Agent for controlling cell competition

Abstract

The present invention provides a cell competition inhibitor comprising an agonist antibody, which is directed against a protein containing the α3 domain of MHC class Ia or LILRB3, or a substance which inhibits the binding of LILRB3 to MHC class Ia.

Description

Technical Field

[0001] The present invention relates to a novel agent for controlling cell competition.

Background Art

[0002] Cell competition was originally discovered in Drosophila in 1975 (Non-Patent Document 1) and has been studied by Drosophila researchers. Although it has not been clarified until recently whether the same competitive phenomenon exists in mammalian cells, in 2009, the group of the present inventors reported for the first time in the world that cell competition occurs in mammals (Non-Patent Document 2), and it is now clear that cell competition is a necessary and universal phenomenon for maintaining the homeostasis of the cell society, occurring in various physiological and pathological processes. Also, recent studies have gradually accumulated knowledge about the molecular mechanism by which cells that become losers in cell competition are excluded from tissues (Non-Patent Document 3).

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, the recognition mechanism between cells, namely, the mechanism of inducing the elimination ability of epithelial cells by antigen presentation, and the intercellular communication molecules have been completely unknown until now. Therefore, an object of the present invention is to elucidate the molecular entity responsible for the mechanism of inducing the elimination ability of epithelial cells by antigen presentation, which is involved in mammalian cell competition, and to provide a controlling agent for cell competition using the mechanism and a method for controlling cell competition using the agent.

[0005] As described above, more than 80% of the cancers that occur in our bodies are derived from epithelial cells. Therefore, by applying the mechanism of inducing the elimination ability of epithelial cells by antigen presentation to medical treatment, it may be possible to develop preventive medicine targeting the treatment of ultra-early stage cancers, that is, removing mutant cells before carcinogenesis. Also, for advanced cancers, the idea was obtained that it may be applicable to cancer treatment by removing cancer cells using the above mechanism. To elucidate the above mechanism, the present inventor first focused on the cancer gene RasV12, which is known to promote cell competition when introduced into epithelial cells. When this gene was introduced into a human skin-derived cell line and RasV12 was expressed in the cells, it was found that the cell membrane expression of major histocompatibility complex (hereinafter referred to as "MHC") class I was promoted. From this finding, the present inventor considered that MHC class I and cell competition might be involved. When RasV12 was expressed in TAP1-deficient cells, which are known to suppress the expression of MHC-I, it was shown that cell competition was not promoted, that is, the cell membrane expression of MHC class I is necessary for cell competition. Next, focusing on DLA, which is canine MHC-I, when individual DLAs were deleted in the RasV12-introduced strain, it was shown that DLA88, which is MHC-Ia, plays an essential role in cell competition. Therefore, even when the full-length peptide of DLA88 was administered to cells, cell competition was not promoted. However, when a peptide fragment consisting of the α3 domain, which constitutes the extracellular domain, was administered to cells, unexpectedly, cell competition was promoted. That is, a new finding was obtained that the α3 domain of MHC-Ia is important for cell competition.

[0006] Based on the above findings, under the presumption that the interaction between MHC class I and its receptor might be involved in cell competition, attempts were made to identify the receptor for MHC class I. As a result, it was found that the immunoglobulin-like receptor LILRB3 (leukocyte immunoglobulin-like receptor, subfamily B, member 3) functions as a receptor for MHC class I. Therefore, focusing on LILRB3 and proceeding with research, it was found that (i) knocking out the immunoglobulin-like receptor LILRB3 (leukocyte immunoglobulin-like receptor, subfamily B, member 3) on the epithelial cell membrane reduces the ability to eliminate abnormal cells, and (ii) a recombinant protein containing the D1-D2 domain among the four immunoglobulin domains D1-D4 of LILRB3 binds to a recombinant protein containing the α3 domain of MHC class 1a, and furthermore, (iii) a recombinant protein containing the D1-D2 domain of LILRB3 exhibits an inhibitory effect on abnormal cells due to cell competition. Based on these findings, further research was carried out, and as a result, the present invention was completed.

Means for Solving the Problems

[0007] That is, the present invention relates to the following. [1] A controller for cell competition, comprising a protein containing the α3 domain of MHC class Ia or an agonist antibody for LILRB3, or a substance that inhibits the binding between MHC class Ia and LILRB3. [2] The agent according to [1], comprising a protein containing the α3 domain of MHC class Ia, wherein the control of cell competition is the promotion of cell competition. [3] The agent according to [2], wherein MHC class Ia is HLA-B or DLA-88. [4] The protein containing the α3 domain of MHC class Ia is as follows: a) A protein having the amino acid sequence shown in SEQ ID NO: 1, b) A protein having an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 1, and specifically binds to a protein containing the D1 and D2 domains of LILRB3, c) A protein having an amino acid sequence having at least 90% similarity to the amino acid sequence shown by SEQ ID NO: 1, and specifically binds to a protein containing the D1 and D2 domains of LILRB3, and d) A protein containing the α3 domain of an ortholog of DLA-88 The agent according to [2] or [3], comprising at least one protein selected from the group consisting of [5] The agent according to [4], wherein the protein containing the D1 and D2 domains of LILRB3 is a protein having the amino acid sequence shown by SEQ ID NO: 8. [6] The agent according to any one of [2] to [5], wherein the cell competition is the elimination of abnormal cells by normal non-immune cells. [7] The agent according to [6], wherein the normal non-immune cells are epithelial cells. [8] The agent according to [6] or [7], wherein the abnormal cells are mutant cells, cancer cells or virus-infected cells. [9] The agent according to [8], wherein the cell competition regulator is a prophylactic or therapeutic agent for cancer or virus infection.

