Anti-cat antibody in which cytotoxic activity and cell phagocytic activity are reduced

Abstract

The present invention addresses the problem of providing an anti-cat antibody in which cytotoxic activity and cell phagocytic activity are reduced without adversely affecting the function of a variable region or binding to FcRn.　The anti-cat antibody comprises a heavy chain into which an amino acid substitution capable of reducing cytotoxic activity and cell phagocytic activity has been introduced, and a light chain.

Description

Anti-cat antibodies with reduced cytotoxic and phagocytic activities

[0001] The present invention relates to anti-feline antibodies and the like comprising a light chain and a heavy chain into which amino acid substitutions that reduce cytotoxic activity or cytophagic activity have been introduced.

[0002] In humans, antibody drugs targeting immune checkpoint molecules, which are immunoregulatory molecules, have recently attracted attention, and many clinical trials are being conducted. In particular, clinical trials targeting PD-1 (Programmed cell death 1) and PD-L1 (Programmed cell death 1 ligand-1) in human cancers have achieved excellent results, and are attracting attention as next-generation cancer treatments.

[0003] PD-1 is a receptor present on the surface of T cells. It has been shown to have the function of suppressing T cell activation and to suppress immune responses against the self. This suppression of immune responses is achieved by the binding of PD-1's ligand, PD-L1, to PD-1. On the other hand, 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 of PD-1 and PD-L1 is thought to be effective in treating cancer.

[0004] To date, there have been proposed a cancer therapeutic agent containing an anti-PD-L1 antibody as an active ingredient and having the effect of suppressing cancer cell proliferation in vivo (Patent Document 1), and an anti-cancer agent containing an anti-PD-1 antibody or an anti-PD-L1 antibody that restores the reactivity of iNKT cells in which allergy has been induced by the administration of an iNKT cell ligand (Patent Document 2). Furthermore, anti-PD-1 antibodies are being developed as therapeutic agents for melanoma, non-small cell lung cancer, and renal cell cancer (Non-Patent Document 1).

[0005] Meanwhile, as pets have become longer-lived, the number of cases of pet cancer has been increasing rapidly in recent years. As with humans, cancer treatment for pets has also progressed, with the three main cancer treatments being actively used: surgery, radiation therapy, and chemotherapy (anticancer drugs).

[0006] Dogs and cats are the most commonly kept pets, and according to a 2020 nationwide survey of dog and cat ownership conducted by the Pet Food Association, there are approximately 8.5 million dogs and 9.6 million cats in Japan. As the number of dogs and cats kept as pets increases, the incidence of cancer in these animals is also on the rise. However, the three major cancer therapies mentioned above have limitations, and new treatments are needed. Potential treatments targeting immune checkpoint molecules such as PD-1 and PD-L1 hold promise, but anti-human PD-1 and anti-human PD-L1 antibodies have the problem of not working in dogs or cats.

[0007] The present inventors have discovered 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).More recently, the present inventors have also discovered an anti-feline PD-1 antibody that inhibits the binding of PD-1 to PD-L1 in cats (Patent Document 4).

[0008] On the other hand, feline IgG mainly exists in only two types, IgG1 and IgG2, and both feline IgG1 and IgG2 have been reported to have effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) (Non-Patent Document 2). Therefore, even if an anti-feline immune checkpoint molecule antibody such as an anti-feline PD-1 antibody is administered to a cat, cytotoxicity and phagocytosis of feline T cells are induced, resulting in the disappearance of feline T cells, and it is thought that this will not fully activate the immune response. For this reason, there has been a demand for anti-feline antibodies with reduced effector functions.

[0009] It has been reported that amino acid substitutions can be introduced into specific sites in the IgG1 heavy chain constant region of an anti-feline antibody to produce an anti-feline antibody with reduced effector function (Patent Document 5). However, it was not previously known that introducing a combination of four amino acid substitutions (M119A, L120A, P214G, and S215A) or a combination of three amino acid substitutions (M119S, L120T, and G121R) into the IgG1 heavy chain constant region of an anti-feline antibody reduces effector function.

[0010] Japanese Patent Application Laid-Open No. 2014-65748 Japanese Patent Application Laid-Open No. 2010-83863 International Publication No. 2016 / 006241 Pamphlet International Publication No. 2024 / 150525 Pamphlet Special Publication No. 2024-530036

[0011] Suzanne L. et al., The New England Journal of Medicine 2012 366: 2443-2454. Strietzel CJ et al., Vet Immunol Immunopathol. 2014 158: 214-223.

[0012] An object of the present invention is to provide an anti-feline antibody that has reduced cytotoxic activity and cytophagocytic activity without adversely affecting the function of the variable region.

[0013] In order to solve the above-mentioned problems, the inventors, relying on their experience and intuition, diligently investigated various mutations in the IgG1 heavy chain constant region of an anti-feline antibody (a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4). After much trial and error, they discovered that introducing a combination of four amino acid substitutions (M119A, L120A, P214G, and S215A) or a combination of three amino acid substitutions (M119S, L120T, and G121R) reduces the cytotoxic activity and cytophagocytic activity of the feline antibody without adversely affecting the function of the variable region, and thus completed the present invention.

[0014] That is, the present invention is as follows: [1] An anti-cat antibody comprising a light chain and a heavy chain, wherein the constant region of the heavy chain consists of an amino acid sequence having an alanine residue at position 119, an alanine residue at position 120, a glycine residue at position 214, and an alanine residue at position 215 of the amino acid sequence shown in SEQ ID NO: 1, and having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having a serine residue at position 119, a threonine residue at position 120, and an arginine residue at position 121 of the amino acid sequence shown in SEQ ID NO: 2, and having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having an alanine residue at position 119, an alanine residue at position 120, a glycine residue at position 214, and an alanine residue at position 215 of the amino acid sequence shown in SEQ ID NO: 5, and having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 5, or

[0014]

[0015] The anti-feline antibody, which comprises an amino acid sequence having the serine residue at position 119, the threonine residue at position 120, and the arginine residue at position 121 of the amino acid sequence set forth in SEQ ID NO: 6, and which has at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 6. [2] The anti-feline antibody according to [1] above, which specifically binds to an immune checkpoint molecule. [3] The anti-feline antibody according to [2] above, wherein the immune checkpoint molecule is PD-1. [4] A polynucleotide encoding the anti-feline antibody according to any of [1] to [3] above. [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 according to [5] above has been introduced. [7] An agent for activating immune responses in cats, comprising the anti-feline antibody according to [2] or [3] above, or a vector comprising a promoter and a polynucleotide encoding the anti-feline antibody according to [2] or [3] above, operably linked downstream of the promoter.[8] A method for activating an immune response in a cat, comprising the step of administering to the cat the anti-cat antibody described in [2] or [3] above, or a vector comprising a promoter and a polynucleotide encoding the anti-cat antibody described in [2] or [3] above, operably linked downstream of the promoter.

[0015] Other embodiments of the present invention include: the anti-feline immune checkpoint molecule antibody or the anti-feline immune checkpoint molecule antibody expression vector for use in activating immune responses in cats; use of the anti-feline immune checkpoint molecule antibody or the anti-feline immune checkpoint molecule antibody expression vector for producing an activator of immune responses in cats; the anti-feline immune checkpoint molecule antibody or the anti-feline immune checkpoint molecule antibody expression vector for use in immunotherapy (e.g., cancer immunotherapy) in cats, and use of the anti-feline immune checkpoint molecule antibody or the anti-feline immune checkpoint molecule antibody expression vector for producing a formulation for immunotherapy (e.g., cancer immunotherapy) in cats.

[0016] In another embodiment of the present invention, there is provided an anti-cat antibody comprising a light chain and a heavy chain, wherein the constant region of the heavy chain comprises one, two, or three amino acid residues selected from the alanine residue at position 119, the alanine residue at position 120, the glycine residue at position 214, and the alanine residue at position 215 of the amino acid sequence shown in SEQ ID NO: 1, and consists of an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or one or two amino acid residues selected from the serine residue at position 119, the threonine residue at position 120, and the arginine residue at position 121 of the amino acid sequence shown in SEQ ID NO: 2, and consists of an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, Examples of the anti-cat antibody include an amino acid sequence having one, two, or three amino acid residues selected from an alanine residue at position 119, an alanine residue at position 120, a glycine residue at position 214, and an alanine residue at position 215 of the amino acid sequence shown in SEQ ID NO: 5, and having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having one or two amino acid residues selected from a serine residue at position 119, a threonine residue at position 120, and an arginine residue at position 121 of the amino acid sequence shown in SEQ ID NO: 6, and having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 6.

[0017] The present anti-feline antibody has reduced cytotoxic activity and cytophagocytic activity without adversely affecting the function of the variable region or binding to neonatal Fc receptors (FcRn). Therefore, by adopting an antibody that specifically binds to an immune checkpoint molecule as the present anti-feline antibody, it is possible to reduce cytotoxicity and induction of cytophagocytosis in feline T cells and more effectively activate the immune response by feline T cells, making it useful for immunotherapy (e.g., cancer immunotherapy) in cats.

