Canine PDL-1 antibody and use thereof

Abstract

The present invention relates to a monoclonal antibody specific to canine PDL-1 or an antigen-binding fragment thereof, a pharmaceutical composition comprising same, and a use thereof. When using a novel canine PDL-1 antibody according to the present invention having high specificity for canine PDL-1 and having a tumor inhibitory effect in a canine osteosarcoma mouse model, a high therapeutic effect can be expected for canine cancers for which conventional immunotherapy has not been able to be performed.

Description

Canine PDL-1 antibody and uses thereof

[0001] The present invention relates to a monoclonal antibody or antigen-binding fragment thereof specific for PDL-1, and more particularly, to a monoclonal antibody or antigen-binding fragment thereof specific for canine PDL-1, a pharmaceutical composition comprising the same, and a use thereof.

[0002]

[0003] Cancer is a common disease in dogs, just as it is in humans, and can manifest in various types depending on factors such as personality and gender. Common tumors in dogs include histiocytomas, lipomas, adenomas, and mammary cell tumors. Current treatment methods for these canine cancers include surgical removal of tumor tissue, radiation therapy, and chemotherapy.

[0004] The regulation of our body's immune system involves complex immune checkpoint mechanisms that regulate T lymphocyte function, antigen recognition, and the balance of stimulatory and inhibitory signals. Detection of tumor-specific new antigens induced by tumor cell mutations helps eliminate tumors and viral sources. However, some tumor cells evade immune attack by modulating the tumor microenvironment to induce immune tolerance or by evading immune cell attacks through immunoediting. Alterations in the binding of immune checkpoint inhibitory ligands / receptors significantly influence tumor-specific T lymphocyte activity. Monoclonal antibodies that inhibit PD-1 or PD-L1 ligand function can enhance tumor-specific T lymphocyte activity and have been shown to have anticancer effects, significantly changing cancer treatment (Curiel TJ et al, Nature Medicine, 9: 562, 2003).

[0005] Programmed death-ligand 1 (PD-L1) has a unique structural feature consisting of two Ig-like domains in the extracellular domain, a transmembrane domain, and a short cytoplasmic domain without known signaling motifs. PD-L1, encoded by the 'CD274' gene, 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 receptor expressed on various immune cells, and influences T-cell immune activation. PD-L1 mediates immune protection against CTL apoptosis and regulates chronic immune responses through upregulation that interferes with immune protective pathways. PD-L1 also interacts with B7.1, potentially affecting immunoregulation, particularly immune evasion by tumor cells.

[0006] Despite the proven efficacy of immune checkpoint inhibitors in cancer treatment in human oncology, their use has not been extensively studied in veterinary medicine. Consequently, the lack of canine-specific therapeutic antibodies poses a significant limitation in the application of immunotherapy strategies for canine cancer treatment.

[0007] Several studies have confirmed that PD-L1 expression is increased in dogs with tumors (Maekawa Net et al, NPJ Precision Oncoloy, 5: 10, 2021). Furthermore, blocking the PD-1 / PD-L1 interaction improves T-cell function in dogs with cancer (Choi JW et al, PLoS One, 15: e0235518, 2020). None of the antibodies developed to date have been approved for canine cancer treatment.

[0008] Accordingly, the present inventors selected a specific scFv (single-chain variable fragment) for canine PD-L1 from a synthetic scFv library using phage display technology for use in the treatment of canine cancer using an immune checkpoint inhibitor, developed an IgG type antibody by fusing the selected scFv to a human IgG1 constant region sequence, and confirmed the binding affinity and anticancer performance of the developed canine PD-L1 binding antibodies in in vitro and in vivo models, thereby completing the present invention.

[0009]

[0010] Summary of the invention

[0011] The purpose of the present invention is to provide an antibody to canine PDL-1, a pharmaceutical composition containing the same, and a method for treating a tumor using the same.

[0012] In order to achieve the above purpose, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof,

[0013] The monoclonal antibody comprises a heavy chain variable region (VH) comprising HCDR1 as set forth in SEQ ID NO: 14, HCDR2 as set forth in SEQ ID NO: 16, and HCDR3 as set forth in SEQ ID NO: 18; and

[0014] A monoclonal antibody or antigen-binding fragment thereof is provided, having a light chain variable region (VL) comprising LCDR1 as set forth in SEQ ID NO: 21, LCDR2 as set forth in SEQ ID NO: 23, and LCDR3 as set forth in SEQ ID NO: 25.

[0015] The present invention also provides a monoclonal antibody or an antigen-binding fragment thereof,

[0016] The monoclonal antibody comprises a heavy chain variable region (VH) comprising HCDR1 as set forth in SEQ ID NO: 28, SEQ ID NO: 42, SEQ ID NO: 56, SEQ ID NO: 70 or SEQ ID NO: 84, HCDR2 as set forth in SEQ ID NO: 30, SEQ ID NO: 44, SEQ ID NO: 58, SEQ ID NO: 72 or SEQ ID NO: 86 and HCDR3 as set forth in SEQ ID NO: 32, SEQ ID NO: 46, SEQ ID NO: 60, SEQ ID NO: 74 or SEQ ID NO: 88; and

[0017] A monoclonal antibody or antigen-binding fragment thereof is provided, having a light chain variable region (VL) comprising LCDR1 as set forth in SEQ ID NO: 35, SEQ ID NO: 49, SEQ ID NO: 63, SEQ ID NO: 77, or SEQ ID NO: 91, LCDR2 as set forth in SEQ ID NO: 37, SEQ ID NO: 51, SEQ ID NO: 65, SEQ ID NO: 79, or SEQ ID NO: 93, and LCDR3 as set forth in SEQ ID NO: 39, SEQ ID NO: 53, SEQ ID NO: 67, SEQ ID NO: 81, or SEQ ID NO: 95.

[0018] The present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain variable region (VH) of the monoclonal antibody or an antigen-binding fragment thereof.

[0019] The present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain variable region (VH) of the monoclonal antibody or an antigen-binding fragment thereof.

[0020] The present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a light chain variable region (VL) of the monoclonal antibody or an antigen-binding fragment thereof.

[0021] The present invention also provides a vector comprising the isolated nucleic acid molecule.

[0022] The present invention also provides a host cell comprising the isolated nucleic acid molecule or the vector.

[0023] The present invention also provides a pharmaceutical composition for treating a tumor, comprising the monoclonal antibody or an antigen-binding fragment thereof; and a pharmaceutically acceptable carrier or excipient.

