Canine PDL-1 antibody and its uses

Abstract

This invention relates to monoclonal antibodies specific to canine PDL-1 or antigen-binding fragments thereof, pharmaceutical compositions comprising the same, and their uses. When using novel canine PDL-1 antibodies according to the invention, which exhibit high specificity for canine PDL-1 and tumor-suppressive effects in a mouse model of canine osteosarcoma, high therapeutic efficacy can be expected for canine cancers that are untreatable with conventional immunotherapy.

Description

Technical Field

[0001] This invention relates to monoclonal antibodies specific to PDL-1 or antigen-binding fragments thereof, and more specifically, to monoclonal antibodies specific to canine PDL-1 or antigen-binding fragments thereof, pharmaceutical compositions comprising the same, and uses thereof. Background Technology

[0002] Cancer is a prevalent disease in both dogs and humans, and it manifests in various forms depending on factors such as breed and sex. Common tumors in dogs include histiocytomas, lipomas, adenomas, and mammary tumors. Current treatments for canine cancer include surgical removal of the tumor tissue, radiation therapy, and chemotherapy.

[0003] The regulation of the immune system involves complex immune checkpoint mechanisms that control T lymphocyte function, antigen recognition, and the balance between stimulatory and inhibitory signals. Detecting tumor-specific neoantigens induced by mutations in tumor cells helps eliminate tumor and viral sources. However, some tumor cells evade the immune system by modulating the tumor microenvironment to induce immune tolerance or by immune editing to escape immune cellular responses. Alterations in immune checkpoint ligand / receptor binding have a significant impact on the activity of tumor-specific T lymphocytes. Monoclonal antibodies that inhibit PD-1 or PD-L1 ligand function have been reported to enhance the activity of tumor-specific T lymphocytes, thereby providing anti-cancer effects, leading to significant advances in cancer therapy (Curiel TJ et al., Nature Medicine, 9:562, 2003).

[0004] PD-L1 (programmed death-ligand 1) possesses unique structural features, including two Ig-like domains in its extracellular region, a transmembrane domain, and a short cytoplasmic domain lacking a known signaling motif. Encoded by the CD274 gene, PD-L1 shares approximately 20% amino acid sequence identity with B7.1 and B7.2, which belong to the B7 protein family. PD-L1 binds to PD-1, an immune checkpoint regulatory receptor expressed on various immune cells, thereby influencing T-cell immune activity. PD-L1 mediates immune protection against cytotoxic T lymphocyte (CTL)-mediated killing and modulates chronic immune responses through upregulation, which interferes with immune protective pathways. PD-L1 can also interact with B7.1, thus potentially playing a role in immunomodulation, particularly in tumor immune escape.

[0005] Although the therapeutic efficacy of immune checkpoint inhibitors in cancer treatment has been demonstrated in human oncology, cancer therapies using this approach have not been extensively studied in the veterinary field. Therefore, the lack of canine-specific therapeutic antibodies for immunotherapy strategies in treating canine cancer presents a significant limitation.

[0006] Several studies have shown increased PD-L1 expression in dogs with tumors (Maekawa N). et al., NPJ Precision Oncology ( , 5:10, 2021). Furthermore, blocking the PD-1 / PD-L1 interaction has been shown to enhance T-cell function in dogs with cancer (Choi JW et al., PLoS One, 15:e0235518, 2020). To date, no antibodies currently developed have been approved as therapeutics for cancer in dogs.

[0007] Therefore, for canine cancer treatment using immune checkpoint inhibitors, the inventors used phage display technology to select scFv (single-chain variable fragment) specific to canine PD-L1 from a synthetic scFv library, fused the selected scFv with a human IgG1 constant region sequence to develop an antibody in IgG form, and identified the binding affinity and anticancer activity of the developed canine PD-L1 binding antibody in in vitro and in vivo models, thus completing the present invention. Summary of the Invention

[0008] The object of this invention is to provide an antibody against canine PDL-1, a pharmaceutical composition comprising the same, and a method of using the same to treat tumors.

[0009] According to one aspect of the present invention, the above and other objectives can be achieved by providing a monoclonal antibody or an antigen-binding fragment thereof. The monoclonal antibody includes: a heavy chain variable region (V... H The heavy chain variable region includes HCDR1 shown in SEQ ID NO:14, HCDR2 shown in SEQ ID NO:16, and HCDR3 shown in SEQ ID NO:18; and The light chain variable region (VL) includes LCDR1 shown in SEQ ID NO: 21, LCDR2 shown in SEQ ID NO: 23 and LCDR3 shown in SEQ ID NO: 25.

[0010] According to another approach, a monoclonal antibody or its antigen-binding fragment is provided. The monoclonal antibody includes a heavy chain variable region (V). HThe heavy chain variable region includes HCDR1 shown in SEQ ID NO:28, SEQ ID NO:42, SEQ ID NO:56, SEQ ID NO:70 or SEQ ID NO:84; HCDR2 shown in SEQ ID NO:30, SEQ ID NO:44, SEQ ID NO:58, SEQ ID NO:72 or SEQ ID NO:86; and HCDR3 shown in SEQ ID NO:32, SEQ ID NO:46, SEQ ID NO:60, SEQ ID NO:74 or SEQ ID NO:88; and Light chain variable region (V L The light chain variable region includes LCDR1 shown in SEQ ID NO:35, SEQ ID NO:49, SEQ ID NO:63, SEQ ID NO:77 or SEQ ID NO:91; LCDR2 shown in SEQ ID NO:37, SEQ ID NO:51, SEQ ID NO:65, SEQ ID NO:79 or SEQ ID NO:93; and LCDR3 shown in SEQ ID NO:39, SEQ ID NO:53, SEQ ID NO:67, SEQ ID NO:81 or SEQ ID NO:95.

[0011] According to another approach, an isolated nucleic acid molecule is provided, comprising a heavy chain variable region (V0) encoding the monoclonal antibody or its antigen-binding fragment thereof. H The nucleotide sequence of ).

[0012] According to another approach, an isolated nucleic acid molecule is provided, comprising a heavy chain variable region (V0) encoding the monoclonal antibody or its antigen-binding fragment thereof. H The nucleotide sequence of ).

[0013] According to another approach, an isolated nucleic acid molecule is provided, comprising a light chain variable region (V0) encoding the monoclonal antibody or its antigen-binding fragment thereof. L The nucleotide sequence of ).

[0014] According to another aspect, a carrier containing the isolated nucleic acid molecules is provided.

[0015] According to another aspect, a host cell containing the isolated nucleic acid molecule or the vector is provided.

[0016] According to another aspect, a pharmaceutical composition for treating tumors is provided, the pharmaceutical composition comprising: the monoclonal antibody or its antigen-binding fragment, and a pharmaceutically acceptable load or excipient.