[0018] FIG. 1 shows the results of Western blotting analysis of protein extracts prepared from four types of cell lines (NIH3T3 cell line ["mock" in the figure], NIH3T3 / fCD16 ["fCD16" in the figure], NIH3T3 / fCD32 ["fCD32" in the figure], and NIH3T3 / fCD64 ["fCD64" in the figure]) using an anti-FLAG antibody (upper panel) and an anti-Vinculin antibody (lower panel). Two types of stably expressing cell lines (NIH3T3 / fCD16 [Fig. 2A] or NIH3T3 / fCD32 [Fig. 2B]) were incubated either 1) in the absence of a primary antibody or a secondary antibody, 2) in the absence of a primary antibody but in the presence of a secondary antibody (IgG-Alexa (registered trademark) 647-labeled anti-cat [anti-cat IgG-Alexa647]), or 3) in the presence of three types of primary antibodies (i.e., three types of antibodies [fIgG, "ch-1A1-2," or "ch-1A1-2 M1"]) and the above-mentioned secondary antibodies), and then the Alexa647-derived fluorescence intensity (horizontal axis) in the above-mentioned cell lines was analyzed by flow cytometry. The vertical axis represents the cell number. Four types of cell lines (NIH3T3 cell line [Figure 3A], NIH3T3 / fCD16 [Figure 3B], NIH3T3 / fCD32 [Figure 3C], or NIH3T3 / fCD64 [Figure 3D]) were incubated either 1) in the absence of a primary antibody and a "biotin-labeled secondary antibody / Dylight649-labeled streptavidin," or 2) in the absence of a primary antibody but in the presence of a "biotin-labeled secondary antibody / Dylight649-labeled streptavidin," or 3) in the presence of four types of primary antibodies (two types of comparative antibodies [fIgG or "ch-1A1-2"], or two types of the present anti-cat antibodies ["ch-1A1-2 M2" or "ch-1A1-2 This figure shows the results of flow cytometry analysis of the fluorescence intensity (horizontal axis) derived from Dylight649 in the cell line after incubation in the presence of a primary antibody (antibody labeled with β-glucan-1-phosphate dehydrogenase (B2) or β-glucan-1-phosphate dehydrogenase (M3)) and in the presence of "biotin-labeled secondary antibody / Dylight649-labeled streptavidin." The vertical axis shows the cell number.FIG. 1 shows the results of analyzing the binding of three types of antibodies ("ch-1A1-2," "ch-1A1-2 M2," and "ch-1A1-2 M3") to complement (human C1q) by enzyme-linked immunosorbent assay (ELISA). Two types of cell lines (NIH3T3 / fPD-1 [FIG. 5B] or NIH3T3 [FIG. 5A]) were incubated either 1) in the absence of a primary antibody and in the presence of a secondary antibody (anti-cat IgG-Alexa647), or 2) in the presence of four types of primary antibodies (two types of comparative antibodies [fIgG or "ch-1A1-2"], or two types of the present anti-cat PD-1 antibodies ["ch-1A1-2 M2" or "ch-1A1-2 M3"]) in the presence of the secondary antibody, and then the fluorescence intensity (horizontal axis) derived from Alexa647 in the above cell lines was analyzed by flow cytometry. The vertical axis represents the number of cells. FIG. 6A shows the results of flow cytometry analysis after incubating NIH3T3 / fPD-1 1) in the absence of comparative antibody ("ch-1A1-2") and in the presence of hIg, 2) in the presence or absence of comparative antibody ("ch-1A1-2") and in the presence of fPD-L1-hIg, or 3) in the presence of comparative antibody ("ch-1A1-2"), followed by further incubation in the presence of anti-cat IgG-Alexa647. The horizontal axis, "Alexa647," shows the results of flow cytometry analysis of the fluorescence intensity (horizontal axis) derived from Alexa647 in NIH3T3 / fPD-1. The vertical axis shows the number of cells. FIG. 6B shows the results of flow cytometry analysis of NIH3T3 / fPD-1 cells incubated 1) in the absence of the present anti-feline PD-1 antibody ("ch-1A1-2 M2") and in the presence of hIg, 2) in the presence or absence of the present anti-feline PD-1 antibody ("ch-1A1-2 M2") and in the presence of fPD-L1-hIg, or 3) in the presence of the present anti-feline PD-1 antibody ("ch-1A1-2 M2"), followed by further incubation in the presence of anti-feline IgG-Alexa647.The horizontal axis indicates "Alexa647," and the figure shows the results of flow cytometry analysis of Alexa647-derived fluorescence intensity (horizontal axis) in NIH3T3 / fPD-1. The vertical axis indicates cell number. Figure 6C shows the results of flow cytometry analysis of NIH3T3 / fPD-1 cells incubated 1) in the absence of the present anti-feline PD-1 antibody ("ch-1A1-2 M3") and in the presence of hIg, 2) in the presence or absence of the present anti-feline PD-1 antibody ("ch-1A1-2 M3") and in the presence of fPD-L1-hIg, or 3) in the presence of the present anti-feline PD-1 antibody ("ch-1A1-2 M3"), followed by incubation in the presence of anti-feline IgG-Alexa647. The horizontal axis indicates "Alexa647," and the vertical axis indicates the number of cells. Peripheral blood mononuclear cells (PBMCs) from healthy cats stimulated with concanavalin A (ConA) were either 1) cultured, 2) cultured in the presence of hIg, or 3) cultured in the presence of fPD-L1-hIg and various concentrations (0 μg / mL, 10 μg / mL, or 40 μg / mL) of four types of antibodies (two types of comparative antibodies [fIgG and "ch-1A1-2"] and two types of the present anti-feline PD-1 antibodies ["ch-1A1-2 M2" and "ch-1A1-2 M3"]), and the concentrations of feline IFN-γ (interferon-γ, hereinafter sometimes referred to as "fIFNγ") in the culture medium were measured. Figure 8A shows the results of Western blotting analysis of protein extracts prepared from five types of cell lines (NIH3T3 cell line ["mock" in the figure], NIH3T3 / fCD64 ["fCD64" in the figure], NIH3T3 / fCD32 ["fCD32" in the figure], NIH3T3 / fCD16 ["fCD16" in the figure], and NIH3T3 / fFcRn ["fFcRn" in the figure]) using an anti-FLAG antibody (upper panel) and an anti-Vinculin antibody (lower panel).Figure 8B shows the results of flow cytometric analysis of the Dylight649-derived fluorescence intensity (horizontal axis) in NIH3T3 / fFcRn after incubating NIH3T3 / fFcRn 1) in the absence of a primary antibody and a "biotin-labeled secondary antibody / Dylight649-labeled streptavidin," 2) in the absence of a primary antibody but in the presence of a "biotin-labeled secondary antibody / Dylight649-labeled streptavidin," or 3) in the presence of three types of primary antibodies (one type of comparative antibody ["ch-1A1-2"] or two types of the present anti-cat antibodies ["ch-1A1-2 M2" or "ch-1A1-2 M3"]) and in the presence of a "biotin-labeled secondary antibody / Dylight649-labeled streptavidin."

[0019] 1. The Anti-Feline Antibody of the Present Invention The antibody of the present invention is an anti-feline antibody (i.e., an antibody that specifically binds to a feline antigen) comprising a light chain and a heavy chain, wherein the constant region in the heavy chain is: 1) a constant region consisting of an amino acid sequence having an alanine residue at position 119, an alanine residue at position 120, a glycine residue at position 214, and an alanine residue at position 215 of the amino acid sequence shown in SEQ ID NO: 1, and having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; 2) a constant region consisting of an amino acid sequence having a serine residue at position 119, a threonine residue at position 120, and an arginine residue at position 121 of the amino acid sequence shown in SEQ ID NO: 2, and having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; 3) a constant region consisting of an amino acid sequence having a serine residue at position 119, a threonine residue at position 120, and an arginine residue at position 121 of the amino acid sequence shown in SEQ ID NO: 5, and having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 6; or 4) a constant region consisting of an amino acid sequence having an alanine residue at position 119, an alanine residue at position 120, a glycine residue at position 214, and an alanine residue at position 215 of the amino acid sequence shown in SEQ ID NO: 5, and having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 6 (hereinafter, these heavy chain constant regions may be collectively referred to as the "heavy chain constant region of the present invention"). By containing these specific amino acid residues, the present anti-cat antibody has reduced cytotoxic activity and / or cytophagocytic activity, and specifically, compared to an anti-cat antibody that does not contain these specific amino acid residues, it has reduced one or more effector functions selected from antibody-dependent cell-mediated cytotoxicity (ADCC) activity, antibody-dependent cellular phagocytosis (ADCP) activity, and complement-dependent cytotoxicity (CDC) activity.

