Canine monoclonal antibody 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 of related applications This application is entitled to priority to U.S. Provisional Patent Application No. 63 / 177,692, filed on 21 April 2021, which is incorporated herein by reference in its entirety under 35 U.S.C. §119(e).

[0002] Description of research and development funded by the federal government. This invention was developed with government support under HHSN 261201800042C, awarded by the U.S. National Institutes of Health. The government has certain rights with respect to this invention. [Background technology]

[0003] Background of the Invention Advances in understanding the interaction 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 achieved great clinical success and are increasingly becoming cutting-edge therapies. In fact, human cancer immunotherapy is now recognized as one of the pillars of treatment, alongside surgery, radiation therapy, and chemotherapy. The field of veterinary cancer immunotherapy has also advanced rapidly in the last decade, but the availability of animal-specific therapies and a complete understanding of their specific effects remain challenges.

[0004] Checkpoint inhibitor therapy is a promising immunotherapy strategy that works by inhibiting receptors that negatively regulate T cells. In effect, these therapies remove natural "brakes" called immune checkpoints that suppress T cell function. While these negative receptors normally play a crucial role in preventing immune responses from becoming toxic, many cancers utilize this negative signaling to blunt the T cell-based immune response directed at them. One key immune checkpoint receptor 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 to 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 excessive activation of the immune system. Antibodies that block CTLA-4, 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] Therefore, in the art, there is a need for the development of canine antibodies that can be used to treat veterinary cancers in dogs by blocking CTLA-4 on tumor-specific T cells. The present invention addresses and satisfies this need. [Overview of the Initiative]

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

[0007] In one aspect, the present invention relates to an antibody or an 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 is i. A heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, and 99% identity with the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 6, 25, 40, or 74; and ii. Light chain variable regions containing amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences described in SEQ ID NO: 8, 27, or 42. Includes; The heavy chain variable region comprises three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 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 NO: 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 NO: 5, 24, and 39). The present invention provides an antibody or an antigen-binding fragment thereof.

[0008] In a particular embodiment, the antibody or its antigen-binding fragment is selected from the group consisting of full-length antibodies, Fabs, and single-chain variable fragments (scFv).

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

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

[0011] In certain embodiments, the antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding domain comprises a heavy-chain variable region comprising the 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 certain embodiments, 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 the amino acid sequence set forth in SEQ ID NO: 8, 27, or 42.

[0014] In certain embodiments, 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 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 comprising an isolated antibody or antigen-binding fragment thereof.

[0016] In another aspect, the present invention 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 NO: 1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 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 NO: 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 NO: 5, 24, and 39). Includes, The heavy chain variable region and the light chain variable region are separated by a linker. It contains a single-chain variable fragment (scFv) that includes an antigen-binding domain that specifically binds to the epitope of canine cytotoxic T lymphocyte-associated protein 4 (CTLA-4).

[0017] In another aspect, the present invention is i. Heavy chain variable region containing amino acid sequences described in SEQ ID NO: 6, 25, 40, or 74; and ii. Light chain variable region containing the amino acid sequence described in SEQ ID NO: 8, 27, or 42 Includes, 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 present invention includes a single-chain variable fragment (scFv) comprising the amino acid sequence described in SEQ ID NO: 29, 44, or 76.

[0019] In another aspect, the present invention includes a single-chain variable fragment (scFv) comprising the amino acid sequence described in SEQ ID NO: 29, 44, or 76.

[0020] In another aspect, the present invention relates to a full-length antibody comprising an antigen-binding domain that specifically binds to the epitope of canine cytotoxic T lymphocyte-associated protein 4 (CTLA-4), The antigen-binding domain is 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 NO: 1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 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 NO: 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 NO: 5, 24, and 39). Contains full-length antibodies.

[0021] In another aspect, the present invention is i. Heavy chain variable region containing amino acid sequences described in SEQ ID NO: 6, 25, 40, or 74; and ii. Light chain variable region containing the amino acid sequence described in SEQ ID NO: 8, 27, or 42 Contains full-length antibodies.

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

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

[0024] In another aspect, the present invention comprises an isolated nucleic acid encoding any of the scFv or full-length antibodies described in any of the preceding claims.

[0025] In another aspect, the present invention relates to an isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof, comprising an antigen-binding domain that specifically binds to the epitope of canine cytotoxic T lymphocyte-associated protein 4 (CTLA-4), The antigen-binding domain is i. Heavy chain variable regions encoded by nucleic acids containing polynucleotide sequences having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, and 99% identity to SEQ ID NO: 7, 26, 41, or 75; and ii. Light chain variable region encoded by a nucleic acid containing a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the light chain variable region described in SEQ ID NO: 9, 28, or 43. Includes; The heavy chain variable region comprises three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 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 NO: 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 NO: 5, 24, and 39). Contains isolated nucleic acids.

[0026] In a particular embodiment, the antibody or its antigen-binding fragment is selected from the group consisting of full-length antibodies, Fabs, and single-chain variable fragments (scFv).

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

[0028] In certain aspects, 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 described 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 a polynucleotide sequence described 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 described 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 a polynucleotide sequence described in SEQ ID NO: 9, 28, or 43.

[0033] In another aspect, the present invention is i. Heavy chain variable regions encoded by nucleic acid sequences including polynucleotide sequences described in SEQ ID NO: 7, 26, 41, or 75; and ii. Light chain variable regions encoded by nucleic acid sequences containing polynucleotide sequences described in SEQ ID NO: 9, 28, or 43. It contains isolated nucleic acids encoding an antibody or its antigen-binding fragment.

[0034] In another aspect, the present invention is 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 NO: 1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 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 NO: 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 NO: 5, 24, and 39). Contains isolated nucleic acids encoding single-chain variable fragments (scFv) including [specific component].

[0035] In another aspect, the present invention is i. Heavy chain variable regions containing nucleotide sequences described in SEQ ID NO: 7, 26, 41, or 75; and ii. Light chain variable region containing the nucleotide sequence described in SEQ ID NO: 9, 28, or 43 Includes, The heavy chain variable region and the light chain variable region are separated by a linker. Contains isolated nucleic acids encoding single-chain variable fragments (scFv).

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

[0037] In another aspect, the present invention includes isolated nucleic acids encoding single-chain variable fragments (scFv) consisting of polynucleotide sequences described in SEQ ID NO: 30, 45, or 77.

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

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

[0040] In certain embodiments, the vector is selected from the group consisting of DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and retroviral vectors.

[0041] In another aspect, the present invention includes a host cell comprising a vector in 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 aspects, the host cells are of mammalian origin.

[0044] In certain aspects, 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 the step of culturing host cells in any one of the above aspects or any aspect or embodiment disclosed herein.

[0046] In another aspect, the present invention includes a pharmaceutical composition comprising a full-length antibody or scFv in any of the above aspects or embodiments disclosed herein, and a pharmaceutically acceptable carrier.

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

[0048] In certain aspects, 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 contexts, cancer-associated cells are T lymphocytes.

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

[0052] In a particular embodiment, the antibody or its antigen-binding fragment is selected from the group consisting of full-length antibodies, Fabs, and single-chain variable fragments (scFv).

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

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

[0055] In certain aspects, the subject is a dog. [Brief explanation of the drawing]

[0056] The following detailed description of preferred embodiments of the present invention will be better understood in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, currently preferred embodiments are shown in the drawings. However, it should be understood that the present invention is not limited to the exact arrangement and means of the embodiments shown in the drawings.

[0057] [Figure 1]This table shows the phage input and output results from library panning against canine CTLA-4 (cCTLA-4). A comprehensive canine IgM / IgG / λ / κscFv 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 phages in the input and output (eluate) are reported along with the percentage of phages bound to the target and the enrichment of antigen-specific conjugates after each panning round. [Figure 2] This shows the enrichment of canine phage display libraries after panning for canine CTLA-4 antigen. Biotinylated avitag-cCTLA-4 was captured overnight at 4°C on streptavidin-coated microtiter plates. Wells were washed with PBS and blocked with 2% milk in PBS (MPBS) at 37°C for 1 hour. 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 at 37°C for 1 hour. Plates were washed with PBS supplemented with 0.5% Tween (PBST), and bound phages were detected using HRP-conjugated anti-M13 mAb (GE Healthcare) at 1:5000 dilution in 2% milk. Plates were washed again, and bound phages were detected by 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 phages from the fourth round of panning against canine CD19, derived from a parallel panning operation, were used as positive controls. [Figure 3]This report describes the screening of canine scFv antibodies from panning rounds 3 and 4 for binding to recombinant canine CTLA-4. Twelve clones from panning round 3 and 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 scFv revealed 20 unique antibodies, the majority of which (17) contained the lambda light chain. Phage-free and unrelated MERS (Middle East Respiratory Syndrome Virus) specific phages were used as negative controls. Polyclonal phages from the fourth round of panning against canine CTLA-4 were used as positive controls. [Figure 4] This shows the binding of clone P4-8 and additional soluble CTLA-4 scFv (identified via high-throughput screening (HTS) expression assay) to cCTLA-4 using ELISA. Unique scFv clones that bind to cCTLA-4 were identified via HTS assay (A1, B10, D5, and G11) in addition to clone P4-8 (Figure 3). Unique, soluble, HA-tagged, purified scFv were tested by ELISA for their ability to bind to increasing concentrations of cCTLA-4. 0.25 ug / ml of soluble scFv was added to each well, and bound scFv was detected using AP-conjugated anti-HA antibody. All clones bound to cCTLA-4 with varying affinities. Soluble single chains against unrelated MERS proteins were used as negative controls. [Figure 5]Figures 5A–5B show that CTLA-4-specific soluble scFv inhibits the binding of cCTLA-4 to its congener ligands, CD80 and CD86. 1.0 and 3.0 pmol of recombinant human CD86-Fc chimeras (Figure 5A) or human CD80-Fc chimeras (Figure 5B) were conjugated overnight to ELISA plates. Wells were blocked with 5% milk / PBS 0.05% Tween 20. Biotinylated cCTLA-4 extracellular domains (ECDs) were pre-incubated with each soluble scFv in the indicated molar ratios at RT for 1 hour, then added to the plates and incubated for 2 hours. After washing, cCTLA-4 bound to CD80 or CD86 was detected using streptavidin-AP conjugate and subsequent AP chromometric substrates. Biotinylated cCTLA-4 and unbiotinylated cCTLA-4, both lacking any antibodies, were used as positive and negative controls. "na" = no antibody. [Figure 6] This table shows the expression levels of anti-canine IgG. [Figure 7] The ELISA analysis of full-length canine anti-canine CTLA-4 mAb is shown. [Figure 8] Figures 8A and 8B show assays for the inhibition of CTLA-4 binding to its congener ligands CD80 (Figure 8A) and CD86 (Figure 8B) as purified full-length monoclonal dog IgG. [Figure 9] This table shows that substitution in the B10 VH framework region of the A1 VH framework region rescues the generation of anti-canine CTLA-4 clone A1 (modified clones are labeled mut2). [Figure 10]This report describes the evaluation of anti-CTLA-4 mAb clones that bind to membrane-expressed canine CTLA-4. K562 cells lacking FcγRII(CD32) were genetically engineered to express canine CTLA-4 (KT32δ.cCTLA4). KT32δ cells (top panel) and KT32δ.cCTLA4 cells (middle panel) 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, the anti-CTLA-4 clones were first incubated with soluble cCTLA-4 protein to block the antigen-binding site, and then used for cell surface labeling (bottom panel). [Figure 11] Figures 11A–11C show that scFv clones reformatted as complete canine IgG molecules retain their ability to bind to cCTLA-4 and inhibit its interaction with recombinant human and canine CD80 and CD86. Full-length bivalent IgG antibodies were prepared from isolated scFv by cloning the VH and VL chains of selected scFv into separate heavy and light chain expression plasmids. Adhered 293T cells were transiently co-transfected with the light and heavy chain plasmids, and mAbs were purified from the plate supernatant after 3 days using protein A affinity chromatography. Figure 11A. mAbs were evaluated by ELISA for their binding to increasing concentrations of cCTLA-4. Figure 11B. mAbs were evaluated for their ability to inhibit the interaction between cCTLA-4 and recombinant human CD80 (left graph) and rhuCD86 (right graph) at the indicated molar ratios. Similarly, Figure 11C shows the inhibition of the interaction between cCTLA-4 and recombinant canine CD80 (left graph) and CD86 (right graph) at the molar ratios shown. [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] This figure shows the normal function of CTLA-4 when it binds to its homologous ligand on antigen-presenting cells, and strategies for antibody-based blockade of CTLA-4 signaling. [Figure 14] This study demonstrates the 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 by ELISA for their ability to bind to increasing concentrations of cCTLA-4. 0.25 ug / ml of soluble scFv was added to each well, and bound scFv was detected using AP-conjugated anti-HA antibody. All clones bound to cCTLA-4 with varying affinities. Soluble single chains against unrelated MERS protein were used as a negative control. A commercially available polyclonal anti-CTLA-4 antibody was used as a positive control in one assay (upper left graph). [Figure 15] This shows the binding of anti-CTLA-4 mAb clones to activated canine T cells. Canine PBMCs were activated with 2.5 ug / ml concanavalin A, collected at 48 and 72 hours post-activation, and labeled with each HA-tagged anti-CTLA-4 mAb clone as shown. Binding anti-CTLA-4 antibody was detected using anti-HA antibody. The plot is gated to CD5+, 7AAD- cells. [Figure 16]This shows the binding of A1mut2 mAb to canine regulatory T cells. Canine PBMCs derived from dogs with T-cell lymphoma were first surface-labeled with either A1mut2 or an unrelated MERS mAb, then permeabilized and labeled with an anti-FOXP3 mAb (top panel). In parallel, after permeabilization, the cells were relabeled with either an anti-MERS antibody or either A1mut2 and Foxp3 to detect intracellular stores of CTLA-4 (bottom panel). The plots are gated to 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 either CD5 and HA-tagged A1mut2 or an unrelated MERS mAb (Figure 17A). The labeled cells were then permeabilized and re-labeled with MERS or A1mut2 to detect intracellular CTLA-4 stores (Figure 17B). Anti-HA secondary antibody alone was used as an additional negative control. The plots are gated to CD5+ lymphocytes. [Figure 18] Figures 18A-18B show that A1mut2 expressed as canine isotypes IgGC (canine IgG3) and IgGB (canine IgG2) immobilizes human complement. Figure 18A. Serial dilutions of HA-tagged A1mut2 reformatted as each of four different IgG subclasses were conjugated to SA-captured biotinylated cCTLA-4. Human serum complement or heat-inactivated (HI) human serum complement was added to the plate and subsequently detected with anti-human C1q IgG HRP conjugate and HRP substrate. Figure 18B. Confirmation of the presence of A1mut2 IgG subclasses conjugated to cCTLA-4. Serial dilutions of A1mut2 IgG conjugated 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 proliferative 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 unrelated MERS antibody. Cells were collected at 72 or 96 hours and labeled with anti-CD5 mAb and viability dye 7-AAD. Cells were captured with FACS Canto II and analyzed using FlowJo software. Responder frequency (number of cells that have experienced at least one division) and proliferative capacity (average number of daughter cells produced per cell) were determined. Figure 19A. Representative histogram from one canine. Figure 19B. Responder frequency and proliferative capacity of nine healthy canines, calculated at 96 hours post-stimulation. [Figure 20] This study demonstrates that A1mut2 increases IFN-γ production from canine T cells. Canine PBMCs were stimulated with 2.5 ug / ml ConA in the presence of either 10 ug / ml or 20 ug / ml of A1mut2 or an unrelated MERS antibody. The supernatant was collected after 96 hours, and the IFN-γ present in the supernatant was measured by ELISA. Data from three experiments from one healthy donor dog are shown. Horizontal bars represent the median. [Figure 21] This table shows the kinetic rate constant, dissociation equilibrium constant, and antibody / antigen interaction half-life of selected full-length canine mAbs for canine anti-cCTLA-4 binding, as determined by surface plasmon resonance (SPR). [Modes for carrying out the invention]

