that binds CD4 + T cells and CD8 + It is upregulated on the surface of T cells after activation and acts as a negative regulator of effector T cell responses. CTLA-4 is also upregulated on exhausted CD8 +It is overexpressed on the surface of T cells. CTLA-4 binds with high affinity to CD80 and CD86, outcompeting CD28 for interaction with these receptor ligands, inhibiting T cell activation. Inhibition of lymphocyte activation by CTLA-4 is achieved not only by outcompeting CD28, but also through recruitment of the tyrosine phosphatase SHP2 and the serine-threonine phosphatase PP2A, which dephosphorylate the TCR ζ chain and target downstream effectors of PI3K, respectively. CTLA-4 can further inhibit effector T cell responses indirectly through its action on antigen-presenting cells (APCs). The high binding affinity of CTLA-4 to CD80 / 86 on APCs can result in the removal of these costimulatory ligands via transendocytosis, promoting a tolerogenic APC phenotype. In contrast to effector T cells, regulatory T cells constitutively express CTLA-4 on their surface, which is essential for their suppressor activity, especially their ability to inhibit dendritic cell maturation. Monoclonal antibodies that bind to CTLA-4 and block its interaction with CD80 / 86 lower the T cell activation threshold and promote the proliferation of highly activated tumor-specific T cells, thereby enhancing endogenous T cell responses and inhibiting Treg function, promoting antitumor immunity.

[0118] Ipilimumab, also known as Yervoy, was the first anti-CTLA-4 monoclonal antibody approved by the FDA in 2011 for the treatment of melanoma in human patients and has become a frontline therapy in the clinic. Considering the similarities between the human and canine immune systems, the present invention provides a complete canine anti-CTLA-4 that can be used as an antitumor therapy in dogs. Utilizing a recently developed canine scFv phage display library, we identified and reformatted three CTLA-4 specific soluble scFvs into complete canine IgG monoclonal antibodies that can be used as immunotherapy for veterinary cancers.

[0119] Immunotherapy for Cancer in Dogs Dogs are closely related to humans phylogenetically and spontaneously develop cancers that share similar biological, behavioral, and genetic characteristics with their human counterparts. Therefore, canine cancer patients can serve as a relevant parallel patient population that can help elucidate the mechanism of action of anti-CTLA-4 mAbs, identify correlative biomarkers of response, and understand mechanisms of resistance to checkpoint inhibitors. In addition, they may also play an important translational role in informing human clinical trial design for the safety and efficacy of combination therapies. Canine CTLA-4 (NP_001003106.1) shares 88% identity with human CTLA-4 (NP_005205.2) and has been shown to induce tolerance in dogs following administration of sheep red blood cells, suggesting a conserved mechanism of action of CTLA-4 between humans and dogs. In addition, a higher percentage of canine peripheral blood CD4 in canine patients with histiocytic sarcoma and B-cell lymphoma compared to control dogs was found. + T cells and CD8 + T cells expressed CTLA-4, suggesting the presence of an exhausted phenotype in these dogs that adversely affects antitumor immunity. Furthermore, a recent report using RNA sequencing showed that canine CD4 + CD25 hi T cells were confirmed to express a regulatory phenotype, including expression of high levels of CTLA-4 transcripts. Together, these findings provide a rationale for the development of therapeutic canine anti-CTLA-4 antibodies capable of promoting anti-tumor immunity in these aggressive tumor types, and provide correlative biomarkers of clinical response and valuable comparable reagents for investigating optimal combination therapies to inform human clinical trials.

[0120] Binding polypeptides, antibodies, and scFvs The binding polypeptides and antibodies of the present invention are characterized by certain functional features or properties of antibodies. For example, the binding polypeptides and antibodies specifically bind to canine cytotoxic T-lymphocyte associated protein 4 (CTLA-4). Preferably, the binding polypeptides and antibodies of the present invention bind to canine CTLA-4 with high affinity. Preferably, the binding polypeptides and antibodies of the present invention specifically recognize the canine CTLA-4 protein naturally expressed on a cell and do not cross-react with other surface molecules on the cell.

[0121] In certain aspects, the present invention provides isolated binding polypeptides comprising an antigen-binding domain that specifically binds to an epitope of canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). 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).

[0122] In certain aspects, the present invention provides an isolated binding polypeptide comprising an HCDR1 comprising an amino acid sequence (SEQ ID NO:1, 21, or 37). Also provided is an isolated binding polypeptide comprising an HCDR2 comprising an amino acid sequence (SEQ ID NO:2, 22, or 38). Also provided is an isolated binding polypeptide comprising an HCDR3 comprising an amino acid sequence (SEQ ID NO:3, 23, or 39). Also provided is an isolated binding polypeptide comprising a light chain variable region comprising an LCDR1 comprising an amino acid sequence (SEQ ID NO:4, 24, or 40). Also provided is an isolated binding polypeptide comprising an LCDR2 comprising an amino acid sequence (SEQ ID NO:5, 25, or 41). Also provided is an isolated binding polypeptide comprising an LCDR3 comprising an amino acid sequence (SEQ ID NO:6, 26, or 42).

[0123] In one particular aspect, the present invention provides an isolated binding polypeptide comprising an HCDR1 comprising an amino acid sequence (SEQ ID NO:1), an HCDR2 comprising an amino acid sequence (SEQ ID NO:2), an HCDR3 comprising an amino acid sequence (SEQ ID NO:3), an LCDR1 comprising an amino acid sequence (SEQ ID NO:4), an LCDR2 comprising an amino acid sequence (SEQ ID NO:5), and an LCDR3 comprising an amino acid sequence (SEQ ID NO:6).

[0124] Permissible variations in complementarity determining region (CDR) sequences will be known to those of skill in the art. For example, in some embodiments, a 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, 21, 22, 23, 24, 25, 26, 37, 38, 39, 40, 41, or 42.

[0125] In some embodiments, the binding polypeptide binds to cytotoxic T-lymphocyte associated protein 4 (CTLA-4). In some embodiments, the CTLA-4 protein comprises the amino acids set forth in SEQ ID NO:53. In some embodiments, the binding polypeptide comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment is selected from the group consisting of a Fab, a single chain variable fragment (scFv), or a single domain antibody. In further embodiments, the antibody is a full-length antibody. In still other embodiments, the antibody or antigen-binding fragment is a canine antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a caninized antibody or an antigen-binding fragment thereof.

[0126] 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:6, 25, 40, or 74. In certain embodiments, a binding polypeptide comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:6, 25, 40, or 74. In certain embodiments, a binding polypeptide consists of a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:6, 25, 40, or 74.

[0127] 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:8, 27, or 42. In certain embodiments, a binding polypeptide comprises a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:8, 27, or 42. In certain embodiments, a binding polypeptide consists of a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:8, 27, or 42.

[0128] Also provided is an isolated binding polypeptide comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:6, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8.

[0129] In certain embodiments, the present invention includes antibodies that bind to the same epitope on canine CTLA-4 as the antibodies of the present invention (i.e., antibodies that have the ability to cross-compete with any of the antibodies of the present invention for binding to canine CTLA-4). In preferred embodiments, the reference antibody for cross-competition studies can be one of the antibodies described herein (e.g., A1mut2). For example, Biacore analysis, ELISA assay, or flow cytometry may be used to demonstrate cross-competition with the antibodies of the present invention. The ability of a test antibody to inhibit, for example, the binding of A1mut2 to canine CTLA-4 demonstrates that the test antibody can compete with A1mut2 for binding to canine CTLA-4, and therefore is likely to bind to the same epitope of CTLA-4 as A1mut2.

[0130] The antibody of the present invention can be prepared by using an antibody having one or more of the VH and / or VL sequences disclosed herein as starting material to engineer a modified antibody, which may 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.

[0131] Also provided is a single chain variable fragment (scFv) that comprises an antigen-binding domain that specifically binds to an epitope of canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

[0132] 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 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 CTLA-4 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 skilled in the art will be able to select the appropriate configuration for use in the present invention.

[0133] 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:58), (GGGS) n (SEQ ID NO:59), and (GGGGS) n (SEQ ID NO:60), where n represents an integer of at least 1. Exemplary linker sequences include, but are not limited to, TIFF2024516621000001.tif26156 and the like. One of skill in the art will be able to select an appropriate linker sequence for use in the present invention. In one aspect, the scFv of the present invention 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 TIFF2024516621000002.tif12128 TIFF2024516621000003.tif4128.

[0134] 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).

[0135] 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, 17, or 33), and / or HCDR2 comprises the amino acid sequence (SEQ ID NO: 2, 18, or 34), and / or HCDR3 comprises the amino acid sequence (SEQ ID NO: 3, 19, or 35), and / or LCDR1 comprises the amino acid sequence (SEQ ID NO: 4, 20, or 36), and / or LCDR2 comprises the amino acid sequence (SEQ ID NO: 5, 21, or 37), and / or LCDR3 comprises the amino acid sequence (SEQ ID NO: 6, 22, or 38). The heavy chain variable region and the light chain variable region are separated by a linker.

[0136] 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:6, 25, 40, or 74, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8, 27, or 42. The heavy and light chain variable regions are separated by a linker.

[0137] In another aspect, there is provided a single chain variable fragment (scFv) comprising the amino acid sequence set forth in SEQ ID NO: 29, 44, or 76. In another aspect, there is provided a single chain variable fragment (scFv) consisting of the amino acid sequence set forth in SEQ ID NO: 29, 44, or 76.

[0138] 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:29, 44, or 76.

[0139] In another aspect, a full length antibody is provided, comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 10, 12, 14, 16, 31, 46, 71, 72, or 73, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 18, 33, or 48. 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: 10, 12, 14, 16, 31, 46, 71, 72, or 73, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 18, 33, or 48. Permissible variations in full length antibody sequences will be known to those of skill in the art. For example, in some embodiments, the antibody 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:11, 13, 15, 17, 19, 33, 35, 49, and 51.