[0024] The present invention also provides a method for treating or preventing a tumor in a canine animal, comprising administering to the canine animal having the tumor the monoclonal antibody or an antigen-binding fragment thereof or the pharmaceutical composition for treating the tumor.

[0025] The present invention also provides a method for treating or preventing a tumor, comprising administering the monoclonal antibody or antigen-binding fragment thereof or the pharmaceutical composition for treating a tumor.

[0026] The present invention also provides a use of the monoclonal antibody or antigen-binding fragment thereof or the pharmaceutical composition for treating a tumor for the treatment or prevention of a tumor.

[0027] The present invention also provides the use of the monoclonal antibody or antigen-binding fragment thereof or the pharmaceutical composition for treating a tumor for the manufacture of a medicament for treating or preventing a tumor.

[0028]

[0029] Figure 1 shows the results of a method for screening canine PD-L1-specific antibodies and discovering scFv clones for canine PD-L1. Figure 1a schematically shows the phage display method used in the present invention, and Figure 1b shows the results of titer for four rounds of repeated panning, and the titer was determined by the number of ER2738 colonies infected with the phage. Figure 1c shows the results of screening clones from the third and fourth panning output pools by binding analysis of scFv to select specific binders for canine PD-L1, and shows the results of confirming the binding activity of 2×protoplast scFv clones for canine PD-L1 by ELISA through TES protoplast extraction. Figure 1d shows the results of analyzing the thermal stability activity of 35 scFv clones selected by binding analysis, and the results are obtained by detecting the OD value at 450 nm using scFv heated at 70°C.

[0030] Figure 2 shows the results of the in vitro PD-1 / PD-L1 interaction inhibition assay and the verification of the functional activity of scFvs. Figure 2a compares the amino acid sequences of human PD-L1 and canine PD-L1, and the purple highlight indicates the region where the amino acid sequences of the two species are identical, and the red box shows the amino acid sequence of canine PD-L1 in the region known to be important for the binding of human PD-L1 to human PD-L1. Figure 2b shows the results of ELISA detection of the binding of 19 scFvs to human PD-L1, where NC represents a sample treated with only the secondary antibody. Figure 2c is a schematic diagram showing a method for confirming through competitive ELISA whether the 19 scFvs can inhibit the binding of human PD-1 to canine PD-L1. Figure 2d is a graph showing the ELISA results indicating the degree of inhibition of cPD-L1 scFv, where PC represents a sample showing the binding ability of canine PD-L1 protein and human PD-1 protein. NC represents a sample coated only with a secondary antibody to the dog PD-L1 protein.

[0031] Figure 3 shows the results of converting the selected scFv into an IgG form and the results of functional verification of the converted IgG. Figure 3a shows an overall schematic diagram of the IgG conversion method for scFv clones with independent sequences. The antibody heavy chain DNA and light chain DNA fragments were linked to a vector that can be expressed in FreeStyle™293-F cells. Then, the plasmid-type sample was infected into FreeStyle™293-F cells via a two-vector system. Figure 3b shows the results of purifying IgG from FreeStyle™293-F cells infected via the two-vector system, and electrophoresis of the purified IgG on an SDS-PAGE gel. Figure 3c shows the results of ELISA to confirm the binding affinity of anti-canine PD-L1 antibodies for canine PD-L1 and human PD-L1. KL-001 is a positive antibody that binds to both canine and human PD-L1, and atezolizumab is a positive antibody that binds to human PD-L1. Figures 3d and 3e show the results of measuring the binding activity of six antibodies by flow cytometry after treating canine PD-L1-expressing canine cell lines with KL-001 (red line), anti-canine PD-L1 antibody (blue line), and control antibody (secondary anti-human Fc Ab, black line). Figure 3d shows the results for DH82 cells, and Figure 3e shows the results for D17 cells. Flow cytometry was performed using Alexa 488-conjugated anti-human Fc or Alexa 647-conjugated anti-human Fc as a secondary fluorescent antibody.

[0032] Figure 4 shows the results of confirming the in vivo antitumor effect of the canine PD-L1 antibody in a canine osteosarcoma mouse model. Figure 4A shows that the mouse group was injected with D17 cells (4x) as a canine tumor cell line. 6 / mouse) were transplanted into the left flank and observed for 20 days. Ten days after tumor injection, canine PBMCs were intravenously injected (8x 6 / mouse). Antibody #1 or #6 was injected intraperitoneally at a concentration of 1 mg / ml. Tumor growth was measured every two days. *p<0.05, ***p<0.001, and ****p<0.0001 (one-way ANOVA; n=5 / group). Figure 4B shows the results of measuring tumor mass after euthanizing all mouse groups. *ns; not significant and *p<0.05 (one-way ANOVA; n=5 / group). These data are derived from three independent experiments.

[0033]

[0034] Detailed description of the invention and preferred embodiments

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0036] Immunotherapy has emerged as a groundbreaking approach in the treatment of human cancer, particularly targeting immune checkpoint molecules such as PD-1 and PD-L1. Despite its proven efficacy in human oncology, this innovative approach has been understudied in the veterinary field, leading to a lack of canine-specific therapeutic antibodies for the application of immunotherapy strategies to treat canine cancer.

[0037] In the present invention, we developed a therapeutic antibody against canine PD-L1. Phage display technology was used to isolate single-chain variable fragments (scFvs) that specifically bind to canine PD-L1 from a synthetic antibody library. Through phage display screening of the scFv library, more than 35 high-affinity clones were selected, exhibiting high ELISA signals, thermostability, and unique sequences in the complementarity determining regions (CDRs) of the heavy and light chains. Among these, 19 scFv antibodies were identified with independent CDR region sequences. To confirm the function of the 19 screened scFv antibodies, we investigated whether canine PD-L1 was related to human PD-1 / PD-L1. First, the amino acid sequence similarity between human and canine PD-L1 was 76.04%, as determined by UniProt. The binding affinity for human PD-L1 was verified and binding capacity was confirmed through direct ELISA analysis. Functional analysis of PD-1 / PD-L1 inhibition revealed that these candidates effectively blocked the PD-1 / PD-L1 interaction. Consequently, these candidates demonstrate the potential for developing novel therapeutic antibodies targeting the PD-1 / PD-L1 interaction.

[0038] In the present invention, the scFv format of the top six candidates with high binding affinity for the canine PD-L1 antigen was converted to an IgG format.