[0058] Detailed explanation definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in the conduct of the present invention for testing, but preferred materials and methods are described herein. The following terms are used in the description and assertion of the present invention:

[0059] It should be understood that the terminology used herein is intended solely to describe specific aspects and is not intended to be limiting.

[0060] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the articles. For example, “an element” means one element or more than one element.

[0061] When referring to measurable values ​​such as quantity or duration, the term "about" as used herein is intended to include variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as this is appropriate for the manner in which such variations are disclosed.

[0062] As used herein, the term “antibody” refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody may be an intact immunoglobulin derived from a natural or recombinant source, or it may be the immunoreactive portion of an intact immunoglobulin. Typically, an antibody is a tetrameric immunoglobulin molecule containing two heavy-chain polypeptides and two light-chain polypeptides. Each polypeptide chain contains three complementarity-determining regions (CDRs) that bind to the antigen, defining the antigen specificity of the antibody.

[0063] As used herein, the terms “antibody” and “antibodies” may 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 may retain antigen-specific binding ability. The antibodies of the present invention may exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab')2, as well as single-chain antibodies (scFv), canine antibodies, canine antibodies, humanized antibodies, and human antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0064] The term "antibody fragment" refers to a polypeptide that contains or is derived from a portion of an intact antibody, and includes the antigen-binding 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, for example, camelid antibodies consisting of either a VL or VH domain that exhibits sufficient affinity for the target (Riechmann, 1999, Journal of Immunological Methods 231:25-38), and multispecific antibodies formed from antibody fragments. Antibody fragments may also include human antibodies or humanized antibodies, or portions of human antibodies 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 conformation.

[0066] As used herein, “antibody light chain” refers to the smaller of two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. κ and λ light chains refer to the two main antibody light chain isotypes.

[0067] As used herein, the term “synthetic antibody” means an antibody produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage as described herein. The term should also be interpreted as meaning an antibody produced by the synthesis of an antibody-encoding DNA molecule, wherein the DNA molecule expresses an antibody protein or an amino acid sequence that designates the antibody, and the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence technologies that are available and well known in the art.

[0068] As used herein, the terms “antigen” or “Ag” are defined as molecules that elicit an immune response. This immune response may involve either antibody production or activation of specific immune cells, or both. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can act as an antigen. Furthermore, antigens may be recombinant or derived from genomic DNA. Those skilled in the art will understand that, as used herein, any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response, therefore encodes an “antigen.” Furthermore, those skilled in the art will understand that antigens do not have to be encoded by the full-length nucleotide sequence of a gene. It will be readily apparent that the present invention, though not limited to, involves the use of partial nucleotide sequences of one or more genes, 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 antigens do not have to be encoded by a “gene.” It will be readily apparent that antigens may be generated, synthesized, or derived from biological samples. Examples of such biological samples include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0069] As used herein, the term “antitumor effect” refers to a biological effect manifested by a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in lifespan, or an improvement in various physiological symptoms associated with the cancerous condition. The “antitumor effect” may also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to prevent tumor development in the first place.

[0070] As used herein, the term “of its own” is intended to refer to any material originating from the same individual that will later be reintroduced into the individual.

[0071] "Homogenous, dissimilar" refers to grafts originating from different animals of the same species.

[0072] "Heterogeneous" refers to grafts derived from different animal species.

[0073] As used herein, the term “cancer” is defined as a disease characterized by the 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, and lung cancer. In certain embodiments, 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, nor alter, the binding properties of an antibody containing the amino acid sequence. Such conserved 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. A conserved amino acid substitution is one in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, 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 antibody with altered ability to bind to GFRα4 can be tested using the functional assays described herein.

[0075] When the term is used herein, “co-stimulatory ligand” includes molecules on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.) that specifically bind to a related co-stimulatory molecule on a T cell, thereby producing signals that mediate T cell responses, including, but not limited to, proliferation, activation, and differentiation, in addition to the primary signaling mediated by, for example, the binding of the TCR / CD3 complex to a peptide-loaded MHC molecule. Examples of co-stimulatory ligands, though not limited to them, include CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), cell 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 Toll ligand receptors, and ligands that specifically bind to B7-H3. Co-stimulatory ligands include, in particular, antibodies that specifically bind to co-stimulatory molecules presented on T cells, such as, but are not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3, as well as ligands that specifically bind to CD83.

[0076] A "costimulatory molecule" refers to a congenital binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as proliferation, but is not limited to. Examples of 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 CTLA-4 expression or activity levels refers to levels of expression or activity that differ from those in otherwise identical healthy animals, organisms, tissues, cells, or components thereof. The term "dysregulated" also refers to alterations in the regulation of CTLA-4 expression and activity levels compared to regulation in otherwise identical healthy animals, organisms, tissues, cells, or components thereof.

[0078] "Code" refers to the inherent properties of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, which has either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties derived therefrom, and which act as a template for the synthesis of other polymers and macromolecules in biological processes. Thus, a gene codes for a protein if, in a cell or other biological system, the transcription and translation of the mRNA corresponding to that gene produces a protein. A gene or cDNA can be said to code for a protein or other product if its nucleotide sequence is identical to the mRNA sequence and both the coding strand, which is usually provided in the sequence listing, and the non-coding strand, which is used as a template for the transcription of the gene or cDNA.

[0079] Unless otherwise specified, "nucleotide sequences encoding an amino acid sequence" include all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may contain introns.

[0080] "Effective dose" or "therapeutic effective dose" are used interchangeably herein and refer to the amount of a compound, formulation, material, or composition described herein that is effective in achieving a particular biological outcome. Such outcomes include, but are not limited to, inhibition of viral infection determined by any appropriate means in the art.

[0081] As used herein, “intrinsic” means any material that originates from or is generated within an organism, cell, tissue, or system.

[0082] As used herein, the term “external” means any material introduced from or generated outside of 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] An "expression vector" refers to a vector containing recombinant polynucleotides that include an expression regulatory sequence functionally linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, e.g., cosmids, plasmids (e.g., naked or liposome-containing), and viruses incorporating 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, between two DNA molecules or two RNA molecules, or between two polypeptide molecules. If the positions of subunits in both molecules are occupied by the same monomeric subunits; for example, if the positions in each of two DNA molecules are occupied by adenine, then they are homologous in that position. The homology between two sequences is a linear function of the number of matching or homologous positions; for example, if half of the positions in two sequences are homologous (e.g., five positions in a polymer of length 10 subunits), then the two sequences are 50% homologous; if 90% of the positions (e.g., nine out of ten) are matching or homologous, then the two sequences are 90% homologous.

[0086] The “humanized,” “canine,” and “chimeric” forms of non-human (e.g., mouse) antibodies are immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody) that contain minimal sequences derived from non-human immunoglobulins. Mostly, humanized, canine, and chimeric antibodies are human or canine immunoglobulins (recipient antibodies) in which residues in the recipient’s complementarity-determining region (CDR) are replaced with residues in the CDR of a non-human or non-canine species (donor antibody), such as mouse, rat, or rabbit, that possess the desired specificity, affinity, and capability. In some cases, residues in the Fv framework region (FR) of the human or canine immunoglobulin are replaced with corresponding non-human or non-canine residues. Furthermore, humanized, canine, and chimeric antibodies may contain residues not found in either the recipient antibody or the transferred CDR or framework sequence. These modifications are made to refine and optimize antibody performance. Generally, humanized, canine, 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 region corresponding to that of a non-human immunoglobulin and all or substantially all of the FR region belonging to a human, canine, or immunoglobulin sequence. Humanized and chimeric antibodies are also considered to optimally contain at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The World Health Organization (WHO) International General Names (INN) Expert Group has defined the requirements for a non-human antibody to be considered "humanized." In accordance with the guidelines, candidate antibodies must be compared to human sequences using 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, and therefore, CDR3 and parts of the J region are excluded from the analysis.For an antibody to be "humanized," in addition to being "closer to humans than other species," the highest-level "hit" must be human, and its identity to the human sequence must be at least 85%; otherwise, the antibody will be designated as a "chimera." 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] "Completely human" refers to immunoglobulins, such as antibodies, whose entire molecule is of human origin or whose amino acid sequence is identical to that of human-type antibodies.

[0088] As used herein, “Instructional Materials” includes publications, audio recordings, diagrams, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the present invention. Instructional materials for a kit of the present invention may, for example, be affixed to a container containing the nucleic acids, peptides, and / or compositions of the present invention, or shipped together with the container containing the nucleic acids, peptides, and / or compositions. Alternatively, the instructional materials may be shipped separately from the container with the intention that the instructional materials and the compounds will be used together 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. Two amino acid sequences are identical in the same position if they have the same residue; for example, if the position in each of two polypeptide molecules is occupied by arginine, then they are identical in that position. The identity or degree to which two amino acid sequences have the same residue in the same position in alignment is often expressed as a percentage. The identity between two amino acid sequences is a linear function of the number of matching or identical positions; for example, if half of the positions in the two sequences are identical (e.g., five positions in a polymer of length 10 amino acids), then the two sequences are 50% identical; if 90% of the positions are matching or identical (e.g., nine out of ten), then the two amino acid sequences are 90% identical.

[0090] "Isolated" means altered or removed from its natural state. For example, nucleic acids or peptides that are naturally present in a living animal are not "isolated," but the same nucleic acids or peptides that are partially or completely separated from coexisting substances in their natural state are "isolated." Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-natural environment, such as a host cell.

[0091] In this invention, the following abbreviations are used for commonly existing nucleic acid bases: "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, “nucleotide sequences encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase “nucleotide sequences encoding a protein or RNA” may include introns to the extent that protein-coding nucleotide sequences can contain introns in certain versions.

[0093] As used herein, "lentivirus" refers to a genus of the family Retroviridae. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; this makes them one of the most efficient methods of gene delivery vectors, as they can deliver a considerable amount of genetic information to the host cell's DNA. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means to achieve a considerable level of gene transfer in vivo.

[0094] The term "functionally linked" refers to a functional link between a regulatory sequence and a heterogeneous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is functionally linked to a second nucleic acid sequence if the first nucleic acid sequence is positioned in a functional relationship with the second nucleic acid sequence. For example, a promoter is functionally linked to a coding sequence if the promoter influences the transcription or expression of the coding sequence. Generally, functionally linked DNA sequences are adjacent and, if it is necessary to link two protein-coding regions, they are in the same reading frame.

[0095] Parenteral administration of 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, nucleic acids are polymers of nucleotides. Thus, as used herein, nucleic acids and polynucleotides are interchangeable. Those skilled in the art will have general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed to monomeric “nucleotides.” Monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotides include, but are not limited to, recombinant methods, i.e., conventional cloning techniques and PCR (商標)This includes, but is 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 such methods, and by synthetic means.

[0097] As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term generally refers to both short chains, also called peptides, oligopeptides, and oligomers in the art, and long chains, both generally called proteins in the art, of which many types exist. Among the many types of “polypeptides” are, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0098] As used herein, the term “promoter” is defined as a DNA sequence recognized by a cellular synthetic mechanism or introduced synthetic mechanism that is required to initiate the specific transcription of a polynucleotide sequence.

[0099] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence required for the expression of a gene product functionally linked to a promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, it may include enhancer sequences and other regulatory elements required for the expression of the gene product. The promoter / regulatory sequence may, for example, express the gene product in a tissue-specific manner.

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

[0101] An "inducible" promoter is a nucleotide sequence that, when functionally linked to a polynucleotide encoding or designating a gene product, causes the cell to substantially produce the gene product only if the corresponding inducer is present in the cell.