[0140] Table 1: Sequences used in the present invention TIFF2024516621000004.tif87163TIFF2024516621000005.tif225163TIFF2024516621000006.tif229163TIFF2024 516621000007.tif226163TIFF2024516621000008.tif229163TIFF2024516621000009.tif226163TIFF20245166210 00010.tif225163TIFF2024516621000011.tif229163TIFF2024516621000012.tif226163TIFF2024516621000013.t if229163TIFF2024516621000014.tif229163TIFF2024516621000015.tif225163TIFF2024516621000016.tif171163

[0141] Nucleic acids and expression vectors The present disclosure provides an isolated nucleic acid encoding a polypeptide. The nucleic acid of the present disclosure may include a polynucleotide sequence encoding any one of the binding polypeptides, scFvs, or antibodies disclosed herein.

[0142] One aspect of the invention includes an isolated nucleic acid encoding a binding polypeptide comprising an antigen-binding domain that specifically binds to an epitope of canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

[0143] 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, 21, or 37), and / or HCDR2 comprises the amino acid sequence (SEQ ID NO: 2, 22, or 38), and / or HCDR3 comprises the amino acid sequence (SEQ ID NO: 3, 23, or 39), and / or LCDR1 comprises the amino acid sequence (SEQ ID NO: 4, 24, or 40), and / or LCDR2 comprises the amino acid sequence (SEQ ID NO: 5, 25, or 41), and / or LCDR3 comprises the amino acid sequence (SEQ ID NO: 6, 26, or 42).

[0144] 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 a Fab, a single chain variable fragment (scFv), or a single domain antibody. In certain embodiments, the antibody is a full-length antibody. In certain embodiments, the antibody or antigen-binding fragment is a humanized antibody or a fragment thereof.

[0145] 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 NOs:8, 28, and 44. In certain embodiments, the heavy chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence set forth in SEQ ID NOs:8, 28, and 44. In certain embodiments, the heavy chain variable region is encoded by a nucleic acid consisting of a polynucleotide sequence set forth in SEQ ID NOs:8, 28, and 44.

[0146] 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 NOs: 10, 30, and 46. In certain embodiments, the light chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence set forth in SEQ ID NOs: 10, 30, and 46. In certain embodiments, the light chain variable region is encoded by a nucleic acid consisting of the polynucleotide sequence set forth in SEQ ID NOs: 10, 30, and 46.

[0147] 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, and a light chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:10.

[0148] 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, 20, and 35), and / or HCDR2 comprises the amino acid sequence (SEQ ID NO: 2, 22, or 38), and / or HCDR3 comprises the amino acid sequence (SEQ ID NO: 3, 22, or 37), and / or LCDR1 comprises the amino acid sequence (SEQ ID NO: 4, 24, or 40), and / or LCDR2 comprises the amino acid sequence (SEQ ID NO: 5, 25, or 41), and / or LCDR3 comprises the amino acid sequence (SEQ ID NO: 6, 26, or 42).

[0149] 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 and / or a light chain variable region comprising the nucleotide sequence set forth in SEQ ID NO:10. The heavy chain variable region and the light chain variable region are separated by a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:72.

[0150] Also provided is an isolated nucleic acid encoding a single chain variable fragment (scFv) comprising a polynucleotide sequence set forth in SEQ ID NO:30, 45, or 77. Also provided is an isolated nucleic acid encoding a single chain variable fragment (scFv) consisting of a polynucleotide sequence set forth in SEQ ID NO:30, 45, or 77.

[0151] Also provided is an isolated nucleic acid encoding a full-length antibody comprising a heavy chain polynucleotide sequence set forth in SEQ ID NOs:12, 14, 16, 18, 34, and 50, and a light chain polynucleotide sequence set forth in SEQ ID NOs:20, 36, and 52. Also provided is an isolated nucleic acid encoding a full-length antibody consisting of a heavy chain polynucleotide sequence set forth in SEQ ID NOs:12, 14, 16, 18, 34, and 50, and a light chain polynucleotide sequence set forth in SEQ ID NOs:20, 36, and 52.

[0152] Acceptable variations in nucleic acid sequences will be known to those of skill in the art. For example, in some embodiments, the nucleic acid 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:8, 10, 12, 14, 16, 18, 20, 28, 30, 32, 34, 36, 44, 46, 48, 50, or 52.

[0153] In certain embodiments, the nucleic acid of the present disclosure comprises a first polynucleotide sequence and a second polynucleotide sequence. The first and second polynucleotide sequences can be separated by a linker. For example, in certain embodiments, the heavy chain variable region and the light chain variable region of the scFv are separated by a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO.72. 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.

[0154] Another aspect of the present invention 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.

[0155] Also provided is a host cell comprising any of the vectors or nucleic acids disclosed herein.Host cell can be of eukaryotic, prokaryotic, mammalian or bacterial origin.Also provided herein is a method for producing binding polypeptide or scFv that binds to FAP, the method comprising culturing host cell.

[0156] In some aspects, the nucleic acids of the disclosure may 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.

[0157] In certain embodiments, the nucleic acid is operably linked to a promoter. In certain embodiments, the promoter is a phosphoglycerate kinase-1 (PGK) promoter.

[0158] 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 retroviral long terminal repeats; mouse metallothionein-I promoter; and various tissue-specific promoters known in the art. 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, modification of reversible promoters derived 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.

[0159] 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 ordinary skill in the art. Suitable promoters 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, e.g., 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). Suitable strong promoters 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 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).

[0160] 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 invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of an 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, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0161] 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, the 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 disclosure of which is incorporated herein by reference).Any suitable combination of recombinase, endonuclease, ligase, recombination site, etc., known in the art, can be used in the generation of irreversibly switchable promoter. The methods, mechanisms, and requirements for performing site-specific recombination described elsewhere herein are used in the generation 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 disclosures of which are incorporated herein by reference.

[0162] The nucleic acids of the present disclosure may be present in an expression vector and / or a cloning vector. Expression vectors may 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 generating recombinant constructs of interest. The following vectors are provided by way of example and should in no way be construed as limiting: Bacterial: 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).

[0163] 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 may also be present. Suitable expression vectors include, but are not limited to, 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 (see, 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).

[0164] Further suitable expression vectors 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 described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

[0165] Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0166] In some embodiments, 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 invention can include a nucleic acid encoding a polypeptide. In some embodiments, an expression vector (e.g., lentiviral vector) includes additional elements that aid in the functional expression of a polypeptide encoded therein. In some embodiments, an expression vector including a nucleic acid encoding a polypeptide further includes a mammalian promoter. In one embodiment, the vector further includes an elongation factor 1 alpha promoter (EF-1α promoter). Use of the EF-1α promoter can increase the efficiency of expression of downstream transgenes. Physiological promoters (e.g., EF-1α promoter) may be less likely to induce integration-mediated genotoxicity and can abrogate the ability of retroviral vectors 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 invention. In some embodiments, 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 of the present invention (e.g., lentiviral vectors). 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 invention 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 of the present invention (e.g., lentiviral vectors).The vector of the present invention can further comprise additional elements, such as a rev response element (RRE) for RNA transport, a packaging sequence, and 5' and 3' long terminal repeats (LTR). The term "long terminal repeat" or "LTR" refers to a domain of base pairs located at the end of 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 vector of the present invention (e.g., lentiviral vector) comprises a 3'U3 deleted LTR. Thus, the vector of the present invention (e.g., lentiviral vector) can comprise any combination of elements described herein to enhance the efficiency of functional expression of the transgene. For example, the vector of the present invention (e.g., lentiviral vector) can comprise a WPRE sequence, a cPPT sequence, an RRE sequence, a 5'LTR, a 3'U3 deleted LTR' in addition to the nucleic acid encoding the CAR.

[0167] The vector of the present invention may 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). The self-inactivating vector can prevent viral transcription beyond the first round of viral replication. Therefore, the self-inactivating vector may infect and then be integrated into the host genome (e.g., mammalian genome) only once, and cannot be passed on further. Therefore, the self-inactivating vector can greatly reduce the risk of producing a replication-competent virus.

[0168] In some embodiments, the nucleic acid of the present invention may 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 the polypeptide of the present disclosure. Methods for introducing RNA into host cells 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 the polypeptide of the present disclosure into host cells can be performed in vitro, ex vivo, or in vivo. For example, RNA comprising a nucleotide sequence encoding the polypeptide of the present disclosure can be used to electroporate host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) in vitro or ex vivo.

[0169] 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, to facilitate the identification and selection of expressing cells from a population of cells that are to be transfected or infected by the 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 flanked by the appropriate regulatory sequences to allow expression in the host cell. Useful selection markers include, but are not limited to, antibiotic resistance genes.

[0170] Reporter genes are used to identify cells that may be transfected and to evaluate the functionality of regulatory sequences. In general, reporter genes are genes that are not present in or expressed by recipient organisms and tissues, and code for polypeptides whose expression is manifested by some easily detectable characteristic, such as enzyme 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, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).

[0171] In some aspects, the nucleic acids of the disclosure provide for the production of the polypeptides described herein, e.g., in a host cell. In some aspects, the nucleic acids of the disclosure provide for the amplification of the nucleic acids encoding the polypeptides.

[0172] Treatment The antibody, binding polypeptide and scFv described herein can be included in a composition for treating disease or condition in a subject in need thereof.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.

[0173] In one aspect, the present invention provides a method for treating cancer in a subject in need thereof. 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:6, 25, 40, or 74, 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:8, 27, or 42.