[0039] In addition, we evaluated whether the candidate substances converted to IgG format still retained their function against the target antigen. As a result, all candidates in IgG format showed consistent binding affinity for canine PD-L1. Cell-based in vitro assays were performed using canine cells known to express PD-L1, and the results confirmed the presence of candidate antibodies that still retained binding affinity for canine PD-L1 on the cell surface. Furthermore, a tumor xenograft experiment in mice showed a significant reduction in tumor growth, confirming that the selected canine PD-L1 antibody exerts a similar role in vivo.

[0040] Heavy chain variable region (V) of six types of canine PDL-1 antibodies according to the present invention H ) and light chain variable region (V L ) The amino acid sequence and base sequence are shown in Table 1 and Table 14.

[0041] In addition, the heavy chain variable region (V) of the six selected antibodies H ) and light chain variable region (V L ) are shown in Tables 2-13.

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] Therefore, in one aspect, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof,

[0068] The above monoclonal antibody comprises a heavy chain variable region (V) comprising HCDR1 as set forth in SEQ ID NO: 14, HCDR2 as set forth in SEQ ID NO: 16, and HCDR3 as set forth in SEQ ID NO: 18. H ); and

[0069] A light chain variable region (V) comprising LCDR1 as set forth in SEQ ID NO: 21, LCDR2 as set forth in SEQ ID NO: 23, and LCDR3 as set forth in SEQ ID NO: 25 L ) relates to a monoclonal antibody or an antigen-binding fragment thereof.

[0070] The heavy chain variable region (V) of the monoclonal antibody or antigen-binding fragment thereof of the present invention H ) is represented by SEQ ID NO: 1, and the light chain variable region (V) of the monoclonal antibody or antigen-binding fragment thereof L ) can be characterized by the amino acid sequence represented by sequence number 2.

[0071] In the present invention, the monoclonal antibody or antigen-binding fragment thereof may be selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, single-chain antibody, humanized antibody, chimeric antibody and diabody, and the single-chain antibody may be characterized as being scFv.

[0072] The monoclonal antibody or antigen-binding fragment thereof of the present invention may be characterized by specifically binding to PDL-1, and the PDL-1 may be characterized by being of canine or human origin.

[0073] In another aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof,

[0074] The monoclonal antibody comprises a heavy chain variable region (VH) comprising HCDR1 as set forth in SEQ ID NO: 28, SEQ ID NO: 42, SEQ ID NO: 56, SEQ ID NO: 70 or SEQ ID NO: 84, HCDR2 as set forth in SEQ ID NO: 30, SEQ ID NO: 44, SEQ ID NO: 58, SEQ ID NO: 72 or SEQ ID NO: 86 and HCDR3 as set forth in SEQ ID NO: 32, SEQ ID NO: 46, SEQ ID NO: 60, SEQ ID NO: 74 or SEQ ID NO: 88; and

[0075] A monoclonal antibody or antigen-binding fragment thereof having a light chain variable region (VL) comprising LCDR1 as set forth in SEQ ID NO: 35, SEQ ID NO: 49, SEQ ID NO: 63, SEQ ID NO: 77 or SEQ ID NO: 91, LCDR2 as set forth in SEQ ID NO: 37, SEQ ID NO: 51, SEQ ID NO: 65, SEQ ID NO: 79 or SEQ ID NO: 93 and LCDR3 as set forth in SEQ ID NO: 39, SEQ ID NO: 53, SEQ ID NO: 67, SEQ ID NO: 81 or SEQ ID NO: 95.

[0076] In the present invention, the monoclonal antibody or antigen-binding fragment thereof may be characterized by being selected from the following:

[0077] (i) a heavy chain variable region (V) having an amino acid sequence of sequence number 3; H ) and a light chain variable region (V) having the amino acid sequence of SEQ ID NO: 4 L ), a monoclonal antibody or antigen-binding fragment thereof;

[0078] (ii) a heavy chain variable region (V) having an amino acid sequence of sequence number 5 H ) and a light chain variable region (V) having the amino acid sequence of SEQ ID NO: 6 L ), a monoclonal antibody or antigen-binding fragment thereof;

[0079] (iii) a heavy chain variable region (V) having an amino acid sequence of sequence number 7 H ) and a light chain variable region (V) having the amino acid sequence of SEQ ID NO: 8 L ), a monoclonal antibody or antigen-binding fragment thereof;

[0080] (iv) a heavy chain variable region (V) having an amino acid sequence of sequence number 9 H ) and a light chain variable region (V) having an amino acid sequence of SEQ ID NO: 10 L ), a monoclonal antibody or an antigen-binding fragment thereof; and

[0081] (v) a heavy chain variable region (V) having an amino acid sequence of sequence number 11 H ) and a light chain variable region (V) having the amino acid sequence of SEQ ID NO: 12 L ) comprising a monoclonal antibody or antigen-binding fragment thereof.

[0082] The present invention also provides a heavy chain variable region (V) of the monoclonal antibody or antigen-binding fragment thereof H ) relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a nucleic acid.

[0083] In the present invention, the monoclonal antibody or antigen-binding fragment thereof may be selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, single-chain antibody, humanized antibody, chimeric antibody and diabody, and the single-chain antibody may be characterized as being scFv.

[0084] The monoclonal antibody or antigen-binding fragment thereof of the present invention may be characterized by specifically binding to PDL-1, and the PDL-1 may be characterized by being of canine or human origin.

[0085] In another aspect, the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain variable region (VH) of the monoclonal antibody or an antigen-binding fragment thereof.

[0086] In the present invention, the isolated nucleic acid molecule may be characterized by comprising a nucleotide sequence of any one 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.

[0087] In another aspect, the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a light chain variable region (VL) of the monoclonal antibody or an antigen-binding fragment thereof.

[0088] In the present invention, the isolated nucleic acid molecule may be characterized by comprising a nucleotide sequence of any one 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.

[0089] In another aspect, the present invention relates to a vector comprising the isolated nucleic acid molecule.

[0090] In another aspect, the present invention relates to a host cell comprising the isolated nucleic acid molecule or the vector.

[0091] In another aspect, the present invention relates to a pharmaceutical composition for treating a tumor, comprising the monoclonal antibody or an antigen-binding fragment thereof; and a pharmaceutically acceptable carrier or excipient.

[0092] In the present invention, the tumor may be, but is not limited to, osteosarcoma, melanoma, kidney cancer, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, non-small cell lung cancer, breast cancer, esophageal cancer, pancreatic cancer, glioma, ovarian cancer, or leukemia.