[0020] As used herein, "reduced ADCC activity" means, more specifically, that the binding ability of the heavy chain constant region (Fc region) of the anti-feline antibody to Fcγ receptor III (CD16) has been reduced; as used herein, "reduced ADCP activity" means, more specifically, that the binding ability to Fcγ receptor I (CD64) and / or Fcγ receptor II (CD32) has been reduced; and as used herein, "reduced CDC activity" means, more specifically, that the binding ability to complement (e.g., C1q, C1r, C2, C3, C3a, C3b, C5a, etc.) has been reduced.

[0021] In this specification, "cat" refers to any of the domestic cat (Felis silvestris catus), wild cat, and feral cat (domestic cat that has re-reinstated into the wild), which are small mammals classified in the order Carnivora, suborder Felidae, family Felinae, and genus Felis, and does not include other felidae (e.g., lions, tigers, leopards, etc.) other than cats.

[0022] As used herein, "having an alanine residue at position 119 of the amino acid sequence shown in SEQ ID NO: 1" specifically means having an amino acid residue corresponding to a mutation (M119A) in which the methionine residue (M) at position 119 of the feline IgG1 heavy chain constant region (a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 or a polypeptide consisting of amino acid residues 126 to 459 of the amino acid sequence of SEQ ID NO: 8; the same applies hereinafter) is substituted with an alanine residue (A). Furthermore, as used herein, "having an alanine residue at position 120 of the amino acid sequence shown in SEQ ID NO: 1" specifically means having an amino acid residue corresponding to a mutation (L120A) in which the leucine residue (L) at position 120 of the feline IgG1 heavy chain constant region is substituted with an alanine residue (A). Furthermore, as used herein, "having a glycine residue at position 214 of the amino acid sequence shown in SEQ ID NO: 1" specifically means having an amino acid residue corresponding to a mutation (P214G) in which the proline residue (P) at position 214 of the feline IgG1 heavy chain constant region is substituted with a glycine residue (G). Furthermore, as used herein, "having an alanine residue at position 215 of the amino acid sequence set forth in SEQ ID NO: 1" specifically means having an amino acid residue corresponding to a mutation (S215A) in which the serine residue at position 215 of the feline IgG1 heavy chain constant region (S) is replaced with an alanine residue (A). Whether or not a protein contains amino acid residues corresponding to M119A, L120A, P214G, and S215A can be determined by performing an alignment analysis with the amino acid sequence of SEQ ID NO: 4 and / or the amino acid sequence of SEQ ID NO: 8.

[0023] As used herein, "M119A" corresponds to a mutation (M120A) in which M at position 120 in the amino acid sequence of SEQ ID NO: 10 (i.e., an amino acid sequence in which an alanine residue [A] is added to the N-terminus of the amino acid sequence of SEQ ID NO: 4) is replaced with A; "L120A" corresponds to a mutation (L121A) in which L at position 121 in the amino acid sequence of SEQ ID NO: 10 is replaced with A; "P214G" corresponds to a mutation (P215G) in which P at position 215 in the amino acid sequence of SEQ ID NO: 10 is replaced with G; and "S215A" corresponds to a mutation (S216A) in which S at position 216 in the amino acid sequence of SEQ ID NO: 10 is replaced with A.

[0024] As used herein, "having a serine residue at position 119 of the amino acid sequence shown in SEQ ID NO: 2" specifically means having an amino acid residue corresponding to a mutation (M119S) in which the methionine residue (M) at position 119 of the feline IgG1 heavy chain constant region is replaced with a serine residue (S). Also, as used herein, "having a threonine residue at position 120 of the amino acid sequence shown in SEQ ID NO: 2" specifically means having an amino acid residue corresponding to a mutation (L120T) in which the leucine residue (L) at position 120 of the feline IgG1 heavy chain constant region is replaced with a threonine residue (T). Also, as used herein, "having an arginine residue at position 121 of the amino acid sequence shown in SEQ ID NO: 2" specifically means having an amino acid residue corresponding to a mutation (G121R) in which the glycine residue (G) at position 121 of the feline IgG1 heavy chain constant region is replaced with an arginine residue (R). Whether or not a protein contains amino acid residues corresponding to M119S, L120T, and G121R can be determined by performing alignment analysis with the amino acid sequence of SEQ ID NO:4 and / or the amino acid sequence of SEQ ID NO:8.

[0025] As used herein, "M119S" corresponds to a mutation (M120S) in which M at position 120 in the amino acid sequence of SEQ ID NO: 10 (i.e., an amino acid sequence in which A is added to the N-terminus of the amino acid sequence of SEQ ID NO: 4), "L120T" corresponds to a mutation (L121T) in which L at position 121 in the amino acid sequence of SEQ ID NO: 10 is replaced with T, and "G121R" corresponds to a mutation (G122R) in which G at position 122 in the amino acid sequence of SEQ ID NO: 10 is replaced with R.

[0026] As used herein, "having an alanine residue at position 119 of the amino acid sequence shown in SEQ ID NO: 5" specifically means having an amino acid residue corresponding to a mutation (I119A) in which the isoleucine residue (I) at position 119 of the feline IgG2 heavy chain constant region (a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7; the same applies hereinafter) is substituted with an alanine residue (A). Furthermore, as used herein, "having an alanine residue at position 120 of the amino acid sequence shown in SEQ ID NO: 5" specifically means having an amino acid residue corresponding to a mutation (P120A) in which the proline residue (P) at position 120 of the feline IgG2 heavy chain constant region is substituted with an alanine residue (A). Furthermore, as used herein, "having a glycine residue at position 214 of the amino acid sequence shown in SEQ ID NO: 5" specifically means having an amino acid residue corresponding to a mutation (P214G) in which the proline residue (P) at position 214 of the feline IgG2 heavy chain constant region is substituted with a glycine residue (G). Furthermore, as used herein, "having an alanine residue at position 215 of the amino acid sequence shown in SEQ ID NO: 5" specifically means having an amino acid residue corresponding to a mutation (S215A) in which the serine residue at position 215 (S) in the feline IgG2 heavy chain constant region is substituted with an alanine residue (A). Whether or not a protein has amino acid residues corresponding to I119A, P120A, P214G, and S215A can be determined by performing an alignment analysis with the amino acid sequence of SEQ ID NO: 7.

[0027] In this specification, "I119A" corresponds to a mutation (I120A) in which I at position 120 in SEQ ID NO: 11 (i.e., an amino acid sequence in which A is added to the N-terminus of the amino acid sequence of SEQ ID NO: 7) is replaced with A; "P120A" corresponds to a mutation (P121A) in which P at position 121 in SEQ ID NO: 11 is replaced with A; "P214G" corresponds to a mutation (P215G) in which P at position 215 in SEQ ID NO: 11 is replaced with G; and "S215A" corresponds to a mutation (S216A) in which S at position 216 in SEQ ID NO: 11 is replaced with A.

[0028] As used herein, "having a serine residue at position 119 of the amino acid sequence shown in SEQ ID NO: 6" specifically means having an amino acid residue corresponding to a mutation (I119S) in which the isoleucine residue (I) at position 119 of the feline IgG2 heavy chain constant region is substituted with a serine residue (S). Furthermore, as used herein, "having a threonine residue at position 120 of the amino acid sequence shown in SEQ ID NO: 6" specifically means having an amino acid residue corresponding to a mutation (P120T) in which the proline residue (P) at position 120 of the feline IgG2 heavy chain constant region is substituted with a threonine residue (T). Furthermore, as used herein, "having an arginine residue at position 121 of the amino acid sequence shown in SEQ ID NO: 6" specifically means having an amino acid residue corresponding to a mutation (G121R) in which the glycine residue (G) at position 121 of the feline IgG2 heavy chain constant region is substituted with an arginine residue (R). Whether or not a protein has amino acid residues corresponding to I119S, P120T, and G121R can be determined by performing alignment analysis with the amino acid sequence of SEQ ID NO:7.

[0029] As used herein, "I119S" corresponds to a mutation (I120S) in which I at position 120 in SEQ ID NO: 11 (i.e., an amino acid sequence in which A is added to the N-terminus of the amino acid sequence of SEQ ID NO: 7), "P120T" corresponds to a mutation (P121T) in which P at position 121 in SEQ ID NO: 11 is replaced with T, and "G121R" corresponds to a mutation (G122R) in which G at position 122 in SEQ ID NO: 11 is replaced with R.

[0030] As used herein, the term "light chain" refers to a polypeptide chain comprising, in order from the amino terminus, a light chain variable region and a light chain constant region. The length of the light chain is, for example, within the range of 200 to 250 amino acids.

[0031] As used herein, the term "heavy chain" refers to a polypeptide chain comprising, in order from the amino terminus, a light chain variable region and a light chain constant region. The length of the heavy chain is, for example, within the range of 400 to 500 amino acids.

