Anti-PD-L1 antibody for detecting PD-L1

Abstract

Provided is a diagnostic antibody that is capable of detecting PD-L1 in tumors for novel therapies for feline tumors targeting PD-1 / PD-L1.　The anti-PD-L1 antibody contains: (a) an L-chain having CDR1 that has the amino acid sequence ESVDSYGNSF (SEQ ID NO: 1), CDR2 that has the amino acid sequence RAS, and CDR3 that has the amino acid sequence QQSNEDPRT (SEQ ID NO: 2); and (b) an H-chain having CDR1 that has the amino acid sequence GFTFSSYG (SEQ ID NO: 3), CDR2 that has the amino acid sequence ISNGGTYT (SEQ ID NO: 4), and CDR3 that has the amino acid sequence ARLGYGSLNWYFDV (SEQ ID NO: 5). A composition for detecting PD-L1 that contains the anti-PD-L1 antibody as an active ingredient. Also provided is a method for producing the anti-PD-L1 antibody.

Description

[Technical Field]

[0001] The present invention relates to an anti-PD-L1 antibody for detecting PD-L1. [Background technology]

[0002] Because existing therapies for feline tumors are often ineffective, the development of novel therapies is desired. In human medicine, tumor therapies targeting the immunosuppressive factor programmed death ligand 1 (PD-L1) are being clinically applied. Specifically, the interaction between PD-L1 and its receptor programmed death 1 (PD-1) is one of the major molecular mechanisms by which tumors and infectious diseases evade elimination by the immune response. It has been reported that antibodies that specifically bind to these molecules and inhibit the PD-1 / PD-L1 interaction (anti-PD-1 antibodies, anti-PD-L1 antibodies) exert antitumor and antipathogen effects (Non-Patent Documents 1-5). However, no such research trends have been reported in cats. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Bramer J, Reckamp K, et al: Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. N Engl J Med, 373:1627-1639, 2015. [Non-patent document 2] Hamanishi J, Mandai M, Ikeda T, et al: Safety and Antitumor Activity of Anti-PD-1 Antibody, Nivolumab, in Patients With Platinum-Resistant Ovarian Cancer. J Clin Oncol, 33:4015-4022, 2015. [Non-patent document 3] Motzer RJ, Escudier B, McDermott DF, et al: Nivolumab versus Everolimus in Advanced Renal-Cell Carcinoma. N Engl J Med, 373:1803-1813, 2015. [Non-patent document 4] Barber DL, Wherry EJ, Masopust D, et al: Restoring function in exhausted CD8 T cells during chronic viral infection. Nature, 439:682-687, 2006. [Non-patent document 5] Velu V, Titanji K, Zhu B, et al: Enhancing SIV-specific immunity in vivo by PD-1 blockade. Nature 458:206-210, 2009. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention aims to provide a diagnostic antibody capable of detecting PD-L1 in tumors, for use in novel therapeutic approaches to feline tumors that target PD-1 / PD-L1. [Means for solving the problem]

[0005] The present inventors have established a diagnostic antibody capable of detecting PD-L1 in tumors, with the aim of developing a novel treatment for feline tumors that targets PD-1 / PD-L1. Specifically, they produced recombinant feline PD-L1 and established a mouse monoclonal antibody against feline PD-L1. PD-L1 expression analysis in feline tumor tissues using immunohistochemical staining with this antibody revealed a high positive rate in tumors such as mammary adenocarcinoma. These findings led to the completion of the present invention.

[0006] The gist of the present invention is as follows. (1) An anti-PD-L1 antibody comprising: (a) a light chain having a CDR1 having the amino acid sequence of ESVDSYGNSF (SEQ ID NO: 1), a CDR2 having the amino acid sequence of RAS, and a CDR3 having the amino acid sequence of QQSNEDPRT (SEQ ID NO: 2); and (b) a heavy chain having a CDR1 having the amino acid sequence of GFTFSSYG (SEQ ID NO: 3), a CDR2 having the amino acid sequence of ISNGGTYT (SEQ ID NO: 4), and a CDR3 having the amino acid sequence of ARLGYGSLNWYFDV (SEQ ID NO: 5). (2) The antibody according to (1), which is derived from a mouse. (3) The antibody according to (2), which is a mouse anti-cat PD-L1 antibody. (4) The antibody described in (3), wherein the L chain variable region has the amino acid sequence of SEQ ID NO: 6 and the H chain variable region has the amino acid sequence of SEQ ID NO: 7. (5) The antibody according to any one of (1) to (4), wherein the L chain constant region has the amino acid sequence of the Kappa chain constant region. (6) The antibody according to any one of (1) to (5), wherein the H-chain constant region has the amino acid sequence of the constant region of IgG1. (7) The antibody according to (5) or (6), wherein the L-chain constant region has the amino acid sequence of SEQ ID NO: 8 and the H-chain constant region has the amino acid sequence of SEQ ID NO: 9. (8) The antibody according to any one of (1) to (7), which has a four-chain structure consisting of two light chains and two heavy chains. (9) A composition for detecting PD-L1, comprising the antibody according to any one of (1) to (8) as an active ingredient. (10) The composition according to (9), which is used for diagnosing cancer and / or infectious diseases. (11) Cancer and / or infectious diseases include neoplastic disease, leukemia, feline leukemia virus infection, feline immunodeficiency virus infection, feline panleukopenia (feline parvovirus infection), feline infectious peritonitis / feline enteric coronavirus infection, feline calicivirus disease, feline viral rhinotracheitis (feline herpesvirus infection), feline foamy virus infection, poxvirus disease, Borna disease, Aujeszky's disease, severe fever with thrombocytopenia syndrome, feline morbillivirus infection, Campylobacter enteritis, Salmonella infection, Bordetella infection, Pasteurellosis, tetanus, tularemia, and atypical mycobacterium infection. (10) The composition according to (10), wherein the agent is selected from the group consisting of feline hemoplasmosis, feline hemoplasmosis, coxiellosis, chlamydiosis, cryptococcosis, dermatophytosis, histoplasmosis, candidiasis, aspergillosis, blastomycosis, coccidioidomycosis, sporotrichosis, protothecia, malasseziosis, Pneumocystis carinii pneumonia, toxoplasmosis, giardiasis, trichomoniasis, amebiasis, balantidiosis, babesiosis, cryptosporidiosis, intestinal coccidiosis, trypanosomiasis, encephalitozoonosis, and cytozoonosis. (12) The composition according to (9), which is used to select a subject animal suitable for treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody. (13) A DNA encoding the anti-PD-L1 antibody according to (1). (14) A vector containing the DNA according to (13). (15) A host cell transformed with the vector according to (14). (16) A method for producing an antibody, comprising culturing the host cell according to (15) above and collecting an anti-PD-L1 antibody from the culture. (17) A host cell transformed with a vector incorporating DNA encoding an L chain having CDR1 having the amino acid sequence of ESVDSYGNSF (SEQ ID NO: 1), CDR2 having the amino acid sequence of RAS, and CDR3 having the amino acid sequence of QQSNEDPRT (SEQ ID NO: 2), and a vector incorporating DNA encoding an H chain having CDR1 having the amino acid sequence of GFTFSSYG (SEQ ID NO: 3), CDR2 having the amino acid sequence of ISNGGTYT (SEQ ID NO: 4), and CDR3 having the amino acid sequence of ARLGYGSLNWYFDV (SEQ ID NO: 5). (18) A method for producing an antibody, comprising culturing the host cell according to (17) above and collecting an anti-PD-L1 antibody from the culture. (19) A protein defined as (a) or (b) below: (a) A protein consisting of the amino acid sequence of SEQ ID NO: 19. (b) A protein consisting of the amino acid sequence of SEQ ID NO: 19 in which one or more amino acids have been deleted, substituted, or added, and which has the function of PD-L1. (20) A DNA encoding the protein according to (19). [Effects of the Invention]

[0007] The present invention provides a novel anti-PD-L1 antibody capable of staining tumor cells such as feline mammary adenocarcinoma cells. The present invention makes it possible to diagnose whether a patient is suitable for immunotherapy with an anti-PD-1 antibody or an anti-PD-L1 antibody. This specification includes the contents as disclosed in the specification and / or drawings of Japanese Patent Application No. 2022-135595, which is a priority document of this application. [Brief explanation of the drawings]

