WO2024150525A1 - 抗ネコｐｄ－１抗体 - Google Patents

抗ネコｐｄ－１抗体 Download PDF

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WO2024150525A1
WO2024150525A1 PCT/JP2023/041176 JP2023041176W WO2024150525A1 WO 2024150525 A1 WO2024150525 A1 WO 2024150525A1 JP 2023041176 W JP2023041176 W JP 2023041176W WO 2024150525 A1 WO2024150525 A1 WO 2024150525A1
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feline
antibody
amino acid
acid sequence
fpd1
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French (fr)
Japanese (ja)
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拓也 水野
翔真 西堀
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Yamaguchi University NUC
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Yamaguchi University NUC
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Priority to EP23915192.1A priority Critical patent/EP4650450A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to an antibody that specifically binds to feline PD-1 (Programmed Cell Death 1), comprising specific heavy chain complementarity determining regions (CDRs) 1-3 and specific light chain CDRs 1-3 (hereinafter sometimes referred to as "the anti-feline PD1 antibody”); a polynucleotide that encodes the anti-feline PD1 antibody (hereinafter sometimes referred to as “the anti-feline PD1 antibody coding polynucleotide”); a vector that comprises the anti-feline PD1 antibody coding polynucleotide operably linked downstream of a promoter (hereinafter sometimes referred to as "the anti-feline PD1 antibody expression vector”); host cells into which a D1 antibody expression vector has been introduced (hereinafter, sometimes referred to as "the subject host cells”); a binding inhibitor between feline PD-1 and feline PD-L1 (hereinafter, sometimes referred to as "the subject inhibitor”), which contains the subject anti-feline
  • PD-1 is a receptor that exists on the surface of T cells. It has the function of suppressing the activation of T cells, and has been shown to have functions such as suppressing immune responses against oneself. Such immune response is suppressed when PD-L1, a ligand of PD-1, binds to PD-1. Meanwhile, cancer cells express PD-L1, and the expressed PD-L1 binds to PD-1, thereby suppressing T cell activation and acquiring the ability to escape immune responses. Therefore, inhibiting the binding between PD-1 and PD-L1 is thought to be effective in treating cancer.
  • Patent Document 1 a cancer treatment agent that contains an anti-PD-L1 antibody as an active ingredient and has the effect of suppressing the proliferation of cancer cells in vivo
  • Patent Document 2 an anti-cancer agent that contains an anti-PD-1 antibody or an anti-PD-L1 antibody that restores the reactivity of iNKT cells in which allergies have been induced by administration of an iNKT cell ligand
  • Patent Document 1 anti-PD-1 antibodies are being developed as treatments for melanoma, non-small cell lung cancer, and renal cell cancer.
  • Patent Document 3 anti-canine PD-1 antibodies and anti-canine PD-L1 antibodies that inhibit the binding of PD-1 to PD-L1 in dogs.
  • Patent Document 3 On the other hand, in cats, there are few researchers conducting research on cats, and research on PD-1 and PD-L1 has not progressed, so there have been no reports of anti-feline PD-1 antibodies or anti-feline PD-L1 antibodies.
  • the objective of the present invention is to provide an antibody or the like that specifically binds to feline PD-1 and has the effect of inhibiting the binding between feline PD-1 and feline PD-L1.
  • the present inventors have been working diligently to solve the above problems. In the process, they first confirmed that the anti-canine PD-1 antibodies (i.e., "3B7-D9" and “4F12-E6” in Patent Document 3) previously produced by the present inventors could not detect recombinant feline PD-1 protein. Therefore, in order to produce a mouse monoclonal antibody against feline PD-1, they first produced a mouse cell line expressing feline PD-1 (feline PD-1-expressing cell line), immunized the mouse, and then produced cells (hybridomas) in which B cells (lymph node monocytic cells) and myelomas (P3U1 cells) were fused.
  • B cells lymph node monocytic cells
  • P3U1 cells myelomas
  • an antibody that specifically binds to feline PD-1 comprising the amino acid sequence shown in SEQ ID NO: 11, a heavy chain complementarity determining region (CDR) 1 consisting of the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3;
  • the antibody comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6.
  • [2] The antibody described in [1] above, comprising a heavy chain variable region consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8.
  • [3] The antibody described in [1] or [2] above, comprising a heavy chain consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9, and a light chain consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 10.
  • [5] A vector comprising a promoter and the polynucleotide according to [4] above operably linked downstream of the promoter.
  • [6] A host cell into which the vector described in [5] above has been introduced.
  • [8] A method for inhibiting binding between feline PD-1 and feline PD-L1, comprising the step of administering to a cat the antibody according to any one of [1] to [3] above.
  • inventions of the present invention include the use of the anti-feline PD1 antibody for use in immunotherapy (e.g., cancer immunotherapy) in cats, and the use of the anti-feline PD1 antibody for the manufacture of a preparation for immunotherapy (e.g., cancer immunotherapy) in cats.
  • an antibody that specifically binds to feline PD-1 and has the effect of inhibiting the binding between feline PD-1 and feline PD-L1 can release the inhibition of T cell activation caused by the binding between fPD1 and fPDL1 in cats and activate T cells, and is therefore useful for immunotherapy (e.g., cancer immunotherapy) in cats.
  • the figure shows the results of flow cytometry analysis of an NIH3T3 cell line (hereinafter sometimes referred to as "NIH3T3/fPD1”) (hereinafter sometimes referred to as “fPD1” in the figure) stably expressing feline (Felis silvestris catus) PD-1 (hereinafter sometimes referred to as “fPD1”) or an NIH3T3 cell line (hereinafter sometimes referred to as "NIH3T3”) (hereinafter sometimes referred to as "mock” in the figure) in the presence of four types of anti-fPD1 mouse monoclonal antibodies (1A1 [Figure 1A], 1A11 [Figure 1B], 1D3 [Figure 1C], and 1H11 [ Figure 1D]), incubated in the presence of anti-mouse IgG-PE.
  • NIH3T3/fPD1 stably expressing feline (Felis silvestris catus) PD-1
  • NIH3T3 NIH3T3
  • FIG. 2A shows the results of Western blotting using anti-human IgG for the fusion protein (hereinafter, sometimes referred to as "fPDL1”) of the extracellular domain of feline PD-L1 (hereinafter, sometimes referred to as "fPDL1”) and the human IgG2 Fc domain (hereinafter, sometimes referred to as "hIg").