[10] The agent according to [1], comprising a substance that inhibits the binding between MHC class Ia and LILRB3, and the control of cell competition is the suppression of cell competition.

[11] The agent according to

[10] , wherein the substance that inhibits the binding between MHC class Ia and LILRB3 is at least one substance selected from the group consisting of a protein containing the D1 and D2 domains of LILRB3, an antibody against the D1 and D2 domains of LILRB3, an aptamer, and an inhibitor of LILRB3 expression.

[12] The protein containing the D1 and D2 domains of LILRB3 is as follows: e) A protein having the amino acid sequence shown by SEQ ID NO: 8, f) A protein having an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 8, and which specifically binds to a protein containing the α3 domain of MHC class Ia, and g) A protein having an amino acid sequence having at least 80% similarity to the amino acid sequence represented by SEQ ID NO: 8, and which specifically binds to a protein containing the α3 domain of MHC class Ia h) A protein containing the D1 and D2 domains of an ortholog of ENSCAFG00000028453 The agent according to

[11] , comprising at least one protein selected from

[13] The agent according to any one of

[10] to

[12] , wherein the cell competition is the elimination of transplanted cells by normal non-immune cells.

[14] The agent according to any one of

[10] to

[12] , wherein the suppression of cell competition is the improvement of engraftment failure in organ transplantation, tissue transplantation or cell transplantation, or the suppression of symptom progression of neurodegenerative diseases, myopathic diseases and other various degenerative diseases.

[15] The agent according to

[14] , wherein the organ transplantation is transplantation of at least one organ selected from the group consisting of the liver, kidney, heart, lung, pancreas, thyroid gland, parathyroid gland, thymus, adrenal cortex and adrenal medulla, and organs containing stem cells.

[16] The agent according to

[14] , wherein the tissue transplantation is transplantation of at least one tissue selected from the group consisting of skin, tissues containing stem cells, and tissues containing immune cells.

[17] The agent according to

[14] , wherein the cell transplantation is transplantation of at least one cell selected from the group consisting of bone marrow cells, non-adherent bone marrow cells, peripheral blood cells, cord blood cells, Wharton's jelly-derived cells, placenta-derived cells, hair follicle-derived cells, adipose tissue-derived cells, lymphocytes, monocytes and macrophages.

[18] The agent according to

[14] , wherein the various degenerative diseases are cervical spondylotic myelopathy and / or lumbar spinal stenosis.

[19] The agent according to

[14] , wherein the neurodegenerative disease is at least one neurodegenerative disease selected from the group consisting of Alzheimer's disease, mild cognitive impairment, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, Creutzfeldt-Jakob disease, trauma-induced neurodegeneration, high-pressure neuropathy syndrome, dystonia, olivopontocerebellar atrophy, amyotrophic lateral sclerosis, multiple sclerosis, epilepsy, dementia, senile dementia, AIDS dementia complex, and AIDS-induced encephalopathy.

[20] The agent according to

[14] , wherein the myopathic disease is at least one myopathic disease selected from the group consisting of muscular dystrophy, distal myopathy, congenital myopathy, thyrotoxic myopathy, glycogenosis, mitochondrial myopathy, steroid myopathy, alcoholic myopathy, inflammatory myopathy, endocrine myopathy, lipid storage disorder myopathy, myopathy associated with infectious diseases such as HIV, vitreous myopathy, and myopathy associated with autoimmune diseases such as myasthenia gravis.

[21] The agent according to

[14] , which is an agent for improving the engraftment failure of organ transplantation, tissue transplantation or cell transplantation, or an agent for suppressing the progression of symptoms of neurodegenerative diseases, myopathic diseases and other various degenerative diseases.

[22] The agent according to any one of [1] to

[21] , which is used in combination with other drugs and / or other treatment methods.

[23] The agent according to

[22] , wherein the other drug is an anticancer agent or an antiviral agent.

[24] The agent according to

[23] , wherein the anticancer agent is at least one anticancer agent selected from the group consisting of alkylating agents, cytotoxic antibiotics, platinum preparations, antimetabolites, kinase inhibitors, angiogenesis inhibitors, hormonal agents, DNA modifying enzyme inhibitors, proteasome inhibitors, alkaloid agents, type I and type II topoisomerase inhibitors, histone deacetylase inhibitors, cytokine preparations, hormonal agents, immune checkpoint inhibitors, natural killer cell activators, indoleamine 2,3-dioxygenase (IDO) inhibitors, monoclonal antibodies, and other molecular target therapeutic agents.