[0008] [Figure 1]Identification of the feline PD-L1 gene. The feline PD-L1 gene was identified. (a) A comparative analysis of the predicted amino acid sequences of feline PD-L1 with those of canine, bovine, human, mouse, and rat PD-L1 was performed. (b) Phylogenetic tree analysis of the predicted amino acid sequences of PD-L1 from various animals was performed. GenBank accession numbers are listed in parentheses to the right of the common names. The scale bar indicates the branch length. [Figure 2] Preparation of PD-1-Ig and PD-L1-Ig. PD-1-Ig and PD-L1-Ig were purified and subjected to SDS-PAGE followed by CBB staining. (a) Electrophoresis images under non-reducing conditions and (b) under reducing conditions are shown. [Figure 3] Binding study of CL1Mab-7 to PD-L1. Binding of CL1Mab-7 to PD-L1-expressing CHO-CG44 cells was measured by flow cytometry. EGFP indicates EGFP-expressing CHO-CG44 cells, and PD-L1-EGFP indicates PD-L1-EGFP-expressing CHO-DG44 cells. The vertical axis indicates cell number, and the horizontal axis indicates fluorescence intensity (amount of antibody binding). [Figure 4] Analysis of PD-L1 expression in a feline macrophage-derived cell line (Fcwf-4). Flow cytometry analysis showed that Fcwf-4 expressed PD-L1 under unstimulated conditions, and that expression was upregulated under IFN-γ stimulation. [Figure 5] Analysis of PD-L1 expression in feline mammary adenocarcinoma-derived cell lines (FKNp, FMCp, FMCm, FYMp, FONp, and FONm). Flow cytometry analysis showed that FKNp, FMCm, FYMp, FONp, and FONm expressed PD-L1 under unstimulated conditions, and this expression was upregulated under IFN-γ stimulation. FMCp did not express PD-L1. [Figure 6] PD-L1 expression analysis in feline tumor tissues. Representative staining-positive images of mammary adenocarcinoma, squamous cell carcinoma, fibrosarcoma, and renal cell carcinoma are shown. As a negative control, a representative staining image of squamous cell carcinoma stained with mouse IgG1 is shown. [Figure 7a]The full-length nucleotide sequence and amino acid sequence of the light chain of the mouse anti-feline PD-L1 monoclonal antibody (clone name CL1Mab-7) are shown. [Figure 7b] The full-length nucleotide sequence and amino acid sequence of the heavy chain of the mouse anti-feline PD-L1 monoclonal antibody (clone name CL1Mab-7) are shown. [Figure 8] SDS-PAGE image of the recombinant mouse anti-feline PD-L1 antibody rCL1Mab-7. The recombinant mouse anti-feline PD-L1 antibody rCL1Mab-7 showed a migration pattern similar to that of the hybridoma-derived mouse anti-feline PD-L1 antibody CL1Mab-7, with no bands other than those of interest visible. [Figure 9] Binding of recombinant murine anti-cat PD-L1 antibody rCL1Mab-7 to feline PD-L1-expressing CHO-DG44 cells. Flow cytometry analysis showed that the recombinant murine anti-cat PD-L1 antibody rCL1Mab-7 bound to feline PD-L1-expressing CHO-DG44 cells to a similar extent as the hybridoma-derived murine anti-cat PD-L1 antibody CL1Mab-7. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention will be described in detail below.

[0010] The present invention provides anti-PD-L1 antibodies comprising: (a) an L chain (light chain) having a CDR1 having the amino acid sequence of ESVDSYGNSF (SEQ ID NO: 1), a CDR2 having the amino acid sequence of RAS, and a CDR3 having the amino acid sequence of QQSNEDPRT (SEQ ID NO: 2); and (b) a H chain (heavy chain) having a CDR1 having the amino acid sequence of GFTFSSYG (SEQ ID NO: 3), a CDR2 having the amino acid sequence of ISNGGTYT (SEQ ID NO: 4), and a CDR3 having the amino acid sequence of ARLGYGSLNWYFDV (SEQ ID NO: 5).

[0011] CDRs 1 to 3 in the L-chain variable region of the mouse anti-feline PD-L1 monoclonal antibody (clone name CL1Mab-7) established by the present inventors are a region consisting of the amino acid sequence of ESVDSYGNSF (SEQ ID NO: 1), a region consisting of the amino acid sequence of RAS, and a region consisting of the amino acid sequence of QQSNEDPRT (SEQ ID NO: 2), respectively (see Figure 7a).

[0012] Furthermore, CDRs 1 to 3 in the H-chain variable region of the mouse anti-feline PD-L1 monoclonal antibody (clone name CL1Mab-7) are a region consisting of the amino acid sequence GFTFSSYG (SEQ ID NO: 3), a region consisting of the amino acid sequence ISNGGTYT (SEQ ID NO: 4), and a region consisting of the amino acid sequence ARLGYGSLNWYFDV (SEQ ID NO: 5), respectively (see Figure 7b).

[0013] The amino acid sequence of ESVDSYGNSF (SEQ ID NO: 1), the amino acid sequence of RAS, and the amino acid sequence of QQSNEDPRT (SEQ ID NO: 2), as well as the amino acid sequence of GFTFSSYG (SEQ ID NO: 3), the amino acid sequence of ISNGGTYT (SEQ ID NO: 4), and the amino acid sequence of ARLGYGSLNWYFDV (SEQ ID NO: 5), may have one, two, three, four, or five amino acids deleted, substituted, or added, and even with these mutations, the CDRs of the light chain variable region or heavy chain variable region of the PD-L1 antibody can still function as CDRs.

[0014] In the present specification, the term "antibody" refers to a whole antibody as well as Fab, F(ab)'2, ScFv, diabody, V H , V L This concept also includes smaller molecules such as Sc(Fv)2, bispecific sc(Fv)2, minibody, ScFv-Fc monomer, and ScFv-Fc dimer.

[0015] The anti-PD-L1 antibodies of the invention may be of murine origin, for example, murine anti-feline PD-L1 monoclonal antibodies.

[0016] The amino acid sequences of the light chain variable region and heavy chain variable region of a mouse anti-feline PD-L1 monoclonal antibody (clone name CL1Mab-7) are set forth in SEQ ID NOs: 6 and 7, respectively; however, one or more (for example, up to five, or at most about ten) amino acids may be deleted, substituted, or added to the amino acid sequences of SEQ ID NOs: 6 and 7, and even if these mutations are introduced, the antibody will still function as the light chain variable region or heavy chain variable region of a PD-L1 antibody. Preferably, the anti-PD-L1 antibody of the present invention has an light chain variable region having the amino acid sequence of SEQ ID NO: 6 and an heavy chain variable region having the amino acid sequence of SEQ ID NO: 7.

[0017] Antibody light chains include kappa chains and lambda chains. In the anti-PD-L1 antibodies of the present invention, the light chain constant region may have the amino acid sequence of either the kappa chain or the lambda chain constant region; however, lambda chains are more abundant in sheep, cats, dogs, and horses, while kappa chains are more abundant in mice, rats, humans, and pigs. The mouse anti-cat PD-L1 monoclonal antibody (clone name CL1Mab-7) is a mouse-derived IgG1, and its light chain constant region has the amino acid sequence of a kappa chain constant region. In the anti-PD-L1 antibodies of the present invention, the light chain constant region preferably has the amino acid sequence of a kappa chain constant region.

[0018] The heavy chain constant region of the anti-PD-L1 antibodies of the invention preferably has the amino acid sequence of the heavy chain constant region of mouse IgG1. Heavy chains are divided into gamma, mu, alpha, delta, and epsilon chains based on differences in their constant regions, and these differences result in the formation of five different immunoglobulin classes (isotypes): IgG, IgM, IgA, IgD, and IgE.

[0019] Immunoglobulin G (IgG) accounts for 70-75% of human immunoglobulins and is the most abundant monomeric antibody in plasma. It has a four-chain structure consisting of two light chains and two heavy chains. Human IgG1, IgG2, and IgG4 have a molecular weight of approximately 146,000, while human IgG3 has a longer hinge region connecting the Fab and Fc regions and a larger molecular weight of 170,000. Human IgG1 accounts for approximately 65% ​​of human IgG, human IgG2 approximately 25%, human IgG3 approximately 7%, and human IgG4 approximately 3%. It is distributed evenly both intravascularly and extravascularly. Human IgG1 has a strong affinity for Fc receptors and complement factors on the surface of effector cells, thereby inducing antibody-dependent cellular cytotoxicity (ADCC) and activating complement to induce complement-dependent cytotoxicity (CDC). Human IgG2 and human IgG4 have low affinity for Fc receptors and complement factors, resulting in low ADCC and CDC activities.

[0020] Immunoglobulin M (IgM) accounts for approximately 10% of human immunoglobulins. It is a pentamer antibody consisting of five basic four-chain structures. Its molecular weight is 970,000. It is normally found only in the blood and is the first immunoglobulin produced against infectious microorganisms, responsible for early immunity.

[0021] Immunoglobulin A (IgA) accounts for 10-15% of human immunoglobulins. Its molecular weight is 160,000. Secretory IgA is a dimeric antibody formed by the combination of two IgAs. IgA1 is present in serum, nasal secretions, saliva, and breast milk, while IgA2 is abundant in intestinal fluids.

[0022] Immunoglobulin D (IgD) is a monomeric antibody that accounts for less than 1% of human immunoglobulins. It is present on the surface of B cells and is involved in the induction of antibody production.

[0023] Immunoglobulin E (IgE) is a monomeric antibody that exists in extremely small amounts, accounting for less than 0.001% of human immunoglobulins. It is thought to be involved in the immune response to parasites, and in developed countries where parasites are rare, it is particularly involved in bronchial asthma and allergies.