  • fPDL1 anti-human IgG for the fusion protein
  • fPDL1 extracellular domain of feline PD-L1
  • hIg human IgG2 Fc domain
  • FIG. 2B shows the results of flow cytometry analysis after incubating NIH3T3/fPD1 ("fPD1" in the figure) or NIH3T3 ("mock” in the figure) in the presence of fPDL1-hIg and in the presence of anti-human IgG-PE.
  • PE on the horizontal axis indicates the PE-derived fluorescence intensity in anti-human IgG-PE, and the vertical axis indicates the cell count.
  • NIH3T3/fPD1 was incubated 1) in the presence of four types of anti-fPD1 mouse monoclonal antibodies (1A1 [FIG. 3A], 1A11 [FIG. 3B], 1D3 [FIG. 3C], and 1H11 [FIG.
  • FIG. 4B 1A1-3 [FIG. 4C], 1D3-1 [FIG. 4D], 1D3-2 [FIG. 4E], and 1D3-3 [FIG. 4F]) (arrowhead in the figure), 2) in the presence of fPDL1-hIg (arrow in the figure), or 3) in the presence of the six types of anti-fPD1 mouse monoclonal antibodies and fPDL1-hIg (open arrow in the figure), and then incubated in the presence of anti-human IgG-PE, and the results of flow cytometry analysis were then obtained.
  • the horizontal axis "PE” indicates the fluorescence intensity derived from PE in anti-human IgG-PE, and the vertical axis indicates the number of cells.
  • FIG. 5A shows the results of detecting NIH3T3/fPD1 by Western blotting using anti-FLAG antibody (left) and "1A1-2" (right).
  • FIG. 5B shows the results of incubating NIH3T3/fPD1 ("fPD1" in the figure) or NIH3T3 ("mock” in the figure) in the presence of "1A1-2", respectively, and incubating in the presence of anti-mouse IgG-PE, followed by flow cytometry analysis.
  • FIG. 5B shows the results of incubating NIH3T3/fPD1 ("fPD1" in the figure) or NIH3T3 ("mock” in the figure) in the presence of "1A1-2", respectively, and incubating in the presence of anti-mouse IgG-PE, followed by flow cytometry analysis.
  • FIGS. 5C shows the results of incubating a feline lymphoma cell line (FT-1) (hereinafter sometimes referred to as "FT-1/k-fPD1") or FT-1 in which the fPD1 gene has been knocked out in the presence of "1A1-2", respectively, and incubating in the presence of anti-mouse IgG-PE, followed by flow cytometry analysis.
  • FT-1 feline lymphoma cell line
  • FT-1 in which the fPD1 gene has been knocked out in the presence of "1A1-2”, respectively
  • FIGS. 5B and 5C "PE” on the horizontal axis indicates the fluorescence intensity derived from PE in anti-mouse IgG-PE, and the vertical axis indicates the number of cells.
  • This figure shows the results of flow cytometry analysis after incubating NIH3T3/fPD1 1) in the absence of "1A1-2” and in the presence of hIg ((1) in the figure), 2) in the presence or absence of "1A1-2” and in the presence of fPDL1-hIg ((2) in the figure), or 3) in the presence of "1A1-2” ((3) in the figure), each in the presence of anti-human IgG-PE.
  • PE on the horizontal axis indicates the PE-derived fluorescence intensity in anti-human IgG-PE, and the vertical axis indicates the cell number.
  • PBMCs Peripheral blood mononuclear cells from three healthy cats (Cat 1, Cat 2, and Cat 3) were cultured in the presence of concanavalin A (ConA) and an isotype control (fake antibody) or "1A1-2," and the concentration of feline IFN- ⁇ (interferon- ⁇ , hereafter sometimes referred to as "fIFN ⁇ ") in the culture medium was measured.
  • ConA concanavalin A
  • Fake antibody isotype control
  • fIFN ⁇ feline IFN- ⁇
  • 8A shows the results of flow cytometry analysis after incubating NIH3T3/fPD1 ("fPD1" in the figure) or NIH3T3 ("mock” in the figure) in the presence of "ch-1A1-2” (i.e., a chimeric antibody consisting of the heavy chain variable region of "1A1-2” and the constant region of feline IgG1, and the light chain variable region of "1A1-2” and the constant region of feline IgG1), and incubating in the presence of anti-feline IgG-PE.
  • ch-1A1-2 i.e., a chimeric antibody consisting of the heavy chain variable region of "1A1-2” and the constant region of feline IgG1, and the light chain variable region of "1A1-2” and the constant region of feline IgG1
  • PE on the horizontal axis indicates the fluorescence intensity derived from PE in anti-feline IgG-PE, and the vertical axis indicates the cell count.
  • FIG. 8B shows the results of flow cytometry analysis after incubating NIH3T3/fPD1 1) in the absence of "ch-1A1-2” and in the presence of hIg ((1) in the figure), or 2) in the presence or absence of "ch-1A1-2” and in the presence of fPDL1-hIg ((2) in the figure), respectively, in the presence of anti-human IgG-PE.
  • the horizontal axis "PE” indicates the fluorescence intensity derived from PE in anti-human IgG-PE, and the vertical axis indicates the number of cells.
  • 8C shows the results of measuring the concentration of fIFN ⁇ in the culture medium after culturing PBMCs derived from four healthy cats (cat 1, cat 2, cat 3, and cat 4) in the presence of ConA and isotype control (fake antibody) or "ch-1A1-2".
  • the present anti-feline PD1 antibody is an antibody that specifically binds to feline PD-1 consisting of the amino acid sequence of SEQ ID NO: 11 (i.e., an antibody that recognizes and binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 11 by a highly specific antigen-antibody recognition mechanism), and comprises a heavy (H) chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, an H chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and an H chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3, and a light (L) chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, an L chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and an L chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6 (i.e., the present anti-feline PD1 antibody), and has an effect of inhibiting the binding
  • the above-mentioned H chain CDR1 to 3 are usually contained in the H chain variable region, and the above-mentioned L chain CDR1 to 3 are usually contained in the L chain variable region.