[0093] In the present invention, the tumor may be characterized as being a canine or human tumor.

[0094] In another aspect, the present invention relates to a method for treating or preventing a tumor in a canine animal, comprising administering to the canine animal having the tumor the monoclonal antibody or an antigen-binding fragment thereof or the pharmaceutical composition for treating the tumor.

[0095] In the present invention, the tumor may be, but is not limited to, osteosarcoma, melanoma, kidney cancer, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, non-small cell lung cancer, breast cancer, esophageal cancer, pancreatic cancer, glioma, ovarian cancer, or leukemia.

[0096] In another aspect, the present invention relates to a method for treating or preventing a tumor, comprising administering the monoclonal antibody or antigen-binding fragment thereof or the pharmaceutical composition for treating a tumor.

[0097] In another aspect, the present invention relates to the use of the monoclonal antibody or antigen-binding fragment thereof or the pharmaceutical composition for treating tumors for the treatment or prevention of tumors.

[0098] In another aspect, the present invention relates to the use of the monoclonal antibody or antigen-binding fragment thereof or the pharmaceutical composition for treating tumors for the manufacture of a medicament for treating or preventing tumors.

[0099] In the present invention, the term "PDL-1 protein" should encompass all sequences, including the aforementioned sequences and natural or artificial variants thereof. Furthermore, when referring to a sequence fragment of the PDL-1 protein, this refers not only to the aforementioned sequence fragments but also to corresponding sequence fragments of natural or artificial variants.

[0100] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair consisting of a "light" (L) chain and a "heavy" (H) chain. Antibody light chains can be classified as κ and λ chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and the corresponding antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 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 region of an antibody can mediate the binding of the immunoglobulin 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 highly variable regions (complementarity determining regions, CDRs) and conserved regions, referred to as the framework (FR), within which the CDRs are arranged. Each VH and VL is composed of three CDRs and four FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxy terminus. The variable regions (VH and VL) of the heavy and light chains form the antigen-binding site. The amino acid assignments for each domain are based on the Kabat sequences of proteins of immunological interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (J. Mol. Biol. 196:901-917(1987); Chothia et al.According to the definition in Nature 342:878-883(1989)).

[0101] The term "antibody" is not limited to any specific antibody production method. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies, among others. Antibodies may be of different isotypes or subisotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0102] In the present invention, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a full-length antibody fragment, which retains the ability to specifically bind to the same antigen as the full-length antibody and / or the ability to compete with the full-length antibody for antigen-specific binding, known as the "antigen-binding portion." Fundamental Immunology, Ch. 7 (Paul, W., ed., second edition, Raven Press, NY (1989)), which is incorporated herein by reference for all purposes. Antigen-binding fragments can be prepared by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. 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, diabodies, and polypeptides comprising at least a portion of a polypeptide sufficient to confer antigen-specific binding affinity to the antibody.

[0103] As used herein, the term "Fd fragment" refers to an antibody fragment consisting of VH and CH1 domains; the term "Fv fragment" refers to an antibody fragment consisting of single-chain VL and VH domains of an antibody; the term "dAb fragment" refers to an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" refers to an antibody fragment consisting of VL, VH, CL and CH1 domains; and the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bond within the hinge region.

[0104] In some cases, the antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv) in which the VL and VH domains are linked by a linker to form a single polypeptide chain (e.g., Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such scFv molecules may have the general structure: NH2-VL-Linker-VH-COOH or NH2-VH-Linker-VL- COOH. A suitable modern linker technology consists of a repeating GGGGS amino acid sequence or variants thereof. For example, (GGGGS)4 can be used, as can variants thereof (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448).

[0105] Other linkers that can be used in the present invention are described 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.

[0106] In some cases, the antigen-binding fragment of the antibody is a diabody, i.e. a bivalent antibody in which the VH and VL are expressed as a single polypeptide chain, in which a very short linker is used to prevent the two domains from pairing from the same chain, i.e., to force the domains to pair with complementary domains from the other chain, forming two antigen-binding sites (e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA90:6444-6448 (1993), and Poljak RJ et al., Structure2:1121-1123 (1994)).

[0107] In other cases, the antibody antigen-binding fragment is a "bispecific antibody" defined as a first antibody (fragment) and a second antibody (fragment) or antibody mimetic coupled by coupling arms, wherein coupling methods include, but are not limited to, chemical reactions, genetic fusions, and enzymatic reactions. The antibody antigen-binding fragment may be a "multispecific antibody" such as a trispecific antibody or a tetraspecific antibody, the former specifically binding to three antigens and the latter specifically binding to four antigens. For example, a designated ankyrin repeat protein (DARPin, designed ankyrin repeat protein) is linked to or combined with an IgG antibody, a scFv-Fc antibody fragment, as in CN104341529A; an anti-IL-17a fynomer is linked to or combined with an anti-IL-6R antibody, as in WO2015141862A1.

[0108] In another case, the antibody antigen binding fragment is defined as a "bispecific antibody conjugate" in which a first antibody (fragment) and a second biologically functional fragment (which is neither an antibody nor a mimetic thereof) are coupled by a coupling arm, wherein the coupling method includes, but is not limited to, a chemical reaction, a genetic fusion, and an enzymatic reaction, and the second biologically functional fragment is a peptide, protein, polyethylene glycol (PEG), a radionuclide, a nucleic acid, a small molecule toxin, a receptor, or a ligand having binding activity, and the conjugate retains the activity of each fragment and thus has dual functionality / bispecificity.

[0109] Antigen-binding fragments of the present invention (e.g., antibody fragments described above) can be obtained from the corresponding antibodies (e.g., 5C10, 5C10H1L1, 5C10H1L2, 5C10H2L1 and 5C10H2L2 in the present invention) by conventional techniques known to those skilled in the art (e.g., recombinant DNA or enzymatic or chemical cleavage methods), and the same specific screening methods can be applied to the antigen-binding fragments as intact antibodies.

[0110] In the present invention, unless explicitly stated, the term “antibody” includes not only a complete antibody but also an antigen-binding fragment of an antibody.