[0024] In mice, the IgG heavy chain sequences identified are IgG1, IgG2a, IgG2b, IgG2c, and IgG3. The mouse anti-feline PD-L1 monoclonal antibody (clone name CL1Mab-7) has the amino acid sequence of the IgG1 heavy chain constant region.

[0025] More preferably, the antibody of the present invention is an anti-PD-L1 antibody whose light chain constant region has the amino acid sequence of a kappa chain constant region and whose heavy chain constant region has the amino acid sequence of an IgG1 heavy chain constant region.

[0026] The amino acid sequence and nucleotide sequence of the light chain variable region of the mouse anti-feline PD-L1 monoclonal antibody (clone name CL1Mab-7) identified by the present inventors are shown in SEQ ID NOs: 6 and 12, respectively.

[0027] The amino acid sequence and nucleotide sequence of the heavy chain variable region of the mouse anti-feline PD-L1 monoclonal antibody (clone name CL1Mab-7) identified by the present inventors are shown in SEQ ID NOs: 7 and 13, respectively.

[0028] The amino acid sequence and nucleotide sequence of the light chain constant region (kappa chain) of the mouse anti-feline PD-L1 monoclonal antibody (clone name CL1Mab-7) identified by the present inventors are shown in SEQ ID NOs: 8 and 14, respectively. These sequences are the same as the sequences registered in GenBank under accession numbers AAB53776.1 and U56411.1.

[0029] The amino acid sequence and nucleotide sequence of the heavy chain constant region (IgG1) of the mouse anti-feline PD-L1 monoclonal antibody (clone name CL1Mab-7) identified by the present inventors are shown in SEQ ID NOs: 9 and 15, respectively. These sequences are the same as the sequences registered in GenBank under accession numbers ABQ85914.1 and EF392839.1.

[0030] In the anti-PD-L1 antibodies of the invention, the light chain constant region may have the amino acid sequence of SEQ ID NO:8, and the heavy chain constant region may have the amino acid sequence of SEQ ID NO:9.

[0031] The amino acid sequence and nucleotide sequence of the full-length L chain of the mouse anti-feline PD-L1 monoclonal antibody (clone name CL1Mab-7) identified by the present inventors are shown in SEQ ID NOs: 10 and 16, respectively.

[0032] The amino acid sequence and nucleotide sequence of the full-length heavy chain of the mouse anti-feline PD-L1 monoclonal antibody (clone name CL1Mab-7) identified by the present inventors are shown in SEQ ID NOs: 11 and 17, respectively.

[0033] In the anti-PD-L1 antibodies of the invention, the light chain preferably has the amino acid sequence of SEQ ID NO: 10 and the heavy chain preferably has the amino acid sequence of SEQ ID NO: 11.

[0034] The anti-PD-L1 antibody of the present invention preferably has a four-chain structure consisting of two light chains and two heavy chains.

[0035] Anti-PD-L1 antibodies of the invention can be produced as follows: An artificial gene containing the light chain sequence (variable region sequence and constant region sequence) and heavy chain sequence (variable region sequence and constant region sequence) of an anti-PD-L1 antibody of the invention is synthesized, and the artificial gene is inserted into a vector (e.g., a plasmid), which is then introduced into host cells (e.g., mammalian cells such as CHO cells). The host cells are then cultured, and the antibody is collected from the culture. When synthesizing the artificial gene, the codons in the nucleotide sequence may be optimized.

[0036] The present invention provides DNA encoding an anti-PD-L1 antibody comprising: (a) an L chain having a CDR1 having the amino acid sequence of ESVDSYGNSF (SEQ ID NO: 1), a CDR2 having the amino acid sequence of RAS, and a CDR3 having the amino acid sequence of QQSNEDPRT (SEQ ID NO: 2); and (b) an H chain having a CDR1 having the amino acid sequence of GFTFSSYG (SEQ ID NO: 3), a CDR2 having the amino acid sequence of ISNGGTYT (SEQ ID NO: 4), and a CDR3 having the amino acid sequence of ARLGYGSLNWYFDV (SEQ ID NO: 5). The DNA of the present invention can be synthesized using a commercially available synthesizer. Restriction enzyme recognition sites, a KOSAK sequence, a poly(A) addition signal sequence, a promoter sequence, an intron sequence, etc. may be added to this DNA.

[0037] The present invention also provides vectors containing DNA encoding the anti-PD-L1 antibodies.

[0038] Examples of vectors that can be used include Escherichia coli-derived plasmids (e.g., pBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5, pC194), yeast-derived plasmids (e.g., pSH19, pSH15), bacteriophages such as λ phage, retroviruses, animal viruses such as vaccinia virus, and insect pathogenic viruses such as baculovirus. In the examples described below, the expression vector pCXN2.1(+) (provided by Professor Takehiko Yokomizo, Juntendo University (Niwa et al., Gene. 1991;108(2):193-199.) was used.

[0039] The vector may contain a promoter, an enhancer, a splicing signal, a polyA addition signal, an intron sequence, a selection marker, an SV40 replication origin, and the like.

[0040] The present invention also provides host cells transformed with the vector. Anti-PD-L1 antibodies can be produced by culturing these host cells and collecting the antibodies from the culture. Therefore, the present invention also provides a method for producing antibodies, which comprises culturing the host cells and collecting the anti-PD-L1 antibodies from the culture. In the antibody production methods of the present invention, host cells may be transfected with a vector incorporating DNAs containing an L chain-encoding DNA and an H chain-encoding DNA, or host cells may be co-transfected with a vector incorporating an L chain-encoding DNA and a vector incorporating an H chain-encoding DNA. In Example 2 described below, host cells (Expi293F cells (Life Technologies)) were co-transfected with a vector incorporating an L chain-encoding DNA and a vector incorporating an H chain-encoding DNA to produce anti-PD-L1 antibodies.

[0041] The present invention also provides host cells transformed with a vector incorporating DNA encoding an L chain having CDR1 having the amino acid sequence of ESVDSYGNSF (SEQ ID NO: 1), CDR2 having the amino acid sequence of RAS, and CDR3 having the amino acid sequence of QQSNEDPRT (SEQ ID NO: 2), and a vector incorporating DNA encoding an H chain having CDR1 having the amino acid sequence of GFTFSSYG (SEQ ID NO: 3), CDR2 having the amino acid sequence of ISNGGTYT (SEQ ID NO: 4), and CDR3 having the amino acid sequence of ARLGYGSLNWYFDV (SEQ ID NO: 5).The present invention also provides a method for producing an anti-PD-L1 antibody, comprising culturing the host cell and collecting the antibody from the culture.

[0042] Examples of host cells include bacterial cells (e.g., Escherichia, Bacillus, Bacillus subtilis, etc.), fungal cells (e.g., yeast, Aspergillus, etc.), insect cells (e.g., S2 cells, Sf cells, etc.), animal cells (e.g., CHO cells, COS cells, HeLa cells, C127 cells, 3T3 cells, BHK cells, HEK293 cells, etc.), and plant cells. Among these, dihydrofolate reductase-deficient CHO-DG44 cells (CHO-DG44(dhfr- / -)) are preferred. In Example 2 described below, Expi293F cells (Life Technologies) were used.

[0043] The recombinant vector can be introduced into a host by the method described in Molecular Cloning 2nd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989 (e.g., calcium phosphate method, DEAE-dextran method, transfection method, microinjection method, lipofection method, electroporation method, transduction method, scrape-loading method, shotgun method, etc.) or by infection.

[0044] The transformants can be cultured in a medium, and the anti-PD-L1 antibodies of the invention can be collected from the culture medium. If the antibody is secreted into the medium, the medium can be collected and the antibody can be isolated and purified from the medium. If the antibody is produced within the transformed cells, the cells can be lysed, and the antibody can be isolated and purified from the lysate.

[0045] Examples of media include OptiCHO medium, Dynamis medium, CD CHO medium, ActiCHO medium, FortiCHO medium, Ex-Cell CD CHO medium, BalanCD CHO medium, ProCHO 5 medium, Cellvento CHO-100 medium, etc. In Example 2 described below, Expi293 Expression Medium (Life Technologies) was used.

[0046] The pH of the medium varies depending on the cells being cultured, but generally it is pH 6.8 to 7.6, and in most cases pH 7.0 to 7.4.

[0047] When the cells to be cultured are CHO cells, they can be cultured using methods known to those skilled in the art. For example, they can usually be cultured in an atmosphere with a CO concentration in the gas phase of 0-40%, preferably 2-10%, at 30-39°C, preferably about 37°C.

[0048] An appropriate culture period is usually 1 day to 3 months, preferably 1 day to 3 weeks.

[0049] Antibody separation and purification can be carried out by known methods, including methods that utilize differences in solubility, such as salting out and solvent precipitation, methods that utilize differences in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, methods that utilize differences in charge, such as ion exchange chromatography, methods that utilize specific affinity, such as affinity chromatography, methods that utilize differences in hydrophobicity, such as reversed-phase high-performance liquid chromatography, and methods that utilize differences in isoelectric point, such as isoelectric focusing.