[0102] A "tissue-specific" promoter is a nucleotide sequence that, when functionally linked to a polynucleotide encoding or specified by a gene, causes a cell to substantially produce a gene product only if the cell is of the tissue type corresponding to the promoter.

[0103] A "signaling pathway" refers to the biochemical relationships between various signaling molecules that play a role in the transmission of signals from one part of a cell to another. The phrase "cell surface receptor" includes molecules and complexes of molecules that can receive signals and transmit those signals across the cell's plasma membrane. An example of a "cell surface receptor" is human GFRα4.

[0104] A "single-chain antibody" refers to an antibody formed by recombinant DNA techniques in which the heavy and light chain fragments of an immunoglobulin are linked together using engineered amino acid spans to reproduce the Fv region of the antibody as a single polypeptide. Various methods for generating 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; and Skerra et al. (1988) Science 242:1038-1041.

[0105] The term “subject” is intended to include living organisms (e.g., mammals) from which an immune response can be induced. As used herein, “subject” or “patient” may be a human or a non-human mammal. Examples of non-human mammals include livestock and pets, such as sheep, cattle, pigs, dogs, cats, and mice. Preferably, the subject is a human.

[0106] As used herein, “substantially purified” cells are cells that essentially contain no other cell types. Substantially purified cells also refer to cells isolated from other cell types that are normally associated with them in their natural state. In some cases, a population of substantially purified cells refers to a homogeneous population of cells. In other cases, the term simply refers to cells isolated from cells that are naturally associated with them in their 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" means treatment and / or prevention. Therapeutic effects are obtained by suppression, remission, or eradication of a disease.

[0108] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process by which a foreign nucleic acid is transferred to or introduced into a host cell. A “transfected,” “transformed,” or “transduced” cell is a cell that has been transfected, transformed, or transduced with a foreign nucleic acid. Examples of such cells include primary target cells and their progeny.

[0109] As used herein, the phrases “transcriptionally controlled” or “functionally 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 the expression of the polynucleotide.

[0110] A “vector” is a composition of substances that contains isolated nucleic acids and can be used to deliver isolated nucleic acids into the interior of a cell. Many vectors are known in the art, including, but are not limited to, linear polynucleotides, polynucleotides conjugated to ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and lentiviral vectors.

[0111] As used herein, the term “specifically binds” means an antibody or ligand that recognizes and binds to a congenerally binding partner protein (e.g., an irritant and / or co-irritant molecule present on T cells) in the sample, but substantially does not recognize or bind to any other molecules in the sample.

[0112] The term "stimulation" refers to a primary response induced by the binding of a stimulating molecule (e.g., the TCR / CD3 complex) and its homologous ligand, thereby mediating a signaling event, e.g., but not limited to signaling via the TCR / CD3 complex. Stimulation can mediate changes in the expression of certain molecules, such as the downregulation of TGF-β and / or rearrangement of the cytoskeleton.

[0113] When used herein, "irritant molecule" means a molecule on a T cell that specifically binds to a homologous irritant ligand present on antigen-presenting cells and / or tumor cells.

[0114] As used herein, “irritating ligand” means a ligand that, when present on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.) or tumor cells, specifically binds to a congenital binding partner on T cells (referred to herein as “irritating molecule”), thereby mediating a primary response by T cells, including but not limited to activation, initiation of an immune response, and proliferation. Irritating ligands are well known in the art and include, in particular, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0115] Scope: Throughout this disclosure, various aspects of the invention may be presented in range form. It should be understood that descriptions in range form are merely for convenience and brevity and should not be interpreted as immutable limitations on the scope of the invention. Therefore, range descriptions should be considered to specifically disclose not only the individual numbers within the range, but also all possible subranges. For example, a range description 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 width of the range.

[0116] explanation The present invention is based on the discovery that antigen-specific single-chain variable fragment (scFv) fusion proteins that specifically bind to canine cytotoxic T lymphocyte protein 4 (CTLA-4) can be identified using a canine antibody phage display library. These canine CTLA-4 specific scFvs can then be converted into full-length antibodies having a canine constant region to make 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 CD4 + T cells and CD8 + CTLA-4 is upregulated on the surface of T cells after activation and acts as a negative regulator of the effector T cell response. +CTLA-4 is overexpressed on the surface of T cells. CTLA-4 binds to CD80 and CD86 with high affinity, defeating CD28 in its interaction with these receptor ligands and inhibiting T cell activation. The inhibition of lymphocyte activation by CTLA-4 is achieved not only by defeating CD28, but also through the recruitment of tyrosine phosphatase SHP2 and serine threonine phosphatase PP2A, which dephosphorylate the TCRζ chain and target downstream effectors of PI3K, respectively. CTLA-4 can also indirectly inhibit effector T cell responses through its action on antigen-presenting cells (APCs). The high binding affinity of CTLA-4 to CD80 / 86 on APCs can lead to 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, particularly their ability to inhibit dendritic cell maturation. Monoclonal antibodies that bind to CTLA-4 and inhibit its interaction with CD80 / 86 enhance the endogenous T cell response and inhibit Treg function by lowering the T cell activation threshold and promoting the proliferation of highly activated tumor-specific T cells, thereby promoting anti-tumor 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 treatment in clinical practice. Considering the similarities between the human and canine immune systems, this invention provides a complete canine anti-CTLA-4 that can be used as an antitumor therapy in dogs. Using a recently developed canine scFv phage display library, three CTLA-4 specific soluble scFvs that can be used as immunotherapies for veterinary cancers were identified and reformatted into complete canine IgG monoclonal antibodies.

[0119] Immunotherapy for cancer in dogs Canines 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, helping to elucidate the mechanisms of action of anti-CTLA-4 mAbs, identify correlated biomarkers of the response, and understand the mechanisms of resistance to checkpoint inhibitors. Furthermore, they can also play a crucial translational role in informing the design of human clinical trials regarding 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 after administration of sheep red blood cells, suggesting a conserved mechanism of action between humans and dogs. In addition, canine patients with histiocytic sarcoma and B-cell lymphoma showed a higher percentage of canine peripheral blood CD4 compared to control dogs. + T cells and CD8 + The T cells expressed CTLA-4, suggesting the presence of an exhausted phenotype in these dogs that negatively impacts antitumor immunity. Furthermore, recent reports using RNA sequencing have shown that canine CD4 + CD25 hi T cells were found to express a regulatory phenotype, including high levels of CTLA-4 transcript expression. Together, these findings provide a basis for the development of therapeutic canine anti-CTLA-4 antibodies that can promote antitumor immunity in these invasive tumor forms, and offer valuable, comparable reagents for investigating correlated biomarkers of clinical response and optimal combination therapies to inform human clinical trials.

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

[0121] In certain aspects, the present invention provides an isolated binding polypeptide 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 isolated bound polypeptides comprising HCDR1 comprising an amino acid sequence (SEQ ID NO: 1, 21, or 37). Isolated bound polypeptides comprising HCDR2 comprising an amino acid sequence (SEQ ID NO: 2, 22, or 38) are also provided. Isolated bound polypeptides comprising HCDR3 comprising an amino acid sequence (SEQ ID NO: 3, 23, or 39) are also provided. Isolated bound polypeptides comprising a light chain variable region comprising LCDR1 comprising an amino acid sequence (SEQ ID NO: 4, 24, or 40) are also provided. Isolated bound polypeptides comprising LCDR2 comprising an amino acid sequence (SEQ ID NO: 5, 25, or 41) are also provided. Isolated bound polypeptides comprising LCDR3 comprising an amino acid sequence (SEQ ID NO: 6, 26, or 42) are also provided.

[0123] In certain contexts, the present invention provides isolated conjugated polypeptides comprising HCDR1 containing an amino acid sequence (SEQ ID NO:1), HCDR2 containing an amino acid sequence (SEQ ID NO:2), HCDR3 containing an amino acid sequence (SEQ ID NO:3), LCDR1 containing an amino acid sequence (SEQ ID NO:4), LCDR2 containing an amino acid sequence (SEQ ID NO:5), and LCDR3 containing an amino acid sequence (SEQ ID NO:6).

[0124] Acceptable variations in complementarity-determining region (CDR) sequences will be known to those skilled in the art. For example, in some embodiments, the polypeptide includes a complementarity-determining region (HCDR or LCDR) comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of the amino acid sequences described in SEQ ID NO: 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 contains amino acids as described in SEQ ID NO:53. In some embodiments, the binding polypeptide comprises an antibody or its antigen-binding fragment. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, single-chain variable fragments (scFv), or single-domain antibodies. In further embodiments, the antibody is a full-length antibody. In yet another embodiment, the antibody or antigen-binding fragment is a canine antibody or its antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is a canine antibody or its antigen-binding fragment.

[0126] In certain embodiments, the conjugated polypeptide includes a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 6, 25, 40, or 74. In certain embodiments, the conjugated polypeptide includes a heavy chain variable region comprising an amino acid sequence described in SEQ ID NO: 6, 25, 40, or 74. In certain embodiments, the conjugated polypeptide comprises a heavy chain variable region consisting of an amino acid sequence described in SEQ ID NO: 6, 25, 40, or 74.

[0127] In certain embodiments, the conjugated polypeptide includes a light chain variable region containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 8, 27, or 42. In certain embodiments, the conjugated polypeptide includes a light chain variable region containing an amino acid sequence described in SEQ ID NO: 8, 27, or 42. In certain embodiments, the conjugated polypeptide consists of a light chain variable region containing an amino acid sequence described in SEQ ID NO: 8, 27, or 42.

[0128] Isolated bound polypeptides are also provided, each containing a heavy chain variable region with an amino acid sequence described in SEQ ID NO:6 and a light chain variable region with an amino acid sequence described in SEQ ID NO:8.

[0129] In certain embodiments, the present invention includes an antibody that binds to the same epitope on canine CTLA-4 as the antibody of the present invention (i.e., an antibody that has 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 may 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 antibody of the present invention. The ability of the test antibody to inhibit the binding of, for example, A1mut2 to canine CTLA-4 demonstrates that the test antibody can compete with A1mut2 for binding to canine CTLA-4 and is therefore considered to bind to the same CTLA-4 epitope as A1mut2.

[0130] The antibodies of the present invention can be prepared using an antibody having one or more of the VH and / or VL sequences disclosed herein as a starting material for manipulating a modified antibody, which may have altered properties compared to the starting antibody. The antibody can be manipulated by modifying one or more amino acids within one or both of the variable regions (i.e., VH and / or VL), for example, within one or more CDR regions and / or one or more framework regions. Additionally or alternatively, the antibody can be manipulated by modifying residues within the constant region, for example, to alter the effector function of the antibody.

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

[0132] As used herein, the term "single-chain variable fragment" or "scFv" is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin (e.g., mouse or human) that are covalently linked to form a VH::VL heterodimer. The heavy (VH) and light (VL) chains are either directly linked or linked by a linker encoding a peptide, and the linker connects the N-terminus of VH to the C-terminus of VL, or the C-terminus of VH to the N-terminus of VL. In some embodiments, the antigen-binding domain (e.g., CTLA-4 binding domain) comprises an scFv having the VH-linker-VL arrangement from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain comprises an scFv having the VL-linker-VH arrangement from the N-terminus to the C-terminus. One of ordinary skill in the art will be able to select an appropriate arrangement for use in the present invention.

[0133] Linkers are typically rich in glycine for flexibility and serine or threonine for solubility. Linkers can connect the heavy-chain variable region and the light-chain variable region of an 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 hereby incorporated by reference in their entirety. A variety of linker sequences are known in the art, including, without limitation, glycine-serine (GS) linkers, such as, for example, (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, without limitation, It may include amino acid sequences such as TIFF0007868811000001.tif26156. Those skilled in the art will be able to select a suitable linker sequence for use in the present invention. In one embodiment, the scFv of the present invention includes a heavy chain variable region (VH) and a light chain variable region (VL), where VH and VL are nucleic acid sequences The amino acid sequence that can be encoded by TIFF0007868811000002.tif12128 It is separated by a linker sequence containing TIFF0007868811000003.tif4128.

[0134] Despite the removal of the constant region and the introduction of a 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. Patents 5,091,513, 5,132,405, and 4,956,778; and U.S. Patent Application Publications 20050196754 and 20050196754. Inhibitory antagonist scFvs have been described (see, for example, 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 eta., 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)). Agonist scFvs with stimulating activity have been described (see, for example, 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 scFv includes a heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs) and a light chain variable region containing three light chain complementarity-determining regions (LCDRs). HCDR1 contains an amino acid sequence (SEQ ID NO: 1, 17, or 33), and / or HCDR2 contains an amino acid sequence (SEQ ID NO: 2, 18, or 34), and / or HCDR3 contains an amino acid sequence (SEQ ID NO: 3, 19, or 35), and / or LCDR1 contains an amino acid sequence (SEQ ID NO: 4, 20, or 36), and / or LCDR2 contains an amino acid sequence (SEQ ID NO: 5, 21, or 37), and / or LCDR3 contains an 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] A single-chain variable fragment (scFv) is also provided, comprising a heavy-chain variable region containing the amino acid sequence described in SEQ ID NO: 6, 25, 40, or 74, and a light-chain variable region containing the amino acid sequence described in SEQ ID NO: 8, 27, or 42. The heavy-chain variable region and the light-chain variable region are separated by a linker.

[0137] In another context, a single-chain variable fragment (scFv) is provided, comprising the amino acid sequence described in SEQ ID NO: 29, 44, or 76.

[0138] Acceptable variations in scFv sequences will be known to those skilled in the art. For example, in some embodiments, the scFv includes 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 with any of the amino acid sequences described in SEQ ID NO: 29, 44, or 76.