[0111] As used herein, the terms "mAb" and "monoclonal antibody" refer to a fragment derived from an antibody or antibody molecule of high homology, which group comprises a group of antibody molecules that are identical unless naturally occurring mutations occur. Monoclonal antibodies have a high degree of specificity for a single epitope on an antigen. Polyclonal antibodies differ from monoclonal antibodies in that polyclonal antibodies typically comprise two or more different antibodies that recognize different epitopes on the same antigen. Monoclonal antibodies are typically obtained through the hybridoma technique first described by Kohler et al. (Nature, 256:495, 1975), but can also be obtained through recombinant DNA techniques (U.S. Patent No. 4,816,567).

[0112] As used herein, the term "chimeric antibody" refers to an antibody composed of light and / or heavy chain portions of an antibody (which may be derived from a particular species or may belong to a particular antibody class or subclass) and light and / or heavy chain portions of another antibody (which may be derived from the same or a different species or may belong to the same or a different antibody class or subclass), but which still retains binding activity to a target antigen (U.S. Patent No. 4,816,567, Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).

[0113] In the present invention, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. An expression vector is a vector capable of expressing a protein encoded by an inserted polynucleotide. The vector can be introduced into a host cell by transformation, transformation, or transfection, and can express the genetic element transferred to the host cell. Vectors are well known to those skilled in the art, and non-limiting examples include plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); phages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (e.g., lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), and the like. Vectors can contain various expression-regulating elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Vectors can also contain an origin of replication.

[0114] In the present invention, the term "host cell" refers to a cell that can be used to introduce a vector, and non-limiting examples thereof include prokaryotic cells such as Escherichia coli or bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as Drosophila cells S2 or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NS0 cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0115] In the present invention, the term "specific binding" refers to a non-random binding reaction between two molecules, for example, a reaction between an antibody and its target antigen. In some embodiments, specific binding of an antibody to an antigen (or an antibody having specificity for an antigen) is defined herein as having a binding affinity (KD) for the antigen of 10 -5 Less than M, for example, 10 -6 Less than M, 10 -7 Less than M, 10 -8 Less than M, 10 -9 Less than M, 10 -10 Refers to antibodies with levels below M or even lower.

[0116] As used herein, the term "pharmaceutically acceptable vehicle and / or excipient" refers to a vehicle and / or excipient well known in the art for use with the recipient entity and active ingredient in the pharmaceutical and physiological fields (e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), including but not limited to pH adjusters, surfactants, adjuvants, ionic strength enhancers, and the like. For example, a pH adjuster includes but not limited to phosphate buffered saline; a surfactant includes but not limited to a cationic, anionic, or non-ionic surfactant such as Tween-80; and an ionic strength enhancer includes but not limited to sodium chloride.

[0117] As used herein, the term "adjuvant" refers to a non-specific immunostimulatory substance that, when delivered to the body prior to or together with an antigen, enhances or alters the type of immune response to an antigen. There are many types of adjuvants, including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharides, and cytokines. Freund's adjuvant is the most commonly used adjuvant in animal studies. Aluminum hydroxide adjuvant is much more commonly used in clinical trials.

[0118] As used herein, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. For example, an effective amount for preventing a disease such as a tumor refers herein to an amount sufficient to prevent, inhibit, or delay the onset of a disease such as a tumor. An effective amount for treating a disease refers herein to an amount sufficient to cure or at least inhibit a patient suffering from a disease and its complications. Determination of such an effective amount is within the ability of those skilled in the art, and for example, an effective amount for treating a disease will be determined based on the severity of the disease, the overall state of the patient's immune system, the patient's overall condition, such as age, weight, and sex, the drug delivery, and other concurrent treatments.

[0119]

[0120] [Example]

[0121] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0122] The cell lines and experimental methods used in the examples below are shown below.

[0123]

[0124] Source of cell lines used and method of cell line infection

[0125] The canine macrophage cell line DH82 and the canine osteosarcoma cell line D17 used in the examples of the present invention were purchased from ATCC, Manassas, Virginia, USA. The cells were cultured in DMEM containing 4.5 g / L glucose, L-glutamine, and sodium pyruvate (Corning, USA), 10% v / v fetal bovine serum, FBS (Gibco, USA), and 1% v / v antibiotic-antimycotic solution (Welzen, Korea). HEK-293F cells were cultured in FreeStyle™293 expression medium (Gibco, USA). FreeStyle™293-F cells (Gibco, USA) were infected using Fectopro infection reagent (Polyplus, Grafenstaden, France).

[0126]

[0127] Phage display panning

[0128] To conduct phage display panning antibody screening, we obtained canine antibody clones that bind to canine PD-L1 using a synthetic antibody library (provided by Professor Hyun-Bo Shim of Ewha Womans University). This antibody library had a diversity of 7.6 x 109, and the scFv was tagged with HA at the C-terminus. The scFv display phage for each panning round was produced using ER2738 cells (NEB, New England Biolabs, USA) and VCSM13 helper phage. Library re-amplification and phage display panning were performed according to the protocol described in the phage display manual book (Phage Display: A Laboratory Manual, ISBN 978-087969740-2).

[0129] In detail, scFv-displaying phages were obtained by thawing frozen library stocks, inoculated into Luria-Bertani (LB) medium (BD Biosciences, USA), and then panned onto LB Agar Miller (BD Biosciences, USA) plates containing 1% D-Glucose (Duchefa Biochemie, Netherlands) and 100 μg / ml Carbenicillin (GoldBio, USA) for panning. 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 h. After incubation, approximately 1012 CFU of VCSM13 helper phage (Strategene, San Diego, CA, USA) was added to the cultured cells, which were transferred to fresh SB medium.

[0130] After shaking culture at 37°C and 210 rpm for 2 h, an equal volume of fresh medium (final 100 μg / ml carbenicillin, 70 μg / ml kanamycin) was added to the entire growing medium. After overnight culture, scFv display phages were obtained using the PEG precipitation method, and then PD-L1-binding antibody phages were panned using ImmunoTubes (Thermo Fisher Scientific) coated with canine PD-L1 antigen protein.

[0131] To reduce nonspecific scFv antibody binding, the antigen-bound ImmunoTube was coated with a blocking solution containing 0.1% PBS-T (containing 0.1% Tween 20, iNtRON biotechnology, Korea) and 5% w / v nonfat milk (BD Biosciences) or 5% w / v bovine serum albumin (HanLAB, Korea), and the scFv phage sample was mixed with the antigen protein for 2 h at room temperature. To obtain high-affinity conjugates, the ImmunoTube that had interacted with the phage was washed with 0.1% PBS-T, and the number of washes increased as the panning round increased. After washing, the phage bound to canine PD-L1 was eluted by treating with 0.25% Trypsin (Gibco) solution, and the eluted phage was reinfected into ER2738 cells for amplification and used for the next round of panning. A total of four rounds of panning were performed to select PD-L1-binding scFv phages, and the PD-L1 antigen binding of the selected scFv antibodies was verified using an ELISA assay using phage-infected E. coli colonies obtained from the third and fourth rounds of panning.