[25] The agent according to

[23] , wherein the antiviral agent is at least one antiviral agent selected from the group consisting of interferon, nucleoside and nucleotide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, fusion inhibitors, maturation inhibitors, guanosine analogs, purine analogs, pyrimidine analogs, and other "unclassified" antiviral agents recognized in the art that are not included in any of the above classes (e.g., foscarnet and milphosohine).

[26] The agent according to

[22] , wherein the other treatment method is at least one method selected from the group consisting of cancer surgery, radiation therapy, laser irradiation therapy, and hyperthermia therapy.

[27] The agent according to

[22] , wherein the other agent is at least one agent selected from the group consisting of immunosuppressants, inhibitors of transplant organ rejection, and promoters of engraftment after transplantation.

[28] The agent according to

[27] , wherein the immunosuppressant is at least one immunosuppressant selected from the group consisting of steroids, cyclosporine, cyclosporine analogs, cyclophosphamide, methylprednisolone, prednisone, azathioprine, tacrolimus hydrate, 15-deoxyspergualin, natalizumab, rapamycin, and etanercept.

[29] The agent according to any one of [1] to

[28] , wherein the agent is a pharmaceutical composition.

[30] A method for controlling cell competition, comprising administering a therapeutically effective amount of an agonist antibody against a protein containing the α3 domain of MHC class Ia or LILRB3, or a substance that inhibits the binding of MHC class Ia and LILRB3, to a subject in need thereof.

[31] The method according to

[30] , wherein an agonist antibody against a protein containing the α3 domain of MHC class Ia or LILRB3 is administered, and the control of cell competition is the promotion of cell competition.

[32] The method according to

[31] , wherein MHC class Ia is HLA-B or DLA-88.

[33] The protein containing the α3 domain of MHC class Ia is as follows: a) A protein having the amino acid sequence shown in SEQ ID NO: 1, b) A protein having an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, and which specifically binds to a protein containing the D1 and D2 domains of LILRB3, c) A protein having an amino acid sequence having at least 90% similarity to the amino acid sequence shown in SEQ ID NO: 1, and which specifically binds to a protein containing the D1 and D2 domains of LILRB3, and d) A protein containing the α3 domain of an ortholog of DLA-88 The method according to

[31] or

[32] , comprising at least one protein selected from the group consisting of:

[34] The method according to

[33] , wherein the protein containing the D1 and D2 domains of LILRB3 is a protein having the amino acid sequence shown in SEQ ID NO: 8.

[35] The method according to any one of

[31] to

[34] , wherein the cell competition is the elimination of abnormal cells by normal non-immune cells.

[36] The method according to

[35] , wherein the normal non-immune cells are epithelial cells.

[37] The method according to

[35] or

[36] , wherein the abnormal cells are mutant cells, cancer cells or virus-infected cells.

[38] The method according to

[37] , wherein the control of cell competition is the prevention or treatment of cancer or virus infection.

[39] The method according to

[30] , wherein a substance that inhibits the binding of MHC class Ia and LILRB3 is administered, and the control of cell competition is the suppression of cell competition.

[40] The method according to

[39] , wherein the substance that inhibits the binding of MHC class Ia and LILRB3 is at least one substance selected from the group consisting of a protein containing the D1 and D2 domains of LILRB3, an antibody against the D1 and D2 domains of LILRB3, an aptamer, and an inhibitor of LILRB3 expression.

[41] The protein containing the D1 and D2 domains of LILRB3 is as follows: e) A protein having the amino acid sequence shown in SEQ ID NO: 8, f) A protein having an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 8, and which specifically binds to a protein containing the α3 domain of MHC class Ia, g) A protein having an amino acid sequence having at least 80% similarity to the amino acid sequence shown in SEQ ID NO: 8, and which specifically binds to a protein containing the α3 domain of MHC class Ia, and h) A protein containing the D1 and D2 domains of an ortholog of ENSCAFG00000028453 The method according to

[39] or

[40] , comprising at least one protein selected from the group consisting of:

[42] The method according to any one of

[39] to

[41] , wherein the cell competition is the elimination of transplanted cells by non-immune normal cells.

[43] The method according to any one of

[39] to

[42] , wherein the suppression of cell competition is the improvement of engraftment failure in organ transplantation, tissue transplantation or cell transplantation, or the suppression of symptom progression of neurodegenerative diseases, myopathic diseases and other various degenerative diseases.

[44] The method according to

[43] , wherein the organ transplantation is transplantation of at least one organ selected from the group consisting of the liver, kidney, heart, lung, pancreas, thyroid gland, parathyroid gland, thymus, adrenal cortex and adrenal medulla, and organs containing stem cells.