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

[0140] (Table 1) Sequence used in the present invention TIFF0007868811000004.tif87163TIFF0007868811000005.tif225163TIFF0007868811000006.tif229163TIFF0007 868811000007.tif226163TIFF0007868811000008.tif229163TIFF0007868811000009.tif226163TIFF00078688110 00010.tif225163TIFF0007868811000011.tif229163TIFF0007868811000012.tif226163TIFF0007868811000013.t if229163TIFF0007868811000014.tif229163TIFF0007868811000015.tif225163TIFF0007868811000016.tif171163

[0141] Nucleic acids and expression vectors This disclosure provides isolated nucleic acids encoding polypeptides. The nucleic acids of this disclosure may comprise a polynucleotide sequence encoding any one of the conjugated polypeptides, scFvs, or antibodies disclosed herein.

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

[0143] In a particular embodiment, the nucleic acid comprises an antigen-binding domain comprising a heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs) and a light chain variable region containing three light chain complementarity-determining regions (LCDRs). HCDR1 comprises an amino acid sequence (SEQ ID NO: 1, 21, or 37), and / or HCDR2 comprises an amino acid sequence (SEQ ID NO: 2, 22, or 38), and / or HCDR3 comprises an amino acid sequence (SEQ ID NO: 3, 23, or 39), and / or LCDR1 comprises an amino acid sequence (SEQ ID NO: 4, 24, or 40), and / or LCDR2 comprises an amino acid sequence (SEQ ID NO: 5, 25, or 41), and / or LCDR3 comprises an amino acid sequence (SEQ ID NO: 6, 26, or 42).

[0144] In certain embodiments, the conjugated polypeptide comprises an antibody or its antigen-binding fragment. In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, single-chain variable fragments (scFv), or single-domain antibodies. 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 containing polynucleotide sequences having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, and 99% identity with SEQ ID NO: 8, 28, and 44. In certain embodiments, the heavy chain variable region is encoded by a nucleic acid containing polynucleotide sequences described in SEQ ID NO: 8, 28, and 44. In certain embodiments, the heavy chain variable region is encoded by a nucleic acid consisting of polynucleotide sequences described in SEQ ID NO: 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%, and 99% identity with the amino acid sequence of the light chain variable region described in SEQ ID NO: 10, 30, and 46. In certain embodiments, the light chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence described in SEQ ID NO: 10, 30, and 46. In certain embodiments, the light chain variable region is encoded by a nucleic acid consisting of a polynucleotide sequence described in SEQ ID NO: 10, 30, and 46.

[0147] Isolated nucleic acids are also provided that encode a binding polypeptide comprising a heavy chain variable region encoded by a nucleic acid sequence containing the polynucleotide sequence described in SEQ ID NO:8, and a light chain variable region encoded by a nucleic acid sequence containing the polynucleotide sequence described in SEQ ID NO:10.

[0148] Also provided are isolated nucleic acids encoding single-chain variable fragments (scFv) containing a heavy-chain variable region with three heavy-chain complementarity-determining regions (HCDRs) and a light-chain variable region with three light-chain complementarity-determining regions (LCDRs). HCDR1 comprises amino acid sequences (SEQ ID NO: 1, 20, and 35), and / or HCDR2 comprises amino acid sequences (SEQ ID NO: 2, 22, or 38), and / or HCDR3 comprises amino acid sequences (SEQ ID NO: 3, 22, and 37), and / or LCDR1 comprises amino acid sequences (SEQ ID NO: 4, 24, or 40), and / or LCDR2 comprises amino acid sequences (SEQ ID NO: 5, 25, or 41), and / or LCDR3 comprises amino acid sequences (SEQ ID NO: 6, 26, or 42).

[0149] Also provided are isolated nucleic acids encoding a single-chain variable fragment (scFv) comprising a heavy-chain variable region containing the nucleotide sequence described in SEQ ID NO:8 and / or a light-chain variable region containing the nucleotide sequence described 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 described in SEQ ID NO:72.

[0150] Isolated nucleic acids encoding single-chain variable fragments (scFv) comprising polynucleotide sequences described in SEQ ID NO: 30, 45, or 77 are also provided.

[0151] Isolated nucleic acids encoding full-length antibodies are also provided, comprising the heavy chain polynucleotide sequences described in SEQ ID NO: 12, 14, 16, 18, 34, and 50, and the light chain polynucleotide sequences described in SEQ ID NO: 20, 36, and 52. Isolated nucleic acids encoding full-length antibodies are also provided, comprising the heavy chain polynucleotide sequences described in SEQ ID NO: 12, 14, 16, 18, 34, and 50, and the light chain polynucleotide sequences described in SEQ ID NO: 20, 36, and 52.

[0152] Acceptable variations in nucleic acid sequences will be known to those skilled 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 with respect to any of the nucleotide sequences described in SEQ ID NO: 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 this disclosure comprises a first polynucleotide sequence and a second polynucleotide sequence. The first and second polynucleotide sequences may be separated by a linker. For example, in certain embodiments, the heavy chain variable region and the light chain variable region of scFv are separated by a linker. In certain embodiments, the linker comprises the amino acid sequence described in SEQ ID NO. 72. In certain embodiments, the nucleic acid comprises, from 5' to 3', a first polynucleotide sequence, a linker, and a second polynucleotide sequence. In certain embodiments, the nucleic acid comprises, from 5' to 3', a second polynucleotide sequence, a linker, and a 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 vectors, RNA vectors, plasmids, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and retroviral vectors. In certain embodiments, the vector is an expression vector.

[0155] Host cells containing either the vector or nucleic acid disclosed herein are also provided. The host cells may be of eukaryotic, prokaryotic, mammalian, or bacterial origin. Methods for producing a binding polypeptide or scFv that binds to FAP are also provided herein, the methods comprising the step of culturing the host cells.

[0156] In some embodiments, the nucleic acids of this disclosure can be functionally ligated to transcriptional regulatory elements, such as promoters and enhancers. Suitable promoter and enhancer elements are known to those skilled in the art.

[0157] In certain embodiments, nucleic acids are functionally 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, lambdaP, and trc. For expression in eukaryotic cells, suitable promoters include, but are not limited to, light and / or heavy chain immunoglobulin gene promoters and enhancer elements; cytomegalovirus early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; 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 from and derived from many organisms, eukaryotes and prokaryotes. Modifications of reversible promoters derived from a first organism for use in a second organism, such as a first prokaryote and a second eukaryote, or a first eukaryote and a second prokaryote, are well known in the art.Such reversible promoters and systems, which are based on such reversible promoters but also include further regulatory proteins, include, but are not limited to, alcohol-regulating promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, alcohol transactivator protein (A1cR) responsive promoter, etc.), tetracycline-regulating promoters (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid-regulating 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-regulating promoters (e.g., metallothionein promoter system, etc.), pathogenicity-related regulatory promoters (e.g., salicylic acid-regulating promoter, ethylene-regulating promoter, benzothiadiazole-regulating promoter, etc.), temperature-regulating promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.)), light-regulating promoters, and synthesis-inducible promoters.

[0159] For expression in yeast cells, suitable promoters are constitutive promoters, such as the ADH1 promoter, PGK1 promoter, ENO promoter, and PYK1 promoter; or moduloable promoters, such as the GAL1 promoter, GAL10 promoter, ADH2 promoter, PHOS promoter, CUP1 promoter, GALT promoter, MET25 promoter, MET3 promoter, CYC1 promoter, HIS3 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, ADC1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TP1 promoter, and AOX1 (for example, for use in Pichia). The selection of appropriate vectors and promoters is well within the realm of those skilled 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, e.g., the lac / tac hybrid promoter, the tac / trc hybrid promoter, the trp / lac promoter, the T7 / lac promoter; the trc promoter; the tac promoter, etc.; the araBAD promoter; in vivo regulatory promoters, e.g., the ssaG promoter or related promoters (see, for example, U.S. Patent Application Publication No. 20040131637); the pagC promoter (Pulkkinen and Miller, J. Bacteriol. (1991) 173(1): 86-93; Alpuche-Aranda et al., Proc. Natl. Acad. Sci. USA (1992) 89(21): 10079-83); and the nirB promoter (Harborne et al. Mol. Micro. (1992)). 6:2805-2813) etc. (e.g., Dunstan et al., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine (2004) 22:3243-3255; and Chatfield et al., Biotechnol.)See (1992) 10:888-892); Sigma 70 promoters, e.g., consensus Sigma 70 promoters (see, e.g., GenBank accession numbers AX798980, AX798961, and AX798183); quiescent promoters, e.g., dps promoter, spv promoter, etc.; promoters derived from pathogenic 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 See Molecular and Structural Biology, Protein--Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162; SP6 promoter (e.g., Melton et al., Nucl. Acids Res.)Examples include (see 1984) 12:7035). Suitable potent promoters for use in prokaryotes such as Escherichia coli include, but are not limited to, Trc, Tac, T5, T7, and P-lambda. Non-limited examples of operators for use in bacterial host cells include the lactose promoter operator (the LacI repressor protein changes conformation when in contact with lactose, thereby preventing the Lad repressor protein from binding to the operator), the tryptophan promoter operator (the TrpR repressor protein has a conformation that binds to the operator when complexed with tryptophan; 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 earliest cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence that can drive high levels of expression of any polynucleotide sequence functionally ligated to it. Other constitutive promoter sequences may also be used, but are 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 leukemia virus promoter, Epstein-Barr virus earliest promoter, Roussarcoma virus promoter, EF-1 alpha promoter, and human gene promoters, such as, but are not limited to, the 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 inducible promoters provides a molecular switch that can turn on the expression of a functionally ligated polynucleotide sequence when such expression is desired, or turn off the expression when such expression is undesirable. Examples of inductive promoters include, but are not limited to, metallothione promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0161] In some embodiments, a locus or construct or transgene containing a suitable promoter is irreversibly switched through the induction of an inducible system. Suitable systems for inducing irreversible switching are well known in the art, and for example, Cre-lox-mediated recombination can be used to induce irreversible switching (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 recombinases, endonucleases, ligases, recombination sites, etc., known in the art, can be used to generate irreversibly switchable promoters. The methods, mechanisms, and requirements for site-directed recombination described elsewhere in this Specified Use in the Generation of Irreversibly Switchable Promoters are well known in the Art; see, for example, Grindley et al. Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.), the disclosure of which is incorporated herein by reference.

[0162] The nucleic acids of this disclosure may be present in expression vectors and / or cloning vectors. Expression vectors may include selection markers, replication origins, and other features that provide vector replication and / or maintenance. Suitable expression vectors include, for example, plasmids and viral vectors. Numerous suitable vectors and promoters are known to those skilled in the art, and many are commercially available for generating recombinant constructs of interest. The following vectors are provided as examples and should not be construed as limitations: 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 a convenient restriction site located near the promoter sequence to allow for the insertion of nucleic acid sequences encoding heterologous proteins. A functional selection marker may also be present in the expression host. Suitable expression vectors are not limited to viral vectors (e.g., vaccinia virus; poliovirus; adenovirus (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 See 95 / 00655; Adeno-associated viruses (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.See USA (1993) 90: 10613-10617); SV40; herpes simplex virus; viral vectors based on human immunodeficiency virus (e.g., see 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., vectors derived from mouse leukemia virus, splenic necrosis virus, and retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus).

[0164] Further expression vectors suitable for use include, but are not limited to, lentiviral vectors, gamma-retroviral vectors, foamy viral vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpesvirus vectors, engineered hybrid viral vectors, and transposon-mediated vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

[0165] Generally, a suitable vector includes a functional replication origin in at least one organism, a promoter sequence, a convenient restriction enzyme site, and one or more selection 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., a lentiviral vector) can be used to introduce nucleic acids into host cells. Therefore, the expression vector (e.g., a lentiviral vector) of the present invention may contain nucleic acids encoding polypeptides. In some embodiments, the expression vector (e.g., a lentiviral vector) may include further elements that assist in the functional expression of the polypeptide encoded therein. In some embodiments, the expression vector containing nucleic acids encoding polypeptides may further include a mammalian promoter. In one embodiment, the vector may further include an elongation factor 1-alpha promoter (EF-1α promoter). The use of the EF-1α promoter can increase the efficiency of downstream transgene expression. Physiological promoters (e.g., EF-1α promoters) may be less likely to induce integration-mediated genotoxicity and can suppress the ability of retroviral vectors to cancer-inducing stem cells. Other physiological promoters suitable for use in vectors (e.g., lentiviral vectors) are known to those skilled in the art and can be incorporated into the vectors of the present invention. In some embodiments, the vector (e.g., a lentiviral vector) may further include non-essential cis-acting sequences that can enhance titer and gene expression. One non-exclusive example of a non-essential cis-acting sequence is the central polyprint lactate and central termination sequence (cPPT / CTS), which is important for efficient reverse transcription and nuclear translocation. Other non-essential cis-acting sequences are known to those skilled in the art and can be incorporated into the vector of the present invention (e.g., a lentiviral vector). In some embodiments, the vector further includes post-transcriptional regulatory elements. Post-transcriptional regulatory elements can improve RNA translation, enhance transgene expression, and stabilize RNA transcripts. An 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 includes a WPRE sequence. Various post-transcriptional regulatory elements are known to those skilled in the art and can be incorporated into the vector of the present invention (e.g., a lentiviral vector).The vectors of the present invention may further include elements such as a rev response element (RRE) for RNA transport, a packaging sequence, and 5' and 3' long terminal repeats (LTRs). The term “long terminal repeat” or “LTR” refers to a domain of base pairs located at the end of retroviral DNA, including the U3, R, and U5 regions. LTRs generally provide functions required for the expression of retroviral genes (e.g., promotion, initiation, and polyadenylation of gene transcripts) and for viral replication. In one embodiment, the vector of the present invention (e.g., a lentiviral vector) includes a 3'U3 deleted LTR. Thus, the vector of the present invention (e.g., a lentiviral vector) may include any combination of the elements described herein to enhance the efficiency of functional expression of the transgene. For example, the vector of the present invention (e.g., a lentiviral vector) may include a WPRE sequence, a cPPT sequence, a RRE sequence, a 5'LTR, and 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) has been modified (e.g., by deletion or substitution). Self-inactivating vectors can prevent the transcription of the virus beyond the first round of viral replication. Therefore, self-inactivating vectors may infect and then be incorporated into the host genome (e.g., a mammalian genome) only once, and there is no possibility of them being passed on further. Thus, self-inactivating vectors can greatly reduce the risk of producing a virus capable of replication.