[0132]

[0133] scFv expression and TES protoplast extraction

[0134] Among the E. coli colonies obtained from the third and fourth panning rounds, a single colony was randomly selected and cultured overnight in a 96-deep well plate containing 1 ml / well of LB medium (containing 100 µg / ml carbenicillin). The following day, a portion of the culture was diluted and inoculated into fresh LB medium, and the culture was shaken until the cell concentration reached OD600 ~ 0.6. IPTG (Duchefa Biochemie) was added to a final concentration of 1 mM and cultured overnight to induce scFv protein expression. The scFv protein expressed in the periplasmic space was extracted from cultured cells using an osmotic extraction method (DOI: 10.1186 / s13568-020-01063-x) using TES solution (20% w / v sucrose (Duchefa Biochemie), 50 nM Tris-HCl (iNtRON), 1 mM EDTA pH 8.0 (BIONEER, Korea)). The scFv expressed in the periplasmic space was obtained by the osmotic extraction method using TES solution, mixed with the PD-L1 antigen protein, and sequentially mixed with an anti-HA-peroxidase secondary antibody against the HA-tag fused to the C-terminus of the scFv (Sigma-Aldrich, # 12013819001), and used in an enzyme-linked immunosorbent assay (ELISA assay) to verify the PD-L1 antigen binding of individual antibodies. After verifying binding to the PD-L1 antigen protein, scFv samples were incubated at 70°C for 10 minutes to analyze the thermal stability of the scFv antibody, and then incubated on ice for 30 minutes, followed by ELISA analysis with the PD-L1 antigen protein.

[0135]

[0136] IgG antibody expression and IgG antibody purification process using two vector systems

[0137] The 16 unique antibodies whose base sequence information was confirmed were amplified by PCR using primer sets that can bind to individual VH and VL domains and the 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 then ligated into the corresponding restriction sites of the pCEP-VH vector and pCEP-VL vector (doi: 10.3390 / v12060684) digested with the same restriction enzymes using T4 DNA ligase (NEB). All heavy and light chain expression vectors that can express the 16 unique antibodies in the form of IgG in animal cells were obtained. The vector, VH, and VL fragments were fused to the CH1-hinge-CH2-CH3 region for heavy chain expression and to Cκ for light chain expression.

[0138] The secured heavy and light chain expression vectors were co-transfected into FreeStyle™293-F cells at a 2:1 DNA ratio (Light:Heavy) using FectoPRO transfection reagent. After 9 days, the culture medium was harvested, and IgG-format antibodies were purified by column chromatography using protein A agarose beads (Religen, USA). After purification and dialysis in DPBS at pH 7.5, the antibody concentration was quantified using a spectrophotometer, and the degree of purification and purity of the antibodies were assessed by SDS-polyacrylamide gel electrophoresis (PAGE) and Coomassie Brilliant Blue staining (Biosesang, Korea).

[0139] Analysis of binding and inhibitory capacity of scFV and IgG using ELISA

[0140] Analysis of the binding and inhibitory activity of individual scFvs and IgG was performed by ELISA. For binding analysis, canine PD-L1-hFc was coated on a 96-well ELISA plate at a concentration of 500 ng / mL in PBS overnight at 4°C. The well solution was discarded, and 3% w / v skim milk in 0.1% PBS-T solution was added to each well and incubated for 1 h at 37°C for blocking. 100 μL / well of scFv-containing protoplast extract or 200 ng / well of purified IgG was added and incubated for 2 h at room temperature. The plates were washed with tap water, treated with horseradish peroxidase (HRP)-conjugated anti-HA (1 / 5,000, Roche, Basel, Switzerland) in blocking buffer, and incubated at 37°C for 45 min.

[0141] For inhibition assays, canine PD-L1 was coated under the same conditions as described above. 100 μL / well of scFv-containing endoplasmic reticulum extract mixed with 100 ng / well of human Cκ-conjugated human PD-1 was treated and incubated for 2 h at room temperature and then washed with tap water. HRP-conjugated anti-human Cκ (Jackson ImmunoResearch, Pennsylvania, USA) was added in blocking buffer and incubated under the same conditions. For detection, 3,3',5,5'-tetramethylbenzidine (TMB, BD Biosciences) was added at 50 μL / well, and the absorbance was read at 450 nm using a Bio Tek Epoch (Agilent, California, USA).

[0142] Flow cytometry

[0143] To analyze the specific binding of purified IgG to cell surface canine PD-L1, a BD FACSVerse™ Biosciences was used. 1 × 105 DH82 and D17 cells were used in each experiment. Cells were resuspended in a non-enzyme-free PBS-based cell dissociation buffer (Gibco). Cells were washed with FACS buffer containing TBS (iNtRON), 0.5% BSA (HanLAB), and 0.05% NaN3 (Acros Organics) and resuspended in FACS buffer mixed with 25 μg / mL of purified IgG for 1 hour at 4°C. After washing with FACS buffer, each IgG clone was treated with Alexa Fluor 488-conjugated anti-human Fc (1:500, Jackson ImmunoResearch, West Grove, PA, USA) or Alexa Fluor 647-conjugated anti-human Fc (1:500, Jackson ImmunoResearch) for 1 h at 4°C. Finally, all samples were measured on a FACSVerse™ flow cytometer and analyzed with FlowJo software (FlowJo LLC, Oregon, USA).

[0144] Example 1: Discovery of scFv antibodies that specifically bind to canine PD-L1.

[0145] Single-chain variable fragments (scFvs) that bind to PD-L1 in dogs were screened using phage display technology. A synthetic scFv library with a diversity of 7.6 × 109 was used (Fig. 1a). The scFv phage candidates selected through panning were tested to evaluate the diversity of the scFvs. The infectious titer measured after the fourth round of panning using ER2738 cells (New England Biolab) was 4.3 × 10 7 Increased (3.5x10 CFU) compared to 9 CFU) and showed that scFv antibodies binding to canine PD-L1 increased within the panning pool (Fig. 1b).