[45] The method according to

[43] , wherein the tissue transplantation is transplantation of at least one tissue selected from the group consisting of skin, tissues containing stem cells, and tissues containing immune cells.

[46] The method according to

[43] , wherein the cell transplantation is transplantation of at least one cell selected from the group consisting of bone marrow cells, non-adherent bone marrow cells, peripheral blood cells, cord blood cells, Wharton's jelly-derived cells, placenta-derived cells, hair root-derived cells, adipose tissue-derived cells, lymphocytes, monocytes and macrophages.

[47] The method according to

[43] , wherein the various degenerative diseases are cervical spondylotic myelopathy and / or lumbar spinal stenosis.

[48] The method according to

[43] , wherein the neurodegenerative disease is at least one neurodegenerative disease selected from the group consisting of Alzheimer's disease, mild cognitive impairment, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, Creutzfeldt-Jakob disease, trauma-induced neurodegeneration, high-pressure neuropathy syndrome, dystonia, olivopontocerebellar atrophy, amyotrophic lateral sclerosis, multiple sclerosis, epilepsy, dementia, senile dementia, AIDS dementia complex, and AIDS-induced encephalopathy.

[49] The method according to

[43] , wherein the myopathic disease is at least one myopathic disease selected from the group consisting of muscular dystrophy, distal myopathy, congenital myopathy, thyrotoxic myopathy, glycogenosis, mitochondrial myopathy, steroid myopathy, alcoholic myopathy, inflammatory myopathy, endocrine myopathy, lipid storage disorder myopathy, myopathy associated with infectious diseases such as HIV, vitreous myopathy, and myopathy associated with autoimmune diseases such as myasthenia gravis.

[50] The method according to

[30] to

[49] , which is used in combination with the administration of other drugs and / or other treatment methods.

[51] The method according to

[50] , wherein the other drug is an anticancer agent or an antiviral agent.

[52] The method according to

[51] , wherein the anticancer agent is at least one anticancer agent selected from the group consisting of alkylating agents, cytotoxic antibiotics, platinum preparations, antimetabolites, kinase inhibitors, angiogenesis inhibitors, hormonal agents, DNA modifying enzyme inhibitors, proteasome inhibitors, alkaloid agents, type I and type II topoisomerase inhibitors, histone deacetylase inhibitors, cytokine preparations, hormonal agents, immune checkpoint inhibitors, natural killer cell activators, indoleamine 2,3-dioxygenase (IDO) inhibitors, monoclonal antibodies, and other molecular target therapeutic agents.

[53] The method according to

[51] , wherein the antiviral agent is at least one antiviral agent selected from the group consisting of interferon, nucleoside and nucleotide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, fusion inhibitors, maturation inhibitors, guanosine analogs, purine analogs, pyrimidine analogs, and other "unclassified" antiviral agents recognized in the art that are not included in any of the above classes (e.g., foscarnet and miltefosine).

[54] The method according to

[50] , wherein the other treatment method is at least one method selected from the group consisting of cancer surgery, radiotherapy, laser irradiation therapy, and hyperthermia therapy.

[55] The method according to

[50] , wherein the other agent is at least one agent selected from the group consisting of immunosuppressants, inhibitors of transplant organ rejection, and promoters of engraftment after transplantation.

[56] The method according to

[55] , wherein the immunosuppressant is at least one immunosuppressant selected from the group consisting of steroids, cyclosporine, cyclosporine analogs, cyclophosphamide, methylprednisolone, prednisone, azathioprine, tacrolimus hydrate, 15-deoxyspergualin, natalizumab, rapamycin, and etanercept.

[57] A protein containing the α3 domain of MHC class Ia or a protein containing the D1 and D2 domains of LILRB3 for use in controlling cell competition.

[58] The protein according to

[57] , which is a protein containing the α3 domain of MHC class Ia, and wherein the control of cell competition is the promotion of cell competition.

[59] The protein according to

[58] , wherein MHC class Ia is HLA-B or DLA-88.

[60] The protein according to

[57] , which is a protein containing the D1 and D2 domains of LILRB3, and wherein the control of cell competition is the suppression of cell competition.

[61] Use of a protein containing the α3 domain of MHC class Ia or an agonist antibody against LILRB3, or a substance that inhibits the binding of MHC class Ia and LILRB3, in the manufacture of a medicament for controlling cell competition.

[62] The use according to

[61] , wherein the agonist antibody against a protein containing the α3 domain of MHC class Ia or LILRB3 promotes cell competition in controlling cell competition.

[63] The protein according to

[60] , wherein MHC class Ia is HLA-B or DLA-88.

[64] Use of a substance that inhibits the binding of MHC class Ia and LILRB3 in the protein according to

[61] , wherein controlling cell competition is suppressing cell competition.