[0032] The light chain variable region and heavy chain variable region of the present anti-feline antibody each contain light (L) chain complementarity-determining regions (CDRs) 1-3 and heavy (H) chain CDRs 1-3, which are necessary for antigen (epitope) binding. Furthermore, the present anti-feline antibody typically has framework regions (FRs) linked to the amino (N)-terminus and carboxyl (C)-terminus of each of the H chain CDRs 1-3 and L chain CDRs 1-3. Examples of such 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. Furthermore, among the above FRs, examples of the L chain FR include an L chain FR1 linked to the N-terminus of the L chain CDR1, an L chain FR2 linked to the C-terminus of the L chain CDR1 (the N-terminus of the L chain CDR2), an L chain FR3 linked to the C-terminus of the L chain CDR2 (the N-terminus of the L chain CDR3), and an L chain FR4 linked to the C-terminus of the L chain CDR3.

[0033] When the anti-feline antibody is an anti-feline PD-1 antibody, the light chain variable region can be, for example, a polypeptide having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 9, in which case the light chain CDR1 is a polypeptide consisting of the 24th to 34th amino acid residues of the amino acid sequence of SEQ ID NO: 9, the light chain CDR2 is a polypeptide consisting of the 50th to 56th amino acid residues of the amino acid sequence of SEQ ID NO: 9, and the light chain CDR3 is a polypeptide consisting of the 89th to 97th amino acid residues of the amino acid sequence of SEQ ID NO: 9 (see Table 1).

[0034] When the anti-feline antibody is an anti-feline PD-1 antibody, the heavy chain variable region can be, for example, a polypeptide having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 8, in which case the heavy chain CDR1 is a polypeptide consisting of amino acid residues 31 to 35 of the amino acid sequence of SEQ ID NO: 8, the heavy chain CDR2 is a polypeptide consisting of amino acid residues 50 to 66 of the amino acid sequence of SEQ ID NO: 8, and the heavy chain CDR3 is a polypeptide consisting of amino acid residues 99 to 113 of the amino acid sequence of SEQ ID NO: 8.

[0035] The antigen to which the present anti-feline antibody specifically binds may be any antigen (feline antigen) present in the body of a cat, and examples thereof include polypeptides, lipids, carbohydrates, polynucleotides, etc., with polypeptides being preferred. When the present anti-feline antibody is used to activate an immune response in a cat, immune checkpoint molecules are preferred as such polypeptides. In this specification, the present anti-feline antibody that specifically binds to an immune checkpoint molecule may be referred to as the "present anti-feline immune checkpoint molecule antibody."

[0036] As used herein, the term "immune checkpoint molecule" refers to a molecule that negatively or positively regulates immune responses by T cells by transmitting immunoregulatory signals (immune co-inhibitory signals or immune co-stimulatory signals). Examples of immune checkpoint molecules include receptors or their ligands present on the cell membrane of T cells, NK (natural killer) cells, etc., and specifically, PD-1 (Programmed cell death 1) or its ligands (e.g., PD-L1, PD-L2, etc.), CTLA-4 (Cytotoxic T-lymphocyte-associated protein 4) or its ligands (e.g., B7-1, B7-2, etc.), CD28 or its ligands (e.g., B7-1, B7-2, etc.), BTLA (B and T lymphocyte attenuator) or its ligands (e.g., HVEM, etc.), CD96 or its ligands (e.g., Nectin-1, CD155, etc.), DNAM-1 or its ligands (e.g., Nectin-2, CD155, etc.), TIGIT (T cell immunoglobulin and ITIM domain) or its ligands (e.g., Nectin-2, Nectin-3, CD155, etc.), PVRIG (Poliovirus receptor related immunoglobulin domain-containing protein) or its ligand (e.g., Nectin-2, etc.), TIM3 (T cell immunoglobulin and mucin-3) or its ligand (e.g., Galectin-9, etc.), LAG-3 (Lymphocyte activation gene 3) or its ligand (e.g., MHCII, etc.), CD28H or its ligand (e.g., B7-H7, etc.), SIRPα (Signal regulatory protein α) or SIRPγ or their ligand (e.g., CD47, etc.), Siglec-7 or its ligand (e.g., CD43), KIR (killer inhibitory receptor) or its ligand, 2B4 or its ligand (e.g., CD48, etc.), TIM-3 (T cell membrane proteinExamples of such antibodies include PD-3) or its ligand (e.g., galectin-9, CEACAM-1, etc.), VISTA (V-domain Ig-containing suppressor of T cell activation) or its ligand, and GITR (glucocorticoid-induced TNF receptor) or its ligand, and PD-1 is a preferred example because its effects have been demonstrated in the Examples described below.

[0037] The present anti-cat antibody generally has a Y-shaped structure consisting of two light chains and two heavy chains, which are generally linked by one or more disulfide bonds.

[0038] The present anti-feline antibody is preferably isolated. Here, "isolated" means that the antibody exists in a state different from its natural state, such as by artificially removing the antibody from its natural environment or expressing it in an environment different from its natural environment. In other words, "isolated antibody" does not include an antibody derived from an individual that is present in the body of that individual or in tissues or body fluids (blood, plasma, serum, etc.) derived from the body without external (artificial) manipulation. Furthermore, the present anti-feline antibody is preferably an antibody produced by artificial manipulation (e.g., the recombinant antibody described above). Such "antibodies derived from or produced by cells produced by artificial manipulation" does not include antibodies that have not been artificially manipulated, such as antibodies produced from naturally occurring B cells.

[0039] The present anti-cat antibody can be obtained by culturing cells (i.e., the present host cells) produced by genetically introducing the present anti-cat antibody expression vector into host cells in a culture medium appropriate for the host cells, and recovering the present anti-cat antibody produced in the culture medium.

[0040] In addition, using transgenic animal production technology, transgenic animals such as mice, cows, goats, sheep, chickens, and pigs can be produced that incorporate part or all of the present anti-cat antibody expression vector, and the present anti-cat antibody can be produced in large quantities from the blood, milk, etc. of such transgenic animals.

[0041] Furthermore, a non-feline animal (e.g., mouse, rat) is immunized with a non-feline animal (e.g., mouse, rat) cell line expressing a substance containing a feline antigen (a feline antigen-expressing non-feline animal cell line), and antibody-producing hybridomas are prepared using cell fusion technology. The anti-feline antibody-producing hybridomas are then 1) screened by ELISA using a plate immobilized with a feline antigen, and / or 2) hybridomas positive for the feline antigen-expressing non-feline animal cell line are screened by flow cytometry to obtain hybridomas that produce anti-feline antibodies. After that, a polynucleotide encoding a constant region of the anti-feline antibody-encoding polynucleotide contained in the genomic DNA of the hybridoma is cloned into a polynucleotide encoding a constant region of the anti-feline antibody contained in the genomic DNA of the hybridoma, as described in the literature "Sci Adv. 2019 Aug 28;5(8):eaaw1822." and the literature "Vet Comp Oncol. 2020" According to the method described in "December;18(4):739-752," the CRISPR / Cas9 system can be used to substitute a polynucleotide encoding the heavy chain constant region of the present invention, thereby producing a hybridoma that produces a chimeric antibody (i.e., the present anti-feline antibody) whose variable and constant regions are derived from different species. The present anti-feline antibody can be purified from the culture supernatant of the hybridoma using known antibody purification techniques.

[0042] 2. Polynucleotide Encoding the Anti-Feline Antibody of the Present Invention The polynucleotide of the present invention may be a polynucleotide encoding the anti-feline antibody of the present invention (sometimes referred to herein as the "anti-feline antibody-encoding polynucleotide"). The nucleotides constituting the anti-feline antibody-encoding polynucleotide may be DNA or RNA having a natural structure, or nucleotides having a structure in which DNA or RNA having a natural structure has been chemically modified (also referred to as "modified nucleic acids"). For example, when the anti-feline antibody-encoding polynucleotide is in the form of mRNA, modified nucleic acids are used to confer resistance to degradation by RNase. Modified nucleic acids are preferably those in which the base moiety of the nucleotide is modified. Examples of modified nucleic acids include pyrimidine nucleotides substituted at the 5th position and pseudouridines optionally substituted at the 1st position. Specific examples include 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkylpseudouridine. Furthermore, the 1-alkylpseudouridine may be 1-(C1-C6 alkyl)pseudouridine, preferably 1-methylpseudouridine or 1-ethylpseudouridine.

[0043] The polynucleotide encoding the present anti-feline antibody can be easily prepared by conventional methods based on the amino acid sequence of the present anti-feline antibody. 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 present anti-feline antibody can be prepared using standard molecular biological and / or chemical procedures.

[0044] The anti-feline antibody-encoding polynucleotide may contain a nucleotide sequence whose codons have been 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.

[0045] The polynucleotide encoding the anti-feline antibody of the present invention may be a single-stranded (sense strand) polynucleotide encoding the anti-feline antibody of the present invention, or a double-stranded polynucleotide consisting of the sense strand and its complementary antisense strand, and the form of the polynucleotide is selected according to the method of introducing the polynucleotide into cells. For example, in the case of mRNA or lentiviral vectors, the polynucleotide is single-stranded, while in the case of plasmid DNA, the polynucleotide may be double-stranded.