  • Cat refers to any of the following small mammals: domestic cat (Felis silvestris catus), wild cats, and feral cats (domestic cats that have reverted to being wild), all of which are classified in the order Felidae (Carnivora), suborder Felidae, subfamily Felinae, and genus Felis. "Cat” does not include animals of the Felidae family other than cats (e.g. lions, tigers, leopards, etc.).
  • the origin, type, class, form, etc. of the present anti-feline PD1 antibody are not particularly limited, and examples of the present anti-feline PD1 antibody include antibodies derived from mice, rats, cats, etc.; polyclonal antibodies, oligoclonal antibodies (a mixture of several to several tens of antibodies), monoclonal antibodies; chimeric antibodies in which a partial region (e.g., a constant region) of an antibody is replaced with a region derived from a different species (e.g., a cat), F(ab') 2 antibody fragments obtained by digesting a monoclonal antibody with pepsin, Fab' antibody fragments obtained by reducing F(ab') 2 antibody fragments, Fab and other antibody fragments obtained by digesting a monoclonal antibody with papain, and recombinant antibodies such as scFv in which an antibody heavy (H) chain variable region and an antibody light (H) chain variable region are linked by an amino acid bridge; etc.
  • the present anti-feline PD1 antibody is preferably isolated.
  • isolated means that the antibody is present in a state different from the state in which it is naturally present, for example, by artificially removing the antibody from the environment in which it naturally exists or by expressing the antibody in an environment different from the environment in which the antibody naturally exists.
  • isolated antibody does not include an antibody derived from an individual and contained in the body of the individual or in tissues or body fluids (blood, plasma, serum, etc.) derived from the body without being subjected to external manipulation (artificial manipulation).
  • the present anti-feline PD1 antibody is preferably an antibody produced by artificial manipulation (for example, the above-mentioned recombinant antibody).
  • Such "antibodies derived from cells produced by artificial manipulation or antibodies produced from such cells” does not include antibodies that have not been subjected to artificial manipulation, for example, antibodies produced from naturally occurring B cells.
  • framework regions are typically linked to the amino (N) termini and carboxyl (C) termini of each of the H chain CDR1-3 and L chain CDR1-3.
  • H chain FRs include H chain FR1 linked to the N terminus of H chain CDR1, H chain FR2 linked to the C terminus of H chain CDR1 (the N terminus of H chain CDR2), H chain FR3 linked to the C terminus of H chain CDR2 (the N terminus of H chain CDR3), and H chain FR4 linked to the C terminus of H chain CDR3.
  • examples of the L chain FR include L chain FR1 linked to the N-terminus of L chain CDR1, L chain FR2 linked to the C-terminus of L chain CDR1 (N-terminus of L chain CDR2), L chain FR3 linked to the C-terminus of L chain CDR2 (N-terminus of L chain CDR3), and L chain FR4 linked to the C-terminus of L chain CDR3.
  • the above-mentioned H chain FR1 can be, for example, a polypeptide consisting of the 1st to 30th amino acid residues of the amino acid sequence of SEQ ID NO: 7, or a polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence of said polypeptide.
  • the H chain FR2 may be, for example, a polypeptide consisting of the 36th to 49th amino acid residues of the amino acid sequence of SEQ ID NO:7, or a polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence of said polypeptide.
  • the H chain FR3 may be, for example, a polypeptide consisting of the 67th to 98th amino acid residues of the amino acid sequence of SEQ ID NO:7, or a polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence of said polypeptide.
  • the above-mentioned H chain FR4 can be, for example, a polypeptide consisting of the 114th to 124th amino acid residues of the amino acid sequence of SEQ ID NO:7, or a polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence of said polypeptide.
  • L-chain FR1 can be, for example, a polypeptide consisting of the 1st to 23rd amino acid residues of the amino acid sequence of SEQ ID NO:8, or a polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence of said polypeptide.
  • L-chain FR2 can be, for example, a polypeptide consisting of the 35th to 49th amino acid residues of the amino acid sequence of SEQ ID NO:8, or a polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence of said polypeptide.
  • L-chain FR3 can be, for example, a polypeptide consisting of the 57th to 88th amino acid residues of the amino acid sequence of SEQ ID NO:8, or a polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence of said polypeptide.
  • L-chain FR4 can be, for example, a polypeptide consisting of the 98th to 109th amino acid residues of the amino acid sequence of SEQ ID NO:8, or a polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence of said polypeptide.
  • the heavy chain CDR1 in the present anti-feline PD1 antibody is usually located at positions H31-35 in the Kabat numbering (see Kabat, E.A. et al., (1991) NIH Publication No. 91-3242, sequences of proteins of immunological interest).
  • the heavy chain CDR2 in the present anti-feline PD1 antibody is usually located at positions H50-52, H52A, and H53-65 in the Kabat numbering.
  • the heavy chain CDR3 in the present anti-feline PD1 antibody is usually located at positions H95-100, H100A-G, H101, and H102 in the Kabat numbering.
  • the light chain CDR1 in the present anti-feline PD1 antibody is usually located at positions L24-34 in the Kabat numbering.
  • the L chain CDR2 in the present anti-feline PD1 antibody is usually located at positions L50 to L56 in the Kabat numbering.
  • the L chain CDR3 in the present anti-feline PD1 antibody is usually located at positions L89 to L97 in the Kabat numbering.
  • the present anti-feline PD1 antibody preferably comprises an H-chain variable region having an amino acid sequence that has 80% or more sequence identity with the amino acid sequence of SEQ ID NO:7, and an L-chain variable region having an amino acid sequence that has 80% or more sequence identity with the amino acid sequence of SEQ ID NO:8, and more preferably comprises an H-chain having an amino acid sequence that has 80% or more sequence identity with the amino acid sequence of SEQ ID NO:9, and an L-chain having an amino acid sequence that has 80% or more sequence identity with the amino acid sequence of SEQ ID NO:10.
  • the present anti-feline PD1 antibody can be obtained by culturing cells (i.e., the present host cells) produced by genetically introducing the present anti-feline PD1 antibody expression vector into host cells in a culture medium appropriate for the host cells, and recovering the present anti-feline PD1 antibody produced in the culture medium.
  • transgenic animals such as mice, cows, goats, sheep, chickens, and pigs can be produced that incorporate part or all of the anti-feline PD1 antibody expression vector of the present invention, and the anti-feline PD1 antibody of the present invention can be mass-produced from the blood, milk, etc. of such transgenic animals.