[0017] According to another aspect, a method for treating or preventing tumors in dogs is provided, comprising administering the monoclonal antibody or its antigen-binding fragment, or the pharmaceutical composition for treating tumors, to a dog suffering from said tumor.

[0018] According to another aspect, a method for treating or preventing tumors is provided, including administering the monoclonal antibody or its antigen-binding fragment, or the pharmaceutical composition for treating tumors.

[0019] According to another aspect, the monoclonal antibody or its antigen-binding fragment, or the pharmaceutical composition for treating tumors, is provided for use in treating or preventing tumors.

[0020] According to another aspect, the use of the monoclonal antibody or its antigen-binding fragment, or the pharmaceutical composition for treating tumors, in the preparation of a medicament for treating or preventing tumors is provided. Attached Figure Description

[0021] Figure 1 shows the screening method for antibodies specific to canine PD-L1 and the identification results of scFv clones targeting canine PD-L1. More specifically, Figure 1a This invention illustrates the phage display method used in the present invention. Figure 1b The titers obtained from four rounds of panning are shown, where the titer was determined by the number of ER2738 colonies infected with bacteriophage. Figure 1c The results show the screening of clones from the third and fourth rounds of panning libraries using scFv binding analysis to select specific binders to canine PD-L1. The binding activity of 2× periplasmic scFv clones to canine PD-L1 was determined by ELISA using TES periplasmic extraction. Figure 1d The results of thermal stability activity analysis of 35 selected scFv clones by conjugation analysis are shown, in which the OD at 450 nm was measured using scFv heated at 70 °C.

[0022] Figure 2 shows the results of the in vitro PD-1 / PD-L1 interaction inhibition analysis and the validation of scFv functional activity. More specifically, Figure 2a This image shows a comparison of the amino acid sequences of human PD-L1 and canine PD-L1. Regions with identical amino acid sequences in both species are highlighted in purple, while red boxes indicate the amino acid sequence of canine PD-L1 in regions known to be important for the binding of human PD-1 to human PD-L1. Figure 2b The results of ELISA detection of the binding of 19 scFvs to human PD-L1 are shown, where NC indicates samples treated with only the secondary antibody. Figure 2cThis is a schematic diagram illustrating the method of using a competitive ELISA to determine whether these 19 scFvs inhibit the binding of human PD-1 to canine PD-L1. Figure 2d This is a graph showing the ELISA results indicating the inhibitory activity of cPD-L1 scFv, where PC represents the sample indicating the binding ability between canine PD-L1 protein and human PD-1 protein, and NC represents the sample coated with canine PD-L1 protein only with a second antibody.

[0023] Figure 3 shows the results of converting the selected scFv into IgG form and validating the function of the converted IgG. More specifically, Figure 3a This is an overall schematic diagram illustrating the method of converting scFv clones with independent sequences into IgG, wherein the heavy chain DNA and light chain DNA fragments of the antibody are ligated into a vector capable of expression in FreeStyle™ 293-F cells, and FreeStyle™ 293-F cells are infected with plasmid samples using a dual-vector system. Figure 3b The results show the purification of IgG from FreeStyle™ 293-F cells infected using a dual-vector system and the identification of the purified IgG by SDS-PAGE gel electrophoresis. Figure 3c The results show the binding affinity of anti-canine PD-L1 antibodies to canine and human PD-L1 as determined by ELISA. KL-001 was a positive antibody binding to both canine and human PD-L1, while atezolizumab was a positive antibody binding to human PD-L1. Figure 3d and Figure 3e The results show the binding activities of six antibodies measured by flow cytometry after treatment of canine cell lines expressing canine PD-L1 with KL-001 (red line), anti-canine PD-L1 antibody (blue line), or control antibody (secondary anti-human Fc antibody, black line). More specifically, Figure 3d The results for DH82 cells were shown, and Figure 3e Results for D17 cells are shown, where flow cytometry was performed using either Alexa 488-conjugated anti-human Fc or Alexa 647-conjugated anti-human Fc as a second fluorescent antibody for detection.

[0024] Figure 4 The results show the in vivo antitumor efficacy of canine PD-L1 antibody in a canine osteosarcoma mouse model, and more specifically, Figure 4 A shows the use of canine tumor cells (D17 cells, 4 × 10⁻⁶) 6 (Cells / mouse) were implanted into the left flank of mice and observed for 20 days. Ten days after tumor inoculation, canine PBMCs (8×10⁶ cells / mouse) were injected intravenously. 6Cells / mouse), administered antibody #1 or antibody #6 intraperitoneally at a concentration of 1 mg / ml, with tumor growth measured every two days. p<0.05 p<0.001 p<0.0001; one-way ANOVA; n=5 / group), and Figure 4 B shows the results of tumor mass measurements after euthanizing all mouse groups and collecting tumors, where, ns; not significant and p<0.05 (one-way ANOVA; n=5 / group), data obtained from three independent experiments. Detailed Implementation

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art. Generally, the nomenclature used herein is well-known and commonly used in the art.

[0026] Immunotherapy has become a breakthrough approach in the treatment of human cancers, particularly targeting immune checkpoint molecules such as PD-1 and PD-L1. Although its efficacy has been demonstrated in human oncology, this innovative approach has not been fully explored in the veterinary field, and therefore, canine-specific therapeutic antibodies for treating canine cancers using immunotherapy strategies are not yet available.

[0027] In this invention, a therapeutic antibody against canine PDL-1 was developed. Using phage display technology, single-chain variable fragments (scFvs) specifically binding to canine PD-L1 were isolated from a synthetic antibody library. Phage display screening of the scFv library selected over 35 high-affinity clones exhibiting high ELISA signals, thermostability, and unique sequences in the heavy and light chain complementarity-determining regions (CDRs). Nineteen scFv antibodies with independent CDR sequences were identified. To evaluate the function of the selected 19 scFv antibodies, the association between canine PD-L1 and human PD-1 / PD-L1 interactions was assessed. First, as confirmed in UniProt, the amino acid sequence similarity between human PD-L1 and canine PD-L1 was 76.04%. Binding affinity and binding capacity to human PD-L1 were determined by direct ELISA. Functional analysis targeting PD-1 / PD-L1 inhibition showed that these antibodies effectively blocked PD-1 / PD-L1 interactions. Therefore, these candidate antibodies show the potential to develop novel therapeutic antibodies that target canine PD-1 / PD-L1 interaction.

[0028] In this invention, the scFv form of the top six candidates with high binding affinity to canine PD-L1 antigen is converted into the IgG form.

[0029] Furthermore, the efficacy of candidate antibodies converted to IgG form was evaluated to determine whether they retained their functional activity against the target antigen. All candidate antibodies in IgG form exhibited sustained binding affinity for canine PD-L1. Cell-based in vitro analyses were performed using canine-derived cells known to express PD-L1. Results showed that some candidate antibodies retained binding affinity for canine PD-L1 on the cell surface. Additionally, tumor xenograft experiments in mice showed a significant reduction in tumor growth, suggesting that the selected canine PD-L1 antibodies exert similar functional activity in vivo.