[0050] The anti-PD-L1 antibodies of the present invention can also be produced by culturing hybridomas. Hybridomas can be produced by a method previously described (Ikebuchi et al., Immunology. 2014;142(4):551-561). A hybridoma producing a mouse anti-feline PD-L1 monoclonal antibody (clone name CL1Mab-7) is stored in the inventors' laboratory (Department of Infectious Diseases, Department of Pathogen Control, Graduate School of Veterinary Medicine, Hokkaido University).

[0051] The anti-PD-L1 antibodies of the present invention can be used to detect PD-L1. Accordingly, the present invention provides a composition for detecting PD-L1, which comprises the above-mentioned anti-PD-L1 antibodies as an active ingredient.

[0052] PD-L1 can be detected by, for example, immunohistochemical staining, immunocytochemical staining, flow cytometry, enzyme-linked immunosorbent assay (ELISA), Western blotting, or the like.

[0053] Examples of specimens include tissues and body fluids collected from living organisms (for example, blood (whole blood, plasma, serum, and specific cells such as red blood cells, white blood cells, and lymphocytes), urine, saliva, etc.), cell culture fluid, and cultured cells (established cell lines, primary cultured cells, passaged cells, etc.). Specimens may be derived from rats, dogs, sheep, goats, pigs, cats, humans, horses, cows, buffalo, yaks, rabbits, mice, hamsters, guinea pigs, etc., with cats being preferred.

[0054] The anti-PD-L1 antibodies of the present invention may be labeled with a radioisotope, enzyme, luminescent substance, fluorescent substance, biotin, etc. Furthermore, if the target molecule (PD-L1) is to be detected by reacting a primary antibody (the anti-PD-L1 antibody of the present invention) that specifically binds to the target molecule, followed by a secondary antibody that binds to the primary antibody, the secondary antibody may be labeled.

[0055] Because PD-L1 is strongly expressed in cancer cells and infected cells, the compositions of the present invention can be used to diagnose cancer and / or infectious diseases. Typically, the amount (concentration) of PD-L1 in a sample is determined based on the amount (concentration) of the conjugate between PD-L1 and an anti-PD-L1 antibody. If the amount (concentration) of PD-L1 in a sample is higher than that of a negative control (e.g., normal surrounding tissue (connective tissue, blood vessels, etc.)), cancer and / or infectious diseases can be diagnosed. Alternatively, if PD-L1 is detected in a sample, cancer and / or infectious diseases can be diagnosed.

[0056] The cancer and / or infectious disease is preferably a cancer in which PD-L1 is expressed in cancer cells, or an infectious disease in which PD-L1 is expressed in cells infected with pathogens such as bacteria or viruses, and includes tumor diseases, leukemia, feline leukemia virus infection, feline immunodeficiency virus infection, feline panleukopenia (feline parvovirus infection), feline infectious peritonitis / feline enteric coronavirus infection, feline calicivirus disease, feline viral rhinotracheitis (feline herpesvirus infection), feline foamy virus infection, poxvirus disease, Borna disease, Aujeszky's disease, severe fever with thrombocytopenia syndrome, feline morbillivirus infection, Campylobacter enteritis, Salmonella infection, Boehringer Ingelheim disease ... Examples include rudetellasis, pasteurellosis, tetanus, tularemia, atypical mycobacterial infection, feline hemoplasmosis, coxiellosis, chlamydia, cryptococcosis, dermatophytosis, histoplasmosis, candidiasis, aspergillosis, blastomycosis, coccidioidomycosis, sporotrichosis, protothecia, malasseziosis, Pneumocystis carinii pneumonia, toxoplasmosis, giardiasis, trichomoniasis, amebiasis, balantidiosis, babesiosis, cryptosporidiosis, intestinal coccidiosis, trypanosomiasis, encephalitozoonosis, and cytozoonosis.

[0057] The compositions of the present invention can be used to select test animals suitable for treatment with anti-PD-1 antibodies or anti-PD-L1 antibodies. For example, candidate animals for treatment should satisfy the following two criteria: 1. Cases diagnosed as cancer (e.g., breast adenocarcinoma, squamous cell carcinoma, fibrosarcoma, renal cell carcinoma, etc.) or infection by pathological examination 2. Cases that tested positive for anti-PD-L1 antibodies The negative control should be normal surrounding tissue (connective tissue, blood vessels, etc.), and the positive control should be a case of cancer (e.g., breast adenocarcinoma, squamous cell carcinoma, fibrosarcoma, renal cell carcinoma, etc.) or an infectious disease. In principle, the subject of a clinical trial should be one in which almost the entire tumor area is positive by immunohistochemical staining.

[0058] Examples of test animals include rats, dogs, sheep, goats, pigs, cats, humans, horses, cows, buffalo, yaks, rabbits, mice, hamsters, and guinea pigs, with cats being preferred.

[0059] The composition of the present invention may further comprise a reagent for detecting the label, a diluent, a washing solution, an instruction manual describing diagnostic and selection criteria, and the like.

[0060] The present invention also provides feline PD-L1 and its gene. The total length of the feline PD-L1 gene ORF is 876 bp and encodes feline PD-L1 consisting of 291 amino acids. The nucleotide sequence and amino acid sequence of feline PD-L1 are set forth in SEQ ID NOs: 18 and 19, respectively. The feline PD-L1 of the present invention preferably consists of the amino acid sequence of SEQ ID NO: 19. The feline PD-L1 gene of the present invention encodes a protein consisting of the amino acid sequence of SEQ ID NO: 19. As long as the function of PD-L1 is maintained, one or several amino acids (e.g., 1, 2, 3, 4, 5, or less, up to about 10) may be deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 19. PD-L1 functions as an immunosuppressant, suppressing T cell activity upon binding to PD-1 on T cells. Cancer cells and infected cells avoid attack by immune cells by binding to PD-L1 expressed on cancer cells and infected cells. PD-L1 also functions as an antigen.

[0061] The DNA encoding feline PD-L1 of the present invention can be prepared, for example, as follows: mRNA is extracted from feline peripheral blood mononuclear cells (PBMCs), and cDNA is synthesized using reverse transcriptase and oligo-dT primers. The feline PD-L1 gene is amplified by PCR using the synthesized cDNA as a template. The amplified PCR product (DNA encoding feline PD-L1) is then inserted into an appropriate expression vector, which is then introduced into a suitable host for production as a recombinant protein, thereby producing feline PD-L1 (see, for example, CURRENT PROTOCOLS Compact Edition, Molecular Biology Experimental Protocols, I, II, III, co-translated by Saigo Kaoru and Sano Yumiko, Maruzen Co., Ltd.; Ausubel, F. M. et al., Short Protocols in Molecular Biology, Third Edition, John Wiley & Sons, Inc., New York). [Example]

[0062] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples. Example 1 1. Introduction The immunoinhibitory receptor PD-1 and its ligand PD-L1 were identified by Tasuku Honjo et al. at Kyoto University as factors that suppress excessive immune responses and are deeply involved in immune tolerance. Their involvement in immunosuppression in various animal infections and tumor diseases has been revealed in recent years, but little is known about the PD-1 / PD-L1 pathway in cats. In this example, an anti-feline PD-L1 monoclonal antibody was produced by immunizing mice, and a clone (CL1Mab-7) capable of detecting feline PD-L1 was selected. Furthermore, the usefulness of the mouse anti-feline PD-L1 monoclonal antibody CL1Mab-7 for detecting PD-L1 in feline mammary adenocarcinoma, squamous cell carcinoma, fibrosarcoma, and renal cell carcinoma was examined using immunohistochemical staining.

[0063] 2. Materials and Methods 2.1. RNA preparation and cDNA synthesis by reverse transcription Total cellular RNA was extracted from testicular tissue and peripheral blood mononuclear cells (PBMCs) of a hybrid cat using TRI reagent (Molecular Research Center), and the concentration was measured using a NanoDrop8000 (Thermo Scientific). RNA samples were stored at -80°C until further investigation. 1 μg of total cellular RNA was treated with DNase I Reaction buffer and 1 U DNase I (Invitrogen), diluted to a total volume of 10 μl with ultrapure water, and then treated with DNase I at room temperature for 15 minutes to 1 hour. Next, 25 mM ethylenediaminetetraacetic acid (EDTA) was added and the mixture was incubated at 65°C for 10 minutes. To this was added 200 pmol of oligo-dT primer (16-mer, Hokkaido System Science) and treated at 65°C for 5 minutes. After that, the reverse transcription reaction solution (Reverse Transcriptase M-MLV Buffer (Takara), 10 mM dNTPs, 10 U RNase inhibitor (Promega), 50 U RT-M-MLV (Takara)) was added, and the final volume was adjusted to 20 μl. Reverse transcription was carried out at 42°C for 60 minutes and 70°C for 10 minutes to synthesize single-stranded cDNA.