[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 containing 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 containing a nucleotide sequence encoding the polypeptide of the present disclosure into host cells can be carried out in vitro, ex vivo, or in vivo. For example, host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) can be electroporated in vitro or ex vivo using RNA containing a nucleotide sequence encoding the polypeptide of the present disclosure.

[0169] To evaluate the expression of a polypeptide or a portion thereof, the expression vector introduced into cells may include 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 intended to be transfected or infected with the viral vector. In some embodiments, the selection marker may be placed on a separate DNA fragment and used in a cotransfection procedure. Both the selection marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selection markers include, but are not limited to, antibiotic resistance genes.

[0170] Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. Generally, a reporter gene is a gene that encodes a polypeptide that is neither present in nor expressed in the recipient organism and tissue, and whose expression is manifested by certain readily detectable characteristics, such as enzymatic activity. Reporter gene expression is evaluated at an appropriate time after DNA has been introduced into recipient cells. Suitable reporter genes, non-limitingly, include genes encoding 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 embodiments, the nucleic acids of the Disclosure are provided for the production of polypeptides described herein, for example, in host cells. In some embodiments, the nucleic acids of the Disclosure are provided for the amplification of nucleic acids encoding polypeptides.

[0172] Treatment method The antibodies, conjugated polypeptides, and scFv described herein may be included in compositions for treating a disease or condition in a subject where such treatment is needed. The composition may include a pharmaceutical composition and may further include a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition may be administered to the subject.

[0173] In one aspect, the present invention provides a method for treating cancer in subjects where such treatment is needed. The method comprises administering to a subject an isolated conjugated polypeptide comprising a heavy chain variable region containing at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 6, 25, 40, or 74, and a light chain variable region containing at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 8, 27, or 42.

[0174] In certain embodiments, cancer is associated with cells expressing cytotoxic T lymphocyte-associated protein 4 (CTLA-4). In certain embodiments, CTLA-4 expressing cells are cancer-associated cells. In certain embodiments, cancer-associated cells are T lymphocytes. In certain embodiments, 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 its antigen-binding fragment. In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, single-chain variable fragments (scFv), or single-domain antibodies. In certain embodiments, the antibody is a full-length antibody. In certain embodiments, the antibody or antigen-binding fragment is a humanized antibody or its antigen-binding fragment.

[0176] The compositions of the present invention can be administered in dosages, routes, and timings determined by appropriate preclinical and clinical experiments and trials. The compositions can be administered multiple times in dosages within these ranges. Administration of the compositions can be combined with other methods useful for treating the desired disease or condition, as determined by those skilled in the art.

[0177] Pharmaceutical compositions and formulations Pharmaceutical compositions comprising any one of the conjugated polypeptides, scFvs, antibodies, or antigen-binding fragments disclosed herein are also provided. Among these compositions are pharmaceutical compositions and formulations for administration, such as for the treatment of a disease or disorder. Methods of therapy for administering a pharmaceutical composition to a subject, such as a patient, are also provided.

[0178] Pharmaceutical compositions and formulations generally comprise one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition comprises at least one further therapeutic agent.

[0179] The term "pharmaceutical preparation" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain any further ingredients that would be unacceptably toxic to the target to which the preparation will be administered. "Pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation other than the active ingredient that is not toxic to the target. Examples of pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In some cases, the choice of carrier is determined to some extent by the specific composition and / or the method of administration. Therefore, a variety of suitable preparations exist. For example, a pharmaceutical composition may contain a preservative. Suitable preservatives include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some cases, a mixture of two or more preservatives is used. Preservatives, or mixtures thereof, are typically present in an amount of about 0.0001 to about 2% by weight of the total composition. The carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).Pharmacochemically acceptable carriers are generally non-toxic to the recipient at the dosage and concentration used, and include, but are not limited to, the following: buffers, e.g., phosphoric acid, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (approximately Polypeptides (less than 10 residues); proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, e.g., polyethylene glycol (PEG).

[0180] In some cases, the composition includes a buffer. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some cases, a mixture of two or more buffers is used. The buffer, or mixture thereof, is typically present in an amount of about 0.001 to about 4% by weight of the total composition. Methods for preparing administerable pharmaceutical compositions are known. Exemplary methods are described in detail, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0181] The formulation may include an aqueous solution. The formulation or composition may also include more than one active ingredient useful for the specific indication, disease, or condition treated by the composition, preferably those having complementary activities to the composition, where their respective activities do not adversely affect each other. Such active ingredients are appropriately present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine. In some embodiments, the pharmaceutical composition includes the composition in an amount effective to treat or prevent a disease or condition, e.g., a therapeutically effective amount or a prophylactically effective amount. In some embodiments, the therapeutic or prophylactic effectiveness is monitored by periodic evaluation of the subject being treated. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

[0182] Formulations may include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, percutaneous, 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 may, in some aspects, be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are somewhat easier to administer, particularly by injection. Viscous compositions, on the other hand, can be formulated within a viscosity range suitable for providing a longer contact period with specific tissues. The liquid or viscous composition may contain a carrier, which may be a solvent or dispersion medium containing, for example, water, saline solution, phosphate-buffered saline solution, polyols (polyoi) (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 solvent by mixing it with a suitable carrier, diluent, or excipient, such as sterile water, saline, glucose, or dextrose. Depending on the desired route of administration and preparation, the composition may include auxiliary substances, such as wetting agents, dispersants, or emulsifiers (e.g., methylcellulose), pH buffers, gelling or viscosity-enhancing additives, preservatives, flavoring agents, and / or coloring agents. In some cases, 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 antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial activity can be ensured by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. Sustained absorption of injectable pharmaceutical dosage forms can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin.

[0185] Preparations used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration using a sterile filtration membrane.

[0186] The content of any articles, patents, and patent applications, as well as all other documents and electronically available information, referenced or cited herein are incorporated herein in whole by reference to the same extent as each individual publication is specifically and individually indicated to be incorporated by reference. The applicants reserve the right to physically incorporate into this application any and all material and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0187] While the present invention has been described in relation to its particular embodiments, those skilled in the art will understand that various modifications can be made and substituted with equivalents without departing from the true spirit and scope of the 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. Furthermore, many modifications can be made to adapt specific situations, materials, compositions of substances, processes, process steps, or steps to the object, spirit, and scope of the invention. All such modifications are intended to be within the scope of the claims appended herein. While certain embodiments have been described in detail here, the same will be more clearly understood by referring to the following examples, which are included for illustrative purposes only and are not intended to be limiting. [Examples]

[0188] Experimental Examples The present invention will be further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only, unless otherwise specified, and are not intended to be limiting. Accordingly, the present invention should not be construed as being limited to the following examples, but rather as encompassing all possible variations that become apparent as a result of the teachings provided herein.

[0189] Without further explanation, it is conceivable that those skilled in the art can prepare and utilize the compounds of the present invention and carry out the claimed methods using the foregoing description and the following exemplary examples. Accordingly, the following working examples illustrate preferred embodiments of the present invention and should not be construed as limiting the remainder of this 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 healthy donor dog blood or blood from dogs with T-cell lymphoma (Treg stained) by discontinuous density centrifugation on a Ficoll-Paque PLUS (GE Healthcare, Chicago, IL). For feline cells, the remaining heparinized blood left over from the 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% thermo-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. Negatively selected canine T cells were used where indicated. PBMCs were washed in (c)RPMI and labeled with mouse anti-canine CD11b (clone CA16.3E10), CD11c (clone CA11.6A1), mouse anti-human CD14 (clone TuK4), and mouse anti-canine CD21 (clone CA2.1D6) (all from ABD Serotec / Biorad), followed by labeling with goat anti-mouse IgG microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). T cells were negatively selected using a MACS LS column (Miltenyi) as described by the manufacturer. T cell purity was determined by CD5 labeling and flow cytometry analysis. Human tumor cell lines (K562 and 293T cells) were grown in RPMI-1640 (cRPMI) supplemented with HEPES, 1 mM sodium pyruvate (Mediatech), glutamine, penicillin, and streptomycin (Thermo Fisher Scientific), and 10% fetal bovine serum.

[0192] Recombinant protein generation. Total RNA was extracted from canine PBMC cells using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA). Reverse transcription was performed using random hexamer 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: TIFF0007868811000017.tif41164

[0193] The 3' primer was designed to include a mobile linker, a histidine tag, and an Avi (biotinylation) tag. The primer was synthesized by Sigma-Aldrich (St. Louis, MO). Conventional PCR using Q5 Hot Start High Fidelity Polymerase was performed with the following thermal cycling program: 3 minutes at 98°C; 30 cycles of 15 seconds at 98°C, 30 seconds at 65°C, and 1 minute at 72°C; 2 minutes at 72°C, and then 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 HA-tagged expression plasmid pFUSE (Invivogen, San Diego, CA). The nucleotide sequence of the cCTLA-4 ECD was confirmed by DNA sequencing analysis. A pFUSE expression plasmid containing cCTLA-4 ECD was transfected into 293T cells using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions, and the amount of Lipofectamine and collection time used for maximum protein expression were investigated. CTLA-4 polypeptide was collected from cell culture medium by binding to Ni-NTA resin (Qiagen, Hilden, Germany) on a rotator for 2 hours. The centrifuged resin pellet was thoroughly washed with a high-salt buffer at pH 8.0 containing 50 mM Tris-HCl, 300 mM NaCl, and 10 mM imidazole, and a low-salt buffer at pH 8.0 containing 50 mM Tris-HCl, 150 mM NaCl, and 10 mM imidazole. The cCTLA-4 ECD protein was eluted from the Ni-NTA resin with an elution buffer at pH 8.0 containing 50 mM Tris-HCl, 150 mM NaCl, and 300 mM imidazole. The buffer was replaced with 1×PBS, and the cCTLA-4 ECD protein was concentrated using an Amicon ultrafiltration unit (MilliporeSigma, Burlington, MA).Protein synthesis was evaluated using SDS-PAGE and Coomassie Blue staining of the gel, and quantified using Image Lab 6.0 software (Bio-Rad, Hercules, CA) with BSA standards.

[0194] Biotinylation of cCTLA-4 protein and validation of the reaction product. Approximately 0.8 mg of recombinant cCTLA-4 ECD protein (hereinafter referred to as cCTLA-4) was biotinylated in preparation for binding to streptavidin (SA) coated plates and subsequent scFv library panning. The Avi tag acts as the biotinylation recognition site for BirA biotin-protein ligase. cCTLA-4 was biotinylated using the BirA biotin-protein ligase standard reaction kit (Avidity, Aurora, CO) according to the manufacturer's instructions. To confirm high levels of cCTLA-4 biotinylation, samples were evaluated by SDS-PAGE after the addition of 0, 2, 4, and 8 ug of SA. The migration of biotinylated cCTLA-4 + SA was delayed compared to unbiotylated cCTLA-4, indicating that the biotinylation reaction was successful and essentially stoichiometric. To confirm the immunoreactivity of biotinylated cCTLA-4 protein in a 96-well plate format, a capture assay followed by ELISA was performed. Briefly, streptavidin at various concentrations in PBS was coated into the wells of a microassay plate. After O / N incubation at 4°C, 5% milk PBS-Tween was added at RT for 1 hour. A PBS-Tween wash was then performed, and the titrator of biotinylated cCTLA-4 protein was added to the wells for 1 hour. After the PBS-Tween wash, anti-canine CTLA-4 rabbit polyclonal antibody (Sino Biologicals, Chesterbrook, PA) was added at RT for 2 hours in two different dilutions. Detection was performed with a 1 / 5000 dilution of anti-canine (H and L) chain AP conjugate IgG secondary antibody (Jackson Immunoresearch, West Grove, PA) at RT for 1 hour. After TBS-Tween washing, a colorimetric AP substrate was added, and the plates were read at absorbance 650 after 1 hour. The results showed a lack of signal in wells without streptavidin, and that an increase in the amount of plated streptavidin-capturing protein was required for the presence of a signal (data not shown).30 ug / ml of streptavidin was used to immobilize biotinylated cCTLA-4 protein for phage library screening and ELISA analysis.

[0195] Preparation of canine CTLA-4-expressing target cell lines. To prepare 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 using the following primers and RT-PCR as described above: The resulting 1856 bp amplicon was cloned into a pMX-puromycin retrovirus expression vector (Cell Biolabs, Inc. San Diego CA). The retrovirus was generated and used to stably transduce human erythroleukemia K562 cell line (KTδ32), which had been pre-edited using CRISPR / Cas9 to remove FcγRII to reduce nonspecific mAb binding in flow cytometry assays. Transduced cells were selected in 2.5 ug / ml puromycin dihydrochloride (Sigma, St. Louis, MO) to produce KTδ32.cCTLA-4. Expression of the cCTLA-4 transgene was confirmed in these cells by RT-PCR using primers synthesized to amplify cCTLA-4. Untransduced 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 overnight at 4°C with 50 µl of 20 µg / ml streptavidin (SA). (24 wells for panning 1, 16 wells for panning 2, and 8 wells for pannings 3 and 4). The plates were washed in PBS, and the 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 SA bound to the plate. After incubation at 37°C for 1 hour, the plates were washed in PBS to remove free CTLA-4. Equialiquot canine scFv phage display libraries of μκ, μλ, γκ, and γλ were mixed and blocked at RT for 1 hour in 2% milk / PBS, and 50 μl were 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 incubation at 37°C for 2 hours, unbound phages were washed with PBS supplemented with 0.5% Tween 20 (PBST) 5 times during the first panning and 10 times during subsequent panning rounds. To select conjugates with longer off-rates, each wash was accompanied by a 5-minute incubation of the wash buffer in the well. To avoid capturing scFv phages specific to biotinylated avitag on SA or cCTLA-4, prior to positive selection in each round, phages were negatively selected against wells coated with SA and preloaded with an unrelated biotinylated avitag protein (in this case, human biotinylated avitag-CD3ε protein, AcroBiosystems, Newark, DE).Using this two-step negative / positive selection approach, we performed four rounds of selection using acid elution of bound phages in the IgM / IgG / κ / λ canine scFv phage display library, as described (Barbas et al., see above).