[0030] The heavy chain variable region (V) of the six canine PDL-1 antibodies according to the present invention H ) and light chain variable region (V L The amino acid and nucleotide sequences of are shown in Tables 1 and 14.

[0031] In addition, the heavy chain variable region (V) of the six selected antibodies H ) and light chain variable region (V L The Kabat sequences are shown in Tables 2 to 13.

[0032] [Table 1] The amino acid sequences of VH and VL of the six canine PD-L1 antibodies according to the present invention [Table 2] Kabat sequence of the heavy chain variable region (VH) of antibody #1 [Table 3] Kabat sequence of the light chain variable region (VL) of antibody #1 [Table 4] Kabat sequence of the heavy chain variable region (VH) of antibody #2 [Table 5] Kabat sequence of the light chain variable region (VL) of antibody #2 [Table 6] Kabat sequence of the heavy chain variable region (VH) of antibody #3 [Table 7] Kabat sequence of the light chain variable region (VL) of antibody #3 [Table 8] Kabat sequence of the heavy chain variable region (VH) of antibody #4 [Table 9] Kabat sequence of the light chain variable region (VL) of antibody #4 [Table 10] Kabat sequence of the heavy chain variable region (VH) of antibody #5 [Table 11] Kabat sequence of the light chain variable region (VL) of antibody #5 [Table 12] Kabat sequence of the heavy chain variable region (VH) of antibody #6 [Table 13] Kabat sequence of the light chain variable region (VL) of antibody #6 [Table 14] The nucleotide sequences of VH and VL of the six canine PD-L1 antibodies according to the present invention Therefore, in one aspect, the present invention relates to a monoclonal antibody or an antigen-binding fragment thereof. The monoclonal antibody includes: a heavy chain variable region (V... H The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 14, HCDR2 shown in SEQ ID NO: 16, and HCDR3 shown in SEQ ID NO: 18; and The light chain variable region (VL) includes LCDR1 shown in SEQ ID NO: 21, LCDR2 shown in SEQ ID NO: 23 and LCDR3 shown in SEQ ID NO: 25.

[0033] In this invention, the heavy chain variable region (V) of the monoclonal antibody or its antigen-binding fragment H The amino acid sequence of the monoclonal antibody or its antigen-binding fragment can be represented by SEQ ID NO:1, and the light chain variable region (V) of the monoclonal antibody or its antigen-binding fragment is... L The amino acid sequence of ) can be represented by SEQ ID NO:2.

[0034] In this invention, the monoclonal antibody or its antigen-binding fragment may be selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, single-chain antibody, humanized antibody, chimeric antibody and biantibody, wherein the single-chain antibody may be scFv.

[0035] In this invention, the monoclonal antibody or its antigen-binding fragment can specifically bind to PDL-1, wherein the PDL-1 can be derived from dogs or humans.

[0036] In another aspect, the present invention relates to a monoclonal antibody or an antigen-binding fragment thereof. This monoclonal antibody includes a heavy chain variable region (V). H The heavy chain variable region includes HCDR1 shown in SEQ ID NO:28, SEQ ID NO:42, SEQ ID NO:56, SEQ ID NO:70 or SEQ ID NO:84; HCDR2 shown in SEQ ID NO:30, SEQ ID NO:44, SEQ ID NO:58, SEQ ID NO:72 or SEQ ID NO:86; and HCDR3 shown in SEQ ID NO:32, SEQ ID NO:46, SEQ ID NO:60, SEQ ID NO:74 or SEQ ID NO:88; and Light chain variable region (V LThe light chain variable region includes LCDR1 shown in SEQ ID NO:35, SEQ ID NO:49, SEQ ID NO:63, SEQ ID NO:77 or SEQ ID NO:91; LCDR2 shown in SEQ ID NO:37, SEQ ID NO:51, SEQ ID NO:65, SEQ ID NO:79 or SEQ ID NO:93; and LCDR3 shown in SEQ ID NO:39, SEQ ID NO:53, SEQ ID NO:67, SEQ ID NO:81 or SEQ ID NO:95.

[0037] In this invention, the monoclonal antibody or its antigen-binding fragment may be selected from the group consisting of: (i) A monoclonal antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region (V) having the amino acid sequence of SEQ ID NO:3. H ) and the light chain variable region (V) having the amino acid sequence of SEQ ID NO:4 L ); (ii) A monoclonal antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region (V) having the amino acid sequence of SEQ ID NO:5. H ) and the light chain variable region (V) having the amino acid sequence of SEQ ID NO:6 L ); (iii) A monoclonal antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region (V) having the amino acid sequence of SEQ ID NO:7. H ) and the light chain variable region (V) having the amino acid sequence of SEQ ID NO:8 L ); (iv) A monoclonal antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region (V) having the amino acid sequence of SEQ ID NO:9. H ) and the light chain variable region (V) having the amino acid sequence of SEQ ID NO:10 L );as well as (v) A monoclonal antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region (V) having the amino acid sequence of SEQ ID NO:11. H ) and the light chain variable region (V) having the amino acid sequence of SEQ ID NO:12. L ).

[0038] On the other hand, the present invention relates to an isolated nucleic acid molecule comprising a heavy chain variable region (V0) encoding the monoclonal antibody or its antigen-binding fragment. H The nucleotide sequence of ).

[0039] In this invention, the monoclonal antibody or its antigen-binding fragment may be selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, single-chain antibody, humanized antibody, chimeric antibody and biantibody, wherein the single-chain antibody may be scFv.

[0040] In this invention, the monoclonal antibody or its antigen-binding fragment can specifically bind to PDL-1, wherein PDL-1 can be derived from dogs or humans.

[0041] On the other hand, the present invention relates to an isolated nucleic acid molecule comprising a heavy chain variable region (V0) encoding the monoclonal antibody or its antigen-binding fragment. H The nucleotide sequence of ).

[0042] In this invention, the isolated nucleic acid molecule may include a nucleotide sequence selected from the group consisting of SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105 and SEQ ID NO:107.

[0043] According to another approach, an isolated nucleic acid molecule is provided, comprising a light chain variable region (V0) encoding the monoclonal antibody or its antigen-binding fragment. L The nucleotide sequence of ).

[0044] In this invention, the isolated nucleic acid molecule may include a nucleotide sequence selected from the group consisting of SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106 and SEQ ID NO:108.

[0045] In another respect, the present invention relates to a carrier containing the isolated nucleic acid molecule.

[0046] In another respect, the present invention relates to a host cell comprising the isolated nucleic acid molecule or vector.