[0046] As used herein, "at least 80% sequence identity with an amino acid sequence" means that the percentage of amino acids identical to the amino acid sequence being compared is 80% or more, preferably 85% or more, more preferably 87% or more, even more preferably 88% or more, even more preferably 89% or more, particularly preferably 90% or more, especially more preferably 93% or more, especially more preferably 96% or more, especially even more preferably 98% or more, and most preferably 99% or more sequence identity (e.g., 100% sequence identity). Amino acid sequence identity can be determined using known programs such as ClustalW, GENETYX, and BLAST.

[0047] As used herein, "at least 80% sequence identity to an amino acid sequence" means, in other words, an amino acid sequence in which zero, one, or several amino acid residues have been deleted, substituted, inserted, and / or added in the amino acid sequence being compared, and which has the same function as a polypeptide consisting of the amino acid sequence being compared. Here, "an amino acid sequence in which one or several amino acid residues have been deleted, substituted, inserted, and / or added" refers to an amino acid sequence in which, for example, 1 to 30 amino acid residues have been deleted, substituted, inserted, and / or added, preferably 1 to 20 amino acid residues, more preferably 1 to 15 amino acid residues, even more preferably 1 to 10 amino acid residues, even more preferably 1 to 5 amino acid residues, even more preferably 1 to 3 amino acid residues, and even more preferably 1 to 2 amino acid residues have been deleted, substituted, inserted, and / or added. Mutation of these amino acid residues can be carried out by any method known to those skilled in the art, such as chemical synthesis, genetic engineering, or mutagenesis.

[0048] 3. The Anti-Feline Antibody Expression Vector of the Present Invention The vector of the present invention is not particularly limited, as long as it comprises a promoter and a polynucleotide encoding the present anti-feline antibody operably linked downstream of the promoter, and is capable of transcribing mRNA encoded by the polynucleotide encoding the present anti-feline antibody (sometimes referred to herein as the "anti-feline antibody expression vector of the present invention").

[0049] The anti-feline 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 vectors may be circular or linear.

[0050] The promoter in the present anti-feline 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 mRNA encoded by the present anti-feline antibody-encoding polynucleotide located downstream thereof. Examples include the SRα 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, EF1α promoter, metallothionein promoter, and heat shock promoter.

[0051] In addition to a promoter, the anti-feline antibody expression vector may optionally contain an enhancer, a poly(A) addition signal, a marker gene, a replication origin, a gene encoding a polypeptide that binds to the replication origin and controls replication, and the like. Such a marker gene is a gene that enables cell sorting or selection by introducing the marker gene into cells. Specific examples of the marker gene include drug resistance genes, fluorescent protein genes, luciferase genes, and chromogenic enzyme genes. These may be used alone or in combination. Specific examples of the drug resistance gene include neomycin resistance genes, tetracycline resistance genes, kanamycin resistance genes, zeocin resistance genes, and hygromycin resistance genes. Specific examples of the fluorescent protein gene include blue fluorescent protein (BFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and red fluorescent protein (RFP) genes. Specific examples of the luciferase gene include the luciferase gene. Specific examples of the chromogenic enzyme gene include the β-galactosidase gene, the β-glucuronidase gene, the alkaline phosphatase gene, etc. In this specification, the anti-feline antibody expression vector containing a polynucleotide encoding the anti-feline immune checkpoint molecule antibody as the anti-feline antibody-encoding polynucleotide may be referred to as the "anti-feline immune checkpoint molecule antibody expression vector."

[0052] 4. Host Cells of the Present Invention The host cells of the present invention are not particularly limited, as long as they are cells (sometimes referred to herein as "host cells") into which the anti-cat antibody expression vector of the present invention has been introduced and which express the anti-cat antibody of the present invention. The host cells of the present invention are typically maintained in a liquid or in a wet state in a container (e.g., a culture plate or dish, or a tube for cell storage or cell sorting). Such liquids are not particularly limited, as long as they allow the survival and maintenance of the host cells of the present invention. Examples of such liquids include culture media (e.g., serum-containing or serum-free culture media), saline, phosphate-buffered saline, Tris-buffered saline, HEPES-buffered saline, Ringer's solution (e.g., lactate Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution), and 5% aqueous glucose solution. Examples of the serum include 0.1-30% (v / v) serum (e.g., fetal bovine serum (FBS), calf bovine serum (CS), etc.). Examples of the culture medium include culture media for animal cell culture (DMEM, EMEM, IMDM, RPMI1640, αMEM, F-12, F-10, M-199, AIM-V, etc.). Examples of the serum-free culture medium (serum-free culture medium) include the culture medium for animal cell culture supplemented with an appropriate amount (e.g., 1 to 30%) of a serum substitute such as commercially available B27 Supplement (minus insulin) (manufactured by Life Technologies), N2 Supplement (manufactured by Life Technologies), B27 Supplement (manufactured by Life Technologies), or Knockout Serum Replacement (manufactured by Invitrogen).

[0053] The present host cells can be prepared by transfecting the present anti-cat antibody expression vector into host cells. The method for transfecting the present anti-cat antibody expression vector into host cells may be any method suitable for the present anti-cat antibody expression vector and host cells. For example, when a non-viral vector is used as the present anti-cat antibody expression vector, methods using liposomes or microparticles containing cationic lipids, etc., as described in WO 96 / 10038, WO 97 / 18185, WO 97 / 25329, WO 97 / 30170, and WO 97 / 31934 (incorporated herein by reference), methods using episomal vectors expressing scaffold / matrix attachment region elements, etc., as described in Jin et al., EMBO Mol Med. 2016 Jul; 8(7): 702-711, etc., and methods using episomal vectors expressing scaffold / matrix attachment region elements, as described in Mol Ther Methods Clin Dev. 2017 Dec, etc., are also available. 22;8:131-140.), etc., using a shell obtained by irradiating viral particles to destroy their contents, which is employed in transposon-based introduction methods such as the PiggyBac method; and a method in which the present anti-cat antibody expression vector (specifically, the present linear anti-cat antibody expression vector) is introduced into the genome of a target cell by genome editing techniques using, for example, the CRISPR / Cas9 system or zinc finger nucleases (e.g., U.S. Pat. No. 8,956,828).

[0054] Furthermore, when a viral vector (i.e., recombinant virus) is used as the anti-feline antibody expression vector of the present invention, methods for introducing the anti-feline antibody expression vector into host cells include infecting immune cells with the virus using a culture supernatant containing the recombinant virus (i.e., viral particles containing a viral vector plasmid (which may be DNA or RNA) in which a pathogenic gene has been removed from the viral genome and a foreign gene (in this case, a polynucleotide encoding the anti-feline antibody of the present invention) has been incorporated), or a concentrated recombinant virus.

[0055] The host cell may be any cell into which the anti-feline antibody-encoding polynucleotide of the present invention is transcribed and the anti-feline antibody of the present invention is expressed, and examples thereof include yeast, mammalian (e.g., human, mouse, rat, dog, cat, etc.) cells, insect (e.g., Spodoptera frugiperda, Trichoplusia ni, etc.) cells, and plant (e.g., tobacco, potato, tomato, carrot, soybean, rapeseed, alfalfa, rice, wheat, barley, etc.) cells.

[0056] 5. The activator of the present invention for activating immune responses in cats is an agent (sometimes referred to herein as the "activator of the present invention") containing the present anti-feline immune checkpoint molecule antibody or the present anti-feline immune checkpoint molecule antibody expression vector, the use of which is specified as "to activate immune responses in cats." The immune response to be activated may be one in cultured feline cells, or one in the living cat.

[0057] The active ingredient of the present activator may be the present anti-feline immune checkpoint molecule antibody or the present anti-feline immune checkpoint molecule antibody expression vector, which may be used alone as a food or drink for cats or a pharmaceutical product (preparation) for cats, or may be further mixed with additives and used in the form of a composition (a food or drink composition for cats or a pharmaceutical composition for cats).

[0058] Examples of additives for the activator of the present invention include physiologically acceptable conventional carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrants, isotonic agents, coating agents, solubilizers, lubricants, glidants, solubilizers, flavoring agents, sweeteners, solvents, gelling agents, nutrients, etc. Specific examples of such additives include water, physiological saline, animal fats and oils, vegetable oils, lactose, starch, gelatin, crystalline cellulose, gum, talc, magnesium stearate, hydroxypropyl cellulose, polyalkylene glycol, polyvinyl alcohol, and glycerin.

[0059] The present activator may contain components that activate immune responses in cats in addition to the active ingredients, the present anti-feline immune checkpoint molecule antibody or the present anti-feline immune checkpoint molecule antibody expression vector; however, because the present anti-feline immune checkpoint molecule antibody or the present anti-feline immune checkpoint molecule antibody expression vector alone exhibits excellent activating effects, it is preferable that the activator does not contain components that activate immune responses in cats (e.g., compounds, proteins [e.g., antibodies], DNA, RNA) in addition to the present anti-feline immune checkpoint molecule antibody or the present anti-feline immune checkpoint molecule antibody expression vector.