[65] The method according to

[64] , wherein the substance that inhibits the binding of MHC class Ia and LILRB3 is at least one substance selected from the group consisting of a protein containing the D1 and D2 domains of LILRB3, an antibody against the D1 and D2 domains of LILRB3, an aptamer, and an inhibitor of LILRB3 expression. [Effect of the Invention]

[0008] According to the present invention, it is possible to provide a controller for cell competition, a method for controlling cell competition, a protein for use in a method for controlling cell competition, and the use of a protein in the manufacture of a medicament for controlling cell competition. The agent of the present invention controls cell competition by controlling the interaction between MHC class Ia and LILRB3, which is a receptor therefor, and as a result, neurodegenerative diseases (e.g., amyotrophic lateral sclerosis, Alzheimer's disease, etc.), muscle degenerative diseases (e.g., muscular dystrophy), cancers (including precancerous states) (e.g., pancreatic cancer, lung cancer, skin cancer, uterine cancer, etc.), bacterial infections or viral infections (e.g., human papillomavirus (HPV), etc.), and prevention and treatment of cell transplantation engraftment failure (e.g., induced pluripotent stem cell (iPS cell) transplantation, cardiomyocyte transplantation, corneal transplantation, skin transplantation, etc.) become possible. [Brief Description of the Drawings]

[0009]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 2A

Figure 2B

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 4A

Figure 4B

Figure 4C

Figure 4D

Figure 5

Figure 6A

Figure 6B

Figure 7A

Figure 7B

Figure 7C

Figure 7D

Figure 7E

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Mode for Carrying Out the Invention

[0010] (1) The control agent of the present invention Embodiments of the present invention are control agents for cell competition, and more specifically, promoters or inhibitors of cell competition. The promoter includes a protein containing the α3 domain of MHC class Ia or an agonist antibody against LILRB3, and the inhibitor includes a substance that inhibits the binding of MHC class Ia and LILRB3, more specifically, the binding of the α3 domain of MHC class Ia and the D1 and D2 domains of LILRB3.

[0011] In this specification, "cell competition" refers to a phenomenon in which when two types of cells with different fitness levels (states) are in proximity in a tissue, the cell with a higher fitness level survives and the cell with a lower fitness level is eliminated. Although it is known that immune system cells such as lymphocytes eliminate mutant cells, it has also been elucidated that normal cells such as epithelial cells other than immune system cells can also eliminate mutant cells through cell competition.

[0012] It has been clarified that mechanical forces mediated by cytoskeletal molecules such as actinomyosin, filamin, and vimentin function as the driving force for cell elimination of mutant cells by cell competition. In the cell competition of mutant cells by epithelial cells, these cytoskeleton-forming factors accumulate on the side of normal epithelial cells surrounding the mutant cells, causing the mutant cells to be extruded and detached to the apical side (lumen side) of the normal epithelial cells. Since mutant cells need to detach to the basal side, which is opposite to the apical side, in order to invade and metastasize, the deviation of the mutant cells to the apical side is considered an antitumor phenomenon that inhibits metastasis by normal epithelial cells (Miho Kajiita et al., Jpn J Pharmacol 2012; 140: 76-80). Also, when the cells extruded during this cell competition process have not initiated apoptosis, it is known that they undergo programmed cell death called anoikis due to the loss of adhesion (VanHook M. A., Sci. Signal. 2017; Vol. 10, Issue 478, eaan5866 DOI: 10.1126 / scisignal.aan5866).

[0013] As used herein, the "eliminated cell" refers to a cell that loses in cell competition and is eliminated among two types of cells with different fitness levels that are the targets of cell competition, and the "normal cell" refers to a cell that wins in cell competition and eliminates the eliminated cell among two types of cells with different fitness levels that are the targets of cell competition. Also, as used herein, among the cells included in the eliminated cell, cells other than the cells transplanted by organ transplantation, tissue transplantation, cell transplantation, etc. (hereinafter referred to as "transplanted cells") are referred to as "abnormal cells". Abnormal cells include, in addition to mutant cells with different fitness levels from normal cells due to mutations in endogenous genes or expression of foreign genes, cells with different fitness levels from normal cells due to epigenetic changes in gene expression. Also, abnormal cells include cancer cells and cells in which carcinogenesis is progressing (including pre-cancerous cells), cells infected with viruses or other pathogens, cells in which the pathogen infection is progressing, cells with disease-specific protein expression mutations, cells damaged physically, degenerated cells and cells in which degeneration is progressing in neurodegenerative, muscular, spinal and other degenerative diseases, but are not limited thereto. In the present invention, when the control of cell competition is a promoting control, the non-eliminated cell is an abnormal cell. Also, in the present invention, when the control of cell competition is a suppressing control, the non-eliminated cell is a transplanted cell.

[0014] The promoting control of cell competition in the present invention includes, for example, the prevention or treatment of cancer or viral infections, or the promotion or enhancement thereof. Also, the suppressing control of cell competition in the present invention includes, for example, the suppression or reduction of the elimination of transplanted cells by non-immune normal cells in organ transplantation, tissue transplantation or cell transplantation.