[0047] In another aspect, the present invention relates to a pharmaceutical composition for treating tumors, the pharmaceutical composition comprising the monoclonal antibody or its antigen-binding fragment, and a pharmaceutically acceptable load or excipient.

[0048] In this invention, the tumor may be osteosarcoma, melanoma, kidney cancer, prostate cancer, bladder cancer, colorectal cancer, stomach cancer, liver cancer, non-small cell lung cancer, breast cancer, esophageal cancer, pancreatic cancer, glioma, ovarian cancer, or leukemia, but is not limited thereto.

[0049] In this invention, the tumor can be a canine tumor or a human tumor.

[0050] In another aspect, the present invention relates to a method for treating or preventing tumors in dogs, comprising administering the monoclonal antibody or its antigen-binding fragment, or the pharmaceutical composition for treating tumors, to a dog suffering from a tumor.

[0051] In this invention, the tumor may be osteosarcoma, melanoma, kidney cancer, prostate cancer, bladder cancer, colorectal cancer, stomach cancer, liver cancer, non-small cell lung cancer, breast cancer, esophageal cancer, pancreatic cancer, glioma, ovarian cancer, or leukemia, but is not limited thereto.

[0052] In another aspect, the present invention relates to a method for treating or preventing tumors, comprising administering the monoclonal antibody or an antigen-binding fragment thereof, or a pharmaceutical composition for treating tumors.

[0053] In another aspect, the present invention relates to the use of the monoclonal antibody or its antigen-binding fragment or a pharmaceutical composition for treating tumors for the treatment or prevention of tumors.

[0054] In another aspect, the present invention relates to the use of the monoclonal antibody or its antigen-binding fragment or the pharmaceutical composition for treating tumors in the preparation of a medicament for treating or preventing tumors.

[0055] As used herein, the term "PDL-1 protein" encompasses all such sequences, including the sequences described above and their natural or artificial variants. Furthermore, when referring to a sequence fragment of the PDL-1 protein, it refers not only to the stated sequence fragment but also to the corresponding sequence fragment of its natural or artificial variant.

[0056] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each consisting of a light (L) chain and a heavy (H) chain. The antibody light chain can be classified as either a κ (kappa) or λ (lambda) chain. The heavy chain can be classified as μ, δ, γ, α, or ε, with the corresponding antibodies defined as IgM, IgD, IgG, IgA, and IgE, respectively. In both the light and heavy chains, the variable and constant regions are linked by "J" regions consisting of approximately 12 or more amino acids, and the heavy chain also includes "D" regions consisting of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of a single domain (CL). The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, such as various immune system cells (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further divided into hypervariable regions (called "complementarity-determining regions" (CDRs)) and conserved regions (called "frameworks" (FRs)), with the CDRs housed within the framework. Each VH and VL region consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions (VH and VL) of the heavy and light chains form antigen-binding sites. The amino acid assignment of each domain is determined according to Kabat's Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991) or according to the definitions of Chothia & Lesk (J.Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989).

[0057] The term "antibody" is not limited to any particular method of antibody production. For example, antibodies include recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes or subisotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0058] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide comprising a portion of a full-length antibody that retains the ability to specifically bind the same antigen as the full-length antibody and / or competes with the full-length antibody for antigen-specific binding; this is referred to as the “antigen-binding moiety” of the antibody. As described in Fundamental Immunology, Ch. 7 (Paul, W., ed., second edition, Raven Press, NY, 1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of a complete antibody. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibody fragments (e.g., scFv), chimeric antibodies, biantibodies, and polypeptides comprising at least a portion of a polypeptide sufficient to confer antigen-specific binding activity to the antibody.

[0059] As used herein, the term “Fd fragment” refers to an antibody fragment consisting of the VH and CH1 domains; the term “Fv fragment” refers to an antibody fragment consisting of the VL and VH domains of an antibody; the term “dAb fragment” refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term “Fab fragment” refers to an antibody fragment consisting of the VL, VH, CL, and CH1 domains; and the term “F(ab')2 fragment” refers to an antibody fragment comprising two Fab fragments linked by disulfide bonds in a hinge region.

[0060] In some cases, the antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv), where the VL and VH domains are linked by a linker to form a monovalent molecule comprising a single polypeptide chain (e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such scFv molecules can have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable linker sequences may include repeating GGGGS amino acid sequences or variants thereof. For example, (GGGGS)4 can be used, and variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other connectors that can be used in this invention are disclosed in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001) Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol.

[0061] In some cases, the antigen-binding fragment of an antibody can be a biantibody, i.e., a bivalent antibody expressing VH and VL on a single polypeptide chain, wherein a very short linker is used to prevent the pairing of two domains from the same chain, thereby forcing the domain to pair with a complementary domain of another chain to form two antigen-binding sites (e.g., Holliger P. et al.). Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0062] In other cases, the antigen-binding fragment of an antibody can be a "bispecific antibody," defined as a first antibody (fragment) and a second antibody (fragment) or antibody mimic conjugated via a conjugation arm. Non-limiting examples of conjugation methods include chemical reactions, gene fusions, and enzymatic reactions. The antigen-binding fragment can also be a "multispecific antibody," such as a trispecific antibody or a tetraspecific antibody, wherein the former can specifically bind three antigens, and the latter can specifically bind four antigens. For example, a designed ankyrin repeat protein (DARPin) can be linked or combined with an IgG antibody or scFv-Fc antibody fragment, as disclosed in CN104341529A; and an anti-IL-17A fynomer can be linked or combined with an anti-IL-6R antibody, as disclosed in WO2015141862A1.

[0063] In another case, the antigen-binding fragment of an antibody can be a "bispecific antibody conjugate," defined as a conjugate in which a first antibody (fragment) and a second biologically functional fragment (neither an antibody nor an antibody mimic) are coupled via a conjugation arm. Non-limiting examples of conjugation methods include chemical reactions, gene fusions, and enzymatic reactions. Examples of the second biologically functional fragment include peptides, proteins, polyethylene glycol (PEG), radionuclides, nucleic acids, small molecule toxins, receptors, or ligands with binding activity. Because the conjugate retains the activity of each fragment, it exhibits dual function or bispecificity.

[0064] The antigen-binding fragments of the present invention (e.g., the antibody fragments described above) can be obtained from the corresponding antibodies (in this invention, for example, 5C10, 5C10H1L1, 5C10H1L2, 5C10H2L1, and 5C10H2L2) using conventional techniques known to those skilled in the art (such as recombinant DNA technology or enzymatic or chemical cleavage methods). The same specificity screening methods can also be applied to the same antigen-binding fragments as the intact antibodies.