  • a non-human animal e.g., mouse, rat
  • a non-human animal e.g., mouse, rat
  • a cell line non-human animal cell line expressing feline PD-1
  • the hybridoma that produces the anti-feline PD-1 antibody can be obtained by 1) screening the hybridomas by an ELISA method using a plate on which feline PD-1 is immobilized, and/or 2) screening the hybridomas that are positive for the non-human animal cell line expressing feline PD-1 by flow cytometry.
  • the polynucleotide encoding the constant region in the anti-feline PD1 antibody-encoding polynucleotide contained in the genomic DNA of the hybridoma can be replaced with a polynucleotide encoding a constant region derived from a different species (e.g., a cat) using the CRISPR/Cas9 system according to the method described in the literature "Sci Adv. 2019 Aug 28; 5(8): eaaw1822.” and the literature “Vet Comp Oncol. 2020 Dec; 18(4): 739-752.” to produce a hybridoma that produces a chimeric antibody derived from a species having different variable and constant regions.
  • the anti-feline PD1 antibody can be purified from the culture supernatant of the hybridoma using known antibody purification techniques.
  • polynucleotide encoding the present anti-feline PD1 antibody may be a polynucleotide that encodes the present anti-feline PD1 antibody (i.e., the present anti-feline PD1 antibody-encoding polynucleotide), and the nucleotides constituting the present anti-feline PD1 antibody-encoding polynucleotide may be DNA or RNA having a natural structure, or may be nucleotides having a structure in which DNA or RNA having a natural structure has been chemically modified (also referred to as "modified nucleic acid").
  • a modified nucleic acid is used to impart resistance to degradation by RNase.
  • the modified nucleic acid is preferably a nucleotide in which the base moiety is modified, for example, a pyrimidine nucleotide substituted at the 5th position, or a pseudouridine which may be substituted at the 1st position, and specific examples of the modified nucleic acid include 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkylpseudouridine.
  • the 1-alkylpseudouridine may be 1-(C1-C6 alkyl)pseudouridine, and is preferably 1-methylpseudouridine or 1-ethylpseudouridine.
  • the polynucleotide encoding the anti-feline PD1 antibody of the present invention can be easily prepared by standard methods based on the amino acid sequence of the anti-feline PD1 antibody of the present invention.
  • the nucleotide sequence encoding the amino acid sequence can be obtained based on the amino acid sequence listed in the sequence listing, and the polynucleotide encoding the anti-feline PD1 antibody of the present invention can be prepared using standard molecular biological and/or chemical procedures.
  • the polynucleotide encoding the anti-feline PD1 antibody of the present invention may contain a nucleotide sequence in which the codons are optimized for expression in a specific host cell.
  • Such an optimized nucleotide sequence can be obtained by applying known algorithms and software to the amino acid sequence of interest.
  • the polynucleotide encoding the anti-feline PD1 antibody of the present invention may be a single-stranded (sense strand) polynucleotide encoding the anti-feline PD1 antibody of the present invention, or a double-stranded polynucleotide consisting of the sense strand and an antisense strand of its complementary sequence, and the form of the polynucleotide is selected to be appropriate for the method of introducing the polynucleotide into cells.
  • the polynucleotide in a single-stranded form, and in the case of plasmid DNA, the polynucleotide may be in a double-stranded form.
  • the polynucleotide encoding the anti-feline PD1 antibody of the present invention preferably includes a polynucleotide encoding an H-chain variable region having an amino acid sequence that has 80% or more sequence identity with the amino acid sequence of SEQ ID NO:7, and a polynucleotide encoding an L-chain variable region having an amino acid sequence that has 80% or more sequence identity with the amino acid sequence of SEQ ID NO:8, and more preferably includes a polynucleotide encoding an H-chain having an amino acid sequence that has 80% or more sequence identity with the amino acid sequence of SEQ ID NO:9, and a polynucleotide encoding an L-chain having an amino acid sequence that has 80% or more sequence identity with the amino acid sequence of SEQ ID NO:10.
  • 80% or more identity means identity of 80% or more (at least 80%), preferably 85% or more, more preferably 88% or more, even more preferably 90% or more, even more preferably 93% or more, particularly preferably 95% or more, especially more preferably 98% or more, and most preferably 100% identity.
  • identity means the degree of polypeptide or polynucleotide sequence similarity, as determined by matching a query sequence with another sequence, preferably of the same type (nucleic acid or protein sequence).
  • Preferred computer program methods for calculating and determining "identity” include GCG BLAST (Basic Local Alignment Search Tool) (Altschul et al., J. Mol. Biol. 1990, 215:403-410; Altschul et al., Nucleic Acids Res. 1997, 25:3389-3402; Devereux et al., Nucleic Acids Res. 1984, 12:387), and BLASTN 2.0 (Gish W., http:// blast. wustl.
  • an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO:X means, in other words, “an amino acid sequence in which 0, 1 or several amino acid residues are deleted, substituted, inserted, and/or added in the amino acid sequence shown in SEQ ID NO:X (amino acid sequence of SEQ ID NO:X) and which has the same function as the amino acid sequence shown in SEQ ID NO:X (amino acid sequence of SEQ ID NO:X).
  • an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted, and/or added means an amino acid sequence in which, for example, 1 to 30 amino acid residues are deleted, substituted, inserted, and/or added, preferably 1 to 20 amino acid residues are deleted, substituted, inserted, and/or added, preferably 1 to 15 amino acid residues are deleted, substituted, inserted, and/or added, preferably 1 to 10 amino acid residue
  • the present anti-feline PD1 antibody expression vector is not particularly limited as long as it contains a promoter and the present anti-feline PD1 antibody-encoding polynucleotide operably linked downstream of the promoter and is capable of transcribing the mRNA encoded by the present anti-feline PD1 antibody-encoding polynucleotide (i.e., the present anti-feline PD1 antibody expression vector).
  • the anti-feline PD1 antibody expression vector of the present invention can be appropriately selected depending on the purpose, and examples thereof include non-viral vectors (e.g., episomal vectors, artificial chromosome vectors, and plasmid vectors) and viral vectors.
  • the vector may be either circular or linear.