[0060] The present activator has the effect of disabling immune checkpoints and activating T cells in cats, and therefore can be advantageously used in preparations for immunotherapy (e.g., cancer immunotherapy) in cats.

[0061] 6. The activation method of the present invention for activating an immune response in a cat is not particularly limited, as long as it includes the step of administering the present anti-feline immune checkpoint molecule antibody or the present anti-feline immune checkpoint molecule antibody expression vector to a cat and activates an immune response in a cat (sometimes referred to herein as the "activation method of the present invention").

[0062] Cats to which the present anti-feline immune checkpoint molecule antibody or the present anti-feline immune checkpoint molecule antibody expression vector can be administered are any cats in need of activating an immune response, and specific examples include cats in need of deactivating immune checkpoints and activating feline T cells, and more specific examples include cats in need of immunotherapy (e.g., cancer immunotherapy) (e.g., cats with cancer, cats at risk of developing cancer, etc.).

[0063] Methods for administering the present anti-feline immune checkpoint molecule antibody or the present anti-feline immune checkpoint molecule antibody expression vector include, for example, intravenous administration, intraperitoneal administration, and administration to a tissue or organ.

[0064] The dosage of the present anti-feline immune checkpoint molecule antibody or the present anti-feline immune checkpoint molecule antibody expression vector is determined appropriately depending on the cat's age, body weight, sex, symptoms, drug sensitivity, etc., and is, for example, in the range of 1 μg to 100 mg / kg (body weight) / day. Furthermore, the present anti-feline immune checkpoint molecule antibody or the present anti-feline immune checkpoint molecule antibody expression vector is administered once or multiple times (for example, 2 to 4 times) per day, and the dosage may be adjusted depending on the progress of improvement in the cat's symptoms.

[0065] The present activation method can be advantageously applied to immunotherapy (e.g., cancer immunotherapy) in cats, because it can release immune checkpoints and activate T cells in cats by administering the present anti-feline immune checkpoint molecule antibody or the present anti-feline immune checkpoint molecule antibody expression vector to cats.

[0066] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. The cell lines used in the following examples were cultured and maintained in a DMEM culture medium containing 10% FBS, 100 units / mL penicillin, 100 μg / mL streptomycin, and 55 μM 2-mercaptoethanol (hereinafter simply referred to as "DMEM culture medium") under conditions of 5% CO2 and 37°C.

[0067] 1. Isolation of Stably Expressing Cell Lines To generate three types of stably expressing cell lines (NIH3T3 / fCD16 [i.e., an NIH3T3 cell line stably expressing fCD16], NIH3T3 / fCD32 [i.e., an NIH3T3 cell line stably expressing fCD32], and NIH3T3 / fCD64 [i.e., an NIH3T3 cell line stably expressing fCD64]), 3.5 × 10 5NIH3T3 cells were seeded into a 6-well dish. One day later, three vectors (pMXs-IP vector containing fCD16 cDNA [pMX-IP-fCD16], pMXs-IP vector containing fCD32 cDNA [pMX-IP-fCD32], and pMXs-IP vector containing fCD64 cDNA [pMX-IP-fCD64]) were transfected into the NIH3T3 cells using PEI Max (Polysciences) according to the manufacturer's protocol and cultured for 48 hours. Subsequently, the cells were cultured in the presence of 10 μg / mL puromycin (Sigma-Aldrich) to generate the three stable-expressing cell lines.

[0068] Protein extracts were prepared from the three stable cell lines and the NIH3T3 cell line using a solution containing 1% NP40. SDS-PAGE and Western blotting were then performed using primary antibodies (anti-FLAG antibody [M2, SIGMA] and anti-Vinculin antibody [Santa Cruz Biotechnology]) and secondary antibodies (anti-mouse IgG conjugated with HRP [anti-mouse IgG-HRP] [Invitrogen]) according to standard methods. The housekeeping protein Vinculin was used as an internal control.

[0069] As a result, no bands were detected with anti-FLAG antibodies in the NIH3T3 cell line (see "mock" in Figure 1), whereas specific bands were detected with anti-FLAG antibodies in the three stable expression cell lines (see "fCD16," "fCD32," and "fCD64" in Figure 1). These results indicate that 2xFLAG-fCD16, 2xFLAG-fCD32, and 2xFLAG-fCD64 were stably expressed in the three stable expression cell lines (NIH3T3 / fCD16, NIH3T3 / fCD32, and NIH3T3 / fCD64), respectively.

[0070] 2. Preparation of anti-cat antibodies with modified feline IgG1 constant regions In an attempt to prepare anti-cat antibodies with reduced cytotoxic activity and cytophagocytic activity, three types of variants were prepared by introducing amino acid substitutions into the feline IgG1 heavy chain constant region of "ch-1A1-2," i.e., a chimeric antibody (see Patent Document 4) consisting of a heavy chain consisting of the heavy chain variable region of "1A1-2" (anti-fPD-1 mouse monoclonal antibody) and the feline IgG1 heavy chain constant region (see Table 1), and a light chain consisting of the light chain variable region of "1A1-2" and the feline IgG1 light chain constant region (see Table 2). Specifically, the following antibodies were prepared: 1) "ch-1A1-2 M1" (see Table 3), in which five amino acid substitutions (M119I, L120P, G122A, P216A, and I217M) were introduced into the feline IgG1 heavy chain constant region (a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4); 2) "ch-1A1-2 M2" (see Table 4), in which four amino acid substitutions (M119A, L120A, P214G, and S215A) were introduced into the feline IgG1 heavy chain constant region; and 3) "ch-1A1-2 M3" (see Table 5), in which three amino acid substitutions (M119S, L120T, and G121R) were introduced into the feline IgG1 heavy chain constant region.

[0071] In the table, the amino acid residues enclosed in boxes represent, in order from the amino-terminus, the H chain CDR1, H chain CDR2, and H chain CDR3; amino acid residues other than those single-underlined represent the H chain variable region; and amino acid residues single-underlined represent the H chain constant region. In the table, the amino acid residues enclosed in boxes represent, in order from the amino-terminus, the L chain CDR1, L chain CDR2, and L chain CDR3; amino acid residues other than those single-underlined represent the L chain variable region; and amino acid residues single-underlined represent the L chain constant region.

[0072] In the table, "I １ " indicates M119I, and "P ２ " indicates L120P, and "A ３ " indicates G122A, and "A ４ " indicates P216A, and "M ５ " indicates I217M.

[0073] In the table, "A １ " indicates M119A, and "A ２ " indicates L120A, and "G ３ " indicates P214G, and "A ４ " indicates S215A.

[0074] In the table, "S １ " indicates M119S, and "T ２ " indicates L120T, and "R ３ " indicates G121R.

[0075] 3. Confirmation that amino acid substitutions in the heavy chain constant region of "ch-1A1-2 M1" do not reduce cytotoxic activity or cytophagocytic activity Whether or not amino acid substitutions in the heavy chain constant region of "ch-1A1-2 M1" reduce cytotoxic activity or cytophagocytic activity was analyzed using binding to fCD16 and fCD32 as an indicator. Specifically, flow cytometry analysis was performed using three types of antibodies (fIgG, "ch-1A1-2," and "ch-1A1-2 M1") and two types of stably expressing cell lines (NIH3T3 / fCD16 or NIH3T3 / fCD32) according to the method described in the literature (Mizuno et al., J Vet Med Sci, 71(12):1561-1568, 2009). More specifically, 2 x 10 5 The two types of stable-expressing cell lines were incubated on ice for 30 minutes in the presence of 10 μg / mL of each of the three antibodies. After washing, the cells were incubated on ice for 30 minutes in the presence of anti-cat IgG-Alexa (registered trademark) 647 (anti-cat IgG manufactured by Jackson ImmunoResearch, labeled with Alexa 647). The fluorescence intensity derived from Alexa 647 was then detected using a flow cytometer (CytoFLEX [manufactured by Beckman Coulter]). The results were analyzed using FlowJo software (manufactured by Treestar).

[0076] As a result, when two types of stably expressing cell lines (NIH3T3 / fCD1 and NIH3T3 / fCD32) were incubated in the presence of "ch-1A1-2 M1," the fluorescence intensity derived from Alexa647 was at the same level as that when they were incubated in the presence of fIgG or "ch-1A1-2" (see Figure 2).

[0077] These results indicate that the binding of "ch-1A1-2 M1" to fCD16 and fCD32 is almost the same as that of fIgG and "ch-1A1-2," and that the amino acid substitutions in the heavy chain constant region of "ch-1A1-2 M1" (i.e., five amino acid substitutions: M119I, L120P, G122A, P216A, and I217M) do not reduce the cytotoxic activity or phagocytic activity.