[0065] Unless explicitly stated otherwise, as used herein, the term "antibody" includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0066] As used herein, the terms “mAb” and “monoclonal antibody” refer to antibodies or antibody-derived fragments from a highly homologous population, wherein the population consists of identical antibody molecules unless a naturally occurring mutation is present. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies differ from monoclonal antibodies in that they typically comprise two or more different antibodies that recognize multiple epitopes on the same antigen. Monoclonal antibodies can generally be obtained using hybridoma technology first described by Kohler et al. (Nature, 256:495, 1975) and can also be obtained using recombinant DNA technology (US Patent No. 4,816,567).

[0067] As used herein, the term "chimeric antibody" refers to an antibody comprising a portion of the light and / or heavy chain of one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass) and another portion of the light and / or heavy chain of another antibody (which may be derived from the same or different species or belong to the same or different antibody classes or subclasses), while retaining binding activity to a target antigen (US Patent Nos. 4,816,567, Cabilly et al.; Morrison et al.). Proc. Natl. Acad. Sci. USA , 81:6851-6855 (1984)).

[0068] As used herein, the term "vector" refers to a nucleic acid vector into which a polynucleotide can be inserted. An expression vector is a vector capable of expressing a protein encoded by the inserted polynucleotide. Vectors can be introduced into host cells through transformation, transduction, or transfection, thereby enabling the expression of genetic elements delivered to the host cells. Vectors are well known to those skilled in the art, and non-limiting examples include plasmids, phages, granules, artificial chromosomes (e.g., yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC)), bacteriophages (e.g., λ phage or M13 phage), and animal viruses. Non-limiting examples of animal viruses that can be used as vectors include retroviruses (e.g., lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and multivacuolar papillomaviruses (e.g., SV40). Vectors may include various expression control elements, and non-limiting examples include promoter sequences, transcription initiation sequences, enhancer sequences, selection markers, and reporter genes. Furthermore, vectors may include origins of replication.

[0069] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, and non-limiting examples include: prokaryotic cells, such as *Escherichia coli* (E. coli). Escherichia coli ) or Bacillus subtilis ( Bacillus subtilis ); fungal cells, such as yeast cells or Aspergillus ( Aspergillus Insect cells, such as Drosophila S2 or Sf9 cells; or animal cells, such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0070] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the interaction between an antibody and its target antigen. In some embodiments, specific binding of an antibody to an antigen (or antigen-specific antibody) means that the binding affinity (KD) of the antibody to the antigen is less than 10. -5 M, for example, less than 10 -6 M, less than 10 -7 M, less than 10 -8 M, less than 10 -9 M, less than 10 -10 M or even lower.

[0071] As used herein, the term "pharmaceutically acceptable loading and / or excipient" means a loading and / or excipient known in the art and suitable for use with subjects and active ingredients in pharmaceutical and physiological fields (e.g., Remington's Pharmaceutical Sciences, Edited by Gennaro AR, 19th ed., Pennsylvania: Mack Publishing Company, 1995). Non-limiting examples include pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, non-limiting examples of pH adjusters include phosphate-buffered saline (PBS); non-limiting examples of surfactants include cationic, anionic, or nonionic surfactants such as Tween-80; and non-limiting examples of ionic strength enhancers include sodium chloride.

[0072] As used herein, the term "adjuvant" refers to a nonspecific immunostimulant that enhances or alters the type of immune response to an antigen before or when administered to a subject in combination with an antigen. Various types of adjuvants are known, and non-limiting examples include aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), and Corynebacterium pumilus (…). Corynebacterium parvum ), lipopolysaccharides, and cytokines. Freund's adjuvant is one of the most commonly used adjuvants in animal experiments, while aluminum hydroxide adjuvant is more frequently used in clinical studies.

[0073] As used herein, the term "effective amount" means an amount sufficient to achieve or at least partially achieve the desired effect. For example, an effective amount for preventing a disease such as a tumor means an amount sufficient to prevent, suppress, or delay the onset of a disease (such as a tumor). An effective amount for treating a disease means an amount sufficient to cure or at least suppress the disease and its complications in a subject suffering from the disease. Such effective amounts are determined by those skilled in the art. For example, an effective amount for treating a disease can be determined based on factors such as the severity of the disease, the patient's overall immune system status, and the patient's general condition (including age, weight, and sex), as well as the method of drug delivery and other concurrent treatments.

[0074] [Example] The present invention will now be described in more detail with reference to the embodiments. However, it will be apparent to those skilled in the art that these embodiments are merely illustrative of the invention and should not be construed as limiting the scope of the invention.

[0075] The cell lines and experimental methods used in the following examples are described below.

[0076] Source of cell lines and methods of cell infection used The canine macrophage cell line DH82 and the canine osteosarcoma cell line D17 used in these embodiments were purchased from ATCC (Manassas, VA, USA). Cells were cultured in DMEM containing 4.5 g / L glucose, L-glutamine, and sodium pyruvate (Corning, USA), supplemented with 10% v / v fetal bovine serum (FBS) (Gibco, USA) and 1% v / v antibiotic-antifungal solution (Welgene, Korea). HEK-293F cells were cultured in FreeStyle™ 293 expression medium (Gibco, USA). FreeStyle™ 293-F cells (Gibco, USA) were transfected using FectoPRO transfection reagent (Polyplus, Ilkisch-Grafenstaden, France).

[0077] Phage display selection To screen phage display panning antibodies, canine antibody clones binding to canine PD-L1 were obtained using a synthetic antibody library (provided by Professor Hyun-Bo Shim, Ewha Womans University). This antibody library has a density of 7.6 × 10⁻⁶. 9 The diversity of scFvs was observed, and the scFvs were tagged with HA at the C-terminus. ER2738 cells (NEB, New England Biolabs, USA) and VCSM13 helper phages were used to generate phages displaying scFvs for each panning round. Library amplification and phage display panning were performed according to the protocol disclosed in Phage Display: A Laboratory Manual (ISBN 978-087969740-2).

[0078] Specifically, frozen library stock was thawed to obtain phages displaying scFv, which were then inoculated into Luria-Bertani (LB) medium (BD Biosciences, USA) and subsequently panned onto LB Agar Miller plates (BD Biosciences, USA) containing 1% D-glucose (Duchefa Biochemie, Netherlands) and 100 μg / ml carbenicillin (GoldBio, USA). After overnight incubation, cells containing phage DNA were inoculated into SB medium containing 1% w / v MOPS (Thermo Fisher Scientific, USA), 2% w / v yeast extract (Thermo Fisher Scientific), and 3% tryptone (Thermo Fisher Scientific) and incubated at 37°C for 2 hours. After incubation, the cells were transferred to fresh SB medium, and approximately 10 μg / ml of scFv was added to the cultured cells. 12 CFU's VCSM13 helper phage (Stratagene, San Diego, CA, USA).

[0079] Cells were incubated at 37°C and 210 rpm with shaking for 2 hours, and then an equal volume of fresh culture medium (final concentration: 100 μg / ml carbenicillin and 70 μg / ml kanamycin) was added to the entire culture. After overnight incubation, phages displaying scFv were harvested using PEG precipitation, and phages with PD-L1 binding antibodies were pan-selected using ImmunoTubes (Thermo Fisher Scientific) coated with canine-derived PD-L1 antigen protein.