  • the promoter in the present anti-feline PD1 antibody expression vector is not particularly limited as long as it is a region to which RNA polymerase (preferably RNA polymerase and general transcription factor) binds and initiates transcription of the mRNA encoded by the present polynucleotide located downstream thereof, and examples thereof include the SR ⁇ promoter, SV40 early promoter, viral LTR (Long Terminal Repeat), CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, HSV-TK (herpes simplex virus thymidine kinase) promoter, EF1 ⁇ promoter, metallothionein promoter, heat shock promoter, etc.
  • RNA polymerase preferably RNA polymerase and general transcription factor
  • the anti-feline PD1 antibody expression vector may contain an enhancer, a polyA addition signal, a marker gene, a replication origin, a gene encoding a polypeptide that binds to the replication origin and controls replication, etc., as desired.
  • a marker gene refers to a gene that allows the selection or selection of cells by introducing the marker gene into cells.
  • Specific examples of the marker gene include drug resistance genes, fluorescent protein genes, luminescent enzyme genes, and chromogenic enzyme genes. These may be used alone or in combination of two or more.
  • Specific examples of the drug resistance genes include neomycin resistance genes, tetracycline resistance genes, kanamycin resistance genes, zeocin resistance genes, and hygromycin resistance genes.
  • fluorescent protein genes include blue fluorescent protein (BFP), green fluorescent protein (GFP) genes, yellow fluorescent protein (YFP) genes, and red fluorescent protein (RFP) genes.
  • BFP blue fluorescent protein
  • GFP green fluorescent protein
  • YFP yellow fluorescent protein
  • RFP red fluorescent protein
  • luminescent enzyme genes include luciferase genes.
  • chromogenic enzyme gene include the ⁇ -galactosidase gene, the ⁇ -glucuronidase gene, and the alkaline phosphatase gene.
  • the host cell of the present invention is not particularly limited as long as it is a cell (i.e., the present host cell) into which the present anti-feline PD1 antibody expression vector is introduced and in which the present anti-feline PD1 antibody is expressed.
  • the host cells survive and are maintained in a liquid or in a moist (wet) state in a liquid (e.g., a culture plate or dish, a tube for cell storage or cell sorting).
  • a liquid e.g., a culture plate or dish, a tube for cell storage or cell sorting.
  • Such liquids include liquids in which the host cells survive and are maintained.
  • the medium is not particularly limited as long as it can be used, and examples of the medium include culture medium (e.g., culture medium containing or not containing serum), saline, phosphate buffered saline, Tris buffered saline, HEPES buffered saline, etc.
  • examples of the liquid include saline, Ringer's solution (lactate Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, etc.), and 5% glucose aqueous solution.
  • the serum may be, for example, 0.1 to 30 (v/v)% serum.
  • examples of the culture medium include culture media for animal cell culture (DMEM, EMEM, IMDM, RPMI1640, ⁇ MEM, F-12, F-10, M-199, AIM-V, etc.).
  • examples of the serum-free culture medium include commercially available B27 supplement (minus insulin) (Life Technologies), N2 supplement (Life Technologies), and B27 supplement (Life Technologies).
  • examples of the culture medium for animal cell culture include the above-mentioned culture medium supplemented with an appropriate amount (for example, 1 to 30%) of a serum substitute such as Serum Replacement (manufactured by Invitrogen) or Knockout Serum Replacement (manufactured by Invitrogen).
  • the host cell of the present invention can be prepared by genetically introducing the anti-feline PD1 antibody expression vector into the host cell.
  • the method of genetically introducing the anti-feline PD1 antibody expression vector into the host cell may be any method suitable for the anti-feline PD1 antibody expression vector and the host cell.
  • a non-viral vector when used as the anti-feline PD1 antibody expression vector, a method using liposomes or microparticles containing cationic lipids or the like, as described in WO 96/10038, WO 97/18185, WO 97/25329, WO 97/30170, and WO 97/31934 (herein incorporated by reference), may be used; see Jin et al, EMBO Mol Med. 2016 Jul; 8(7): 702-711.
  • the method of gene transfer of the anti-feline PD1 antibody expression vector of the present invention into host cells can be exemplified by a method of infecting immune cells with the virus using a culture supernatant containing the recombinant virus (i.e., a viral particle containing a viral vector plasmid [which may be DNA or RNA] in which a foreign gene [in the present application, a polynucleotide encoding the anti-feline PD1 antibody of the present invention] has been incorporated by removing a gene related to pathogenicity from the viral genome) or a concentrated recombinant virus.
  • a culture supernatant containing the recombinant virus i.e., a viral particle containing a viral vector plasmid [which may be DNA or RNA] in which a foreign gene [in the present application, a polynucleotide encoding the anti-feline PD1 antibody of the present invention] has been incorporated by removing a gene
  • the host cell may be any cell into which the anti-feline PD1 antibody-encoding polynucleotide is transcribed and into which the anti-feline PD1 antibody is expressed, and examples of such cells include yeast, mammalian (e.g., human, mouse, rat, dog, cat, etc.) cells, insect (e.g., Spodoptera frugiperda, Trichoplusiani, etc.) cells, and plant (e.g., tobacco, potato, tomato, carrot, soybean, rapeseed, alfalfa, rice, wheat, barley, etc.) cells.
  • yeast mammalian cells
  • insect e.g., Spodoptera frugiperda, Trichoplusiani, etc.
  • plant e.g., tobacco, potato, tomato, carrot, soybean, rapeseed, alfalfa, rice, wheat, barley, etc.
  • the present binding inhibitor between feline PD-1 and feline PD-L1 of the present invention is an agent (i.e., the present inhibitor) containing the present anti-feline PD-1 antibody, which is specified for the use "to inhibit the binding between feline PD-1 and feline PD-L1.”
  • the "binding between feline PD-1 and feline PD-L1" to be inhibited may be in cultured feline cells or in the living cat.
  • the subject inhibitor may be used as a cat food or drink or a cat medicine (formulation) by itself, consisting of the subject anti-feline PD1 antibody, which is the active ingredient, or may be used in the form of a composition (feline food or drink composition or feline medicinal composition) by further mixing with additives.
  • Additives for the inhibitor of the present invention include physiologically acceptable typical carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrants, isotonicity agents, coating agents, solubilizers, lubricants, glidants, dissolution aids, lubricants, flavorings, sweeteners, solvents, gelling agents, nutrients, etc.