[0146] The binding of individual scFv clones selected from the clones obtained through the panning process to canine PD-L1 was confirmed by ELISA analysis. Thirty-five scFv clones exhibiting high affinities of 0.2 or higher for canine PD-L1 were obtained and are indicated by red arrows (Figure 1c). Next, the thermal stability of the 35 selected clones was evaluated. The evaluation method involved heat treatment at 70°C to induce structural change, followed by lowering the temperature to allow the structure to reform.

[0147] ELISA results demonstrated that the thermal stability of the scFv clone was maintained even after heat treatment, demonstrating that binding to dog PD-L1 was maintained (Fig. 1d).

[0148] The scFv DNA was sequenced for all 35 clones. The analysis revealed distinct sequences in the CDR region for 19 scFv clones.

[0149] In conclusion, we screened a diverse scFv library using phage display to generate 19 unique high-affinity and thermostable scFv clones that specifically target canine PD-L1.

[0150]

[0151] Example 2: Screening of scFvs for inhibition of canine PD-L1 binding to human PD-1.

[0152] In this example, an analysis system was constructed to evaluate the inhibitory function of a canine PD-L1 neutralizing antibody clone.

[0153] Although the nucleotide sequence homology between canine PD-L1 and human PD-L1 is 76.04%, identical nucleotide sequences were confirmed, as highlighted in purple in Figure 2a. Notably, the region boxed in red indicates the essential binding site for human PD-1, suggesting that the binding sites between canine and human PD-L1 are similar (Figure 2a). Therefore, ELISA was performed to verify the binding of the scFv candidate to human PD-L1, and the results confirmed that a specific scFv bound to human PD-L1 (Figure 2b).

[0154] In the present invention, the function of scFv was verified by assuming that the desired antibody should bind to PD-L1 in a manner similar to human PD-1. In this example, the potential of an HA-conjugated canine PD-L1 scFv to interfere with the interaction between human Fc-conjugated canine PD-L1 and human Cκ-conjugated human PD-1 was evaluated. The effect of the scFv in inhibiting the interaction between canine PD-L1 and human PD-1 was quantified using an HRP-conjugated anti-human Cκ secondary antibody bound to human PD-1 (Fig. 2c).

[0155] While analyzing the results of the competitive ELISA, we observed a decrease in OD450 values ​​when scFvs were introduced compared to samples treated with only canine PD-L1 and human PD-1 proteins. These results strongly suggest that scFvs effectively inhibit the binding between canine PD-L1 and human PD-1 proteins (Figure 2d). After evaluating binding affinity, thermal stability, and PD-1 / PD-L1 blocking activity with canine PD-L1, we prioritized and converted these to IgG form those with superior binding affinity to canine PD-L1.

[0156]

[0157] Example 3: Functional validation of a canine PD-L1-specific scFv-derived IgG antibody.

[0158] Using the result data of Examples 1 and 2, replication was performed to convert six high-priority scFvs (#1 to #6) into IgG form.

[0159] scFv is composed of the variable regions of immunoglobulins, the heavy (VH) and light (VL) chains, connected by a linker. We cloned the VH and VL and inserted them into an expression vector capable of producing human IgG using a two-vector system (Fig. 3a). We confirmed that the expressed IgG antibody was properly assembled by disulfide bonds between two heavy and two light chains. This was confirmed by SDS-PAGE under reducing and non-reducing conditions. The protein bands observed in all six samples confirmed the presence of intact IgG (Fig. 3b).

[0160] Because antibodies converted from scFv to IgG form likely lost binding activity to canine PD-L1, we performed an in vitro binding assay using these IgG antibodies. We evaluated the binding activity of the antibodies to both canine and human PD-L1. KL-001 (KR102357951B1), an anti-human PD-L1 antibody that binds to both human and canine PD-L1, and atezolizumab (Genentech, US8217149) were used as positive controls. Measurement of OD values ​​at 450 nm confirmed that the binding affinity to canine PD-L1 was consistently maintained (Fig. 3c).

[0161] Additionally, cell-based in vitro binding assays were performed using canine PD-L1-expressing canine cell lines, specifically DH82 macrophages and D17 canine osteosarcoma cells, followed by flow cytometry analysis. Six IgG antibodies were bound to DH82 and D17 cells to detect fluorescent signals and assess binding. KL-001 was used as a positive control in these assays.

[0162] As a result, it was confirmed that in the DH82 cell line, strong binding signals were observed in all six samples, whereas in the D17 cell line, binding signals were observed in all clones except #3 and #5 (Fig. 3d and Fig. 3e).

[0163] Therefore, the results of this example confirmed that IgG antibodies extracted from scFv exhibited significant functional binding to canine PD-L1. Based on binding affinity and sequence specificity, antibodies 1 and 6 were selected as candidates for further analysis.

[0164]

[0165] Example 4: Selection of αPD-L1 antibody candidates showing antitumor effects against canine osteosarcoma cancer.

[0166] In our previous study, we developed a novel αPD-L1 antibody candidate that can bind to canine and human PD-L1. After confirming its affinity for canine PD-L1 in vitro, we conducted a study on its in vivo effect. The canine osteosarcoma cell line D17 (4 × 10) was injected into NOG mice (KOATECH, Korea) deficient in T cells, B cells, and NK cells. 6 / mouse) was xenografted to create a mouse model of canine osteosarcoma.

[0167] Ten days after tumor cell xenografting in the above mouse model, 8x of dog PBMCs were injected with #1 antibody or #6 antibody to evaluate antitumor efficacy. 6was intravenously administered to NOG mice that had grown D17 tumors. Mice administered PBMCs treated with antibodies #1 and #6 showed reduced tumor size compared to the control group treated with PBMCs (Fig. 4A). In particular, mice administered PBMCs treated with antibody #1 showed the smallest tumor volume compared to the other groups. In addition, when the tumor weights were measured on the day of sacrifice, the group administered PBMCs treated with antibody #1 showed the smallest tumor growth compared to the other groups (Fig. 4B). These results demonstrate that antibodies #1 and #6 exhibit antitumor effects by binding to canine PD-L1.

[0168]

[0169] Electronic file attached.

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof, The above monoclonal antibody comprises a heavy chain variable region (V) comprising HCDR1 as set forth in SEQ ID NO: 14, HCDR2 as set forth in SEQ ID NO: 16, and HCDR3 as set forth in SEQ ID NO:

18. H ); and A light chain variable region (V) comprising LCDR1 as set forth in SEQ ID NO: 21, LCDR2 as set forth in SEQ ID NO: 23, and LCDR3 as set forth in SEQ ID NO: 25 L ) having a monoclonal antibody or an antigen-binding fragment thereof.