[0015] Cell competition in this specification is not limited to the elimination of abnormal cells and transplanted cells by immune system cells, and may be the elimination of abnormal cells and transplanted cells by non-immune normal cells.

[0016] As used herein, the "immune system cells" refer to lymphocytes such as T cells, B cells and NK (natural killer) cells, and cells involved in immune functions such as dendritic cells and macrophages.

[0017] In this specification, examples of the organ to be transplanted include the liver, kidney, heart, lung, pancreas, or endocrine organs including the thyroid gland, parathyroid gland, thymus, adrenal cortex, or adrenal medulla, organs including stem cells, and the like.

[0018] In this specification, examples of the tissue to be transplanted include the skin, tissue including stem cells, tissue including immune cells, and the like.

[0019] In this specification, examples of the cells to be transplanted include bone marrow cells, non-adherent bone marrow cells, peripheral blood cells, cord blood cells, cells derived from Wharton's jelly, placenta-derived cells, hair follicle-derived cells, adipose tissue-derived cells, lymphocytes, monocytes, macrophages, and the like. Alternatively, cells including one or more selected from the group consisting of bone marrow cells, non-adherent bone marrow cells, peripheral blood cells, cord blood cells, cells derived from Wharton's jelly, placenta-derived cells, hair follicle-derived cells, and adipose tissue-derived cells; cells including one or more selected from the group consisting of lymphocytes, monocytes, and macrophages; cells selected therefrom.

[0020] In this specification, examples of neurodegenerative diseases include Alzheimer's disease, mild cognitive impairment, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, Creutzfeldt-Jakob disease, trauma-induced neurodegeneration, high-pressure neuropathy syndrome, dystonia, olivopontocerebellar atrophy, amyotrophic lateral sclerosis, multiple sclerosis, epilepsy, dementia, senile dementia, AIDS dementia complex, AIDS-induced encephalopathy, and the like.

[0021] In this specification, examples of myopathic diseases include muscular dystrophy, distal myopathy, congenital myopathy, thyrotoxic myopathy, glycogenosis, mitochondrial myopathy, steroid myopathy, alcoholic myopathy, inflammatory myopathy, endocrine myopathy, lipid storage disorder myopathy, myopathy associated with infectious diseases such as HIV, vitreous myopathy, and myopathy associated with autoimmune diseases such as myasthenia gravis, and the like.

[0022] MHC is a gene family of many polymorphic proteins necessary for immune responses encoded in the major histocompatibility complex (MHC) region on the chromosomes of higher animals. The proteins of this MHC family are involved in the elimination of infectious pathogens such as bacteria and viruses, the rejection of cancer cells, and the rejection reaction during organ transplantation by presenting antigens, and play a very important role in immunity. In addition, various protein groups related to immunity, such as TAP (transporter associated with antigen processing) involved in peptide transport, are also encoded in this MHC region.

[0023] MHC class I proteins are present and expressed in all nucleated cells. And MHC class I proteins bind to endogenous antigens within cells. That is, for pathogens that proliferate within infected cells such as viruses or cancer antigens produced within cancer cells, an immune response is induced through antigen presentation via MHC class I. MHC class I proteins can be further divided into classical class I proteins (class Ia) and non-classical class I proteins (class Ib). MHC class I proteins are dimers in which a 45 kDa heavy chain (α chain) with attached sugar chains and a 12 kDa β2-microglobulin light chain are non-covalently bound, and peptide antigens bind to this to be expressed on the cell surface as a trimer. The class I heavy chain consists of three extracellular domains α1-α3, a transmembrane domain, and an intracellular domain. There is a large groove-like structure between the α1 region and the α2 region, where antigens bind and are presented.

[0024] Intracellular pathogens that proliferate within infected cells like viruses, or proteins produced within cancer cells, etc. After being ubiquitinated, cytoplasmic proteins are degraded by the proteasome into peptides approximately 5 to 15 amino acids in length. The degraded peptides are transported into the endoplasmic reticulum (ER) by an ATP-driven transporter called TAP (transporter associated with antigen processing) located on the endoplasmic reticulum (ER) membrane. Note that the structure of TAP is transmembrane and is a heterodimer consisting of TAP1 and TAP2. MHC class I α-chain and β2-microglobulin are synthesized within the endoplasmic reticulum (ER), and within the endoplasmic reticulum (ER), the three components, MHC class I α-chain, β2-microglobulin, and the peptide, bind to form an MHC-peptide complex. Subsequently, the MHC-peptide complex is placed inside a smaller endoplasmic reticulum, passes through the Golgi apparatus during transport towards the cell membrane by vesicular transport, undergoes sugar chain modification, and then reaches and is expressed on the cell membrane.

[0025] In the present invention, examples of proteins containing the α3 domain of MHC class Ia involved in the control of cell competition include the α3 domain of MHC class I and fragments of the α3 domain of MHC class I. The fragment of the α3 domain of MHC class I can be any fragment excluding the full length of the α3 domain of MHC class I. In this specification, the terms "protein" and "peptide" may be used interchangeably.