  • Specific examples of such additives include water, saline, animal fats and oils, vegetable oils, lactose, starch, gelatin, crystalline cellulose, gum, talc, magnesium stearate, hydroxypropyl cellulose, polyalkylene glycol, polyvinyl alcohol, and glycerin.
  • the subject inhibitor may contain, in addition to the subject anti-feline PD1 antibody, which is the active ingredient, a component that inhibits the binding between feline PD-1 and feline PD-L1.
  • a component that inhibits the binding between feline PD-1 and feline PD-L1 e.g., a compound, a protein [e.g., an antibody], DNA, RNA).
  • the present inhibitor inhibits the binding of feline PD-1 and feline PD-L1, removes the inhibition of T cell activation in cats, and activates T cells in cats, making it advantageously applicable to formulations for immunotherapy (e.g., cancer immunotherapy) in cats.
  • immunotherapy e.g., cancer immunotherapy
  • the present inhibition method for inhibiting the binding between feline PD-1 and feline PD-L1 is not particularly limited, so long as it comprises a step of administering the present anti-feline PD1 antibody to a cat and inhibits the binding between PD-1 and PD-L1 in the living body of a cat (i.e., the present inhibition method).
  • Cats that can be administered with the present anti-feline PD1 antibody may be any cat that requires inhibition of the binding between feline PD-1 and feline PD-L1, specifically cats that require the activation of T cells to be activated by removing the inhibition of T cell activation caused by the binding between PD1 and PDL1, and more specifically cats that require immunotherapy (e.g. cancer immunotherapy) (e.g. cats with cancer, cats at risk of developing cancer, etc.).
  • immunotherapy e.g. cancer immunotherapy
  • the administration form of the anti-feline PD1 antibody in this case can be oral administration in the form of powder, granules, tablets, capsules, syrup, etc., or parenteral administration in the form of topical skin preparation, transdermal preparation, transmucosal preparation, nasal preparation, enteral preparation, injection, suppository, inhalant, patch, etc.
  • the dosage of the present anti-feline PD1 antibody is determined appropriately depending on the cat's age, weight, sex, symptoms, drug sensitivity, etc., and is, for example, in the range of 1 ⁇ g to 100 mg/kg (body weight)/day.
  • the present anti-feline PD1 antibody is administered once or multiple times (for example, 2 to 4 times) per day, and the dosage may be adjusted depending on the improvement of the cat's symptoms.
  • the present inhibitory method inhibits the binding between feline PD-1 and feline PD-L1 by administering the present anti-feline PD1 antibody to a cat, thereby releasing the inhibition of T cell activation in the cat and activating the T cells in the cat, and therefore can be advantageously applied to immunotherapy (e.g., cancer immunotherapy) in cats.
  • immunotherapy e.g., cancer immunotherapy
  • the present inhibition kit is a kit (i.e., the present inhibition kit) containing the present anti-feline PD1 antibody or a labeled product thereof, or the present anti-feline PD1 antibody expression vector, specified for the use "to inhibit the binding between feline PD-1 and feline PD-L1", and such a kit usually contains the items generally contained in this type of kit, such as a carrier, a pH buffer, a stabilizer, an instruction manual, and instructions for inhibiting the binding between feline PD-1 and feline PD-L1.
  • the "binding between feline PD-1 and feline PD-L1" to be inhibited may be in cultured feline cells or in the living cat.
  • Examples of the labeling substance in the label of the anti-feline PD1 antibody of the present invention include enzymes such as peroxidase (e.g., horseradish peroxidase [HRP]), alkaline phosphatase, ⁇ -D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apo-glucose oxidase, urease, luciferase, and acetylcholinesterase; fluorescent substances such as fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelates, dansyl chloride, and tetramethylrhodamine isothiocyanate; green fluorescent protein (GFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), red fluorescent protein (Red Fluorescence Protein), and the like.
  • fluorescent protein examples include fluorescent protein such as radioisotope (RFP), luciferase, and the like; radioisotopes such as 3 H, 14 C, 125 I, and 131 I, biotin, avidin, and chemiluminescent substances.
  • fluorescent protein such as radioisotope (RFP), luciferase, and the like
  • radioisotopes such as 3 H, 14 C, 125 I, and 131 I, biotin, avidin, and chemiluminescent substances.
  • the primers used in PCR in the following examples are shown in Table 1.
  • the NIH3T3 cell line which is a mouse fibroblast cell line used in the following examples, was cultured and maintained in a DMEM culture medium containing 10% FBS, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin, and 55 ⁇ M 2-mercaptoethanol (hereinafter simply referred to as "DMEM culture medium") under conditions of 5% CO 2 and 37° C.
  • DMEM culture medium containing 10% FBS, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin, and 55 ⁇ M 2-mercaptoethanol
  • PCR was performed under the conditions of pre-denaturation at 94 ° C for 2 minutes, followed by denaturation at 98 ° C for 10 seconds, annealing reaction at 58 ° C for 30 seconds, and extension reaction at 68 ° C for 60 seconds for 35 cycles, and then a final extension reaction was performed at 68 ° C for 10 minutes.
  • the following PCR was also performed under the above conditions.
  • PCR product i.e., cDNA of fPD1
  • cDNA of fPD1 was gel-purified, cleaved with BamHI and EcoRI, and then inserted between two restriction enzyme sites (BamHI and EcoRI) in the pMXs-IP vector to prepare pMX-IP-fPD1.
  • PCR was performed using forward primer 2 (single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 15) and reverse primer 2 (single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 16) and BigDye (registered trademark) Termination v3.1 Cycle Sequencing Kit (manufactured by Perkin-Elmer) according to the protocol attached to the product, and the amplified PCR product was confirmed to be a nucleotide sequence (feline PD-1 gene consisting of the nucleotide sequence of SEQ ID NO: 12) encoding fPD1 (feline PD-1 consisting of the amino acid sequence of SEQ ID NO: 11) using an ABI Prism (registered trademark) 377 automated DNA sequencer (manufactured by Applied Biosystems).
  • fPD1-expressing retroviral vector expressing a protein (FLAG-fused fPD1) in which two FLAG tags (2xFLAG) are fused to the C-terminus of fPD1
  • pMX-IP-fPD1 was used as a template, and PCR was performed using forward primer 1, reverse primer 3 (a single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 17 [i.e., a nucleotide sequence encoding a polypeptide containing a FLAG tag at the C-terminus of fPD1]), and KOD-Plus-Neo according to the protocol attached to the product to obtain a primary PCR product.