[0174] In certain embodiments, the cancer is associated with cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expressing cells. In certain embodiments, the CTLA-4 expressing cells are cancer-associated cells. In certain embodiments, the cancer-associated cells are T-lymphocytes. In certain embodiments, the CTLA-4 expressing cancer-associated cells are CTLA-4 expressing T-lymphocytes.

[0175] In certain embodiments, the binding polypeptide specifically binds to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). 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 a Fab, a single chain variable fragment (scFv), or a single domain antibody. In certain embodiments, the antibody is a full-length antibody. In certain embodiments, the antibody or antigen-binding fragment is a humanized antibody or an antigen-binding fragment thereof.

[0176] The composition of the present invention can be administered at a dosage and route and time determined by appropriate preclinical and clinical experiments and tests.The composition can be administered multiple times at dosages within these ranges.The administration of the composition can be combined with other methods useful for treating the desired disease or condition, as determined by those skilled in the art.

[0177] Pharmaceutical Compositions and Formulations Also provided is a pharmaceutical composition comprising any one of the binding polypeptides, scFvs, 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, such as a patient.

[0178] 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.

[0179] 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 have unacceptable toxicity to the subject to which the formulation will be administered. "Pharmaceutically acceptable carrier" refers to ingredients other than the active ingredient in a pharmaceutical formulation that are not 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 include 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 phosphate, citric acid, 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 (approximately polypeptides (fewer than 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; 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., zinc-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG).

[0180] In some aspects, the composition includes a buffering agent. 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).

[0181] The formulation may comprise an aqueous solution. The formulation or composition may comprise more than one active ingredient useful for a particular indication, disease, or condition treated with the composition, preferably one that has complementary activities in the composition, where the respective activities do not adversely affect each other. Such active ingredients are suitably present in combination in an amount effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharma-ceutical 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. In some embodiments, the pharmaceutical composition comprises the composition in an amount effective to treat or prevent a disease or condition, such as a therapeutically or prophylactically effective amount. In some embodiments, therapeutic or prophylactic effectiveness is monitored by periodic evaluation of the subject undergoing treatment. 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 administration of a continuous infusion of the composition.

[0182] 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. As used herein, the term "parenteral" 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. In some embodiments, the composition is provided as a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which in some aspects may be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to provide a longer contact period with a particular tissue. The liquid or viscous composition may contain a carrier, which may 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.

[0183] Sterile injectable solutions can be prepared, for example, by incorporating the composition into a vehicle by mixing 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 agents, 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 appropriate preparations.

[0184] Various additives can be added to enhance the stability and sterility of the composition, including antibacterial preservatives, antioxidants, chelating agents, and buffers.Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as paraben, chlorobutanol, phenol, and sorbic acid.Prolonged absorption of injectable pharmaceutical dosage forms can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0185] Formulations to be used for in vivo administration are generally sterile. Sterility is readily accomplished, for example, by filtration through sterile filtration membranes.

[0186] The contents of the articles, patents, and patent applications, and all other literature and electronically available information mentioned or cited in this specification 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.

[0187] Although the present invention has been described in relation to its specific embodiments, those skilled in the art will understand that various modifications can be made and equivalents can be substituted without departing from the true spirit and scope of the present invention. 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 appropriate 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 invention. 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

[0188] Experimental Example The present invention 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 invention should not be interpreted as being limited to the following examples in any way, but rather as embracing any and all variations that become evident as a result of the teachings provided herein.

[0189] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. Accordingly, the following working examples specifically demonstrate preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0190] The materials and methods used in the experimental examples are described below.

[0191] Cells and cell lines: Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized blood of healthy donor dogs or blood from dogs with T-cell lymphoma (Treg staining) by discontinuous density centrifugation over Ficoll-Paque PLUS (GE Healthcare, Chicago, IL). For feline cells, residual heparinized blood left over from clinical hematological evaluation of two cats with hematological malignancies was first subjected to ACK lysis (ThermoFisher Scientific, Waltham, MA) to remove red blood cells. Cells were washed twice with complete (c)RPMI medium (RPMI 1640 containing 2 mM L-glutamine (Mediatech, Manassas, VA), 10% heat-inactivated fetal bovine serum (Atlanta Biologicals, Flowery Branch, Georgia), 10 mM HEPES (Gibco, Grand Island, NY), and 100 U / ml penicillin and 100 μg / ml streptomycin (Gibco)) before use. Where indicated, negatively selected canine T cells were used. PBMCs were washed in (c)RPMI and labeled with mouse anti-dog CD11b (clone CA16.3E10), CD11c (clone CA11.6A1), mouse anti-human CD14 (clone TuK4), and mouse anti-dog CD21 (clone CA2.1D6) (all from ABD Serotec / Biorad) followed by goat anti-mouse IgG Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). T cells were negatively selected using MACS LS columns (Miltenyi) as described by the manufacturer. T cell purity was determined by CD5 labeling and flow cytometric analysis. Human tumor cell lines (K562 and 293T cells) were grown in RPMI-1640 (cRPMI) supplemented with HEPES, 1 mM sodium pyruvate (Mediatech), glutamine, and penicillin and streptomycin (Thermo Fisher Scientific) and supplemented with 10% fetal bovine serum.

[0192] Production of recombinant proteins. Total RNA was extracted from canine PBMC cells using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA). Reverse transcription was performed using random hexamers and Superscript III reverse transcriptase according to the manufacturer's instructions (Life Technologies), followed by RNAse H digestion to remove any remaining RNA. Primers to amplify the extracellular, N-terminal domain of canine CTLA-4 (NCBI reference sequence: NM_001003106.1) were designed using Primer3 software (http: / / frodo.wi.mit.edu / primer3 / ). The primer sequences were as follows: TIFF2024516621000017.tif41164

[0193] The 3' primer was designed to contain a flexible linker, a histidine tag, and an Avi (biotinylated) tag. Primers were synthesized by Sigma-Aldrich (St. Louis, MO). Conventional PCR using Q5 Hot Start High Fidelity Polymerase was used with the following thermal cycling program: 98°C for 3 min; 30 cycles of 98°C for 15 s, 65°C for 30 s, and 72°C for 1 min; 72°C for 2 min, and cooled to 4°C. The expected band size of the amplified extracellular domain (ECD) of cCTLA-4 is 590 bp. The cCTLA-4 ECD amplicon was cloned into the expression plasmid pFUSE (Invivogen, San Diego, CA) containing an HA tag. The nucleotide sequence of cCTLA-4 ECD was confirmed by DNA sequence analysis. The pFUSE expression plasmid containing cCTLA-4 ECD was transfected into 293T cells using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions to determine the amount of Lipofectamine and time of harvest used for maximum protein expression. CTLA-4 polypeptide was harvested from cell culture medium by binding to Ni-NTA resin (Qiagen, Hilden, Germany) for 2 hours on a rotator. The centrifuged resin pellet was washed extensively in high salt buffer, pH 8.0, containing 50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, and low salt buffer, pH 8.0, containing 50 mM Tris-HCl, 150 mM NaCl, 10 mM imidazole. cCTLA-4 ECD protein was eluted from the Ni-NTA resin with elution buffer, pH 8.0, containing 50 mM Tris-HCl, 150 mM NaCl, 300 mM imidazole. The buffer was exchanged with 1×PBS and the cCTLA-4 ECD protein was concentrated in an Amicon ultrafiltration unit (MilliporeSigma, Burlington, Mass.).Protein production was assessed using SDS-PAGE and Coomassie Blue staining of the gels and quantified by Image Lab 6.0 software (Bio-Rad, Hercules, Calif.) using BSA standards.

[0194] Biotinylation of cCTLA-4 protein and validation of reaction products. Approximately 0.8 mg of recombinant cCTLA-4 ECD protein (hereafter referred to as cCTLA-4) was biotinylated in preparation for binding to streptavidin (SA)-coated plates and subsequent scFv library panning. The Avi tag serves as a biotinylation recognition site for BirA biotin-protein ligase. cCTLA-4 was biotinylated using the BirA biotin-protein ligase standard reaction kit (Avidity, Aurora, CO) following the manufacturer's instructions. To confirm the high degree of cCTLA-4 biotinylation, 0, 2, 4, and 8 ug of SA were added and samples were assessed by SDS-PAGE. Migration of biotinylated cCTLA-4 + SA was delayed compared to non-biotinylated cCTLA-4, indicating that the biotinylation reaction was successful and essentially stoichiometric. To confirm that biotinylated cCTLA-4 protein was immunoreactive in a 96-well plate format, a capture assay followed by ELISA was performed. Briefly, various concentrations of streptavidin in PBS were coated into the wells of a microassay plate. After O / N incubation at 4°C, 5% milk PBS-Tween was added for 1 hour at RT. A PBS-Tween wash was then performed and titrations of biotinylated cCTLA-4 protein were added to the wells for 1 hour. After a PBS-Tween wash, anti-canine CTLA-4 rabbit polyclonal antibody (Sino Biologicals, Chesterbrook, PA) was added at two different dilutions for 2 hours at RT. Detection was with a 1 / 5000 dilution of anti-canine (heavy and light) chain AP-conjugated IgG secondary antibody (Jackson Immunoresearch, West Grove, PA) for 1 hour at RT. After a TBS-Tween wash, colorimetric AP substrate was added and at 1 hour the plates were read at an absorbance of 650. Results showed a lack of signal in wells where no streptavidin was added and that the presence of a signal required increasing amounts of streptavidin-captured protein plated (data not shown).Streptavidin at 30 ug / ml was used for immobilization of biotinylated cCTLA-4 protein for phage library screening and ELISA analysis.