[0078] 4. Confirmation that amino acid substitutions in the heavy chain constant regions of "ch-1A1-2 M2" and "ch-1A1-2 M3" reduce cytotoxic activity and cytophagocytic activity To investigate whether amino acid substitutions in the heavy chain constant regions of "ch-1A1-2 M2" and "ch-1A1-2 M3" reduce cytotoxic activity and cytophagocytic activity, the binding of "ch-1A1-2 M2" and "ch-1A1-2 M3" to fCD16, fCD32, and fCD64 was analyzed. Specifically, flow cytometry analysis was performed using four types of antibodies (fIgG, "ch-1A1-2," "ch-1A1-2 M2," and "ch-1A1-2 M3") and four types of cell lines (NIH3T3 cell line, NIH3T3 / fCD16, NIH3T3 / fCD32, or NIH3T3 / fCD64) according to the method described in the literature "Mizuno et al., J Vet Med Sci, 71(12):1561-1568, 2009." More specifically, 2 × 10 5Each of the four cell lines was incubated on ice for 30 minutes in the presence of 10 μg / mL of each of the four antibodies. After washing, the cells were incubated on ice for 30 minutes in the presence of anti-cat IgG F(ab)'2-biotin (biotin-labeled secondary antibody) (Rockland Immunochemicals), washed, and then incubated on ice for 30 minutes in the presence of Dylight649-labeled streptavidin (StAv-Dylight649) (BioLegend). Fluorescence intensity derived from Dylight649 was detected using a flow cytometer (CytoFLEX [Beckman Coulter]). The results were analyzed using FlowJo software (Treestar).

[0079] As a result, when three types of stably expressing cell lines (NIH3T3 / fCD16, NIH3T3 / fCD32, and NIH3T3 / fCD64) were incubated in the presence of fIgG or "ch-1A1-2," the fluorescence intensity derived from Dylight649 increased compared to when incubated in the absence of these antibodies, whereas when incubated in the presence of the two types of anti-cat antibodies of the present invention ("ch-1A1-2 M2" and "ch-1A1-2 M3"), no such increase in fluorescence intensity was observed (see Figure 3).

[0080] These results indicate that "ch-1A1-2 M2" and "ch-1A1-2 M3" hardly bind to fCD16, fCD32, and fCD64, and that the amino acid substitutions in the heavy chain constant region of "ch-1A1-2 M2" (i.e., four amino acid substitutions: M119A, L120A, P214G, and S215A) and the amino acid substitutions in the heavy chain constant region of "ch-1A1-2 M3" (i.e., three amino acid substitutions: M119S, L120T, and G121R) reduce cytotoxic activity and cytophagocytic activity.

[0081] 5. Confirmation that amino acid substitutions in the heavy chain constant regions of "ch-1A1-2 M2" and "ch-1A1-2 M3" reduce cytotoxic activity To investigate whether amino acid substitutions in the heavy chain constant regions of "ch-1A1-2 M2" and "ch-1A1-2 M3" reduce cytotoxic activity, the complement binding of "ch-1A1-2 M2" and "ch-1A1-2 M3" was analyzed by ELISA. Specifically, various concentrations (0.39 [μg / mL], 0.78 [μg / mL], 1.56 [μg / mL], 3.12 [μg / mL], 6.25 [μg / mL], 12.5 [μg / mL], 25 [μg / mL], and 50 [μg / mL]) of three types of antibodies ("ch-1A1-2," "ch-1A1-2 M2," and "ch-1A1-2 M3") were added in 100 μL volumes to a Maxisorp (registered trademark) ELISA plate (manufactured by Nunc) and allowed to stand overnight at 4 ° C., thereby carrying out antibody immobilization treatment. After washing the plate, 100 μL of PBS (Phosphate Buffered Saline) containing 6% BSA (Bovine Serum Albumin) was added to each plate, and blocking treatment was carried out by incubating at 37 ° C. for 1 hour. After washing the plate, 100 μL of 2% BSA / PBS containing 10 μg / mL human C1q (QuidelOrtho) was added to each plate and incubated at 37°C for 1 hour to allow antibody-complement binding. After washing the plate, 100 μL of mouse anti-human C1q antibody (A201, QuidelOrtho) diluted 3000-fold with 2% BSA / PBS was added to each plate and incubated at 37°C for 1 hour to allow primary antibody reaction. After washing the plate, 100 μL of anti-mouse IgG-HRP (Invitrogen) diluted 10000-fold with 2% BSA / PBS was added to each plate and incubated at 37°C for 1 hour to allow secondary antibody reaction. After washing the plate, 100 μL of ABTS substrate (SeraCare) was added to each well and incubated at 37° C. for 40 minutes. The absorbance (OD value) at 405 nm was then measured using a microplate reader (ARVO X4, PerkinElmer).

[0082] As a result, when plates immobilized with "ch-1A1-2 M2" or "ch-1A1-2 M3" were used, the OD value (405 nm) was lower at all concentrations compared to when plates immobilized with "ch-1A1-2" were used (see Figure 4).

[0083] These results indicate that the binding of "ch-1A1-2 M2" and "ch-1A1-2 M3" to complement (human C1q) is reduced, and that the amino acid substitutions in the heavy chain constant region of "ch-1A1-2 M2" (i.e., four amino acid substitutions: M119A, L120A, P214G, and S215A) and the amino acid substitutions in the heavy chain constant region of "ch-1A1-2 M3" (i.e., three amino acid substitutions: M119S, L120T, and G121R) reduce cytotoxic activity.

[0084] 6. Confirmation that the amino acid substitutions in the heavy chain constant region of the present anti-feline antibody do not adversely affect the function of the variable region of the present anti-feline antibody 1 To confirm that the amino acid substitutions in the heavy chain constant region of the present anti-feline antibodies "ch-1A1-2 M2" and "ch-1A1-2 M3" do not adversely affect the function of the variable region of the present anti-feline antibody, the binding ability of the anti-feline PD-1 antibody to the antigen (fPD-1) was analyzed. Specifically, three types of anti-fPD-1 antibodies ("ch-1A1-2," "ch-1A1-2 M2," and "ch-1A1-2 M3") and NIH3T3 / fPD-1 (NIH3T3 cell line stably expressing fPD-1; see Patent Document 4) were used, and flow cytometry analysis was performed according to the method described in the literature "Mizuno et al., J Vet Med Sci, 71(12):1561-1568, 2009." More specifically, 2 × 10 5Each NIH3T3 / fPD-1 or NIH3T3 cell line was incubated on ice for 30 minutes in the presence of 10 μg / mL of each of the three types of anti-fPD-1 antibodies or fIgG, washed, and then incubated on ice for 30 minutes in the presence of the above-mentioned anti-cat IgG-Alexa647. Fluorescence intensity derived from Alexa647 was then detected using a flow cytometer (CytoFLEX [manufactured by Beckman Coulter]), and the results obtained were analyzed using FlowJo software (manufactured by Treestar).

[0085] As a result, when the NIH3T3 cell line was incubated in the presence of three types of anti-fPD-1 antibodies (see FIG. 5A), or when NIH3T3 / fPD-1 was incubated in the presence of fIgG (see FIG. 5B), no increase in fluorescence intensity derived from Alexa647 was observed. However, when NIH3T3 / fPD-1 was incubated in the presence of "ch-1A1-2 M2" or "ch-1A1-2 M3", an increase in fluorescence intensity derived from Alexa647 was observed, and the level was the same as when incubated in the presence of "ch-1A1-2" (see FIG. 5B).

[0086] These results indicate that the two types of anti-cat antibodies of the present invention ("ch-1A1-2 M2" and "ch-1A1-2 M3"), in which amino acid substitutions were introduced into the feline IgG1 heavy chain constant region to reduce cytotoxic activity and cytophagocytic activity, can bind to the antigen (fPD-1) to the same extent as the anti-fPD-1 antibody ("ch-1A1-2") before the amino acid substitutions were introduced, and that the introduction of amino acid substitutions in the heavy chain constant region has almost no adverse effect on antigen (fPD-1) binding.

[0087] 7. Confirmation that the amino acid substitutions in the heavy chain constant region of the present anti-feline antibody do not adversely affect the function of the variable region of the present anti-feline antibody 2 To confirm that the amino acid substitutions in the heavy chain constant region of the present anti-feline antibody do not adversely affect the function of the variable region of the present anti-feline antibody, the inhibitory effect of anti-feline PD-1 antibodies on the binding of fPD-1 and fPD-L1 was analyzed. 5NIH3T3 / fPD-1 cells were 1) incubated on ice for 30 minutes in the absence of three types of anti-fPD-1 antibodies ("ch-1A1-2," "ch-1A1-2 M2," and "ch-1A1-2 M3") and in the presence of hIg, or 2) incubated on ice for 30 minutes in the presence of various concentrations (0 μg / mL, 0.16 μg / mL, 0.63 μg / mL, 2.5 μg / mL, 10 μg / mL, or 40 μg / mL) of the three types of anti-fPD-1 antibodies and 10 μg / mL of fPD-L1-hIg, or 3) incubated on ice for 30 minutes in the presence of 40 μg / mL of the three types of anti-fPD-1 antibodies. After washing, the cells were incubated on ice for 30 minutes in the presence of anti-human IgG-PE, and then analyzed using a flow cytometer (CytoFLEX [Beckman The fluorescence intensity derived from PE was detected using a fluorescent dye (manufactured by Coulter), and the results were analyzed using FlowJo software (manufactured by Treestar).