  • PCR was performed using the primary PCR product as a template, forward primer 1, reverse primer 4 (a single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 18 [i.e., a nucleotide sequence encoding a 2xFLAG tag]), and KOD-Plus-Neo according to the protocol attached to the product to obtain a secondary PCR product.
  • forward primer 1 a single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 18 [i.e., a nucleotide sequence encoding a 2xFLAG tag]
  • KOD-Plus-Neo according to the protocol attached to the product to obtain a secondary PCR product.
  • the obtained secondary PCR product was introduced into the restriction enzyme site SmaI of the pbluescript SK(-) vector, and then further cleaved with EcoRI and NotI, and then inserted between the two restriction enzyme sites (EcoRI and NotI) of the pMXs-IP vector to prepare pMX-IP-fPD1-FL (i.e., an fPD1-expressing retroviral vector).
  • fPD1-expressing mouse cell line 3.5 ⁇ 10 5 NIH3T3 cell lines were seeded in a 6-well dish, and one day later, pMX-IP-fPD1-FL was transfected into the NIH3T3 cell line using PEI Max (Polysciences) according to the protocol attached to the product, and incubated for 48 hours. Then, the cells were cultured in the presence of 10 ⁇ g/ml puromycin (Sigma-Aldrich) to prepare NIH3T3/fPD1 (i.e., an NIH3T3 cell line stably expressing fPD1).
  • fPD1 in the above NIH3T3/fPD1 was confirmed by immunofluorescence using anti-FLAG M2 antibody (1:1000 dilution, Sigma-Aldrich) as the primary antibody and IgG-Alexa (registered trademark) 647-labeled anti-mouse (Jackson Immunoresearch) as the secondary antibody.
  • mice with NIH3T3/fPD1 and subsequent screening of NIH3T3/fPD1 positive hybridomas 500 ⁇ L of DMEM culture solution containing 1 ⁇ 10 7 NIH3T3/fPD1 was emulsified with an equal amount of Titer Max (registered trademark) Gold (manufactured by CytRx) and administered intradermally to the hind footpad of a 5-week-old Balb/c mouse (manufactured by SLC). A total of four administrations were performed every other week, and 10 days after the last administration, popliteal lymph node cells were isolated and fused with P3U1 cells.
  • Titer Max registered trademark
  • SLC 5-week-old Balb/c mouse
  • lymph node monocytic cells (1 ⁇ 10 8 cells) were mixed with P3U1 cells (2 ⁇ 10 7 cells) and washed twice with serum-free RPMI1640 culture solution. Then, following the instructions of the ClonaCell-HY Hybridoma Kit (Veritas), the cells were fused and seeded (100 ⁇ L/well) into four 96-well tissue culture plates. After colonies were obtained, NIH3T3 cell-positive hybridomas expressing fPD1 were screened by ELISA and flow cytometry, and 288 types of NIH3T3/fPD1-positive hybridomas were obtained.
  • hybridomas producing anti-fPD1 mouse monoclonal antibodies were screened by ELISA, and as a result, four types of hybridomas (hybridoma 1A1, hybridoma 1A11, hybridoma 1D3, and hybridoma 1H11) were obtained (in this specification, the anti-fPD1 mouse monoclonal antibody produced by hybridoma 1A1 may be referred to as "1A1", the anti-fPD1 mouse monoclonal antibody produced by hybridoma 1A11 may be referred to as "1A11", the anti-fPD1 mouse monoclonal antibody produced by hybridoma 1D3 may be referred to as "1D3”, and the anti-fPD1 mouse monoclonal antibody produced by hybridoma 1H11 may be referred to as "1H11").
  • hybridomas producing anti-fPD1 antibodies were screened by ELISA, and as a result, four types of hybridomas (hybridoma 1A1, hybridoma 1A11,
  • 2 x 10 5 NIH3T3/fPD1 or NIH3T3 were incubated in the presence of 10 ⁇ g/mL of each of the above four types of mouse monoclonal antibodies, and then incubated in the presence of anti-mouse IgG-PE (manufactured by Southern Biotech), and the fluorescence intensity derived from PE was detected using a flow cytometer (CytoFLEX [manufactured by Beckman Coulter]), and the obtained results were analyzed using FlowJo software (manufactured by Treestar).
  • fPDL1 expression retroviral vector In order to amplify the cDNA of fPDL1 (the nucleotide sequence of NCBI Reference Sequence: XM_006939039.3), PCR was performed using the cDNA of the feline lymphoblast cell line FL-4 as a template, forward primer 3 (single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 19), reverse primer 5 (single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 20), and KOD-Plus-Neo (manufactured by Toyobo Co., Ltd.) according to the protocol attached to the product.
  • forward primer 3 single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 19
  • reverse primer 5 single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 20
  • KOD-Plus-Neo manufactured by Toyobo Co., Ltd.
  • the amplified PCR product i.e., the cDNA of fPDL1
  • the amplified PCR product was gel-purified, cleaved with EcoRI and XhoI, and then inserted between the two restriction enzyme sites (EcoRI and XhoI) in the pMXs-IP vector to prepare pMX-IP-fPDL1-K.
  • PCR was performed using forward primer 2 (single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 15) and reverse primer 2 (single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 16) and BigDye (registered trademark) Termination v3.1 Cycle Sequencing Kit (manufactured by Perkin-Elmer) in accordance with the protocol attached to the product, and then the amplified PCR product was confirmed to be a nucleotide sequence (feline PD-L1 gene consisting of the nucleotide sequence of SEQ ID NO: 12) encoding fPDL1 (feline PD-L1 consisting of the amino acid sequence of SEQ ID NO: 11) using an ABI Prism (registered trademark) 377 automated DNA sequencer (manufactured by Applied Biosystems).
  • fPDL1-hIg Expression Vector In order to amplify a polynucleotide encoding the extracellular region of fPDL1, pMx-IP-fPDL1 was used as a template, and PCR was performed using forward primer 3 (single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 19) and reverse primer 6 (single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 22) and KOD-Plus-Neo (manufactured by Toyobo Co., Ltd.) according to the protocol attached to the product to obtain a primary PCR product.