[0197] scFv phage ELISA. For an ELISA to detect the binding of phage display scFv, microplates were coated with streptavidin and biotinylated avitag CTLA-4 and a control antigen, similar to the library panning described above. Polyclonal phage samples from the PEG-precipitated initial library (P0) and libraries obtained after each round of panning (P1-P4), diluted 1:1000 in MPBS, or monoclonal phages (not PEG-precipitated, diluted 1:100 in milk / PBS) prepared from phage clones randomly selected from the output plates of the third round (P3) and fourth round (P4) of panning, were added to the antigen-coated plates and incubated at 37°C for 1 hour. The plates were washed with (PBST) and HRP-conjugated anti-M13 mAb (GE Healthcare, Chicago, IL) diluted 1:5000 in MPBS was added. The plates were washed again, and the conjugated HRP-conjugated secondary antibody was detected by ABTS. OD was read at 405 nm after 30 minutes using a Molecular Devices SpectraMax 340 spectrophotometer. Plates coated with no antigen, canine CD19, streptavidin only, and streptavidin with unrelated 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 with FACS buffer (1% thermally inactivated FBS in PBS containing calcium and magnesium). Cells were blocked with 10 ug of canine IgG at RT for 10 minutes, and then the cell surface was labeled with 5 ug of canine scFv or 500 ng of canine monoclonal antibody (mAb). mAb binding specificity was evaluated in blocking experiments where 250 ng of mAb was pre-incubated with 1.25 μg of cCTLA-4 ECD protein at RT for 1 hour before incubation with target cells. Cells were washed with FACS buffer and incubated with either mAb alone or mAb pre-incubated 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 the cells were incubated at RT for 30 minutes. For experiments using PBMCs, the cells shown were also labeled with rat anti-canine CD45 (clone YKIX716.13, BioRad, Hercules, CA), rat anti-canine CD5 mAb (clone: ​​YKIX 322.3, ThermoFisher Scientific), or mouse anti-feline CD5 mAb (clone: ​​FE1.1B11, BioRad), and / or rat anti-canine CD4 (clone YKIX302.9, BioRad). After cell surface labeling, the cells were washed twice with FACS buffer and fixed with 1% paraformaldehyde (Thermo Fisher Scientific, Waltham, MA). The cells were washed again, resuspended in FACS buffer, and then captured using a FACS Canto II flow cytometer (BD Biosciences). The data were analyzed using FlowJo software version X (Treestar, Ashland, OR). All plots shown are gated to 7AAD-negative cells.For intracellular staining of canine PBMCs or regulatory T cells, cells were surface-stained where applicable, washed twice in FACS buffer, and resuspended in the 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 on ice for 30 minutes with APC conjugate 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). Cells were washed once with permeabilization buffer and twice with 1× FACS buffer, and then captured on a FACS Canto II flow cytometer.

[0199] Preparation of soluble scFv. Phage clones confirmed to bind to cCTLA-4 by phage ELISA were used to infect TOP10 F' Escherichia coli (Invitrogen, Carlsbad, CA) at various dilutions and plated onto LB agar plates containing 100 ug / ml carbenicillin and 1% glucose. Single colonies were inoculated into starter culture flasks containing 20 ml of LB containing 50 ug / ml carbenicillin and 1% glucose. The cultures were grown overnight at 37°C with shaking at 225 rpm. 10 ml of saturated starter culture was inoculated into an expression culture containing 200 ml of SB containing 50 ug / ml carbenicillin and 0.1% glucose. The flasks were grown with shaking at room temperature for approximately 5 hours until the OD reached 0.5 OD600, and then induced with 0.5 mM IPTG. Growth was over-the-clock (RT) and a total of 22-24 hours after induction. The culture was centrifuged, and the medium was gently transferred from the cell pellet. The cell pellet was successively extracted with two periplasmic extraction buffers. Buffer I contained 100 mM Tris-HCl, 20% sucrose, and 1 mM EDTA, with a pH of 8.0, while Buffer II contained 5 mM MgCl2. The extraction was carried out with a pipette to break up any clumps, and the extracts were kept on ice for 20–30 minutes. After each extraction, the extracts were centrifuged at 3400 × g for 15 minutes, and the two types of extracts were combined and mixed. For purification by immobilized metal affinity chromatography, NaCl (300 mM) and imidazole (10 mM) were added, and the combined extracts were recentrifuged at 3400 × g for 15 minutes. The supernatant was then conjugated to Ni-NTA agarose, and scFv was purified in the same manner as the CTLA-4 protein described above.

[0200] High-throughput expression extracts were prepared according to a two-step growth protocol similar to that used for preparing E. coli cells for scFv purification (described above). A single TOP10F' colony resulting from infection with a polyclonal phage derived from a cCTLA-4 panning round was placed in a 96-well round-bottom cell culture plate containing LB with 50 ug / ml carbenicillin and 1% glucose. The plate was shaken over-temperature at 37°C. Five ul of the confluent wells were inoculated into a second set of 96-well plates containing SB with 50 ug / ml carbenicillin, 0.1% glucose, and 0.5 mM IPTG. The colonies were grown over-temperature in a bacterial shaker. Expression extracts were prepared by adding 1 / 4 volume of BEL buffer to the wells of the expression plate and shaking over temperature for 1 hour. The BEL buffer consisted of 320 mM NaCl, 400 mM H3BO3, and pH 8.0. Lysozyme was added to a final concentration of 2 mg / ml, EDTA to 4 mM, and benzonase nuclease to 12.5 units / ml. The lysozyme and benzonase were derived from Sigma Aldrich. The extracts were blocked with 2.5% milk / PBS-Tween and shaken for 30 minutes. The expression extracts were used directly in the anti-cCTLA-4 ELISA.

[0201] Soluble scFv ELISA: 30 ug / ml streptavidin was added to the ELISA plate and incubated overnight at 4°C. The plate was then blocked with 5% milk / PBS-Tween. A titrant of biotinylated CTLA-4 was added, and after washing, 0.25 ug / ml soluble scFv was added and conjugated at RT for 2 hours. For expression extract ELISA, 10–100 ug of extract was added to the ELISA plate and incubated at RT for 2 hours. The conjugated scFv was incubated at RT for 1 hour, washed, and detected using an AP colorimetric substrate. Detection was performed using anti-HA mouse IgG AP conjugate (or anti-rabbit AP conjugate for positive polyclonal rabbit anti-canine CTLA-4 mAb control (Sino Biological)). The plate was read at 650 nm. In some cases, bivalent scFv was prepared from monovalent scFv for use in ELISA and inhibition studies. In short, 2.5 ug of soluble scFv was incubated for 2 hours in RT with either 1 ug / ml (for ELISA) or 5 ug / ml (for blocking studies) of anti-HA alkaline phosphatase antibody or anti-HA-FITC antibody (clone: ​​HA-7, Sigma Aldrich), conjugated to the C-terminal HA molecule on each scFv, to prepare bivalent scFv preparations.

[0202] ELISA-based interaction assay. 0.3, 1.0, and 3.0 pmol of recombinant human CD86-Fc chimeras (carrier-free) or human CD80-Fc chimeras (carrier-free) (BioLegend, San Diego, CA) were conjugated overnight to ELISA plates. 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 were added to the wells and shaken at RT for 2 hours. For experiments aimed at evaluating the ability of canine scFv or canine mAb to inhibit the interaction between plate-bound CD80 and CD86 and cCTLA-4 ECD, cCTLA-4 ECD was pre-incubated with scFv or mAb in the indicated molar ratios at RT for 1 hour, then added to plates and incubated on a shaker at RT for 2 hours. After washing, streptavidin-AP conjugate (Jackson Immunoresearch, West Grove, PA) was added under RT for 1 hour. Three TBS-Tween washes were performed, and cCTLA-4 bound to CD80 or CD86 was detected using an AP colorimetric substrate (Invitrogen).

[0203] Preparation of complete canine anti-CTLA-4 mAbs. The VH and VL chains of the selected scFv are converted to either the canine constant light kappa (CLκ) domain or the canine constant light lambda (CLλ) domain, or the canine constant IgG single heavy chain domain (IgG). A Full-length bivalent IgG antibodies were prepared from isolated scFv by cloning them into separate expression plasmids containing ). Canine CLλ and CLκ were cloned into pFUSE2ss(Invitrogen) to prepare pFUSE2ss-CLIg-dλ3, pFUSE2ss-CLIg-dλ5, and pFUSE2ss-CLIg-dκ, which can be selected using blastosidine resistance. Similarly, canine VH chain (IgG A-DIgG1-IgG4 (also known as IgG1-IgG4) was cloned into pFUSEss to create pFUSEss-CHIg-dG1 (Invivogen), which can be selected in zeocin. Plasmids for cloning the variable region were selected based on the subtype (λ vs. κ) of the isolated VL region. Adhered 293T cells were transiently cotransfected with light chain plasmids and heavy chain plasmids in a 1.5:1 ratio 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 of streptavidin was added to an ELISA plate and incubated overnight at 4°C. The plate was then blocked with 5% milk / PBS-Tween. 200 ug / 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 from four different subclasses (A, B, C, and D) ranging from 0.03 to 10 ug / ml were added to the plate and incubated at RT for 1 hour. The plate was washed three times with PBS-Tween, and then a 1:35 dilution of normal human complement serum was added to one set of wells. The plate was incubated at RT for 2 hours. As a negative control, thermo-inactivated complement (HI) was added to a parallel set of wells. After incubation, the wells were washed three times with PBS-Tween, and the bound complement was detected with anti-human C1q IgG HRP conjugate antibody (BioRad, Hercules, CA). The plates were incubated in RT for 1 hour, washed three times with PBS-Tween, and then HRP substrate (R&D Systems, Minneapolis, MN) was added for colorimetric analysis. To confirm antibody binding to the plates, the bound antibodies were detected using the same canine antibody dilution with AP conjugate anti-HA IgG (Sigma) and then detected using 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 at RT for 20 minutes. Cells were washed in complete IMDM and 1 × 10⁶ cells were removed.6 Cells were resuspended in 1 / ml and cultured in triplicate in 96-well round-bottom plates with 2.5 mg / ml concanavalin A (Sigma-Aldrich) in or without 10 mg / ml anti-canine CTLA-4 mAb or negative anti-MERS mAb control. On day 3 of culture, the supernatant was collected and the amount of IFN-γ present in the supernatant was measured by canine IFN-γ ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. For experiments to detect cell surface expression of activated CTLA-4, canine PBMCs were cultured at 1 × 10⁶ 6 The cells were resuspended in cells / ml and cultured in triplicate in 96-well round-bottom plates with 2.5 mg / ml concanavalin A for 48 and 72 hours prior to analysis.

[0206] Surface plasmon resonance coupling 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 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 pH 5.0 and injected into an experimental flow cell over 60 seconds. For B10, 440–475 RU were captured; for D5, 400–500 RU were captured; and for A1mut2, 485–535 RU were captured. 10 × 1:2 serial dilutions of cCTLA-4 ECD in the range of 200 nM to 0 nM were prepared in double-row running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, and 0.05% Tween20). The flow rate across the tip was 30 μL / min, and the contact time between the cCTLA-4 ECD sample and the tip 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 in each analysis cycle was reported in relative response units (RUs). Response vs. antigen concentration plots were prepared using the BIAcore Wizard program. After each injection of CTLA-4, the RUs for each concentration of analyte were recorded, and the tip surface was regenerated with 20 mM glycine at 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. on , k off , and K D This was reported.

[0207] Example 1: Preparation 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 synthesis was confirmed by SDS-PAGE. SDS-PAGE analysis revealed a band of approximately 23 kDa, which was higher than the predicted molecular weight of ECD alone (16.4 kDa), likely due to differences in protein glycosylation. Mass spectrometry verified the identity of the recombinant protein (data not shown). To confirm the appropriate stoichiometry of biotin bound to cCTLA-4 for SA binding, increasing concentrations of SA were added to biotinylated cCTLA-4, and SDS-PAGE analysis was repeated. After SA addition, biotinylated cCTLA-4 lagged in the gel, and the band was no longer identified at 23 kDa, confirming the biotinylated protein's ability to bind to SA. To confirm the immunoreactivity of biotinylated cCTLA-4 ECD protein in a 96-well plate format, a capture assay and subsequent ELISA were performed. The results indicated that the immunoreactivity of biotinylated cCTLA-4 required the presence of plate-bound SA.

[0208] Example 2: Isolation of canine anti-canine CTLA-4 scFv A comprehensive canine IgM / IgG / λ / κ 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 phages for cCTLA-4-specific conjugates began in the second round of panning (P2) and increased to the fourth round of panning (P4) (Figure 1). To confirm that the selection of the scFv phage library against cCTLA-4 produced antigen-specific scFv phage particles, polyclonal scFv phages from each round of panning were evaluated by scFv phage ELISA using cCTLA-4 as the target antigen (Figure 2). scFv phages captured through positive selection against cCTLA-4 reacted only with wells loaded with cCTLA-4 and not with wells coated with SA alone or with SA loaded with unrelated biotinylated avitag-binding proteins. Recombinant canine (rc)CD19 antigen acted as a negative control target antigen, while canine scFv phage (Pan 4) and rcCD19 selected for CD19 acted as positive controls. These results demonstrate that the phages 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 and tested for their ability to bind to cCTLA-4 by scFv phage ELISA. All 24 clones bound to cCTLA-4 with varying affinities (Figure 3). Nucleotide sequencing of these 24 scFv revealed 20 unique antibodies, 17 of which possessed a lambda light chain and 3 possessed a kappa light chain.