[0064] 2.2. Identification of the PD-L1 gene To determine the full-length feline PD-L1 cDNA, primers (fePD-L1_5'GSP1: 5'-CTA GAA TCA TGA AGT GA-3' (SEQ ID NO: 20) and fePD-L1_3'GSP1: 5'-GCT GCT TGA TTG GCT ATG GC-3' (SEQ ID NO: 21)) were designed based on the partial feline PD-L1 nucleotide sequence (GenBank accession number: EU246348) already registered with the National Center for Biotechnology Information (NCBI). 5' and 3' RACE were performed using RNA derived from feline testis tissue using the 5' RACE System for Rapid Amplification of cDNA Ends and the 3' RACE System for Rapid Amplification of cDNA Ends (Invitrogen) to determine the nucleotide sequence of feline PD-L1 cDNA.

[0065] 2.3. Generation of PD-1-Ig and PD-L1-Ig (a) Construction of PD-1-Ig and PD-L1-Ig expression plasmids To construct expression plasmids for recombinant soluble fusion proteins composed of the extracellular domain of feline PD-1 or PD-L1 and the Fc domain of rabbit IgG, i.e., PD-1-Ig and PD-L1-Ig, respectively, the signal peptide (SignalP, http: / / www.cbs.dtu.dk / services / SignalP / ) and transmembrane domain (TMHMM, http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ) in the predicted amino acid sequences of feline PD-1 (GenBank accession number: EU295528.2) and PD-L1 were first predicted using an analysis tool. The cells were cultured for 10 hours after the addition of 5 μg / ml of concanavalin A (ConA; Sigma). PCR was performed using feline PBMC-derived cDNA synthesized according to the method described in Materials and Methods, 2.1, as a template and primers amplifying the full-length open reading frame (ORF) (fePD-1_ORF_F: 5′-ATG GGG ACC CCA CGG GC-3′ (SEQ ID NO: 22) and fePD-1_ORF_R: 5′-CAT GTG TGG AGG TGC AGA GCA G-3′ (SEQ ID NO: 23) or fePD-L1_ORF_F: 5′-CTC CCC GCC GGC AGA AAA-3′ (SEQ ID NO: 24) and fePD-L1_ORF_R: 5′-CTG GTC ATG CTT ACC CCT GAC G-3′ (SEQ ID NO: 25)), and the fragments were cloned into the pGEM-T Easy vector (Promega).A plasmid containing the target gene sequence was extracted using a FastGene Plasmid Mini Kit (Nippon Genetics Co., Ltd.), and the plasmid was used as a template to amplify the predicted extracellular domain. Primers (fePD-1-Ig_F: 5'-CGC GGC TAG CAT GGG GAC CCC ACG GGC GC-3' (SEQ ID NO: 26) and fePD-L1-Ig_F: 5'-CGC GGC TAG CAT GAG GAT ATT TAG TGT CT-3' (SEQ ID NO: 27) or fePD-1-Ig_R: 5'-CGC GGA TAT CCA GCC CCT GGC CTT GGC CG-3' (SEQ ID NO: 28) and fePD-L1-Ig_R: 5'-CGC GGA TAT CCC TCT CAT TTG CTG GAA) were added to the 5' end of the primers to amplify the predicted extracellular domain. PCR was performed using the pCXN2.1-Rabbit IgG Fc vector (p ... N Cloning was performed using the pCXN2.1-PD-1-Ig and pCXN2.1-PD-L1-Ig vectors (Iwa et al., Gene. 1991;108(2):193-199; Zettlmeissl et al., DNA Cell Biol. 1990;9(5):347-353; provided by Dr. Takehiko Yokomizo, Juntendo University; modified in our laboratory to include a gene sequence encoding the rabbit IgG Fc region downstream of the multiple cloning site). Expression plasmids were purified using the FastGene Xpress Plasmid PLUS Kit (Nippon Genetics Co., Ltd.) and stored at -30°C until use in experiments. Hereafter, the constructed expression plasmids are referred to as pCXN2.1-PD-1-Ig and pCXN2.1-PD-L1-Ig. (b) Expression and purification of PD-Ig and PD-L1-Ig in Expi293F cells 7.5 × 10 7Expi293F cells (Life Technologies) were suspended in 25.5 mL of Expi293 Expression Medium (Life Technologies) and plated at 125 cm 3 Cells were cultured in flasks (Corning). 30 μg of pCXN2.1-PD-1-Ig or pCXN2.1-PD-L1-Ig was transfected into the cells using the ExpiFectamine 293 Transfection Kit (Life Technologies) to express the target proteins. PD-1-Ig and PD-L1-Ig were purified from the resulting culture supernatant using Ab-Capcher ExTra (ProteNova). After purification, the solvent was replaced with phosphate-buffered saline (PBS) (pH 7.2; Fujifilm Wako Pure Chemical Industries) using a PD midiTrap G-25 (GE Healthcare) and stored at 4°C or 30°C until further study. Protein concentration was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).

[0066] 2.4. Generation of feline PD-L1-expressing CHO-DG44 cells To prepare an expression plasmid for a recombinant protein fused to full-length feline PD-L1 and enhanced green fluorescent protein (EGFP), PCR was performed using the pGEM-T easy vector containing the full-length feline PD-L1 ORF sequence as a template and primers containing the BglII (forward) or EcoRI (reverse) restriction enzyme recognition site at the 5' end (fePD-L1-EGFP_F: 5'-GAA GAT CTA TGA GGA TAT TTA GTG TCT T-3' (SEQ ID NO: 30) or fePD-L1-EGFP_R: 5'-CGG AAT TCC GTC TCC TCA AAT TGT AGA T-3' (SEQ ID NO: 31)). The resulting PCR product was digested with BglII (Takara) and EcoRI (Takara), purified using the FastGene Gel / PCR Extraction Kit (Nihon Genetics), and cloned into pEGFP-N2 vector (Clontech) that had been similarly digested with restriction enzymes. The resulting expression plasmid was purified using the FastGene Xpress Plasmid PLUS Kit (Nihon Genetics) and stored at -30°C until use in the experiment. 4 × 10 6CHO-DG44 cells (Life Technologies) were transfected with 2.5 μg of the expression plasmid using Lipofectamine LTX (Invitrogen). After 48 hours, the medium was replaced with CD-DG44 medium (Life Technologies) containing 800 μg / ml G418 (Enzo Life Sciences), 20 ml / l GlutaMAX supplement (Life Technologies), and 18 ml / l 10% Pluronic F-68 (Life Technologies). Cells were then seeded into flat-bottom 96-well plates (Corning) using limiting dilution to obtain a large number of stable expression clones. Flow cytometry analysis was performed using a FACS Verse (Becton, Dickinson and Company), and clones with high PD-L1-EGFP expression were selected for further experiments.

[0067] 2.5. Establishment of mouse anti-feline PD-L1 antibodies (a) Generation of mouse anti-feline PD-L1 monoclonal antibody (CL1Mab-7) 100 μg of the resulting feline PD-L1-Ig was intraperitoneally administered to 4-week-old female BALB / c mice (CLEA Japan) with Imject Alum (Thermo Fisher Scientific). After two booster immunizations, spleens were harvested and fused with mouse myeloma cells (P3U1 cells, ATCC) using PEG1500 (Roche Diagnostics) to establish a hybridoma pool. The resulting hybridoma pool was cloned by limiting dilution, and a monoclonal antibody (CL1Mab-7) was purified from the culture supernatant using Protein G Sepharose 4 Fast Flow (GE Healthcare). The isotype of the resulting monoclonal antibody was determined using an isotype-specific secondary antibody (SouthernBiotech). (b) Examination of the binding ability of mouse anti-cat PD-L1 antibody (CL1Mab-7) to PD-L1 The binding ability of CL1Mab-7 to PD-L1 was examined by flow cytometry. 5 PD-L1-EGFP-expressing CHO-DG44 cells and EGFP-expressing CHO-DG44 cells similarly established using the pEGFP-N2 vector (Clontech) were blocked with 200 μl of 10% goat serum (Gibco) in PBS (Fujifilm Wako Pure Chemical Industries, Ltd.) for 15 minutes at room temperature. After washing, 50 μl of 1 μg / mL CL1Mab-7 was added to each plate and incubated for 30 minutes at room temperature. After washing, Alexa Fluor 647-conjugated anti-mouse IgG (H+L) goat F(ab')2 (Thermo Fisher Scientific) was added as a secondary antibody and incubated for 30 minutes at room temperature. Analysis was performed using a FACS Verse (Becton, Dickinson and Company). All washing procedures and antibody dilutions were performed using PBS (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 1% bovine serum albumin (BSA, Sigma).