[0195] Generation of canine CTLA-4 expressing target cell lines. To generate cCTLA-4 expressing target cell lines for antibody validation, full-length cCTLA-4 (NCBI reference sequence: NM_001003106.1) was amplified from cDNA derived from canine PBMCs by RT-PCR as described above using the following primers: TIFF2024516621000018.tif19152The resulting 1856 bp amplicon was cloned into pMX-puromycin retroviral expression vector (Cell Biolabs, Inc. San Diego CA). Retrovirus was generated and used to stably transduce human erythroleukemia K562 cell line (KTδ32) that had been previously edited with CRISPR / Cas9 to remove FcγRII to reduce non-specific binding of mAb in flow cytometry assays. Transduced cells were selected in 2.5 ug / ml puromycin dihydrochloride (Sigma, St. Louis, MO) to generate KTδ32.cCTLA-4. Expression of the cCTLA-4 transgene was confirmed in these cells by RT-PCR using primers synthesized to amplify cCTLA-4. Non-transduced KTδ32 cells were used as negative control target cells.

[0196] Canine scFv phage display panning. For each round of panning, wells of a 96-well Costar 3490 1 / 2 area plastic microplate were coated with 50 ul of 20 ug / ml streptavidin (SA) overnight at 4° C. (24 wells for panning 1, 16 wells for panning 2, 8 wells for panning 3 and 4). Plates were washed in PBS and wells were blocked with 2% milk in PBS (MPBS) and incubated at 37° C. for 1 hour. Biotinylated avitag-cCTLA-4 (10 pmol) was added to each streptavidin-coated well and captured by plate-bound SA. After 1 hour incubation at 37° C., plates were washed with PBS to remove free CTLA-4. Equal aliquots of μκ, μλ, γκ, and γλ canine scFv phage display libraries were mixed and blocked in 2% milk / PBS for 1 hour at RT, and 50 ul was added to each CTLA-4-coated well. Panning was performed as previously described for other libraries and targets (e.g., Barbas et al. Phage Display: A Laboratory Manual, Cold Spring Harbor Press, 2001), and after 2 hours of incubation at 37° C., unbound phages were washed away with PBS supplemented with 0.5% Tween 20 (PBST) five times during the first panning and ten times during subsequent panning rounds. To select for binders with longer off-rates, each wash was accompanied by a 5 minute incubation of wash buffer in the wells. To avoid capturing scFv phages specific for biotinylated avitag on SA or cCTLA-4, before each round of positive selection, phages were negatively selected against wells coated with SA and preloaded with an irrelevant biotinylated avitag protein (in this case human biotinylated avitag-CD3ε protein, AcroBiosystems, Newark, Del.).Using this two-step negative / positive selection approach, an IgM / IgG / κ / λ canine scFv phage display library was subjected to four rounds of selection with acid elution of binding phage as described (Barbas et al., supra).

[0197] scFv phage ELISA. For ELISA to detect binding of phage-displayed scFvs, microplates were coated with streptavidin and biotinylated avitag CTLA-4 and control antigens, similar to library panning as described above. Samples of polyclonal phages from the PEG-precipitated initial library (P0) and libraries obtained after each round of panning (P1-P4) were added to the antigen-coated plates at a 1:1000 dilution in MPBS, or monoclonal phages (not PEG-precipitated, 1:100 dilution in milk / PBS) prepared from randomly picked phage clones from the output plates of the third (P3) and fourth (P4) rounds of panning were added to the coated plates and incubated for 1 h at 37°C. Plates were washed with (PBST) and HRP-conjugated anti-M13 mAb (GE Healthcare., Chicago, IL) at a 1:5000 dilution in MPBS was added. Plates were washed again and bound HRP-conjugated secondary antibody was detected with ABTS. OD was read at 405 nm after 30 min using a Molecular Devices SpectraMax 340 spectrophotometer. Plates coated with no antigen, canine CD19, streptavidin only, and streptavidin with irrelevant human avitag-CD3ε were used as negative controls.

[0198] Flow cytometry. Canine PBMCs or isolated T cells, feline leukocytes, and KTδ32.cCTLA-4 and KTδ32(WT) cell lines were washed twice in FACS buffer (1% heat-inactivated FBS in PBS with calcium and magnesium). Cells were blocked with 10 ug of canine IgG for 10 min at RT, and then cell surface labeled with 5 ug of canine scFv, or 500 ng of canine monoclonal antibody (mAb). Specificity of mAb binding was assessed in blocking experiments, where 250 ng of mAb was preincubated with 1.25 μg cCTLA-4 ECD protein for 1 h at RT before incubation with target cells. Cells were washed in FACS buffer and incubated with either mAb alone or mAb preincubated with soluble cCTLA-4 ECD protein to block the antigen-binding site of the mAb. After washing, APC-labeled anti-HA.11 epitope tag (BioLegend, San Diego, CA) and viability dye 7-AAD (BioLegend) were added and cells were incubated for 30 min at RT. For experiments with PBMCs, the indicated cells were also labeled with rat anti-dog CD45 (clone YKIX716.13, BioRad, Hercules, CA), rat anti-dog CD5 mAb (clone: ​​YKIX 322.3, ThermoFisher Scientific) or mouse anti-cat CD5 mAb (clone: ​​FE1.1B11, BioRad), and / or rat anti-dog CD4 (clone YKIX302.9, BioRad). After cell surface labeling, cells were washed twice in FACS buffer and fixed with 1% paraformaldehyde (Thermo Fisher Scientific, Waltham, MA). Cells were washed again and resuspended in FACS buffer before acquisition on a FACS Canto II flow cytometer (BD Biosciences). Data was analyzed using FlowJo software version X (Treestar, Ashland, OR). All plots shown are gated on 7AAD-negative cells.For intracellular staining of canine PBMCs or regulatory T cells, cells were surface stained, if applicable, washed twice in FACS buffer, and resuspended in fixed / permeabilized FOXP3 / Transcription Factor Staining Buffer Set (ThermoFisher Scientific, Waltham, MA). Cells were incubated on ice in the dark for 30 minutes and then washed once with permeabilization buffer. Cells were then incubated with APC-conjugated anti-mouse FoxP3 (clone: ​​FJK-16s, ThermoFisher Scientific) or rat IgG2a kappa isotype control APC (17-4321-81, ThermoFisher Scientific), anti-CTLA-4 mAb (0.5 ug), or anti-MERS antibody (0.5 ug, negative control) for 30 minutes on ice. Cells were washed once with permeabilization buffer and twice with 1×FACS buffer before being captured on a FACS Canto II flow cytometer.

[0199] Generation of soluble scFv. Phage clones confirmed to bind cCTLA-4 by phage ELISA were used to infect TOP10 F' E. coli (Invitrogen, Carlsbad, CA) at various dilutions and plated on LB agar plates containing 100 ug / ml carbenicillin and 1% glucose. A single colony was used to inoculate a starter culture flask containing 20 ml LB with 50 ug / ml carbenicillin and 1% glucose. Cultures were grown overnight at 37°C with shaking at 225 rpm. 10 ml of the saturated starter culture was used to inoculate an expression culture containing 200 ml SB with 50 ug / ml carbenicillin and 0.1% glucose. Flasks were grown with shaking at room temperature for approximately 5 hours to an OD600 of 0.5 and then induced with 0.5 mM IPTG. Growth was O / N at RT, 22-24 hours total post-induction. The cultures were centrifuged and the medium was decanted from the cell pellet. The cell pellet was extracted successively with two periplasmic extraction buffers. Buffer I contained 100 mM Tris-HCl, 20% sucrose, and 1 mM EDTA, pH 8.0, and buffer II contained 5 mM MgCl2. Extractions were accompanied by a pipette to break up clumps, and the extracts were kept on ice for 20-30 min. After each extraction, the extracts were centrifuged at 3400 x g for 15 min, and the two types of extracts were combined and mixed. For immobilized metal affinity chromatography purification, NaCl (300 mM) and imidazole (10 mM) were added, and the combined extracts were recentrifuged at 3400 x g for 15 min. The supernatants were then bound to Ni-NTA agarose, and the scFvs were purified similarly to the CTLA-4 protein as described above.

[0200] High-throughput expression extracts were generated following a similar two-step growth protocol as for generating E. coli cells for scFv purification (above). Single TOP10F' colonies resulting from infection with polyclonal phages from the cCTLA-4 panning round were placed into 96-well round-bottom cell culture plates containing LB with 50 ug / ml carbenicillin and 1% glucose. Plates were shaken O / N at 37°C. 5 ul of the confluent wells were used to inoculate a second set of 96-well plates containing SB with 50 ug / ml carbenicillin, 0.1% glucose, and 0.5 mM IPTG. Colonies were grown O / N at RT in a bacterial shaker. Expression extracts were generated by adding ¼ volume of BEL buffer to the wells of the expression plate and shaking for 1 hour at RT. BEL buffer consisted of 320 mM NaCl, 400 mM H3BO3, pH 8.0, supplemented with lysozyme at 2 mg / ml, EDTA at 4 mM, and benzonase nuclease at 12.5 units / ml final. Lysozyme and benzonase were from Sigma Aldrich. Extracts were blocked with 2.5% milk / PBS-Tween and shaken for 30 min. Expression extracts were used directly in anti-cCTLA-4 ELISA.

[0201] Soluble scFv ELISA. 30 ug / ml streptavidin was added to the ELISA plate and incubated O / N at 4°C. Plates were then blocked with 5% milk / PBS-Tween. Titrations of biotinylated CTLA-4 were added and after washing, 0.25 ug / ml soluble scFv was added and allowed to bind for 2 hours at RT. For the expressed extract ELISA, 10-100 ul of extract was added to the ELISA plate and incubated for 2 hours at RT. Bound scFv was detected using anti-HA mouse IgG AP conjugate (or anti-rabbit AP conjugate for positive polyclonal rabbit anti-dog CTLA-4 mAb control (Sino Biological)) incubated for 1 hour at RT, washed, and detected by AP colorimetric substrate. Plates were read at 650 nm. In some cases, bivalent scFv were made from monovalent scFv for use in ELISA and inhibition studies. Briefly, bivalent scFv preparations were generated by incubating 2.5 ug of soluble scFv for 2 hours at RT with 1 ug / ml (for ELISA) or 5 ug / ml (for blocking studies) of anti-HA alkaline phosphatase or anti-HA-FITC antibodies (clone: ​​HA-7, Sigma Aldrich), respectively, which bind to the C-terminal HA molecule on each scFv.