[0026] Furthermore, in the present invention, examples of the said MHC class Ia include HLA-A, HLA-B, HLA-C or DLA-88, and preferably, HLA-B or DLA-88.

[0027] Also, examples of the protein containing the α3 domain of MHC class Ia (MHC class Ia α3 domain fragment protein) in the present invention are as follows: a) A protein having the amino acid sequence represented by SEQ ID NO: 1 (the amino acid sequence of the α3 domain of DLA88), b) A protein having an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, and which specifically binds to a protein containing the D1 and D2 domains of LILRB3, c) A protein having an amino acid sequence having at least 90% similarity to the amino acid sequence represented by SEQ ID NO: 1, and which specifically binds to a protein containing the D1 and D2 domains of LILRB3, and d) A protein containing the α3 domain of an ortholog of DLA-88 and is at least one protein selected from the above.

[0028] As used herein, "similarity" means the percentage of identical and similar amino acid residues to all overlapping amino acid residues in an optimal alignment (preferably, the algorithm can consider the introduction of gaps into one or both of the sequences for optimal alignment) when two amino acid sequences are aligned using a mathematical algorithm known in the art. The identity / similarity of amino acid sequences in this specification can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value = 10; allowing gaps; matrix = BLOSUM62; filtering = OFF). Other algorithms for determining the homology of amino acid sequences include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993) [this algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) (Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997))], the algorithm described in Needleman et al., J. Mol. Biol., 48:444-453 (1970) [this algorithm is incorporated into the GAP program in the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4:11-17 (1988) [this algorithm is incorporated into the ALIGN program (version 2.0), which is part of the CGC sequence alignment software package], the algorithm described in Pearson et al., Proc. Natl. Acad. Sci. USA, 85:2444-2448 (1988) [this algorithm is incorporated into the FASTA program in the GCG software package], etc., and these can also be preferably used in the same way.

[0029] In addition, as long as the protein containing the α3 domain of class Ia of the present invention specifically binds to the protein containing the D1 and D2 domains of ILRB3, 1 to 20, preferably 1 to 10, more preferably 1 to several (5, 4, 3, or 2) amino acids in the amino acid sequence of the protein containing the α3 domain (for example, the amino acid sequence of the α3 domain of DLA88 shown in SEQ ID NO: 1) are deleted; 1 to 20, preferably 1 to 10, more preferably 1 to several (5, 4, 3, or 2) amino acids are added to the amino acid sequence of the protein containing the α3 domain (for example, the amino acid sequence shown in SEQ ID NO: 1); 1 to 20, preferably 1 to 10, more preferably 1 to several (5, 4, 3, or 2) amino acids in the amino acid sequence of the protein containing the α3 domain (for example, the amino acid sequence shown in SEQ ID NO: 1) are substituted with other amino acids; or it may be a protein comprising or consisting of an amino acid sequence combining them. Substitutions and the like between amino acids with similar properties (for example, glycine and alanine, valine, leucine and isoleucine, serine and threonine, aspartic acid and glutamic acid, asparagine and glutamine, lysine and arginine, cysteine and methionine, phenylalanine and tyrosine, etc.) may allow for even more substitutions and the like.

[0030] As long as the protein containing the α3 domain of class Ia of the present invention specifically binds to the protein containing the D1 and D2 domains of ILRB3, it may be a protein comprising or consisting of an amino acid sequence having 80% or more similarity to the amino acid sequence of the protein containing the α3 domain of class Ia (for example, the amino acid sequence shown in SEQ ID NO: 1), preferably 85% or more (86%, 87%, 88%, or 89% or more), more preferably 90% or more (91%, 92%, 93%, or 94% or more), still more preferably 95% or more (96%, 97%, or 98% or more), and most preferably 99% or more.

[0031] In the present invention, the agonist antibody against LILRB3 is not particularly limited as long as it binds to LILRB3 (more specifically, the D1 and D2 domains of LILRB3) and can control (more specifically, promote) cell competition. Such an antibody can be appropriately screened and obtained by those skilled in the art based on, for example, the methods described in WO 2003 / 091424, WO 2005 / 056602, etc.

[0032] LILRB3, which functions as a receptor for MHC class Ia, is also called ENSCAFG00000028453, CD85a, ILT5 or LIR3. LILRB3 is a type of immunoglobulin-like receptor, having extracellular immunoglobulin domains D1 - D4, a transmembrane domain, and an intracellular domain. Although its expression in immune professional cells is well known, it is also expressed in epithelial cells. Its function has not necessarily been fully elucidated.

[0033] In the present specification, examples of the protein containing the D1 and D2 domains of LILRB3 include the D1 and D2 domains of LILRB3 and fragments of the D1 and D2 domains of LILRB3. The fragment of the D1 and D2 domains of LILRB3 may be any fragment excluding the full length of the D1 and D2 domains of LILRB3.