  • forward primer 3 single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 19
  • reverse primer 6 single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 22
  • KOD-Plus-Neo manufactured by Toyobo Co., Ltd.
  • PCR was performed using the primary PCR product as a template, forward primer 3, reverse primer 4 (single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 18 [i.e., a nucleotide sequence encoding a 2xFLAG tag]), and KOD-Plus-Neo according to the protocol attached to the product to obtain a secondary PCR product.
  • the obtained secondary PCR product was inserted into a pANT vector to prepare pANT-fPD1-FL.
  • PCR was performed using pANT-fPD1-FL as a template, forward primer 4, reverse primer 7 primer (nucleotide sequence of SEQ ID NO: 23), and KOD-Plus-Neo according to the protocol attached to the product to obtain a tertiary PCR product (i.e., a polynucleotide encoding the extracellular domain of fPDL1).
  • pFUSE-hIgG2-Fc2 Exvivogen
  • hIg human IgG2 Fc domain [hereinafter sometimes referred to as "hIg”] expression vector
  • 2 x 10 5 NIH3T3/fPD1 or NIH3T3 were incubated in the presence of fPDL1-hIg at various concentrations (0 ⁇ g/mL, 0.16 ⁇ g/mL, 0.63 ⁇ g/mL, 2.5 ⁇ g/mL, or 10 ⁇ g/mL), and then incubated in the presence of anti-human IgG-PE (Southern Biotech).
  • the fluorescence intensity derived from PE was detected using a flow cytometer (CytoFLEX [Beckman Coulter]), and the results obtained were analyzed using FlowJo software (Treestar).
  • hybridomas three types of hybridomas (hybridoma 1A1-1, hybridoma 1A1-2, and hybridoma 1A1-3) were isolated from hybridoma 1A1, and three types of hybridomas (hybridoma 1D3-1, hybridoma 1D3-2, and hybridoma 1D3-3) were isolated from hybridoma 1D3.
  • the mouse monoclonal antibody produced by hybridoma 1A1-1 may be referred to as "1A1-1," the mouse monoclonal antibody produced by hybridoma 1A1-2 as “1A1-2,” the mouse monoclonal antibody produced by hybridoma 1A1-3 as "1A1-3,” the mouse monoclonal antibody produced by hybridoma 1D3-1 as "1D3-1,” the mouse monoclonal antibody produced by hybridoma 1D3-2 as "1D3-2,” and the mouse monoclonal antibody produced by hybridoma 1D3-3 as "1D3-3”).
  • the hybridoma 1A1-2 is stored at the Joint Faculty of Veterinary Medicine, Yamaguchi University, a national university corporation, and can be sold under certain conditions.
  • fPD1 gene was knocked out of a feline lymphoma cell line (FT-1) using the CRISPR/Cas9 system described in the literature "Vet Comp Oncol. 2020 Dec;18(4):739-752.” to generate FT-1/ko-fPD1. Then, flow cytometry analysis was performed on FT-1/ko-fPD1 or FT-1 using 10 ⁇ g/mL of "1A1-2" according to the method described in Example 2 above. As a result, it was shown that "1A1-2" specifically binds to fPD1 in FT-1 ( Figure 5C).
  • NIH3T3/fPD1 was 1) incubated in the absence of "1A1-2” and in the presence of hIg, 2) incubated in the presence of "1A1-2" at various concentrations (0 ⁇ g/mL, 0.16 ⁇ g/mL, 0.63 ⁇ g/mL, 2.5 ⁇ g/mL, 10 ⁇ g/mL, or 40 ⁇ g/mL) and fPDL1-hIg, or 3) incubated in the presence of 40 ⁇ g/mL of "1A1-2", and then incubated in the presence of anti-human IgG-PE, and then flow cytometry analysis was performed according to the method described in "3-5" above.
  • the antibody heavy chain CDR1 is located at positions H31-35 in the Kabat numbering
  • the antibody heavy chain CDR2 is located at positions H50-52, H52A, and H53-65 in the Kabat numbering
  • the antibody heavy chain CDR3 is located at positions H95-100, H100A-G, H101, and H102 in the Kabat numbering
  • the antibody light chain CDR1 is located at positions L24-34 in the Kabat numbering
  • the antibody light chain CDR2 is located at positions L50-56 in the Kabat numbering
  • the antibody light chain CDR3 is located at positions L89-97 in the Kabat numbering.
  • hybridoma ch-1A1-2 was prepared that produces a chimeric antibody consisting of the heavy chain variable region of "1A1-2” and the constant region of feline IgG1, and the light chain variable region of "1A1-2” and the constant region of feline IgG1 (in this specification, the mouse monoclonal chimeric antibody produced by hybridoma ch-1A1-2 may be referred to as "ch-1A1-2").
  • the amino acid sequences of the heavy and light chains of "ch-1A1-2” are shown in Table 4.
  • the hybridoma ch-1A1-2 is kept at the Joint School of Veterinary Medicine, Yamaguchi University, a national university corporation, and can be sold under certain conditions.
  • NIH3T3/fPD1 was 1) incubated in the absence of "ch-1A1-2” and in the presence of hIg, or 2) incubated in the presence of various concentrations (0 ⁇ g/mL, 0.16 ⁇ g/mL, 0.63 ⁇ g/mL, 2.5 ⁇ g/mL, 10 ⁇ g/mL, or 40 ⁇ g/mL) of "ch-1A1-2” and fPDL1-hIg, and each was incubated in the presence of anti-human IgG-PE, and then flow cytometry analysis was performed according to the method described in "3-5" above.
  • PBMCs were isolated from four healthy cats (cat 1, cat 2, cat 3, and cat 4) according to a standard method, and then the PBMCs were seeded in a 96-well round-bottom plate at 2 x 10 5 cells per well, and 10 ⁇ g/mL ConA was added, and at the same time, a pseudo antibody (feline IgG, Jackson Immunoresearch) or "ch-1A1-2" was added. After 72 hours of culture, the culture supernatant was collected, and fIFN ⁇ in the culture supernatant was measured by Feline IFN-gamma DuoSet ELISA (R&D).
  • the present invention is useful for immunotherapy (e.g., cancer immunotherapy) in cats.
  • immunotherapy e.g., cancer immunotherapy

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