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

[0211] Given that 20 unique scFvs were identified from 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 prepared and analyzed by scFv ELISA (data not shown). 41 clones showed a signal greater than 3 times the background. Ten clones showing a greater than 20-fold increase in binding to cCTLA-4 compared to the background 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). Clones C5 could not be generated as a soluble scFv. Therefore, a total of 21 unique fully soluble canine scFvs that bound to the extracellular domain of cCTLA-4 were available for further analysis.

[0212] Next, we 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 could confer therapeutic potential by enhancing the T cell response in patients with tumors. First, we developed an ELISA-based interaction assay focusing 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 clones and 4 / 4 clones from the original and additional screening groups showed varying degrees of inhibition of cCTLA-4 binding to huCD86, respectively. Of the original clones that inhibited cCTLA-4 binding to huCD86, 13 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 compared to P4-8 and P3-7 (Figure 5). In all cases, the use of a higher molar ratio of scFv:cCTLA-4 resulted in greater inhibition (6:1 compared to 2:1). Greater binding inhibition was observed when bivalent scFv (created by crosslinking two scFv via an anti-HA antibody) was compared to monovalent scFv.

[0213] Example 3: Generation of anti-CTLA-4 mAb One of the key features used to determine whether an antibody can progress along the pipeline to an effective therapeutic agent is its "development feasibility." To this end, we assessed the feasibility of reformatting cCTLA-4-specific scFvs that inhibit cCTLA-4 binding to CD80 / CD86 to be reformatted into full-length IgG mAbs. Briefly, we reformatted the VH and VL chains of selected scFvs to either a canine constant light kappa (CLκ) domain or a canine constant light lambda (CLλ) domain, or a canine constant IgG single heavy-chain domain (IgG). A Full-length bivalent IgG antibodies were prepared from isolated scFv by cloning them into separate expression plasmids containing ). Canine CLλ and CLκ were cloned into pFUSE2ss (Invitrogen) to prepare pFUSE2ss-CLIg-dλ3, pFUSE2ss-CLIg-dλ5, and pFUSE2ss-CLIg-dκ, which can be selected via blastosidin resistance. Similarly, canine VH chains were cloned into pFUSEss-CHIg-dG1 purchased from Invivogen to complete the full-range plasmid reformatting. Plasmids for cloning the VL region were selected based on the subtype (λ vs. κ) of the isolated VL region. Adhered 293T cells were transiently co-transfected with lipofectamine L2000 reagent in a 1.5:1 ratio of light chain plasmids and heavy chain plasmids, respectively. The plate supernatant was collected after 3 days, and mAbs were purified using protein A affinity chromatography.

[0214] Based on their ability to inhibit cCTLA-4 binding to ligands CD80 and CD86, clones A1, D5, B10, G11, and P4-8 were selected and reformatted to full-length IgG mAbs for further functional analysis. B10 and D5 were IgG1(IgG AAlthough reformatting as ) was successful, only low levels of clone A1 were produced, and clones G11 and P4-8 could not be produced as full-length canine mAbs (Figure 6). Considering the superior ability of clone A1 to bind to cCTLA-4 (Figure 7) and block the interaction between cCTLA-4 and CD80 / 86 (Figure 8), we investigated whether A1 mAb production could be improved through CDR transplantation experiments. Using chain swapping experiments, the low productivity was localized to the VH chain of A1 (Figure 9). Since clone B10 was easily produced, the CDR region of A1 was transplanted into the VH backbone of clone B10. The resulting chimeric A1 VH chain, transfected together with the original A1 VL chain, resulted in a high yield of full-length canine mA, named A1mut2. Complete canine mAbs A1mut2, B10, and D5 were confirmed to bind to cCTLA-4 in ELISA (Figure 11A) and to inhibit the interaction between cCTLA-4 and CD80 / 86 (Figure 11B). In parallel, the ability of complete canine mAbs to inhibit the binding of cCTLA-4 to canine CD80 / 86 was also confirmed (Figure 11C). For these experiments, hemagglutinin (HA) tags separated by glycine-serine-rich linker sequences were attached to the carboxyl terminus of full-length IgG, and antibodies were detected using secondary antibodies against the tags. This is the amino acid sequence. The nucleotide sequence encoding TIFF0007868811000019.tif4128 The addition of TIFF0007868811000020.tif19163 was required. To obtain 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 tagging 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 sequence was fused to a triple alanine sequence followed by a modified human IgG1 Fc having a C-to-S mutation in the hinge region. Natural canine CD80 and CD86 signal peptides were replaced with mouse IgG heavy chain signal sequences. The construct sequence gBlocks were ordered from IDT (Coralville, Iowa) and cloned into pFUSE plasmids. The pFUSE cCD80-hFc and cCD86-hFc plasmids were transfected into 293T cells with Lipofectamine 2000, and the cell medium was collected after 3 days. Purification of ligand Fc fusion proteins from the cell culture supernatant was performed using protein A agarose (MilliporeSigma, Burlington, MA). Sample concentration, buffer exchange to PBS, SDS-PAGE analysis, and quantification were performed in the same manner as for the generation of recombinant canine CTLA-4 protein.

[0215] Example 4: CTLA-4 specific mAb binds to CTLA-4 expressed on the cell surface. To determine whether the 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 the parental KTδ32 cells. Furthermore, pre-incubation of each mAb with soluble cCTLA-4 to block the CTLA-4 binding site eliminated mAb binding, indicating that membrane binding is antigen-specific. Unrelated MERS mAbs did not bind to KTδ32.cCTLA-4, further suggesting that the selected clones specifically bind 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). + 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 post-activation. At each time point, a larger percentage of CD4 T cells expressed CTLA-4 compared to CD8 T cells (50.7% vs. 29.5% at 48 hours and 56.6% vs. 40.5% at 72 hours, respectively). Next, canine CD5 + 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 the 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 and feline CTLA-4 share 99% identity, feline leukocytes were labeled with A1mut2 mAb to determine antibody cross-reactivity. Feline CD5+ cells showed minimal surface staining with A1mut2, but the antibody bound strongly to intracellular CTLA-4 (Figure 17).

[0216] Example 5: The complete canine anti-CTLA-4 mAb shows high binding affinity to soluble cCTLA-4. The affinity of A1mut2, D5, and B10 for cCTLA-4 was evaluated to determine their suitability 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) of A1mut2 was also evaluated. D ) was in the sub-nanomolar range, showing the highest binding affinity among the three clones. The other two clones showed affinities in the single order of nanomolars. Therefore, all three mAbs showed sufficient binding affinity to cCTLA-4 to serve as candidates to be evaluated as therapeutic checkpoint inhibitors (illustrated in Figure 13).

[0217] Example 6: Complete canine anti-CTLA-4 IgG2 (IgG B ) and IgG3 (IgG C ) fixes the complement Given the increasing recognition that the contribution of CTLA-4 targeted antibodies to antitumor immunity lies in their ability to deplete intratumoral regulatory T cells, we tested the complement fixation ability of the A1mut2 IgG subclass in vitro. As previously demonstrated, IgG B and IgG C Although it effectively fixed the complement, IgG1 (IgG A ) and IgG4 (IgG D ) were not fixed (Figures 18A-18B). This result indicates that these subclasses of A1mut2 Ig have an effect that is almost limited to intratumor Tregs expressing high levels of CTLA-4. + It has been shown that it may have the potential to initiate complement-mediated cytotoxicity in cells.

[0218] Example 7: Clone A1mut2 enhances T cell proliferation and IFN-γ production. To determine whether A1mut2, which exhibited the highest affinity for cCTLA-4 and the greatest inhibition of cCTLA-4 binding to CD80 / 86, could enhance 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 unrelated MERS-specific mAb. Cells were collected at 72 or 96 hours, and the responder frequency and proliferative capacity of T cells activated in the presence of A1mut2 versus MERS were determined by flow cytometry (Figure 19). A1mut2 increased the percentage of mitogen-responsive T cells (responder frequency) and the average number of daughter cells produced per responding cell (proliferative capacity) at 96 hours post-stimulation in 8 / 9 dogs, compared to an unrelated MERS mAb. IFN-γ production was evaluated in the supernatant of ConA-stimulated T cells 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 culture (Figure 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 an mAb that enhances tumor-specific T cell priming and effector responses within the tumor microenvironment.

[0219] Example 8: Discussion CTLA-4 blockade has proven to be a potent strategy for promoting antitumor immunity by inducing the expansion and proliferation of Th1-like CD4 effector T cells and exhausted CD8+ T cells, and by eliminating intratumoral regulatory T cells. These effects have resulted in clinically relevant antitumor immunity, particularly in human patients with malignant melanoma, NSCLC, and renal cell carcinoma. However, a greater understanding of the mechanisms of acquired and / or innate resistance of tumors to anti-CTLA-4 therapy, identification of response biomarkers, optimized protocols and combination approaches to improve outcomes, and understanding and mitigating the mechanisms of toxicity are needed to increase overall response rates and reduce toxicity. The studies presented in this disclosure employed a potent scFv phage display approach to identify multiple unique clones of complete canine scFv that specifically bind to canine CTLA-4 with nanomolar and sub-nanomolar affinity. From these clones, complete canine mAb lead candidates were generated and further selected based on their developability and in vitro functional capability for inclusion in clinical trials in dogs with spontaneously occurring cancers. The use of a complete canine anti-CTLA-4 mAb in immunocompetent canine cancer patients with spontaneously occurring tumors, sharing similar characteristics with its human counterpart, would yield beneficial results for human clinical trial designs.

[0220] This disclosure demonstrates the specific binding of A1mut2 to canine CTLA-4 using flow cytometry, and shows that CTLA-4 is upregulated on the surface of CD4+ 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 findings in human CD4 and CD8 T cells, as well as the differential dynamics of CTLA-4 upregulation on canine T cell subsets, where expression on CD4+ T cells peaks earlier (48 hours) than on CD8+ T cells (over 72 hours). Studies in human T cells have demonstrated that the peak of CTLA-4 expression on all mitogen-activated T cells occurs 72 hours after stimulation.

[0221] In human studies, ipilimumab monotherapy showed minimal effects on IFN-γ production and T cell proliferation in allogeneic T:DC MLR assays, as well as only a slight increase in IL-2 production after mitogen activation in human T cells. Furthermore, responses varied among individual samples tested. While the in vitro assay response in human T cells was modest, the clinical response in a subset of human patients was dramatic, highlighting the multifactorial mechanisms of action of CTLA-4 blockade and the potential influence of patient-specific factors on clinical response. The studies in this disclosure also observed significant variability in T cell responses to CTLA-4 blockade among mitogen-activated T cells derived from healthy donor dogs, as well as moderate 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, while we do not wish to be bound by theory, suggests that the use of A1mut2 in canine cancer patients does not result in inappropriate activation of naive T cells. Furthermore, these studies have confirmed that, at the protein level, CTLA-4 is present / upregulated in regulatory T cells and can act as a target for A1mut2-dependent complement-mediated cytotoxicity. Indeed, in human patients, higher levels of CTLA-4 expression on intratumor Tregs compared to Tregs in general circulation enable selective depletion after anti-CTLA-4 treatment, which leads to an increase in CD8:Tregs after treatment. A1mut2 is currently functionally equivalent to the human IgG1 subtype, which efficiently binds to complement and induces ADCC. B This has been observed in the treatment (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] Since the clinical response in canine malignancies reflects the clinical response in human patients with melanoma, gastric cell carcinoma, and urothelial carcinoma, this treatment is expected to improve outcomes in pet dogs suffering from similar malignancies.