[0068] 2.6. Analysis of feline PD-L1 expression by flow cytometry Feline macrophage-derived cell line Fcwf-4 [Fcwf] (ATCC CRL-2787) and mammary adenocarcinoma-derived cell lines (FKNp, FMCp, FMCm, FYMp, FONp, and FONm; Uyama et al., J Vet Med Sci. 2005;67(12):1273-1276; provided by Professor Takayuki Nakagawa, University of Tokyo) were cultured for 24 hours under unstimulated or feline IFN-γ-stimulated conditions (Kingfisher Biotech, final concentration 100 ng / mL) at 2 × 10 5Each plate was then aliquoted and blocked with 200 μl of 10% goat serum (Gibco) in PBS (Fujifilm Wako Pure Chemical Industries, Ltd.) at 25°C for 20 minutes. After washing, 50 μl of 10 μg / mL CL1Mab-7 was added to each plate and incubated at 25°C for 30 minutes. After washing, Alexa Fluor 647-labeled anti-mouse IgG (H+L) goat F(ab')2 (Thermo Fisher Scientific) was added as a secondary antibody and incubated at 25°C for 30 minutes. Analysis was performed using a FACS Lyric (Becton, Dickinson and Company). All washing procedures and antibody dilutions were performed using PBS containing 1% BSA (Sigma).

[0069] 2.7. Analysis of PD-L1 expression in feline tumor tissues by immunohistochemical staining 10% neutral-buffered formalin-fixed paraffin-embedded sections from feline mammary gland adenocarcinoma (n = 5), squamous cell carcinoma (n = 5), fibrosarcoma (n = 5), and renal cell carcinoma (n = 2) were cut at 4 μm thickness and subjected to antigen retrieval by microwave irradiation twice at 700 W for 5 minutes in pH 9.0 Tris-EDTA (Agilent Technologies). Endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol, and the sections were incubated with the primary antibody CL1Mab-7 at a final concentration of 10 μg / ml for 30 minutes at room temperature. A mouse IgG1 isotype antibody (clone name: MG1-45, BioLegend) was used as a negative control antibody. Histofine MAX-PO (Multi) (Nichirei) was used as a secondary antibody. Color development was performed using diaminobenzidine (3,3'-Diaminobenzidine Tetrahydrochloride; DAB, Nichirei Corporation) and counterstained with hematoxylin. When observed under an optical microscope, staining of tumor cells was judged to be positive.

[0070] 3. Results 3.1. Identification of the feline PD-L1 gene Although the feline PD-1 gene had been identified, only a partial sequence of the feline PD-L1 gene had been reported (Folkl et al., Vet Immunol Immunopathol. 2010;134(1-2):107-114.). Therefore, we attempted to identify the full-length feline PD-L1 gene. Using cDNA derived from feline testis tissue as a template, the feline PD-L1 gene was amplified by PCR, and its nucleotide sequence was identified. The full-length open reading frame of the feline PD-L1 gene was 876 bp (SEQ ID NO: 18).

[0071] When compared with the PD-L1 genes from dog, bovine, human, mouse, and rat genes registered in GenBank to date, feline PD-L1 showed 83% homology with dog, 77% with bovine, 73% with human, 67% with mouse, and 67% with rat in the predicted amino acid sequence (Fig. 1a). Phylogenetic tree analysis using the predicted amino acid sequences of PD-L1 from each species showed that feline PD-L1 was most closely related to canine PD-L1 and relatively distantly related to mouse and rat PD-L1 (Fig. 1b).

[0072] 3.2. Generation of PD-1-Ig and PD-L1-Ig To generate PD-1-Ig and PD-L1-Ig, the signal peptide, extracellular domain, transmembrane domain, and intracellular domain were predicted from the predicted amino acid sequences of the feline PD-1 and PD-L1 genes (Table 1). The gene sequences encoding the signal peptide and extracellular domain of PD-1 and PD-L1 were inserted into the pCXN2.1-Rabbit IgG Fc vector to generate the PD-1-Ig expression plasmid (pCXN2.1-PD-1-Ig) and the PD-L1-Ig expression plasmid (pCXN2.1-PD-L1-Ig). The resulting plasmids were transfected into Expi293F cells, and the culture supernatants were obtained. The purified recombinant proteins were analyzed by SDS-PAGE (Figure 2). The molecular weights of PD-1-Ig and PD-L1-Ig under reducing conditions were approximately 55 kDa and 60 kDa, respectively, as determined by comparison with molecular weight markers. These values ​​were higher than the predicted molecular weights (42 kDa and 50 kDa) calculated from the predicted amino acid sequences. This is thought to be due to post-translational modifications such as glycosylation during expression in mammalian cells. Furthermore, under non-reducing conditions, both molecules exhibited approximately twice the molecular weight (approximately 150 kDa), suggesting that PD-1-Ig and PD-L1-Ig exist as dimers via disulfide bonds in the attached rabbit IgG Fc region.

[0073] [Table 1]

[0074] 3.3. Establishment of mouse anti-feline PD-L1 antibody CL1Mab-7 Mice were immunized with the feline PD-L1-Ig antibody and hybridomas were generated from splenic lymphocytes, yielding multiple mouse anti-feline PD-L1 monoclonal antibody clones (clone names: CL1Mab-6, CL1Mab-7, CL1Mab-8, CL1Mab-9, and CL1Mab-10). These clones were of the mouse IgG1 subclass with κ light chains. Preliminary immunohistochemical staining of feline renal cell carcinoma revealed that CL1Mab-7 showed the best staining, and therefore was used in subsequent experiments. Next, the binding of CL1Mab-7 to feline PD-L1 was examined by flow cytometry. CL1Mab-7 bound to PD-L1-EGFP-expressing CHO-DG44 cells, but not to EGFP-expressing CHO-DG44 cells (negative control), demonstrating its specific binding to feline PD-L1 (Figure 3).

[0075] 3.4. PD-L1 expression analysis in feline macrophage-derived cell lines PD-L1 expression in the feline macrophage-derived cell line Fcwf-4 was examined by flow cytometry using CL1Mab-7. CL1Mab-7 bound to Fcwf-4 cells cultured under unstimulated conditions, and the amount of binding was higher in cells cultured under IFN-γ stimulation (Figure 4). These results demonstrate that PD-L1 is expressed on Fcwf-4 cells and that its expression is upregulated by IFN-γ stimulation.

[0076] 3.5. Analysis of PD-L1 expression in feline mammary adenocarcinoma-derived cell lines Similarly, PD-L1 expression was examined in the feline mammary adenocarcinoma-derived cell lines FKNp, FMCp, FMCm, FYMp, FONp, and FONm by flow cytometry using CL1Mab-7. CL1Mab-7 bound to FKNp, FMCm, FYMp, FONp, and FONm cells cultured without stimulation, and the binding was higher in cells cultured under IFN-γ stimulation. CL1Mab-7 binding was not observed in FMCp cells under either condition (Figure 5). These results demonstrate that PD-L1 expression is upregulated by IFN-γ stimulation in all feline mammary adenocarcinoma-derived cell lines except FMCp.

[0077] 3.6. Analysis of PD-L1 expression in feline tumor tissues by immunohistochemical staining PD-L1 expression was analyzed by immunohistochemical staining using the established murine anti-feline PD-L1 monoclonal antibody CL1Mab-7 in four feline malignant tumors: mammary adenocarcinoma (n = 5), squamous cell carcinoma (n = 5), fibrosarcoma (n = 5), and renal cell carcinoma (n = 2). Staining in tumor cells on sections was considered positive, and the positivity rate for each tumor type was calculated. The positive rates for PD-L1 expression were 80% (4 of 5) for mammary adenocarcinoma, 100% (5 of 5) for squamous cell carcinoma, 100% (5 of 5) for fibrosarcoma, and 100% (2 of 2) for renal cell carcinoma (Table 2, Figure 6).

[0078] 4. Conclusion These results demonstrate that the mouse anti-feline PD-L1 monoclonal antibody CL1Mab-7 specifically binds to feline PD-L1 and is useful for detecting feline PD-L1 by flow cytometry and immunohistochemical staining.

[0079] [Table 2]

[0080] Example 2 1. Introduction Monoclonal antibodies can be produced by culturing hybridomas and purifying the antibody from the culture supernatant. Furthermore, once the antibody's light and heavy chain gene sequences have been identified, antibody-expressing cells can be produced by transfecting cultured cells with a vector that expresses the gene sequences, and these cells can serve as a substitute for hybridomas. In this example, recombinant murine anti-feline PD-L1 antibody rCL1Mab-7 was produced using a protein expression system using an expression vector and mammalian cells.