[0202] ELISA-based interaction assay. 0.3, 1.0, and 3.0 pmol of recombinant human CD86-Fc chimera (carrier-free) or human CD80-Fc chimera (carrier-free) (BioLegend, San Diego, CA) were bound to ELISA plates overnight. Wells were blocked with 5% milk / PBS 0.05% Tween 20. 3 pmol of biotinylated or non-biotinylated cCTLA-4 ECD protein in PBS / Tween was added to wells and shaken at RT for 2 h. For experiments aimed at assessing the ability of canine scFv or canine mAb to inhibit the interaction of plate-bound CD80 and CD86 with cCTLA-4 ECD, cCTLA-4 ECD was preincubated with scFv or mAb at the indicated molar ratio for 1 h at RT and then added to the plate and incubated on a shaker at RT for 2 h. After washing, streptavidin-AP conjugate (Jackson Immunoresearch, West Grove, PA) was added for 1 h at RT. Three TBS-Tween washes were performed and cCTLA-4 bound to CD80 or CD86 was detected using AP colorimetric substrate (Invitrogen).

[0203] Generation of complete canine anti-CTLA-4 mAbs. The VH and VL chains of selected scFvs were cloned using either the canine constant light kappa (CLκ) domain or the constant light lambda (CLλ) domain, or the canine constant IgG1 heavy chain domain (IgG A Full-length bivalent IgG antibodies were generated from the isolated scFvs by cloning into separate expression plasmids containing the canine VH chain (IgG A-DVariable regions (IgG1-IgG4, also called IgG1-IgG4) were cloned into pFUSEss to generate pFUSEss-CHIg-dG1 (Invivogen), which can be selected in zeocin. Plasmids for cloning of the variable regions were selected based on the subtype (lambda vs. kappa) of the isolated VL region. Adherent 293T cells were transiently co-transfected with a 1.5:1 ratio of light and heavy chain plasmids, respectively, using lipofectamine L2000 reagent (ThermoFisher Scientific). Plate supernatants were collected after 3 days and mAbs were purified using Protein A affinity chromatography.

[0204] Complementation fixation assay. 30 ug / ml streptavidin was added to the ELISA plate and incubated O / N at 4°C. The plate was then blocked with 5% milk / PBS-Tween. 200 ul / well of biotinylated cCTLA-4 was added to the plate and after one wash with PBS-T, serial dilutions of HA-tagged canine anti-canine CTLA-4 (A1mut2) antibodies of four different subclasses (A, B, C, and D), ranging from 0.03 to 10 ug / ml, were added to the plate and incubated for 1 hour at RT. The plate was washed three times in PBS-Tween and then a 1:35 dilution of normal human complement serum was added to one set of wells. The plate was incubated for 2 hours at RT. As a negative control, heat-inactivated complement (HI) was added to a parallel set of wells. After incubation, wells were washed three times with PBS-Tween and bound complement was detected with anti-human C1q IgG HRP-conjugated antibody (BioRad, Hercules, CA). Plates were incubated for 1 h at RT and washed three times in PBS-Tween, after which HRP substrate (R&D Systems, Minneapolis, MN) was added and colorimetric analysis was performed. To confirm antibody binding to the plate, bound antibody was detected with AP-conjugated anti-HA IgG (Sigma) using the same canine antibody dilution and detected with AP substrate (InvivoGen, San Diego, CA).

[0205] In vitro stimulation assay. PBMCs from healthy donor dogs were isolated by density gradient centrifugation using Ficoll-Paque Plus (Sigma, St. Louis, MO). Cells were washed once with PBS and RBCs were removed with ACK lysis buffer (ThermoFisher Scientific). Cells were washed once in complete IMDM medium supplemented with 10% FBS, labeled with 0.5 uM Cell Trace Violet (ThermoFisher Scientific) and incubated for 20 min at RT. Cells were washed once in complete IMDM and diluted at 1×106 The canine PBMCs were resuspended at 1x10 cells / ml and cultured in triplicate in 96-well round-bottom plates with 2.5 mg / ml concanavalin A (Sigma-Aldrich) in the presence or absence of 10 mg / ml anti-canine CTLA-4 mAb or a negative anti-MERS mAb control. On day 3 of culture, supernatants were collected and the amount of IFN-γ present in the supernatants was measured by canine IFN-γ ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. For experiments detecting cell surface expression of CTLA-4 after activation, canine PBMCs were cultured at 1x10 6 Cells were resuspended at 2.5 mg / ml and cultured in triplicate in 96-well round-bottom plates with 2.5 mg / ml concanavalin A for 48 and 72 h before analysis.

[0206] Surface Plasmon Resonance Binding Assay. The binding affinity of anti-canine CTLA-4 antibodies to soluble cCTLA-4 ECD was determined using a Biacore T200 version 2.0 and an HC30M (Xantec Bioanalytics, Duesseldorf, Germany) sensor chip. Anti-CTLA-4 antibodies were immobilized on the surface of a linear carboxylic acid SPR sensor chip coated with protein A / G. Briefly, each monoclonal anti-CTLA-4 antibody was diluted to 2.5 ug / ml in 20 mM acetic acid at a pH of 5.0 and injected into the experimental flow cell for 60 seconds. For B10, 440-475 RU were captured, for D5, 400-500 RU were captured, and for A1mut2, 485-535 RU were captured. 10x 1:2 serial dilutions of cCTLA-4 ECD ranging from 200 nM to 0 nM were prepared in duplicate in running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, and 0.05% Tween20). The flow rate across the chip was 30 uL / min and the contact time of the cCTLA-4 ECD sample with the chip surface was 240 seconds. Dissociation was monitored for 720 seconds. Report points were recorded before and after each injection and the amount of antigen binding at each analysis cycle was reported in relative response units (RU). Plots of response versus antigen concentration were generated using the BIAcore Wizard program. After each injection of CTLA-4, the RU for each concentration of analyte was recorded and then the chip surface was regenerated with 20 mM glycine, pH 2.0 for 60 seconds. All experiments were performed at 25°C. Assay data were processed using Biacore Evaluation Software, version 2.0, to obtain kinetic values, k on , k off , and K D reported as.

[0207] Example 1: Generation of recombinant HA-tagged canine CTLA-4 For panning and binding experiments, the extracellular domain of cCTLA-4 was cloned and biotinylated for streptavidin (SA)-mediated plate immobilization. Successful protein production was confirmed by SDS-PAGE. SDS-PAGE analysis revealed a band at approximately 23 kDa, which was higher than the predicted molecular weight of the ECD alone of 16.4 kDa, likely with differences due to protein glycosylation. The identity of the recombinant protein was verified by mass spectrometry (data not shown). To confirm the proper stoichiometry of biotin bound to cCTLA-4 for SA binding, increasing concentrations of SA were added to the biotinylated cCTLA-4 and the SDS-PAGE analysis was repeated. After addition of SA, biotinylated cCTLA-4 was retarded in the gel and a band was no longer identified at 23 kDa, confirming the ability of the biotinylated protein to bind SA. To confirm that biotinylated cCTLA-4 ECD protein was immunoreactive in a 96-well plate format, a capture assay followed by ELISA was performed. The results showed that biotinylated cCTLA-4 immunoreactivity required the presence of plate-bound SA.

[0208] Example 2: Isolation of canine anti-canine CTLA-4 scFv A comprehensive canine IgM / IgG / lambda / Kappa scFv phage display library containing an estimated 40 billion independent canine scFv transformants was subjected to four rounds of solid-phase selection ("panning") against biotinylated cCTLA-4. Substantial enrichment of phage for cCTLA-4 specific binders began with the second round of panning (P2) and increased through the fourth round of panning (P4) (Figure 1). To confirm that selection of the scFv phage library against cCTLA-4 yielded antigen-specific scFv phage particles, polyclonal scFv phage from each round of panning were evaluated by scFv phage ELISA using cCTLA-4 as the target antigen (Figure 2). scFv phage captured through positive selection against cCTLA-4 reacted only with wells loaded with cCTLA-4 and did not react with wells coated with SA alone or with SA loaded with an irrelevant biotinylated avitag binding protein. Recombinant canine (rc)CD19 antigen served as a negative control target antigen, and canine scFv phage selected against CD19 (Pan 4) and rcCD19 served as positive controls. These results demonstrate that phage eluted in the second, third, and fourth rounds of panning contained cCTLA-4-specific scFv.

[0209] In the initial screening, 12 clones from P3 and 12 clones from P4 were randomly selected to test their ability to bind cCTLA-4 by scFv phage ELISA. All 24 clones bound to cCTLA-4 with various affinities (Figure 3). Nucleotide sequencing of these 24 scFvs revealed 20 unique antibodies, 17 of which had lambda light chains and 3 had kappa light chains.

[0210] To prioritize and further develop unique cCTLA-4 specific scFvs, soluble scFvs were first generated, purified, and their ability to bind increasing concentrations of cCTLA-4 was confirmed by ELISA. Sufficient amounts of purified soluble scFv were obtained for analysis of 18 of the 20 clones. 17 / 18 clones were tested for their ability to bind cCTLA-4 (Figure 14). All clones bound cCTLA-4 with a wide range of binding affinities, with clone P4-8 repeatedly demonstrating superior binding compared to the other scFvs.