[0080] To reduce non-specific binding of scFv antibodies, ImmunoTubes coated with the antigen were blocked using a blocking solution containing 0.1% PBS-T (PBS containing 0.1% Tween-20; iNtRON Biotechnology, Korea) and 5% w / v skim milk (BD Biosciences) or 5% w / v bovine serum albumin (BSA; HanLAB, Korea). The scFv phage samples were mixed with the antigen protein at room temperature for 2 hours. To obtain high-affinity phages, ImmunoTubes interacting with the phages were washed with 0.1% PBS-T, with the number of washing steps increasing with each panning round. After washing, phages binding to canine PD-L1 were eluted with 0.25% trypsin solution (Gibco). The eluted phages were then re-infected with ER2738 cells for amplification and used in the next panning round. A total of four panning rounds were performed to select PD-L1-binding scFv phages. The binding of PD-L1 antigen to selected scFv antibodies from phage-infected E. coli colonies obtained in the third and fourth rounds of panning was verified using ELISA.

[0081] scFv expression and TES periphery extraction Single colonies were randomly selected from the *E. coli* colonies obtained after the third and fourth rounds of panning and incubated overnight in 96-well plates containing 1 mL / well of LB medium supplemented with 100 μg / mL carbenicillin. The next day, aliquots of the cultures were diluted, inoculated into fresh LB medium, and cultured with shaking until the cell density reached approximately OD600 ≈ 0.6. IPTG (Duchefa Biochemie) was then added to a final concentration of 1 mM, followed by overnight incubation to induce scFv protein expression. The expressed scFv protein was extracted from the periplasmic space using TES buffer (20% w / v sucrose (Duchefa Biochemie), 50 mM Tris-HCl (iNtRON), and 1 mM EDTA, pH 8.0 (BIONEER, Korea)) via osmotic shock (DOI: 10.1186 / s13568-020-01063-x). scFv protein expressed in the periplasmic space was obtained by osmotic shock extraction using TES buffer, mixed with PD-L1 antigen protein, and then mixed with a second antibody conjugated to anti-HA peroxidase targeting an HA tag fused to the C-terminus of scFv (Sigma-Aldrich, #12013819001). The resulting mixture was used for enzyme-linked immunosorbent assay (ELISA) to evaluate the PD-L1 binding activity of each antibody. After confirming binding to the PD-L1 antigen protein, the scFv sample was incubated at 70°C for 10 min to determine the thermostability of the scFv antibody, followed by incubation on ice for 30 min. ELISA was then performed again to confirm binding to the PD-L1 antigen protein.

[0082] Antibodies in the form of IgG were expressed and purified using a dual-vector system. Sixteen unique antibodies with identified nucleotide sequences were amplified by PCR using a primer set capable of binding to each of the VH and VL domains and a KAPA HiFi HotStart PCR kit (Roche Sequencing, Indianapolis, USA). The VH and VL PCR products of each clone were digested with BamHI / NheI and BamHI / BsiWI restriction enzymes, respectively, and ligated to the corresponding restriction sites in the pCEP-VH and pCEP-VL vectors (doi: 10.3390 / v12060684) digested with the same restriction enzymes using T4 DNA ligase (NEB) to obtain heavy and light chain expression vectors capable of expressing the 16 unique antibodies in IgG form in mammalian cells. The VH fragment was fused to the CH1-hinge-CH2-CH3 region for heavy chain expression, and the VL fragment was fused to the Cκ region for light chain expression.

[0083] The resulting heavy and light chain expression vectors were co-transfected into FreeStyle™ 293-F cells using FectoPRO transfection reagent at a DNA ratio of 2:1 (light chain:heavy chain). Nine days later, the culture supernatant was harvested, and the antibody in IgG form was purified by column chromatography using protein A agarose beads (Repligen, USA). After purification and dialysis in DPBS at pH 7.5, antibody concentration was quantified using a spectrophotometer, and the purification level and purity were evaluated by SDS-polyacrylamide gel electrophoresis (PAGE) followed by Coomassie Brilliant Blue staining (Biosesang, Korea).

[0084] The binding and inhibitory activities of scFv and IgG were analyzed by ELISA. The binding and inhibitory activities of individual scFv and IgG antibodies were analyzed by ELISA. For the binding assay, 96-well ELISA plates were coated with 500 ng / mL canine PD-L1-hFc protein in PBS and incubated overnight at 4°C. The solution in the wells was discarded, and the plates were blocked with 3% (w / v) skim milk in 0.1% PBS-T at 37°C for 1 hour. Subsequently, the wells were treated with 100 μL / well of periplasmic extract containing scFv or 200 ng / well of purified IgG and incubated at room temperature for 2 hours. The plates were washed with tap water and then incubated in blocking buffer with horseradish peroxidase (HRP) conjugated anti-HA antibody (1:5,000; Roche, Basel, Switzerland) at 37°C for 45 minutes.

[0085] For the inhibition assay, canine PD-L1 was coated onto the ELISA plate under the same conditions as described above. The ELISA plate was treated with a mixture of periplasmic extract containing scFv (100 μL / well) and 100 ng / well of human Cκ-tagged human PD-1, incubated at room temperature for 2 hours, and washed with tap water. Then, HRP-conjugated anti-human Cκ antibody (Jackson Immuno Research, PA, USA) diluted in blocking buffer was added, followed by incubation under the same conditions. For the assay, 50 μL / well of 3,3',5,5'-tetramethylbenzidine (TMB; BD Biosciences) was added, and absorbance was measured at 450 nm using a BioTek Epoch microplate reader (Agilent, CA, USA).

[0086] Flow cytometry analysis The specific binding of purified IgG to canine PD-L1 expressed on the cell surface was analyzed using BD FACSVerse™ (BD Biosciences). 1 × 10⁻⁶ cells were used for each experiment. 5DH82 and D17 cells were collected. Cells were resuspended in cell dissociation buffer (Gibco) based on enzyme-free PBS. Cells were washed with FACS buffer containing TBS (iNtRON), 0.5% BSA (HanLAB), and 0.05% NaN3 (Acros Organics), and then resuspended in FACS buffer containing purified IgG (25 μg / mL) and incubated at 4°C for 1 hour. After washing with FACS buffer, each IgG clone was treated with either Alexa Fluor 488-conjugated anti-human Fc antibody (1:500; Jackson ImmunoResearch, West Grove, PA, USA) or Alexa Fluor 647-conjugated anti-human Fc antibody (1:500; Jackson ImmunoResearch) and incubated at 4°C for 1 hour. Finally, all samples were measured using a FACSVerse™ flow cytometer and analyzed using FlowJo software (FlowJo LLC, Oregon, USA).