[0081] 2. Materials and Methods 2.1 Identification of the light and heavy chain variable region gene sequences of the murine anti-feline PD-L1 antibody CL1Mab-7 The antibody light and heavy chain variable region genes were identified from the hybridoma producing the mouse anti-feline PD-L1 antibody CL1Mab-7 by 5' RACE. The complementarity-determining regions (CDRs) of the mouse anti-feline PD-L1 antibody CL1Mab-7 were determined using NCBI IGBLAST (http: / / www.ncbi.nlm.nih.gov / igblast / ) (Figures 7a and 7b). The nucleotide sequences of the antibody light and heavy chain variable region genes are shown in SEQ ID NOs: 12 and 13, respectively. The amino acid sequences of the antibody light and heavy chain variable region genes are shown in SEQ ID NOs: 6 and 7, respectively. (1) Amino acid sequences of L chain CDR1, 2, and 3 of CL1Mab-7 <cdr1>ESVDSYGNSF (SEQ ID NO: 1) <cdr2>RAS <cdr3>QQSNEDPRT (SEQ ID NO: 2) (2) Amino acid sequences of H chain CDR1, 2, and 3 of CL1Mab-7 <cdr1>GFTFSSYG (SEQ ID NO: 3) <cdr2>ISNGGTYT (SEQ ID NO: 4) <cdr3>ARLGYGSLNWYFDV (SEQ ID NO: 5)

[0082] 2.2 Construction of expression vector for mouse anti-cat PD-L1 antibody CL1Mab-7 Based on the identified gene sequences of the light and heavy chain variable regions of the murine anti-feline PD-L1 antibody CL1Mab-7, forward primers CL1Mab-7_LC_FW (5'-CGC GGC TAG CAT GGA GAC AGA CAC ACT CCT-3' (SEQ ID NO: 32)) and CL1Mab-7_HC_FW (5'-CGC GGC TAG CAT GAA CTT CGG GCT CAG CTT-3' (SEQ ID NO: 33)) containing an NheI restriction enzyme recognition sequence were designed, and reverse primers CL1Mab-7_LC_RV (5'-CGC GGA TAT CCT AAC ACT CAT TCC TGT TGA -3' (SEQ ID NO: 34)) and CL1Mab-7_HC_RV (5'-CGG GGT ACC TCA TTT ACC AGG AGA GTG GG -3' (SEQ ID NO: 35)) containing EcoRV or KpnI restriction enzyme recognition sequences were designed. The light and heavy chain genes of the mouse anti-cat PD-L1 antibody CL1Mab-7 were amplified by PCR using cDNA generated from a hybridoma producing the mouse anti-cat PD-L1 antibody CL1Mab-7. These genes were then inserted into the multicloning site of the expression vector pCXN2.1(+) (Niwa et al., Gene. 1991;108(2):193-199; provided by Dr. Takehiko Yokomizo, Juntendo University) using restriction enzyme recognition sequences to generate expression vectors for the mouse anti-cat PD-L1 antibody CL1Mab-7 light chain and mouse anti-cat PD-L1 antibody CL1Mab-7 heavy chain. The expression plasmids were purified using the FastGene Xpress Plasmid PLUS Kit (Nippon Genetics Co., Ltd.) and stored at -30°C until use in experiments.

[0083] 2.3 Expression and purification of recombinant murine anti-feline PD-L1 antibody rCL1Mab-7 The mouse anti-cat PD-L1 antibody CL1Mab-7 light chain expression pCXN2.1 (+) vector and heavy chain expression pCXN2.1 (+) vector prepared in 2.2 were transfected into Expi293F cells (Life Technologies) using the ExpiFectamine 293 Transfection Kit (Life Technologies) to express the target protein. Recombinant mouse anti-cat PD-L1 antibody rCL1Mab-7 was purified from the resulting culture supernatant using Ab-Capcher ExTra (ProteNova). The buffer was replaced with PBS (pH 7.2, Fujifilm Wako Pure Chemical Industries) using a PD midiTrap G-25 (GE Healthcare), and the solution was concentrated using an Amicon Ultra-0.5 Centrifugal Filter Unit (Merck Millipore). The absorbance at 280 nm was measured using a NanoDrop8000 spectrophotometer (Thermo Fisher Scientific) to determine the concentration of the purified protein.

[0084] 2.4 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Five micrograms of the mouse anti-feline PD-L1 antibody CL1Mab-7, derived from hybridoma culture supernatant purified according to the method described in Section 2.5 of Materials and Methods in Example 1, and the recombinant mouse anti-feline PD-L1 antibody rCL1Mab-7 prepared in Section 2.3 were each aliquoted and treated with an equal volume of 2x Laemmli Sample Buffer (Bio-Rad) supplemented with 2-mercaptoethanol at 96°C for 5 minutes (reducing conditions). A sample solution without 2-mercaptoethanol was also prepared (non-reducing conditions). Sample proteins were then separated by electrophoresis using a polyacrylamide gel (SuperSep Ace, 5-20%, 13-well; Fujifilm Wako Pure Chemical Industries, Ltd.). Precision Plus Protein Dual Color Standards (Bio-Rad) were used as molecular weight markers. After electrophoresis, the gel was stained with a Quick-CBB kit (Fujifilm Wako Pure Chemical Industries, Ltd.) and then destained by shaking in heated distilled water.

[0085] 2.5 Examination of the binding ability of the recombinant mouse anti-feline PD-L1 antibody rCL1Mab-7 to PD-L1 The binding ability of the hybridoma-derived mouse anti-feline PD-L1 antibody CL1Mab-7 and the recombinant mouse anti-feline PD-L1 antibody rCL1Mab-7 to PD-L1 was examined by flow cytometry. 2 × 10 CL1Mab-7 cells were prepared according to the method described in 2.4 of Materials and Methods in Example 1. 5 PD-L1-EGFP-expressing CHO-DG44 cells were suspended in PBS supplemented with 10% goat serum (Gibco) and blocked for 15 minutes at room temperature. After washing, the cells were incubated with the hybridoma-derived mouse anti-cat PD-L1 antibody CL1Mab-7 and recombinant mouse anti-cat PD-L1 antibody rCL1Mab-7, both diluted to 10 μg / ml, for 30 minutes at room temperature. A mouse IgG1 κ isotype control (SouthernBiotech) was used as a negative control. After washing, the cells were incubated with Alexa Fluor 647-conjugated anti-mouse IgG (H+L) goat F(ab')2 (Thermo Fisher Scientific) as a secondary antibody for 30 minutes at room temperature. Analysis was performed using a FACS Lyric (Becton, Dickinson and Company). All washing steps and antibody dilutions were performed in PBS supplemented with 1% bovine serum albumin (Sigma).

[0086] 3. Results 3.1 Generation of recombinant mouse anti-feline PD-L1 antibody rCL1Mab-7 The antibody light and heavy chain gene sequences were identified from a hybridoma producing the mouse anti-cat PD-L1 antibody CL1Mab-7, and expression vectors for each were constructed. Recombinant mouse anti-cat PD-L1 antibody rCL1Mab-7 was expressed using a mammalian cell transient expression system, and purified antibody was isolated from the culture supernatant by affinity chromatography using a protein A derivative. SDS-PAGE analysis of the hybridoma-derived mouse anti-cat PD-L1 antibody CL1Mab-7 and the recombinant mouse anti-cat PD-L1 antibody rCL1Mab-7 revealed bands likely representing heterotetramers at 150-250 kDa under non-reducing conditions, while under reducing conditions, bands likely representing the heavy chain at approximately 50 kDa and the light chain at 25 kDa were observed (Figure 8). The full-length nucleotide and amino acid sequences of the light chain of the mouse anti-cat PD-L1 antibody CL1Mab-7 are shown in SEQ ID NOs: 16 and 10, respectively. The full-length nucleotide sequence and amino acid sequence of the heavy chain of the mouse anti-feline PD-L1 antibody CL1Mab-7 are shown in SEQ ID NOs: 17 and 11, respectively.

[0087] 3.2 Binding of recombinant murine anti-feline PD-L1 antibody rCL1Mab-7 to feline PD-L1-expressing CHO-DG44 cells To investigate the binding ability of the recombinant murine anti-feline PD-L1 antibody rCL1Mab-7 to feline PD-L1, flow cytometry analysis was performed using feline PD-L1-expressing CHO-DG44 cells. Both the hybridoma-derived murine anti-feline PD-L1 antibody CL1Mab-7 and the recombinant murine anti-feline PD-L1 antibody rCL1Mab-7 bound to feline PD-L1-expressing CHO-DG44 cells (Figure 9).

[0088] 4. Conclusion These results suggest that the recombinant mouse anti-feline PD-L1 antibody rCL1Mab-7 has the same molecular structure, purity, and binding ability to feline PD-L1 as the hybridoma-derived mouse anti-feline PD-L1 antibody CL1Mab-7, and that the recombinant mouse anti-feline PD-L1 antibody rCL1Mab-7 can be used as a substitute for the hybridoma-derived mouse anti-feline PD-L1 antibody CL1Mab-7. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. [Industrial Applicability]

[0089] The anti-PD-L1 antibodies of the present invention can be used to diagnose cancer and / or infectious diseases, and can also be used to select test animals suitable for treatment with anti-PD-1 or anti-PD-L1 antibodies. [Sequence List Free Text]