[0211] Given that 20 unique scFvs were identified among the 24 randomly selected clones, it was hypothesized that additional unique cCTLA-4 specific scFvs were likely present in P3 and P4. Therefore, an additional 88 clones were randomly selected from both P3 and P4, and expression extracts containing soluble scFvs from these additional 176 clones were generated and analyzed by scFv ELISA (data not shown). 41 clones showed a signal greater than 3-fold above background. Ten clones that showed a greater than 20-fold increase in binding to cCTLA-4 were selected and sequenced. From these, three more unique clones were identified from P3 (A1, B10, and C5) and two newer clones (D5 and G11) were identified from P4. Binding of soluble scFvs (A1, B10, D5, and G11) to cCTLA-4 was confirmed by ELISA (Figure 4). Clone C5 could not be produced as a soluble scFv, therefore, a total of 21 unique complete canine soluble scFvs that bind to the extracellular domain of cCTLA-4 were available for further analysis.

[0212] We next sought to determine whether any of the unique isolated cCTLA-4 specific soluble single chains could block the interaction between cCTLA-4 and CD80 / 86, a property that may confer therapeutic potential by enhancing T cell responses in tumor-bearing patients. First, an ELISA-based interaction assay was developed that focused on the ability of biotinylated cCTLA-4 to bind to commercially available human (hu)CD86-Fc and huCD80-Fc chimeric proteins. Using increasing doses of huCD86-Fc or huCD80-Fc bound to microtiter plates, cCTLA-4 was shown to bind to both huCD86-Fc and huCD80-Fc in a dose-dependent manner (data not shown). Next, the ability of 18 soluble scFvs from the original group of 20 unique clones and 4 unique soluble scFvs (A1, B10, D5, and G11) identified from expression extracts to inhibit cCTLA-4:huCD86 and cCTLA-4:huCD80 interactions was determined (Figures 5A-5B). 17 / 18 and 4 / 4 clones from the original and additional screening groups, respectively, showed different degrees of inhibition of cCTLA-4 binding to huCD86. Thirteen of the original clones that inhibited cCTLA-4 binding to huCD86 also inhibited the higher affinity binding of cCTLA-4 to huCD80, with clones P4-8 and P3-7 showing the greatest inhibition. Clones from additional screening rounds that inhibited cCTLA-4 binding to huCD86 also showed comparable or greater inhibition of cCTLA-4 binding to huCD80 / 86 when compared to P4-8 and P3-7 (Figure 5). In all cases, use of a higher molar ratio of scFv:cCTLA-4 resulted in greater inhibition (6:1 compared to 2:1). Greater inhibition of binding was seen when bivalent scFvs (made by cross-linking two scFvs via an anti-HA antibody) were compared to monovalent scFvs.

[0213] Example 3: Generation of anti-CTLA-4 mAbs One of the main characteristics that determines whether an antibody can progress down the pipeline into an effective therapeutic is its "developability". Towards this end, the feasibility of reformatting a cCTLA-4 specific scFv that inhibits cCTLA-4 binding to CD80 / CD86 was determined so that it could be reformatted into a full-length IgG mAb. Briefly, the VH and VL chains of selected scFvs were transformed into either the canine constant light kappa (CLκ) domain or the constant light lambda (CLλ) domain, or into the canine constant IgG1 heavy chain domain (IgG A Full-length bivalent IgG antibodies were generated from the isolated scFvs by cloning into separate expression plasmids containing the CL and CL genes. Canine CL and CL were cloned into pFUSE2ss (Invitrogen) to generate pFUSE2ss-CLIg-dλ3, pFUSE2ss-CLIg-dλ5, and pFUSE2ss-CLIg-dκ, which can be selected via blasticidin resistance. Similarly, canine VH chains were cloned into pFUSEss-CHIg-dG1, purchased from Invivogen, to complete the reformatting of the full range of plasmids. Plasmids for cloning of the VL regions were selected based on the subtype (lambda vs. kappa) of the isolated VL regions. Adherent 293T cells were transiently co-transfected with a 1.5:1 ratio of light and heavy chain plasmids, respectively, using lipofectamine L2000 reagent. Plate supernatants were harvested after 3 days and the mAbs were purified using Protein A affinity chromatography.

[0214] Based on their ability to inhibit cCTLA-4 binding to its ligands CD80 and CD86, clones A1, D5, B10, G11, and P4-8 were selected to be reformatted into full-length IgG mAbs for further functional analysis. B10 and D5 are IgG1 (IgG A), only low levels of clone A1 could be produced, and clones G11 and P4-8 could not be produced as full-length canine mAbs (Figure 6). Given the superior ability of clone A1 to bind cCTLA-4 (Figure 7) and block the interaction of cCTLA-4 with CD80 / 86 (Figure 8), we investigated whether production of the A1 mAb could be improved through CDR grafting experiments. Using chain swapping experiments, the low productivity was localized to the VH chain of A1 (Figure 9). Clone B10 was readily produced, so the CDR regions of A1 were grafted into the VH backbone of clone B10. The resulting chimeric A1 VH chain transfected together with the original A1 VL chain resulted in high yield production of a full canine mAb, designated A1mut2. The ability of whole canine A1mut2, B10, and D5 to bind cCTLA-4 in ELISA (Figure 11A) and to inhibit the interaction of cCTLA-4 with CD80 / 86 was confirmed (Figure 11B). In parallel, the ability of whole canine mAbs to inhibit cCTLA-4 binding to canine CD80 / 86 was also confirmed (Figure 11C). For these experiments, a hemagglutinin (HA) tag separated by a glycine-serine rich linker sequence was added to the carboxyl terminus of full-length IgG, and the antibody was detected with a secondary antibody against the tag, which has the amino acid sequence Nucleotide sequence encoding TIFF2024516621000019.tif4128 TIFF2024516621000020.tif19163 was required for addition. To obtain the non-commercially available canine CD80 and CD86 proteins, the amino acid sequences of accession numbers NP_001003147.1 and ABH87294.1, respectively, were obtained from the NCBI database. Canine CD80 and CD86 human Fc tagged constructs were designed using the corresponding human CD80-Fc and CD86-Fc constructs (ACROBiosystems, Newark, Delaware; #B71-H5259 and #CD6-H5257, respectively) as guides. The canine extracellular domain sequences were fused to a triple alanine sequence followed by a modified human IgG1 Fc with a C to S mutation in the hinge region. The native canine CD80 and CD86 signal peptides were replaced with mouse IgG heavy chain signal sequences. The gBlocks of the construct sequences were ordered from IDT (Coralville, Iowa) and cloned into the pFUSE plasmid. The pFUSE cCD80-hFc and cCD86-hFc plasmids were transfected into 293T cells with Lipofectamine 2000, and cell media was harvested after 3 days. Purification of the ligand Fc fusion proteins from cell culture supernatants was performed using Protein A agarose (MilliporeSigma, Burlington, MA). Sample concentration, buffer exchange into PBS, SDS-PAGE analysis, and quantification were performed as for the production of recombinant canine CTLA-4 protein.

[0215] Example 4: CTLA-4 specific mAb binds to cell surface expressed CTLA-4 To determine whether A1mut2, B10, and D5 mAbs bind to membrane-expressed cCTLA-4, KTδ32 cells were genetically modified to express cCTLA-4 (KTδ32.cCTLA-4) and used as target cells in flow cytometry experiments (Figure 10). All clones bound to KTδ32.cCTLA-4 but not to parental KTδ32 cells. Furthermore, preincubation of each mAb with soluble cCTLA-4 to block the CTLA-4 binding site abolished mAb binding, indicating that membrane binding was antigen-specific. An irrelevant MERS mAb did not bind to KTδ32.cCTLA-4, further suggesting that the selected clones specifically bound to cCTLA-4. Next, the ability of the three selected mAbs to bind to CTLA-4 on the surface of activated canine T cells was evaluated (Figure 15). CD5 cells prior to activation were stained for CTLA-4. + CD4 + Cells and CD5 + CD4 - Although only trace amounts of CTLA-4 were detected on the cell surface, both T cell subsets showed a significant increase in CTLA-4 expression at 48 and 72 hours after activation. At each time point, a greater percentage of CD4 T cells expressed CTLA-4 compared with CD8 T cells (50.7% vs. 29.5% at 48 hours and 56.6% vs. 40.5% at 72 hours, respectively). + CD4 + FoxP3 + Surface and intracellular expression of CTLA-4 on regulatory T cells was measured using A1mut2 mAb. A1mut2 labeled both surface-expressed and intracellular CTLA-4 on Tregs isolated from peripheral blood of dogs with T cell lymphoma, confirming that CTLA-4 is constitutively expressed by canine peripheral blood Tregs (Figure 16). Finally, considering that canine CTLA-4 and feline CTLA-4 share 99% identity, feline leukocytes were labeled with A1mut2 mAb to determine cross-reactivity of the antibody. Feline CD5+ cells showed minimal surface staining with A1mut2, but the antibody bound strongly to intracellular CTLA-4 (Figure 17).

[0216] Example 5: Whole canine anti-CTLA-4 mAb exhibits high binding affinity to soluble cCTLA-4 The affinity of A1mut2, D5, and B10 for cCTLA-4 was evaluated to determine whether they may be suitable for therapeutic use (Figures 12A-12C). Surface plasmon resonance (SPR) was used to determine the on-rate and off-rate of these three mAbs for cCTLA-4 (Figure 21). The dissociation constant (K D ) exhibited the highest binding affinity of the three clones, in the subnanomolar range. The other two clones exhibited single-digit nanomolar affinities. Thus, all three mAbs exhibited sufficient binding affinity for cCTLA-4 to serve as candidates for evaluation as therapeutic checkpoint inhibitors (depicted in FIG. 13).