[0088] As a result, the fluorescence intensity derived from anti-human IgG-PE, which was increased by incubating NIH3T3 / fPD-1 in the presence of fPD-L1-hIg (but not hIg), was reduced by co-incubation with "ch-1A1-2 M2" or "ch-1A1-2 M3" in a concentration-dependent manner (see Figures 6B and 6C), and the level of reduction was the same as when incubated in the presence of "ch-1A1-2" (see Figure 6A).

[0089] These results indicate that the two types of anti-cat antibodies of the present invention ("ch-1A1-2 M2" and "ch-1A1-2 M3"), in which amino acid substitutions were introduced into the feline IgG1 heavy chain constant region to reduce cytotoxic activity and cytophagocytic activity, inhibit the binding of fPD-1 to fPD-L1 in fPD-L1-hIg to the same extent as the anti-fPD-1 antibody ("ch-1A1-2") before the amino acid substitutions were introduced.

[0090] 8. Confirmation that the amino acid substitutions in the heavy chain constant region of the present anti-feline antibody do not adversely affect the function of the variable region of the present anti-feline antibody 3 To confirm that the amino acid substitutions in the heavy chain constant region of the present anti-feline antibody do not adversely affect the function of the variable region of the present anti-feline antibody, the effect of anti-feline PD-1 antibodies in relieving lymphocyte exhaustion was analyzed. Specifically, fPD-L1-hIg was added simultaneously with PBMC stimulation to reproduce a state in which fIFNγ secretion was suppressed (i.e., a state in which lymphocytes were exhausted). Analysis was performed to determine whether this exhaustion state could be reversed by three types of anti-fPD-1 antibodies ("ch-1A1-2," "ch-1A1-2 M2," and "ch-1A1-2 M3"). More specifically, PBMCs were isolated from healthy cats according to standard methods, and then placed in a 96-well round-bottom plate at 2 x 10 per well. ５ The cells were seeded so that each cell number was 1, and 10 μg / mL of ConA was added (i.e., stimulated with ConA). At the same time, 10 μg / mL of fPD-L1-hIg or hIg and 10 μg / mL or 40 μg / mL of the three types of anti-fPD-1 antibodies or pseudoantibodies (fIgG1, Biolegend) were added. After 48 hours of culture, the culture supernatant was collected, and fIFNγ in the culture supernatant was measured using Feline IFN-gamma DuoSet ELISA (R&D).

[0091] First, we confirmed that when PBMCs stimulated with ConA were cultured in the presence of fPD-L1-hIg, the concentration of fIFNγ produced in the culture medium was reduced compared to when cultured in the presence of hIg (see Figure 7). This result indicates that the exhausted state of lymphocytes was reproduced.

[0092] Next, when PBMCs stimulated with ConA were cultured in the presence of fPD-L1-hIg and fIgG, no increase in the concentration of fIFNγ produced in the culture medium was observed. However, when PBMCs were cultured in the presence of fPD-L1-hIg and "ch-1A1-2 M2" or "ch-1A1-2 M3," the concentration of fIFNγ produced in the culture medium increased, and the level of increase was shown to be at the same level as when cultured in the presence of "ch-1A1-2" (see Figure 7).

[0093] These results indicate that the two types of anti-cat antibodies of the present invention ("ch-1A1-2 M2" and "ch-1A1-2 M3"), in which amino acid substitutions were introduced into the feline IgG1 heavy chain constant region to reduce cytotoxic activity and cytophagocytic activity, relieve the exhausted state of lymphocytes to the same extent as the anti-fPD-1 antibody ("ch-1A1-2") before the amino acid substitutions were introduced.

[0094] 9. Confirmation that amino acid substitutions in the heavy chain constant region of the present anti-cat antibodies do not adversely affect binding to FcRn To confirm that the amino acid substitutions in the heavy chain constant region of the present anti-cat antibodies "ch-1A1-2 M2" and "ch-1A1-2 M3" do not adversely affect binding to FcRn, the binding of the anti-cat PD-1 antibodies to FcRn was analyzed. Specifically, NIH3T3 / fFcRn (i.e., an NIH3T3 cell line stably expressing fFcRn) was prepared according to the method described in Example 1 (see "fFcRn" in Figure 8A), and flow cytometry analysis using the prepared NIH3T3 / fFcRn and three types of antibodies ("ch-1A1-2," "ch-1A1-2 M2," and "ch-1A1-2 M3") was performed according to the method described in Example 4 (see Figure 8B).

[0095] As a result, when NIH3T3 / fFcRn was incubated in the presence of two types of the present anti-cat antibodies ("ch-1A1-2 M2" and "ch-1A1-2 M3"), an increase in fluorescence intensity derived from Dylight649 was observed, and the level was similar to that observed when the antibody was incubated in the presence of "ch-1A1-2" (see Figure 8B).

[0096] These results indicate that the two types of anti-cat antibodies of the present invention ("ch-1A1-2 M2" and "ch-1A1-2 M3"), in which amino acid substitutions were introduced into the feline IgG1 heavy chain constant region to reduce cytotoxic activity and cytophagocytic activity, can bind to fFcRn to the same extent as the anti-fPD-1 antibody ("ch-1A1-2") before the amino acid substitutions were introduced (i.e., the blood half-life of the anti-cat antibodies of the present invention is unchanged and at the same level as the blood half-life of the anti-fPD-1 antibody before the amino acid substitutions were introduced).

[0097] In summary, these results demonstrate that anti-feline antibodies containing a feline IgG1 heavy chain constant region with four amino acid substitutions (M119A, L120A, P214G, and S215A; M2 variant) and anti-feline antibodies containing a feline IgG1 heavy chain constant region with three amino acid substitutions (M119S, L120T, and G121R; M3 variant) have reduced cytotoxic and cytophagic activities without adversely affecting the function of the variable region or binding to FcRn.

[0098] Furthermore, similar effects can be expected for feline IgG2 having the M2 or M3 variant, as with feline IgG1 having the M2 or M3 variant. That is, feline IgG2 having the M2 variant (polypeptide consisting of the amino acid sequence of SEQ ID NO: 5; see Table 6) of the feline IgG2 heavy chain constant region (polypeptide consisting of the amino acid sequence of SEQ ID NO: 7; see Table 8) in which four amino acid substitutions (I119A, P120A, P214G, and S215A) have been introduced, and feline IgG2 having the M3 variant (polypeptide consisting of the amino acid sequence of SEQ ID NO: 6; see Table 7) of the feline IgG2 heavy chain constant region in which three amino acid substitutions (I119S, P120T, and G121R) have been introduced, are also expected to have reduced cytotoxic activity and cytophagocytic activity without adversely affecting the function of the variable region or FcRn binding.

[0099] In the table, "A １ " indicates I119A, and "A ２ " indicates P120A, and "G ３ " indicates P214G, and "A ４ " indicates S215A.

[0100] In the table, "S １ " indicates I119S, and "T ２ " indicates P120T, and "R ３ " indicates G121R.

[0101] The present invention is useful for immunotherapy (e.g., cancer immunotherapy) in cats.

Claims

1. An anti-cat antibody comprising a light chain and a heavy chain, wherein the constant region of the heavy chain consists of an amino acid sequence having an alanine residue at position 119, an alanine residue at position 120, a glycine residue at position 214, and an alanine residue at position 215 of the amino acid sequence shown in SEQ ID NO: 1, and having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having a serine residue at position 119, a threonine residue at position 120, and an arginine residue at position 121 of the amino acid sequence shown in SEQ ID NO: 2, and having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having an alanine residue at position 119, an alanine residue at position 120, a glycine residue at position 214, and an alanine residue at position 215 of the amino acid sequence shown in SEQ ID NO: 5, and having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 5, or The anti-cat antibody comprises an amino acid sequence having a serine residue at position 119, a threonine residue at position 120, and an arginine residue at position 121 of the amino acid sequence shown in SEQ ID NO: 6, and having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

6.

2. The anti-cat antibody of claim 1, which specifically binds to an immune checkpoint molecule.

3. The anti-cat antibody according to claim 2, wherein the immune checkpoint molecule is PD-1.

4. A polynucleotide encoding the anti-cat antibody according to any one of claims 1 to 3.

5. A vector comprising a promoter and the polynucleotide of claim 4 operably linked downstream of said promoter.

6. A host cell into which the vector of claim 5 has been introduced.

7. An agent for activating immune responses in cats, comprising the anti-feline antibody of claim 2 or 3, or a vector comprising a promoter and a polynucleotide encoding the anti-feline antibody of claim 2 or 3, operably linked downstream of the promoter.

8. A method for activating an immune response in a cat, comprising the step of administering to a cat the anti-feline antibody of claim 2 or 3, or a vector comprising a promoter and a polynucleotide encoding the anti-feline antibody of claim 2 or 3, operably linked downstream of the promoter.

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