[0223] Listed aspects The following enumerated aspects are provided, and their numbering should not be interpreted as indicating a level of importance. Embodiment 1 provides the following: An antibody or its antigen-binding fragment comprising an antigen-binding domain that specifically binds to the epitope of canine cytotoxic T lymphocyte-associated protein 4 (CTLA-4), The antigen-binding domain is i. A heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, and 99% identity with the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 6, 25, 40, or 74; and ii. Light chain variable regions containing amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences described in SEQ ID NO: 8, 27, or 42. Includes, The heavy chain variable region comprises three heavy chain complementarity determining regions (HCDRs), where HCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 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 NO: 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 NO: 5, 24, and 39). An antibody or its antigen-binding fragment. Embodiment 2 provides the following: An antibody or antigen-binding fragment thereof according to embodiment 1, selected from the group consisting of a full-length antibody, Fab, and a single-chain variable fragment (scFv). Embodiment 3 provides the following: The antibody or its antigen-binding fragment according to embodiment 2, which is a full-length antibody. Embodiment 4 provides the following: The antibody or its antigen-binding fragment according to embodiment 3, wherein the antibody is a canine antibody. Embodiment 5 provides the following: The antibody or antigen-binding fragment according to embodiment 1, wherein the antigen-binding domain comprises a heavy chain variable region containing an amino acid sequence described in SEQ ID NO: 6, 25, 40, or 74. Embodiment 6 provides the following: The antibody or antigen-binding fragment according to embodiment 1, wherein the antigen-binding domain comprises a heavy chain variable region consisting of an amino acid sequence described in SEQ ID NO: 6, 25, 40, or 74. Embodiment 7 provides the following: The antibody or antigen-binding fragment according to any one of embodiments 1 to 4, wherein the antigen-binding domain includes a light chain variable region comprising the amino acid sequence described in SEQ ID NO: 8, 27, or 42. Embodiment 8 provides the following: The antibody or antigen-binding fragment according to any one of embodiments 1 to 4, wherein the antigen-binding domain comprises a light chain variable region containing the amino acid sequence described in SEQ ID NO: 8, 27, or 42. Embodiment 9 provides the following: i. Heavy chain variable region containing the amino acid sequence described in SEQ ID NO:6; and ii. Light chain variable region containing the amino acid sequence described in SEQ ID NO:8 An isolated antibody or its antigen-binding fragment, containing [the specified substance]. Embodiment 10 provides the following: 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 NO: 1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 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 NO: 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 NO: 5, 24, and 39). Includes, 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 the epitope of canine cytotoxic T lymphocyte-associated protein 4 (CTLA-4). Embodiment 11 provides the following: i. Heavy chain variable region containing amino acid sequences described in SEQ ID NO: 6, 25, 40, or 74; and ii. Light chain variable region containing the amino acid sequence described in SEQ ID NO: 8, 27, or 42 Includes, The heavy chain variable region and the light chain variable region are separated by a linker. Single-chain variable fragment (scFv). Embodiment 12 provides the following: A single-chain variable fragment (scFv) containing the amino acid sequence described in SEQ ID NO: 29, 44, or 76. Embodiment 13 provides the following: A single-chain variable fragment (scFv) consisting of the amino acid sequence described in SEQ ID NO: 29, 44, or 76. Embodiment 14 provides the following: 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), wherein the antigen-binding domain is 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 NO:1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO:2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: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 NO: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 NO:5, 24, and 39) A full-length antibody comprising the above. Aspect 15 provides the following: 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 the above. Aspect 16 provides the following: A full-length antibody comprising the heavy-chain amino acid sequence set forth in SEQ ID NO:10, 12, 14, 16, 31, 46, 71, 72, or 73, and the light-chain amino acid sequence set forth in SEQ ID NO:18, 33, or 48. Aspect 17 provides the following: A full-length antibody consisting of the heavy-chain amino acid sequence set forth in SEQ ID NO:10, 12, 14, 16, 31, 46, 71, 72, or 73, and the light-chain amino acid sequence set forth in SEQ ID NO:18, 33, or 48. Embodiment 18 provides the following: An isolated nucleic acid encoding an scFv or full-length antibody according to any of the above embodiments. Embodiment 19 provides the following: An isolated nucleic acid encoding an antibody or its antigen-binding fragment, which includes an antigen-binding domain that specifically binds to the epitope of canine cytotoxic T lymphocyte-associated protein 4 (CTLA-4), The antigen-binding domain is i. Heavy chain variable regions encoded by nucleic acids containing polynucleotide sequences having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, and 99% identity to SEQ ID NO: 7, 26, 41, or 75; and ii. Light chain variable region encoded by a nucleic acid containing a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the light chain variable region described in SEQ ID NO: 9, 28, or 43. Includes, The heavy chain variable region comprises three heavy chain complementarity determining regions (HCDRs), where HCDR1 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 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 NO: 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 NO: 5, 24, and 39). Isolated nucleic acids. Embodiment 20 provides the following: The nucleic acid according to embodiment 19, wherein the antibody or its antigen-binding fragment is selected from the group consisting of a full-length antibody, Fab, and a single-chain variable fragment (scFv). Embodiment 21 provides the following: The nucleic acid according to embodiment 20, wherein the antibody is a full-length antibody. Embodiment 22 provides the following: The nucleic acid according to embodiment 21, wherein the antibody is a canine antibody. Embodiment 23 provides the following: The nucleic acid according to embodiment 19, wherein the heavy chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence described in SEQ ID NO: 7, 26, 41, or 75. Embodiment 24 provides the following: The nucleic acid according to embodiment 19, wherein the heavy chain variable region is encoded by a nucleic acid consisting of a polynucleotide sequence described in SEQ ID NO: 7, 26, 41, or 75. Embodiment 25 provides the following: The nucleic acid according to embodiment 19, wherein the light chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence described in SEQ ID NO: 9, 28, or 43. Embodiment 26 provides the following: The nucleic acid according to embodiment 19, wherein the light chain variable region is encoded by a nucleic acid consisting of a polynucleotide sequence described in SEQ ID NO: 9, 28, or 43. Embodiment 27 provides the following: i. Heavy chain variable regions encoded by nucleic acid sequences including polynucleotide sequences described in SEQ ID NO: 7, 26, 41, or 75; and ii. Light chain variable regions encoded by nucleic acid sequences containing polynucleotide sequences described in SEQ ID NO: 9, 28, or 43. Isolated nucleic acids encoding an antibody or its antigen-binding fragment. Embodiment 28 provides the following: 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 NO: 1, 20, and 35), HCDR2 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 2, 21, and 36), and HCDR3 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 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 NO: 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 NO: 5, 24, and 39). Isolated nucleic acids encoding single-chain variable fragments (scFv) containing [a specific component]. Embodiment 29 provides the following: i. Heavy chain variable regions containing nucleotide sequences described in SEQ ID NO: 7, 26, 41, or 75; and ii. Light chain variable region containing the nucleotide sequence described in SEQ ID NO: 9, 28, or 43 Includes, The heavy chain variable region and the light chain variable region are separated by a linker. Isolated nucleic acids that encode single-chain variable fragments (scFv). Embodiment 30 provides the following: Single-stranded variable fragments (scFv) containing polynucleotide sequences described in SEQ ID NO: 30, 45, or 77. An isolated nucleic acid that codes for [something]. Embodiment 31 provides the following: Single-chain variable fragments (scFv) consisting of polynucleotide sequences described in SEQ ID NO: 30, 45, or 77. An isolated nucleic acid that codes for [something]. Embodiment 32 provides the following: A vector comprising the isolated nucleic acid according to any one of Aspects 19 to 32. Aspect 33 provides the following: The vector according to Aspect 32, which is an expression vector. Aspect 34 provides the following: The vector according to any one of Aspects 32 and 33, which is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and a retroviral vector. Aspect 35 provides the following: A host cell comprising the vector according to any one of Aspects 32 to 34. Aspect 36 provides the following: The host cell according to Aspect 35, which is of eukaryotic or prokaryotic origin. Aspect 37 provides the following: The host cell according to any one of Aspects 35 and 36, which is of mammalian origin. Aspect 38 provides the following: The host cell according to any one of Aspects 35 and 36, which is of bacterial origin. Aspect 39 provides the following: A method for generating an antibody or an antigen-binding fragment thereof that binds to canine cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the method comprising culturing the host cell according to any one of Aspects 35 to 38. Aspect 40 provides the following: A pharmaceutical composition comprising the full-length antibody or scFv according to any one of Aspects 1 to 17 and a pharmaceutically acceptable carrier. Aspect 41 provides the following: A method for treating cancer in a subject in need thereof, the method comprising administering the antibody or an antigen-binding fragment thereof according to any one of Aspects 1 to 17 to the subject. Aspect 42 provides the following: The method according to Aspect 41, wherein the cancer is associated with cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Aspect 43 provides the following: The method according to embodiment 42, wherein CTLA-4 is expressed on cancer-associated cells. Embodiment 44 provides the following: The method according to embodiment 43, wherein the cancer-related cells are T lymphocytes. Embodiment 45 provides the following: The method according to embodiment 41, wherein the antibody or its antigen-binding fragment specifically binds to canine CTLA-4. Embodiment 46 provides the following: The method according to embodiment 41, wherein the antibody or its antigen-binding fragment is selected from the group consisting of a full-length antibody, Fab, and a single-chain variable fragment (scFv). Embodiment 47 provides the following: The method according to embodiment 46, wherein the antibody or its antigen-binding fragment is a full-length antibody. Embodiment 48 provides the following: The method according to embodiment 47, wherein the antibody is a canine antibody. Embodiment 49 provides the following: The method according to embodiment 41, wherein the subject is a dog.

[0224] Other embodiments Any description of a list of elements in any definition of a variable in this specification includes the definition of that variable as any single element or combination (or partial combination) of the enumerated elements. Any description of an aspect in this specification includes that aspect as any single aspect, or in combination with any other aspect or a part thereof.

[0225] Any patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. While the present invention has been disclosed in relation to particular embodiments, it will be apparent that other embodiments and variations of the present invention can be devised by other persons skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. An antibody or its antigen-binding fragment, comprising an antigen-binding domain that specifically binds to the epitope of canine cytotoxic T lymphocyte-associated protein 4 (CTLA-4), The antigen-binding domain is (i) A heavy chain variable region having at least 90% identity with the amino acid sequence of the heavy chain variable region described in SEQ ID NO:6; and (ii) Light chain variable region containing an amino acid sequence having at least 90% identity with the amino acid sequence described in SEQ ID NO:8 Includes, The heavy chain variable region includes three heavy chain complementarity determining regions (HCDRs), where HCDR1 includes the amino acid sequence of SEQ ID NO:1, HCDR2 includes the amino acid sequence of SEQ ID NO:2, and HCDR3 includes the amino acid sequence of SEQ ID NO:3, and The light chain variable region includes three light chain complementarity determining regions (LCDRs), where LCDR1 includes the amino acid sequence of SEQ ID NO:4, LCDR2 includes the amino acid sequence of VDG, and LCDR3 includes the amino acid sequence of SEQ ID NO:

5. An antibody or its antigen-binding fragment.

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

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a canine antibody.

4. A single-chain variable fragment (scFv) comprising an antigen-binding domain that specifically binds to an epitope of canine cytotoxic T lymphocyte-associated protein 4 (CTLA-4), (i) A heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:1, HCDR2 comprises the amino acid sequence of SEQ ID NO:2, and HCDR3 comprises the amino acid sequence of SEQ ID NO:3; and (ii) A light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence of SEQ ID NO:4, LCDR2 comprises the amino acid sequence of VDG, and LCDR3 comprises the amino acid sequence of SEQ ID NO:

5. Includes, The heavy chain variable region and the light chain variable region are separated by a linker. Single-chain variable fragment (scFv).

5. A full-length antibody comprising an antigen-binding domain that specifically binds to the epitope of canine cytotoxic T lymphocyte-associated protein 4 (CTLA-4), The antigen-binding domain is (i) A heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:1, HCDR2 comprises the amino acid sequence of SEQ ID NO:2, and HCDR3 comprises the amino acid sequence of SEQ ID NO:3; and (ii) A light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence of SEQ ID NO:4, LCDR2 comprises the amino acid sequence of VDG, and LCDR3 comprises the amino acid sequence of SEQ ID NO:

5. A full-length antibody containing this antibody.

6. A full-length antibody comprising an antigen-binding domain that specifically binds to the epitope of canine cytotoxic T lymphocyte-associated protein 4 (CTLA-4), It includes a heavy chain amino acid sequence having at least 95% identity with the amino acid sequence described in SEQ ID NO:10, and a light chain amino acid sequence having at least 95% identity with the amino acid sequence described in SEQ ID NO:

18. The heavy chain contains three heavy chain complementarity-determining regions (HCDRs): HCDR1 contains the amino acid sequence of SEQ ID NO:1, HCDR2 contains the amino acid sequence of SEQ ID NO:2, and HCDR3 contains the amino acid sequence of SEQ ID NO:

3. The light chain contains three light chain complementarity-determining regions (LCDRs): LCDR1 contains the amino acid sequence of SEQ ID NO: 4, LCDR2 contains the amino acid sequence of VDG, and LCDR3 contains the amino acid sequence of SEQ ID NO:

5. Full length antibody.

7. An isolated nucleic acid encoding an antibody or its antigen-binding fragment according to any one of claims 1 to 3, an scFv according to claim 4, or a full-length antibody according to claim 5 or 6.

8. An isolated nucleic acid encoding an antibody or its antigen-binding fragment, which includes an antigen-binding domain that specifically binds to the epitope of canine cytotoxic T lymphocyte-associated protein 4 (CTLA-4), The antigen-binding domain is (i) A heavy chain variable region encoded by a polynucleotide sequence having at least 90% identity with SEQ ID NO:7; and (ii) Light chain variable region encoded by a polynucleotide sequence having at least 90% identity with the amino acid sequence of the light chain variable region described in SEQ ID NO:9 Includes, The heavy chain variable region includes three heavy chain complementarity determining regions (HCDRs), where HCDR1 includes the amino acid sequence of SEQ ID NO:1, HCDR2 includes the amino acid sequence of SEQ ID NO:2, and HCDR3 includes the amino acid sequence of SEQ ID NO:3, and The light chain variable region includes three light chain complementarity determining regions (LCDRs), where LCDR1 includes the amino acid sequence of SEQ ID NO:4, LCDR2 includes the amino acid sequence of VDG, and LCDR3 includes the amino acid sequence of SEQ ID NO:

5. Isolated nucleic acids.

9. A vector comprising an isolated nucleic acid as described in claim 7, wherein the vector is an expression vector.

10. The vector according to claim 9, wherein the vector is selected from the group consisting of DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and retroviral vectors.

11. A host cell comprising the vector according to claim 10.

12. The host cell according to claim 11, wherein the host cell is of eukaryotic or prokaryotic origin.

13. The host cell according to claim 11, wherein the host cell is of mammalian or bacterial origin.

14. A method for producing a full-length antibody that binds to an antibody or its antigen-binding fragment, scFv, or canine cytotoxic T lymphocyte-associated protein 4 (CTLA-4), comprising the step of culturing the host cells described in Claim 11.

15. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, an scFv according to claim 4, or a full-length antibody according to claim 5 or 6, and a pharmaceutically acceptable carrier.

16. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, an scFv according to claim 4, or a full-length antibody according to claim 5 or 6, for use in a method for treating cancer in a subject where such treatment is necessary.

17. The antibody according to claim 16, or its antigen-binding fragment, scFv, or full-length antibody, wherein the subject is a dog.

18. The antibody according to claim 16, or its antigen-binding fragment, scFv, or full-length antibody, wherein the cancer is associated with cytotoxic T lymphocyte-associated protein 4 (CTLA-4).

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