[0217] Example 6: Whole Canine Anti-CTLA-4 IgG2 (IgG B ) and IgG3 (IgG C ) fixes complement Given that a contribution of CTLA-4-targeting antibodies to antitumor immunity is increasingly recognized as their ability to deplete intratumoral regulatory T cells, the ability of the A1mut2 IgG subclass to fix complement was tested in vitro. As previously demonstrated, IgG B and IgG C effectively fixed complement, but IgG1 (IgG A ) and IgG4 (IgG D ) did not bind (Figures 18A-18B). This result suggests that these subclasses of A1mut2 Ig have an effect that is mostly restricted to intratumoral Tregs expressing high levels of CTLA-4, a CTLA-4 + It has been shown to have the potential to initiate complement-mediated cytotoxicity of cells.

[0218] Example 7: Clone A1mut2 increases T cell proliferation and IFN-γ production To determine whether A1mut2, which showed the highest affinity for cCTLA-4 and the greatest inhibition of cCTLA-4 binding to CD80 / 86, could increase T cell proliferation through its ability to block checkpoint signaling, canine PBMCs were labeled with Cell Trace Violet and stimulated with concanavalin A in the presence of either A1mut2 or an irrelevant MERS-specific mAb. Cells were harvested at 72 or 96 hours, and the responder frequency and proliferation potential of T cells activated in the presence of A1mut2 versus MER were determined by flow cytometry (Figure 19). A1mut2 increased the percentage of T cells responding to mitogens (responder frequency) and the average number of daughter cells produced per responder cell (proliferation potential) at 96 hours post-stimulation in 8 / 9 dogs when compared to an irrelevant MERS mAb. IFN-γ production was assessed in the supernatants of T cells stimulated with ConA in the presence of A1mut2. T cell cultures from X / Y dogs showed increased IFN-γ production at 96 hours when A1mut2 was added to the cultures ( FIG. 20 ). Together, these results suggest that A1mut2-mediated checkpoint inhibition promotes canine T cell proliferation and IFN-γ production, and further support its clinical evaluation as a mAb to enhance tumor-specific T cell priming and effector responses within the tumor microenvironment.

[0219] Example 8: Discussion CTLA-4 blockade has proven to be a powerful strategy to promote antitumor immunity by inducing the expansion of Th1-like CD4 effector T cells and exhausted CD8+ T cells, and eliminating intratumoral regulatory T cells. These effects have resulted in clinically relevant antitumor immunity, especially in human patients with malignant melanoma, NSCLC, and renal carcinoma. However, greater understanding of the mechanisms of tumor acquired and / or innate resistance to anti-CTLA-4 therapy, identification of biomarkers of response, optimized protocols and combination approaches to improve outcomes, and understanding and reduction of mechanisms related to toxicity are needed to increase overall response rates and reduce toxicity. The work presented in this disclosure employed a powerful scFv phage display approach to identify multiple unique clones of full canine scFvs that specifically bind with nanomolar and sub-nanomolar affinity to canine CTLA-4. From these clones, full canine mAb lead candidates were generated and further selected based on developability and in vitro functional capacity to be adopted into clinical trials in dogs with spontaneous cancer. The use of fully canine anti-CTLA-4 mAbs in immunocompetent canine cancer patients with spontaneous tumors that share similar characteristics with their human counterparts will yield beneficial results for human clinical trial design.

[0220] The study of the present disclosure confirmed the specific binding of A1mut2 to canine CTLA-4 using A1mut2 in flow cytometry, and demonstrated that CTLA-4 is upregulated on the surface of CD4+T cells and CD8+T cells after mitogen activation.Higher CTLA-4 expression was observed in CD4 T cell subsets compared to CD8 T cells after activation, which is consistent with the findings in human CD4 T cells and CD8 T cells, and the differential kinetics of CTLA-4 upregulation on T cell subsets in dogs, with expression on CD4+T cells peaking earlier (48 hours) than CD8+T cells (72 hours or more).Studies in human T cells have demonstrated that the peak expression of CTLA-4 on all mitogen-activated T cells occurs 72 hours after stimulation.

[0221] In human studies, monotherapy with ipilimumab showed only minimal effects on IFN-γ production and T cell proliferation in allogeneic T:DC MLR assays, and only a slight increase in IL-2 production after mitogen activation of human T cells. Furthermore, responses varied among individual samples tested. While in vitro assay responses in human T cells were modest, clinical responses in a subset of human patients were dramatic, highlighting the multifactorial mechanism of action of CTLA-4 blockade and the impact that patient-specific factors may have on clinical response. The presently disclosed study also observed significant variability in T cell responses to CTLA-4 blockade among mitogen-activated T cells from healthy donor dogs, as well as modest effects on T cell proliferation and cytokine production. Similar to the results from human donors, low levels of CTLA-4 surface expression were observed on resting T cells, which suggests, without wishing to be bound by theory, that the use of A1mut2 in canine cancer patients does not result in inappropriate activation of naive T cells. Furthermore, these studies confirmed that, at the protein level, CTLA-4 is present / upregulated in regulatory T cells and can serve as a target for A1mut2-dependent complement-mediated cytotoxicity. Indeed, in human patients, higher levels of CTLA-4 expression on intratumoral Tregs compared to general circulating Tregs allows selective depletion after anti-CTLA-4 treatment, which results in an expansion of CD8:Tregs after treatment. A1mut2 is now an IgG1 subtype functionally equivalent to the human IgG1 subtype, which efficiently binds complement and induces ADCC. B (Bergeron et al. Vet Immunol Immunopath, 157:31-41, 2014). Therefore, a similar effect is expected to occur on intratumoral regulatory T cells in canine tumors.

[0222] Because clinical responses in canine malignancies mirror those of human patients with melanoma, gastric cell carcinoma, and urothelial carcinoma, it is anticipated that this therapy will improve outcomes in pet dogs suffering from similar malignancies.