[0090] (1) Amino acid sequences of L chain CDR1, 2, and 3 of CL1Mab-7 <cdr1>ESVDSYGNSF (SEQ ID NO: 1) <cdr2>RAS <cdr3>QQSNEDPRT (SEQ ID NO: 2) (2) Amino acid sequences of H chain CDR1, 2, and 3 of CL1Mab-7 <cdr1>GFTFSSYG (SEQ ID NO: 3) <cdr2>ISNGGTYT (SEQ ID NO: 4) <cdr3>ARLGYGSLNWYFDV (SEQ ID NO: 5) (3) Amino acid and nucleotide sequences of the L chain variable region of CL1Mab-7 <Amino acid sequence> METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMNWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPRTFGGGTKLEIK (SEQ ID NO: 6) <Base sequence> ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGTTCCACAGGTGACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAATAGTTTTATGAATTGGTACCAGCAGAAACCAGGACAGCC ACCCAAACTCCTCATCTATCGTGCATCCAACCTAGAATCTGGGATCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGGCTGATGATGTTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 12) (4) Amino acid and nucleotide sequences of the heavy chain variable region of CL1Mab-7 <Amino acid sequence> MNFGLSLIFLVLILKGVQCEVKLVESGGGLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKRLEWVASISNGGTYTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARLGYGSLNWYFDVWGAGTTVTVSS (SEQ ID NO: 7) <Base sequence> ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTAATTTTAAAAGGTGTCCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGGCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAATGGGTCGCAAGCATCAGTAATGGTGGTACTTACACCTACTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACAACCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCTTGTATTACTGTGCAAGACTAGGATACGGTAGCCTTAACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO: 13) (5) Amino acid sequence and nucleotide sequence of the constant region of the light chain of CL1Mab-7 <Amino acid sequence> RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO: 8) <Nucleotide sequence> CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG(SEQ ID NO: 14) (6) Amino acid and nucleotide sequences of the heavy chain constant region of CL1Mab-7 <Amino acid sequence> AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 9) <Base sequence> GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA(SEQ ID NO: 15) (7) Base sequence and amino acid sequence of the full-length CL1Mab-7 light chain <Base sequence> ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGTTCCACAGGTGACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAATAGTTTTATGAATTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCGTGCATCCAACCTAGAATCTGGGATCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGGCTGATGATGTTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG (SEQ ID NO: 16) <Amino acid sequence> METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMNWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPRTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 10) (8) Nucleotide and amino acid sequences of the full-length heavy chain of CL1Mab-7 <Base sequence> <Amino acid sequence> MNFGLSLIFLVLILKGVQCEVKLVESGGGLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKRLEWVASISNGGTYTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARL GYGSLNWYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKK IVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 11) (9) Nucleotide and amino acid sequences of feline PD-L1 <Base sequence> ATGAGGATATTTAGTGTCTTTGCATTCATGGCCTACTGTCATTTGCTGAAAGCGTTTACGATCACAGTGTCCAAGGACCTGTATGTGGTAGAGTACGGCAGCAATGTGACAATGGAGTGCAGATTCCCCGTAGAAGAACAATTAGACCTGGTTTCACTGATCGTCTACTGGGAAATGGAGGATAAGAAAATCATTCAGTTTGTGCAAGGGAAGGAAGACCTGAAAGTTCAGCACAGAAGCTACAGTCAGAGGGCCCAGCTGTTGAAGGACCAGCTCTTCCTGGGGAAGGCCGCGCTTCAGATCACAAACGTGACCCTGGAGGATGCCGGGGTTTACTGCTGCTTGATTGGCTATGGCGGTGCTGACTATAAGCGGATTACTTTGAAAGTTCATGCCCCATATCGAAAAATCAACCAAAGAATTTCTGTGGATCCTGTCACCTCTGAACATGAACTAATGTGTCAGGCTGAGGGTTACCCAACCGCTGAAGTCATCTGGACAAACAGTGCCCATCAAGTCCTGAATGGCAAAACCATCATCTCTGTTTCCAATATGGAGACAAAGCTTTTCAATGTGACCAGCACGCTGAGAATCAACACAACGGCTAACGAGATTTTCTACTGCACTTTTCTTCAAAGATCAAGTCCCGAGGGAAACAGTACTGCTGAGTTGGTCATCCCAGAACCATTTCTGGTTCCAGCAAATGAGAGGACTCACTTCATGATTCTAGGAGCCATCCTGTTGTTTCTTGTCGTGGTCCCGGCTGTCACTTTCTGTCTGAAGAAACGAGATGTACGAACGATGGATGTGGAAAAATGTGACACCGCAGATATGAACTCAAAGAAGCAAAATGATCTACAATTTGAGGAGACGTAA(SEQ ID NO: 18) <Amino acid sequence> MRIFSVFAFMAYCHLLKAFTITVSKDLYVVEYGSNVTMECRFPVEEQLDLVSLIVYWEMEDKKIIQFVQGKEDLKVQHRSYSQRAQLLKDQLFLGKAALQITNVTLEDAGVYCCLIGYGGADYKRITLKVHAPYRKINQRISVDPVTSEHELMCQAEGYPTAEVIWTNSAHQVLNGKTIISVSNMETKLFNVTSTLRINTTANEIFYCTFLQRSSPEGNSTAELVIPEPFLVPANERTHFMILGAILLFLVVVPAVTFCLKKRDVRTMDVEKCDTADMNSKKQNDLQFEET (SEQ ID NO: 19) SEQ ID NOs: 20 to 35: Show the base sequences of the primers used in the examples.

Claims

1. An anti-PD-L1 antibody comprising: (a) an light chain having a CDR1 having the amino acid sequence of ESVDSYGNSF (SEQ ID NO: 1), a CDR2 having the amino acid sequence of RAS, and a CDR3 having the amino acid sequence of QQSNEDPRT (SEQ ID NO: 2); and (b) an heavy chain having a CDR1 having the amino acid sequence of GFTFSSYG (SEQ ID NO: 3), a CDR2 having the amino acid sequence of ISNGGTYT (SEQ ID NO: 4), and a CDR3 having the amino acid sequence of ARLGYGSLNWYFDV (SEQ ID NO: 5), wherein the antibody is capable of specifically binding to feline PD-L1.

2. The antibody of claim 1, which is derived from a mouse.

3. The antibody of claim 2, which is a mouse anti-feline PD-L1 antibody.

4. The antibody of claim 3, wherein the L chain variable region has the amino acid sequence of SEQ ID NO: 6 and the H chain variable region has the amino acid sequence of SEQ ID NO:

7.

5. The antibody according to claim 1, wherein the L chain constant region has the amino acid sequence of the Kappa chain constant region.

6. The antibody according to claim 1, wherein the H chain constant region has the amino acid sequence of the IgG1 constant region.

7. The antibody according to claim 5 or 6, wherein the L chain constant region has the amino acid sequence of SEQ ID NO: 8 and the H chain constant region has the amino acid sequence of SEQ ID NO:

9.

8. The antibody according to claim 1, which has a four-chain structure consisting of two light chains and two heavy chains.

9. A composition for detecting PD-L1, comprising the antibody of claim 1 as an active ingredient.

10. The composition according to claim 9, which is used for diagnosing cancer and / or infectious diseases.

11. Cancer and / or infectious diseases include neoplastic diseases, leukemia, feline leukemia virus infection, feline immunodeficiency virus infection, feline panleukopenia (feline parvovirus infection), feline infectious peritonitis / feline enteric coronavirus infection, feline calicivirus disease, feline viral rhinotracheitis (feline herpesvirus infection), feline foamy virus infection, poxvirus disease, Borna disease, Aujeszky's disease, severe fever with thrombocytopenia syndrome, feline morbillivirus infection, Campylobacter enteritis, Salmonella infection, Bordetella infection, Pasteurellosis, tetanus, tularemia, atypical mycobacterial infection, The composition according to claim 10, wherein the agent is selected from the group consisting of feline hemoplasmosis, coxiellosis, chlamydiosis, cryptococcosis, dermatophytosis, histoplasmosis, candidiasis, aspergillosis, blastomycosis, coccidioidomycosis, sporotrichosis, protothecia, malasseziosis, Pneumocystis carinii pneumonia, toxoplasmosis, giardiasis, trichomoniasis, amebiasis, balantidiosis, babesiosis, cryptosporidiosis, intestinal coccidiosis, trypanosomiasis, encephalitozoonosis, and cytozoonosis.

12. The composition according to claim 9, which is used to select a subject animal suitable for treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody.

13. DNA encoding the anti-PD-L1 antibody of claim 1.

14. A vector comprising the DNA of claim 13.

15. A host cell transformed with the vector of claim 14.

16. 16. A method for producing an antibody, comprising culturing the host cell of claim 15 and harvesting the anti-PD-L1 antibody from the culture.

17. A host cell transformed with a vector incorporating a DNA encoding an L chain having CDR1 having the amino acid sequence of ESVDSYGNSF (SEQ ID NO: 1), CDR2 having the amino acid sequence of RAS, and CDR3 having the amino acid sequence of QQSNEDPRT (SEQ ID NO: 2), and a vector incorporating a DNA encoding an H chain having CDR1 having the amino acid sequence of GFTFSSYG (SEQ ID NO: 3), CDR2 having the amino acid sequence of ISNGGTYT (SEQ ID NO: 4), and CDR3 having the amino acid sequence of ARLGYGSLNWYFDV (SEQ ID NO: 5), wherein the CDR1, CDR2, and CDR3 of the L chain and H chain are capable of specifically binding to feline PD-L1.

18. 20. A method for producing an antibody, comprising culturing the host cell of claim 17 and harvesting the anti-PD-L1 antibody from the culture.

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