[0087] Example 1: Identification of scFv antibodies that specifically bind to canine PD-L1 Single-stranded variable fragments (scFvs) binding to canine PD-L1 were screened using phage display technology. A diversity level of 7.6 × 10⁻⁶ was used. 9 Synthetic scFv library ( Figure 1a The diversity of the selected scFv phage candidates was evaluated by panning the obtained scFv phages. The titer obtained after the third round of panning was compared with that obtained after the third round of panning (4.3 × 10⁻⁶). 7 Compared to CFU, the infection titer measured after the fourth round of screening using ER2738 cells (New England Biolabs) increased to 3.5 × 10⁻⁶. 9 CFU indicates an increase in scFv antibodies binding to canine PD-L1 in the panning library. Figure 1b ).

[0088] The binding of each selected scFv clone from the panning process to canine PD-L1 was identified by ELISA. Thirty-five scFv clones with relatively high binding activity to canine PD-L1 (OD value ≥ 0.2) were obtained and are indicated by red arrows. Figure 1c The thermal stability of 35 selected clones was then evaluated. For this evaluation, the scFv clones were heat-treated at 70 °C to induce structural changes, followed by cooling to allow structural refolding.

[0089] ELISA results showed that the binding to canine PD-L1 was maintained even after heat treatment, demonstrating the thermostability of the scFv clone. Figure 1d ).

[0090] The DNA sequences of all 35 scFv clones were analyzed. As a result of the analysis, distinct sequences in the CDR region were identified for 19 scFv clones.

[0091] As a result, diverse scFv libraries using phage display technology were screened to generate 19 unique scFv clones that specifically target canine PD-L1 with high affinity and thermostability.

[0092] Example 2: Screening for scFv for inhibiting the binding of canine PD-L1 to human PD-1 In this embodiment, an assay system was established to evaluate the inhibitory activity of canine PD-L1 neutralizing antibody clones.

[0093] The amino acid sequence homology between canine PD-L1 and human PD-L1 was 76.04%. However, identical amino acid sequences were observed in several regions, such as... Figure 2a The area highlighted in purple is shown in the image. Specifically, the area indicated by the red box represents a key binding site for human PD-1, indicating that the binding sites for canine and human PD-L1 are similar. Figure 2a Therefore, ELISA was performed to verify the binding of candidate scFv to human PD-L1, and the results showed that specific scFvs could bind to human PD-L1. Figure 2b ).

[0094] In this invention, the function of scFv is evaluated based on the assumption that the desired antibody should bind to PD-L1 in a manner similar to that of human PD-1. In this embodiment, the potential of the HA-tagged canine PD-L1 scFv to interfere with the interaction between human Fc-fused canine PD-L1 and human Cκ-fused human PD-1 was evaluated. The inhibitory effect of scFv on the interaction between canine PD-L1 and human PD-1 was quantified using an HRP-conjugated anti-human Cκ secondary antibody that recognizes human PD-1. Figure 2c ).

[0095] During the analysis of competitive ELISA results, a decrease in OD450 was observed when scFv was introduced compared to samples treated with canine PD-L1 and human PD-1 proteins alone. These results strongly suggest that scFv effectively inhibits the interaction between canine PD-L1 and human PD-1. Figure 2d Based on the evaluation results of canine PD-L1 binding affinity, thermal stability and PD-1 / PD-L1 blocking activity, clones with excellent binding affinity to canine PD-L1 were preferentially selected and then converted into IgG form.

[0096] Example 3: Functional validation of IgG antibody derived from canine PD-L1 specific scFv Based on the results obtained in Examples 1 and 2, the six scFvs with the highest priority (#1~#6) were selected and cloned to convert them into IgG form.

[0097] scFv includes variable regions of the immunoglobulin heavy chain (VH) and light chain (VL) that bind via linkers. The VH and VL domains were cloned and inserted into an expression vector capable of forming human IgG using a dual-vector system. Figure 3a The expressed IgG antibody was confirmed to be correctly assembled via disulfide bonds into a structure consisting of two heavy chains and two light chains. This was verified by SDS-PAGE under both reducing and non-reducing conditions. The protein bands observed in all six samples indicated the presence of intact IgG molecules. Figure 3b ).

[0098] Because the IgG antibody converted from scFv may lose its binding activity against canine PD-L1, in vitro binding assays were performed using these IgG antibodies. The binding activity of the antibodies against both canine and human PD-L1 was evaluated. Anti-human PD-L1 antibodies KL-001 (KR102357951B1), which binds to both human and canine PD-L1, and atezolizumab (Genentech, US8217149) were used as positive controls. OD measurements at 450 nm showed stable binding affinity to canine PD-L1. Figure 3c ).

[0099] In addition, cell-based in vitro binding assays were performed using canine cell lines expressing canine PD-L1 (specifically DH82 macrophages and D17 canine osteosarcoma cells), followed by flow cytometry. To evaluate binding by detecting fluorescence signals, six IgG antibodies were co-incubated with DH82 and D17 cells. KL-001 was used as a positive control in this analysis.

[0100] As a result, strong binding signals were observed in all six samples in the DH82 cell line, while binding signals were observed in all clones except #3 and #5 in the D17 cell line. Figure 3d and Figure 3e ).

[0101] Therefore, the results of this embodiment demonstrate that the IgG antibody derived from scFv exhibits significant functional binding to canine PD-L1. Based on binding affinity and sequence specificity, two antibodies, #1 and #6, were selected as candidate antibodies for further analysis.

[0102] Example 4: Selection of αPD-L1 antibody candidates with anti-tumor activity against canine osteosarcoma In previous studies by the inventors, novel αPD-L1 antibody candidates capable of binding to canine and human PD-L1 were developed. After confirming the affinity of these antibodies for canine PD-L1 in vitro, further studies were conducted to evaluate their in vivo efficacy. This was achieved using D17 cells (4 × 10⁻⁶ cells). 6 A canine osteosarcoma mouse model was established by xenografting (one cell / mouse) (a canine osteosarcoma cell line) into NOG mice (KOATECH, South Korea) that lack T cells, B cells, and NK cells.

[0103] In a mouse model, ten days after tumor xenograft, to evaluate the antitumor efficacy of the antibody, mice that had been administered 8×10⁸ g of the drug were... 6 NOG mice with D17 tumors on canine PBMCs were intravenously administered antibodies #1 or #6. Mice treated with antibodies #1 or #6 showed reduced tumor size compared to the control group that received only PBMCs. Figure 4 A). Specifically, mice treated with PBMCs of antibody #1 had the smallest tumor volume in the experimental group. Furthermore, tumor weight measurements taken on the day of sacrifice showed that the PBMC-treated group with antibody #1 had the least tumor growth compared to other groups. Figure 4 B). These results indicate that antibodies #1 and #6 bind to canine PD-L1 and exert anti-tumor effects.