[0223] Enumerated aspects The following enumerated aspects are provided, the numbering of which should not be construed as designating a level of importance. Aspect 1 provides: An antibody or antigen-binding fragment thereof, comprising an antigen-binding domain that specifically binds to an epitope of canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), The antigen-binding domain comprises: i. 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:6, 25, 40, or 74; and ii. 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:8, 27, or 42; Including, The heavy chain variable region comprises three heavy chain complementarity determining regions (HCDRs), HCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 3, 22, and 37); The light chain variable region comprises three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 4, 23, and 38), LCDR2 comprises an amino acid sequence selected from the group consisting of (VDG, GNY, and GNS), and LCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 5, 24, and 39); An antibody or an antigen-binding fragment thereof. Aspect 2 provides: The antibody or antigen-binding fragment thereof according to embodiment 1, which is selected from the group consisting of a full-length antibody, a Fab, and a single-chain variable fragment (scFv). Aspect 3 provides: The antibody or antigen-binding fragment thereof of embodiment 2, which is a full-length antibody. Aspect 4 provides: The antibody or antigen-binding fragment thereof according to embodiment 3, wherein the antibody is a canine antibody. Aspect 5 provides: 2. The antibody or antigen-binding fragment thereof of embodiment 1, wherein the antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:6, 25, 40, or 74. Aspect 6 provides: The antibody or antigen-binding fragment thereof according to embodiment 1, wherein the antigen-binding domain consists of a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:6, 25, 40, or 74. Aspect 7 provides: 5. The antibody or antigen-binding fragment thereof according to any one of aspects 1 to 4, wherein the antigen-binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8, 27, or 42. Aspect 8 provides: The antibody or antigen-binding fragment thereof according to any one of aspects 1 to 4, wherein the antigen-binding domain consists of a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8, 27, or 42. Aspect 9 provides: i. a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:6; and ii. A light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8. 2. An isolated antibody or antigen-binding fragment thereof comprising: Aspect ten provides: i. a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 3, 22, and 37); and ii. a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 4, 23, and 38), LCDR2 comprises an amino acid sequence selected from the group consisting of (VDG, GNY, and GNS), and LCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 5, 24, and 39). Including, the heavy chain variable region and the light chain variable region are separated by a linker; A single-chain variable fragment (scFv) containing an antigen-binding domain that specifically binds to an epitope of canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Aspect 11 provides: i. a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:6, 25, 40, or 74; and ii. a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8, 27, or 42; Including, the heavy chain variable region and the light chain variable region are separated by a linker; Single chain variable fragment (scFv). Aspect 12 provides: A single-chain variable fragment (scFv) comprising the amino acid sequence set forth in SEQ ID NO:29, 44, or 76. Aspect 13 provides: A single-chain variable fragment (scFv) consisting of the amino acid sequence set forth in SEQ ID NO:29, 44, or 76. Aspect 14 provides: A full-length antibody comprising an antigen-binding domain that specifically binds to an epitope of canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), The antigen-binding domain comprises: i. a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 3, 22, and 37); and ii. a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 4, 23, and 38), LCDR2 comprises an amino acid sequence selected from the group consisting of (VDG, GNY, and GNS), and LCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 5, 24, and 39). A full-length antibody comprising: Aspect 15 provides: i. a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:6, 25, 40, or 74; and ii. a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8, 27, or 42; A full-length antibody comprising: Aspect 16 provides: A full-length antibody comprising a heavy chain amino acid sequence set forth in SEQ ID NO:10, 12, 14, 16, 31, 46, 71, 72, or 73, and a light chain amino acid sequence set forth in SEQ ID NO:18, 33, or 48. Aspect 17 provides: A full-length antibody consisting of a heavy chain amino acid sequence set forth in SEQ ID NO:10, 12, 14, 16, 31, 46, 71, 72, or 73, and a light chain amino acid sequence set forth in SEQ ID NO:18, 33, or 48. Aspect 18 provides: An isolated nucleic acid encoding an scFv or full length antibody according to any preceding embodiment. Aspect 19 provides: An isolated nucleic acid encoding an antibody or antigen-binding fragment thereof comprising an antigen-binding domain that specifically binds to an epitope of canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), The antigen-binding domain comprises: i. 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 SEQ ID NO:7, 26, 41, or 75; and ii. 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 amino acid sequence of the light chain variable region set forth in SEQ ID NO:9, 28, or 43; Including, The heavy chain variable region comprises three heavy chain complementarity determining regions (HCDRs), HCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 3, 22, and 37); The light chain variable region comprises three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 4, 23, and 38), LCDR2 comprises an amino acid sequence selected from the group consisting of (VDG, GNY, and GNS), and LCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 5, 24, and 39); Isolated nucleic acid. Aspect 20 provides: 20. The nucleic acid of embodiment 19, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, a Fab, and a single-chain variable fragment (scFv). Aspect 21 provides: 21. The nucleic acid of embodiment 20, wherein the antibody is a full-length antibody. Aspect 22 provides: 22. The nucleic acid of embodiment 21, wherein the antibody is a canine antibody. Aspect 23 provides: 20. The nucleic acid of embodiment 19, wherein the heavy chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence set forth in SEQ ID NO:7, 26, 41, or 75. Aspect 24 provides: 20. The nucleic acid of embodiment 19, wherein the heavy chain variable region is encoded by a nucleic acid consisting of the polynucleotide sequence set forth in SEQ ID NO:7, 26, 41, or 75. Aspect 25 provides: 20. The nucleic acid of embodiment 19, wherein the light chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence set forth in SEQ ID NO:9, 28, or 43. Aspect 26 provides: 20. The nucleic acid of embodiment 19, wherein the light chain variable region is encoded by a nucleic acid consisting of the polynucleotide sequence set forth in SEQ ID NO:9, 28, or 43. Aspect 27 provides: i. a heavy chain variable region encoded by a nucleic acid sequence comprising a polynucleotide sequence set forth in SEQ ID NO:7, 26, 41, or 75; and ii. a light chain variable region encoded by a nucleic acid sequence comprising a polynucleotide sequence set forth in SEQ ID NO:9, 28, or 43; An isolated nucleic acid encoding an antibody or antigen-binding fragment thereof comprising: Aspect 28 provides: i. a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 3, 22, and 37); and ii. a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 4, 23, and 38), LCDR2 comprises an amino acid sequence selected from the group consisting of (VDG, GNY, and GNS), and LCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NOs: 5, 24, and 39). An isolated nucleic acid encoding a single chain variable fragment (scFv) comprising: Aspect 29 provides: i. a heavy chain variable region comprising the nucleotide sequence set forth in SEQ ID NO:7, 26, 41, or 75; and ii. a light chain variable region comprising the nucleotide sequence set forth in SEQ ID NO:9, 28, or 43. Including, the heavy chain variable region and the light chain variable region are separated by a linker; An isolated nucleic acid encoding a single chain variable fragment (scFv). Aspect 30 provides: A single chain variable fragment (scFv) comprising a polynucleotide sequence set forth in SEQ ID NO: 30, 45, or 77. An isolated nucleic acid encoding Aspect 31 provides: A single chain variable fragment (scFv) consisting of a polynucleotide sequence set forth in SEQ ID NO: 30, 45, or 77. An isolated nucleic acid encoding Aspect 32 provides: A vector comprising the isolated nucleic acid according to any one of aspects 19 to 32. Aspect 33 provides: 33. The vector of embodiment 32, which is an expression vector. Aspect 34 provides: 34. The vector of any one of embodiments 32 and 33, wherein the vector 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. Aspect 35 provides: A host cell comprising the vector according to any one of aspects 32 to 34. Aspect 36 provides: 36. The host cell of embodiment 35, which is of eukaryotic or prokaryotic origin. Aspect 37 provides: 37. The host cell of any one of embodiments 35 or 36, which is of mammalian origin. Aspect 38 provides: 37. The host cell of any one of embodiments 35 or 36, which is of bacterial origin. Aspect 39 provides: A method for producing an antibody or antigen-binding fragment thereof that binds to canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the method comprising culturing a host cell according to any one of aspects 35 to 38. Aspect 40 provides: A pharmaceutical composition comprising the full-length antibody or scFv according to any one of aspects 1 to 17 and a pharma- ceutically acceptable carrier. Aspect 41 provides: A method for treating cancer in a subject in need thereof, comprising the step of administering to the subject the antibody or antigen-binding fragment thereof according to any one of aspects 1 to 17. Aspect 42 provides: The method of embodiment 41, wherein the cancer is associated with cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Aspect 43 provides: The method of embodiment 42, wherein CTLA-4 is expressed on cancer-associated cells. Aspect 44 provides: The method of embodiment 43, wherein the cancer-associated cells are T lymphocytes. Aspect 45 provides: The method of embodiment 41, wherein the antibody or antigen-binding fragment thereof specifically binds to canine CTLA-4. Aspect 46 provides: The method of embodiment 41, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, a Fab, and a single-chain variable fragment (scFv). Aspect 47 provides: The method of embodiment 46, wherein the antibody or antigen-binding fragment thereof is a full-length antibody. Aspect 48 provides: The method of embodiment 47, wherein the antibody is a caninized antibody. Aspect 49 provides: The method of embodiment 41, wherein the subject is a dog.

[0224] Other Aspects The recitation of a list of elements in any definition of a variable herein includes definition 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.

[0225] The disclosures of any and all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed in connection with certain embodiments, it is clear that other embodiments and modifications of the present invention may be devised by others skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

1. An antibody or antigen-binding fragment thereof comprising an antigen-binding domain that specifically binds to an epitope of canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), The antigen-binding domain comprises: (i) 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:6, 25, 40, or 74; and (ii) a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:8, 27, or 42; Including, the heavy chain variable region comprises three heavy chain complementarity determining regions (HCDRs), HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 20, and 35, HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 21, and 36, and HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 22, and 37; the light chain variable region comprises three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 23, and 38, LCDR2 comprises an amino acid sequence selected from the group consisting of VDG, GNY, and GNS, and LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 24, and 39; An antibody or an antigen-binding fragment thereof.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, a Fab, and a single-chain variable fragment (scFv), optionally wherein the antibody or antigen-binding fragment thereof is a full-length antibody, and optionally wherein the antibody or antigen-binding fragment thereof is a canine antibody.

3. The antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 90% identity to an amino acid sequence set forth in SEQ ID NO:6, 25, 40, or 74; and / or the antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 8, 27, or 42; The antibody or antigen-binding fragment thereof described in claim 1. (i) a heavy chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:6; and (ii) a light chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

8.

2. An isolated antibody or antigen-binding fragment thereof comprising:

5. (i) a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 20, and 35, HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 21, and 36, and HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 22, and 37; and (ii) a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 23, and 38; LCDR2 comprises an amino acid sequence selected from the group consisting of VDG, GNY, and GNS; and LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 24, and 39. Including, the heavy chain variable region and the light chain variable region are separated by a linker; A single-chain variable fragment (scFv) containing an antigen-binding domain that specifically binds to an epitope of canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

6. A full-length antibody comprising an antigen-binding domain that specifically binds to an epitope of canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), The antigen-binding domain comprises: (i) a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 20, and 35, HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 21, and 36, and HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 22, and 37; and (ii) a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 23, and 38; LCDR2 comprises an amino acid sequence selected from the group consisting of VDG, GNY, and GNS; and LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 24, and 39. A full-length antibody comprising: (i) a heavy chain variable region comprising an amino acid sequence having at least 95% identity to an amino acid sequence set forth in SEQ ID NO:6, 25, 40, or 74; and (ii) a light chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 8, 27, or 42. A full-length antibody comprising:

8. A full-length antibody comprising a heavy chain amino acid sequence having at least 95% identity to the amino acid sequence described in SEQ ID NO: 10, 12, 14, or 16, and a light chain amino acid sequence having at least 95% identity to the amino acid sequence described in SEQ ID NO:

18.

9. An isolated nucleic acid encoding an scFv or full-length antibody described in any one of claims 1 to 8.

10. 1. An isolated nucleic acid encoding an antibody or antigen-binding fragment thereof comprising an antigen-binding domain that specifically binds to an epitope of canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), The antigen-binding domain comprises: (i) 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 SEQ ID NO:7, 26, 41, or 75; and (ii) 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%, or 99% identity to the amino acid sequence of a light chain variable region set forth in SEQ ID NO:9, 28, or 43. Including, the heavy chain variable region comprises three heavy chain complementarity determining regions (HCDRs), HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 20, and 35, HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 21, and 36, and HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 22, and 37; the light chain variable region comprises three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 23, and 38, LCDR2 comprises an amino acid sequence selected from the group consisting of VDG, GNY, and GNS, and LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 24, and 39; Isolated nucleic acid.

11. 10. A vector comprising the isolated nucleic acid of claim 9, wherein the vector is an expression vector, and optionally the vector 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.

12. 12. A host cell comprising the vector of claim 11, optionally wherein the host cell is of eukaryotic or prokaryotic origin, and optionally wherein the host cell is of mammalian or bacterial origin.

13. A method for producing an antibody or an antigen-binding fragment thereof that binds to canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), comprising culturing the host cell of claim 12.

14. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 4, a single-chain variable fragment (scFv) described in claim 5, or a full-length antibody described in any one of claims 6 to 8, and a pharma- ceutically acceptable carrier.

15. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, the single chain variable fragment (scFv) of claim 5, or the full length antibody of any one of claims 6 to 8 for use in a method for treating cancer in a subject in need thereof, optionally wherein the subject is a dog, and optionally the cancer is associated with cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

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