2. In paragraph 1, The heavy chain variable region (V) of the above monoclonal antibody or antigen-binding fragment thereof H ) is represented by sequence number 1. The light chain variable region (V) of the above monoclonal antibody or antigen-binding fragment thereof L ) is characterized by an amino acid sequence represented by SEQ ID NO: 2, or an antigen-binding fragment thereof.

3. A monoclonal antibody or an antigen-binding fragment thereof according to claim 1, characterized in that the monoclonal antibody or an antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, single-chain antibody, humanized antibody, chimeric antibody and diabody.

4. A monoclonal antibody or an antigen-binding fragment thereof, characterized in that the single-chain antibody in claim 1 is scFv.

5. A monoclonal antibody or an antigen-binding fragment thereof characterized by specifically binding to PDL-1 in claim 1.

6. In paragraph 5, a monoclonal antibody or an antigen-binding fragment thereof, characterized in that the PDL-1 is of canine or human origin.

7. As a monoclonal antibody or an antigen-binding fragment thereof, The monoclonal antibody comprises a heavy chain variable region (VH) comprising an HCDR1 set forth in SEQ ID NO: 28, SEQ ID NO: 42, SEQ ID NO: 56, SEQ ID NO: 70 or SEQ ID NO: 84, an HCDR2 set forth in SEQ ID NO: 30, SEQ ID NO: 44, SEQ ID NO: 58, SEQ ID NO: 72 or SEQ ID NO: 86 and an HCDR3 set forth in SEQ ID NO: 32, SEQ ID NO: 46, SEQ ID NO: 60, SEQ ID NO: 74 or SEQ ID NO: 88; and A light chain variable region (V) comprising an LCDR1 as set forth in SEQ ID NO: 35, SEQ ID NO: 49, SEQ ID NO: 63, SEQ ID NO: 77 or SEQ ID NO: 91, an LCDR2 as set forth in SEQ ID NO: 37, SEQ ID NO: 51, SEQ ID NO: 65, SEQ ID NO: 79 or SEQ ID NO: 93 and an LCDR3 as set forth in SEQ ID NO: 39, SEQ ID NO: 53, SEQ ID NO: 67, SEQ ID NO: 81 or SEQ ID NO: 95 L ) having a monoclonal antibody or an antigen-binding fragment thereof.

8. In paragraph 7, a monoclonal antibody or an antigen-binding fragment thereof selected from the following: (i) a heavy chain variable region (V) having an amino acid sequence of sequence number 3; H ) and a light chain variable region (V) having the amino acid sequence of sequence number 4 L ), a monoclonal antibody or an antigen-binding fragment thereof; (ii) a heavy chain variable region (V) having an amino acid sequence of sequence number 5; H ) and a light chain variable region (V) having the amino acid sequence of sequence number 6 L ), a monoclonal antibody or an antigen-binding fragment thereof; (iii) a heavy chain variable region (V) having an amino acid sequence of sequence number 7; H ) and a light chain variable region (V) having the amino acid sequence of sequence number 8 L ), a monoclonal antibody or an antigen-binding fragment thereof; (iv) a heavy chain variable region (V) having an amino acid sequence of sequence number 9;H ) and a light chain variable region (V) having the amino acid sequence of sequence number 10 L ), a monoclonal antibody or an antigen-binding fragment thereof; and (v) a heavy chain variable region (V) having an amino acid sequence of sequence number 11; H ) and a light chain variable region (V) having the amino acid sequence of sequence number 12. L ) comprising a monoclonal antibody or an antigen-binding fragment thereof.

9. A monoclonal antibody or an antigen-binding fragment thereof, characterized in that the monoclonal antibody or an antigen-binding fragment thereof in paragraph 7 is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, single-chain antibody, humanized antibody, chimeric antibody and diabody.

10. A monoclonal antibody or an antigen-binding fragment thereof, characterized in that the single-chain antibody in claim 7 is scFv.

11. A monoclonal antibody or an antigen-binding fragment thereof characterized by specifically binding to PDL-1 in claim 7.

12. A monoclonal antibody or antigen-binding fragment thereof, characterized in that the PDL-1 in claim 11 is of canine or human origin.

13. The heavy chain variable region (V) of the monoclonal antibody or antigen-binding fragment thereof of paragraph 1 H ) An isolated nucleic acid molecule comprising a nucleotide sequence encoding a ) 14. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 97 in claim 13.

15. The light chain variable region (V) of the monoclonal antibody or antigen-binding fragment thereof of paragraph 1 L ) An isolated nucleic acid molecule comprising a nucleotide sequence encoding a ) 16. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 98 in claim 15.

17. The heavy chain variable region (V) of the monoclonal antibody or antigen-binding fragment thereof of clause 7 H ) An isolated nucleic acid molecule comprising a nucleotide sequence encoding a ) 18. An isolated nucleic acid molecule comprising a nucleotide sequence of any one of SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, and SEQ ID NO: 107, according to claim 17.

19. The light chain variable region (V) of the monoclonal antibody or antigen-binding fragment thereof of clause 7 L ) An isolated nucleic acid molecule comprising a nucleotide sequence encoding a ) 20. An isolated nucleic acid molecule comprising a nucleotide sequence of any one of SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, and SEQ ID NO: 108, according to claim 19.

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

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

23. A pharmaceutical composition for treating a tumor, comprising a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12; and a pharmaceutically acceptable carrier or excipient.

24. A pharmaceutical composition for treating a tumor, characterized in that in claim 23, the tumor is osteosarcoma, melanoma, renal cancer, prostate cancer, bladder cancer, colorectal cancer, gastric cancer, liver cancer, non-small cell lung cancer, breast cancer, esophageal cancer, pancreatic cancer, glioma, ovarian cancer or leukemia.

25. A pharmaceutical composition according to claim 23, characterized in that the tumor is a canine or human tumor.

26. A method for treating or preventing a tumor in a canine, comprising administering to a canine having a 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 a tumor according to claim 23.

27. A method according to claim 26, characterized in that the tumor is selected from the group consisting of osteosarcoma, melanoma, renal cancer, prostate cancer, bladder cancer, colorectal cancer, gastric cancer, liver cancer, non-small cell lung cancer, breast cancer, esophageal cancer, pancreatic cancer, glioma, ovarian cancer, and leukemia.

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