[0104] Sequence List Free Text Please attach the electronic file.

Claims

1. A monoclonal antibody or its antigen-binding fragment, in, The monoclonal antibody includes: Heavy chain variable region (V H The heavy chain variable region includes HCDR1 containing the sequence of SEQ ID NO: 14, HCDR2 containing the sequence of SEQ ID NO: 16, and HCDR3 containing the sequence of SEQ ID NO: 18; and Light chain variable region (V L The light chain variable region includes LCDR1 containing the sequence of SEQ ID NO: 21, LCDR2 containing the sequence of SEQ ID NO: 23, and LCDR3 containing the sequence of SEQ ID NO:

25.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, wherein, The heavy chain variable region (V) of the monoclonal antibody or its antigen-binding fragment H The amino acid sequence of ) includes the sequence of SEQ ID NO: 1, and The light chain variable region (V) of the monoclonal antibody or its antigen-binding fragment L The amino acid sequence of the compound includes the sequence of SEQ ID NO:

2.

3. The monoclonal antibody or its antigen-binding fragment according to claim 1, wherein, The monoclonal antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, single-chain antibody, humanized antibody, chimeric antibody and biantibody.

4. The monoclonal antibody or its antigen-binding fragment according to claim 1, wherein, The single-chain antibody is scFv.

5. The monoclonal antibody or its antigen-binding fragment according to claim 1, wherein, The monoclonal antibody or its antigen-binding fragment specifically binds to PDL-1.

6. The monoclonal antibody or its antigen-binding fragment according to claim 5, wherein, The PDL-1 is derived from dogs or humans.

7. A monoclonal antibody or its antigen-binding fragment, in, The monoclonal antibody includes: Heavy chain variable region (V H The heavy chain variable region includes: HCDR1 containing a sequence of SEQ ID NO: 28, SEQ ID NO: 42, SEQ ID NO: 56, SEQ ID NO: 70, or SEQ ID NO: 84; HCDR2 containing a sequence of SEQ ID NO: 30, SEQ ID NO: 44, SEQ ID NO: 58, SEQ ID NO: 72, or SEQ ID NO: 86; and HCDR3 containing a sequence of SEQ ID NO: 32, SEQ ID NO: 46, SEQ ID NO: 60, SEQ ID NO: 74, or SEQ ID NO: 88; and Light chain variable region (V L The light chain variable region includes: LCDR1 containing a sequence of SEQ ID NO:35, SEQ ID NO:49, SEQ ID NO:63, SEQ ID NO:77 or SEQ ID NO:91; LCDR2 containing a sequence of SEQ ID NO:37, SEQ ID NO:51, SEQ ID NO:65, SEQ ID NO:79 or SEQ ID NO:93; and LCDR3 containing a sequence of SEQ ID NO:39, SEQ ID NO:53, SEQ ID NO:67, SEQ ID NO:81 or SEQ ID NO:

95.

8. The monoclonal antibody or its antigen-binding fragment according to claim 7, wherein, The monoclonal antibody or its antigen-binding fragment is selected from the group consisting of: (i) A monoclonal antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO:

3. H ); and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO:

4. L ); (ii) A monoclonal antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO:

5. H ); and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO:

6. L ); (iii) A monoclonal antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO:

7. H ); and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO:

8. L ); (iv) A monoclonal antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO:

9. H ); and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO:

10. L );as well as (v) A monoclonal antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO:

11. H ); and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO:

12. L ).

9. The monoclonal antibody or its antigen-binding fragment according to claim 7, wherein, The monoclonal antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, single-chain antibody, humanized antibody, chimeric antibody and biantibody.

10. The monoclonal antibody or its antigen-binding fragment according to claim 7, wherein, The single-chain antibody is scFv.

11. The monoclonal antibody or its antigen-binding fragment according to claim 7, wherein, The monoclonal antibody or its antigen-binding fragment specifically binds to PDL-1.

12. The monoclonal antibody or its antigen-binding fragment according to claim 11, wherein, The PDL-1 is derived from dogs or humans.

13. An isolated nucleic acid molecule comprising a heavy chain variable region (V0) encoding a monoclonal antibody or an antigen-binding fragment thereof according to claim 1. H The nucleotide sequence of ).

14. The isolated nucleic acid molecule according to claim 13, wherein, The nucleic acid molecule includes the nucleotide sequence of SEQ ID NO:

97.

15. An isolated nucleic acid molecule, said nucleic acid molecule comprising a light chain variable region (V0) encoding a monoclonal antibody or an antigen-binding fragment thereof according to claim 1. L The nucleotide sequence of ).

16. The isolated nucleic acid molecule according to claim 15, wherein, The nucleic acid molecule includes the nucleotide sequence of SEQ ID NO:

98.

17. An isolated nucleic acid molecule, said nucleic acid molecule comprising a heavy chain variable region (V0) encoding a monoclonal antibody or an antigen-binding fragment thereof according to claim 7. H The nucleotide sequence of ).

18. The isolated nucleic acid molecule according to claim 17, wherein, The nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 99, 101, 103, 105 and 107.

19. An isolated nucleic acid molecule, said nucleic acid molecule comprising a light chain variable region (V0) encoding a monoclonal antibody or an antigen-binding fragment thereof according to claim 7. L The nucleotide sequence of ).

20. The isolated nucleic acid molecule according to claim 19, wherein, The isolated nucleic acid molecules comprise nucleotide sequences selected from the group consisting of SEQ ID NO: 100, 102, 104, 106, and 108.

21. A vector comprising the isolated nucleic acid molecule according to any one of claims 13 to 20.

22. A host cell comprising an isolated nucleic acid molecule according to any one of claims 13 to 20 or a vector according to claim 21.

23. A pharmaceutical composition for treating tumors, said pharmaceutical composition comprising: The monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 12; and Pharmaceutically acceptable loads or excipients.

24. The pharmaceutical composition according to claim 23, wherein, The tumor is osteosarcoma, melanoma, kidney cancer, prostate cancer, bladder cancer, colorectal cancer, stomach cancer, liver cancer, non-small cell lung cancer, breast cancer, esophageal cancer, pancreatic cancer, glioma, ovarian cancer, or leukemia.

25. The pharmaceutical composition according to claim 23, wherein, The tumor originated from a dog or a human.

26. A method of treating or preventing tumors in dogs or animals, comprising administering to the dog or animal suffering from the tumor a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12, or a pharmaceutical composition for treating tumors according to claim 23.

27. The method according to claim 26, wherein, The tumors are selected from the group consisting of: osteosarcoma, melanoma, kidney cancer, prostate cancer, bladder cancer, colorectal cancer, stomach cancer, liver cancer, non-small cell lung cancer, breast cancer, esophageal cancer, pancreatic cancer, glioma, ovarian cancer, and leukemia.

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