[0034] In the present invention, the ortholog of DLA-88 (a protein consisting of the full-length amino acid sequence of DLA-88 shown in SEQ ID NO: 13) and the ortholog of canine LILRB3 (ENSCAFG00000028453) (a protein consisting of the full-length amino acid sequence of ENSCAFG00000028453 shown in SEQ ID NO: 14) are, for example, those in mammals other than dogs, preferably primates (e.g., apes, humans, etc.), more preferably humans (e.g., SEQ ID NO: 11: amino acid sequence of the α3 domain of human MHC Ib, SEQ ID NO: 15: full-length amino acid sequence of human MHC B, SEQ ID NO: 12: amino acid sequence of the D1 and D2 domains of human LILRB3, SEQ ID NO: 16: full-length amino acid sequence of human LILRB3).

[0035] In the present invention, examples of the protein containing the D1 and D2 domains of the LILRB3 are as follows: e) A protein having the amino acid sequence shown in SEQ ID NO: 8 (amino acid sequence of the D1 and D2 domains of ENSCAFG00000028453), f) A protein having an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 8, and which specifically binds to a protein containing the α3 domain of MHC class Ia, g) A protein having an amino acid sequence having at least 80% similarity to the amino acid sequence shown in SEQ ID NO: 8, and which specifically binds to a protein containing the α3 domain of MHC class Ia, and h) At least one protein selected from proteins containing the D1 and D2 domains of the ortholog of ENSCAFG00000028453.

[0036] In addition, as long as the protein containing the D1 and D2 domains of LILRB3 of the present invention specifically binds to the protein containing the α3 domain of MHC class Ia, 1 to 20, preferably 1 to 10, more preferably 1 to several (5, 4, 3, or 2) amino acids in the amino acid sequence of the protein containing the D1 and D2 domains (for example, the amino acid sequence of the D1 and D2 domains of ENSCAFG00000028453 shown in SEQ ID NO: 8), an amino acid sequence in which 1 to 20, preferably 1 to 10, more preferably 1 to several (5, 4, 3, or 2) amino acids are added to the amino acid sequence of the protein containing the D1 and D2 domains (for example, the amino acid sequence shown in SEQ ID NO: 8), 1 to 20, preferably 1 to 10, more preferably 1 to several (5, 4, 3, or 2) amino acids in the amino acid sequence of the protein containing the D1 and D2 domains (for example, the amino acid sequence shown in SEQ ID NO: 8) are substituted with other amino acids, or a protein comprising or consisting of an amino acid sequence combining them may also be used. For substitutions between amino acids with similar properties (for example, glycine and alanine, valine, leucine and isoleucine, serine and threonine, aspartic acid and glutamic acid, asparagine and glutamine, lysine and arginine, cysteine and methionine, phenylalanine and tyrosine, etc.), there can be even more substitutions, etc.

[0037] As long as the protein containing the D1 and D2 domains of LILRB3 of the present invention specifically binds to the protein containing the α3 domain of MHC class Ia, it may be a protein comprising or consisting of an amino acid sequence having 80% or more similarity to the amino acid sequence of the protein containing the D1 and D2 domains of LILRB3 (for example, the amino acid sequence shown in SEQ ID NO: 8), preferably 85% or more (86%, 87%, 88%, or 89% or more), more preferably 90% or more (91%, 92%, 93%, or 94% or more), still more preferably 95% or more (96%, 97%, or 98% or more), and even more preferably 99% or more.

[0038] The proteins containing the α3 domain of MHC class Ia and the proteins containing the D1 and D2 domains of LILRB3 used in the present invention may be their salts, hydrates, solvates, etc.

[0039] The "substance that inhibits the binding between MHC class Ia and LILRB3" (hereinafter sometimes abbreviated as "the substance of the present invention") used in the present invention is not particularly limited as long as it inhibits the binding between MHC class Ia and LILRB3, more specifically, the binding between the protein containing the α3 domain of MHC class Ia and the D1 and D2 domains of (cell-expressed) LILRB3. Specifically, for example, proteins containing the D1 and D2 domains of LILRB3 (including dominant negative mutants, etc.), antibodies against the D1 and D2 domains of LILRB3 (including neutralizing antibodies, etc.), aptamers, LILRB3 expression inhibitors, etc. can be mentioned.

[0040] The antibody against the D1 and D2 domains of LILRB3 used in the present invention can be obtained as a polyclonal or monoclonal antibody using means known per se. Alternatively, commercially available products may be used. The origin of the antibody used in the present invention is not particularly limited, but is preferably derived from a mammal, more preferably a human-derived antibody. As the mammalian-derived monoclonal antibody, either one produced by a hybridoma or one produced by a host transformed with an expression vector containing an antibody gene by genetic engineering techniques may be used. The antibody-producing hybridoma can be prepared by a method known per se. For example, using the D1 and D2 domains of LILRB3 or a part thereof as an antigen, the antigen is immunized according to a normal immunization method, and the obtained immune cells are fused with a known parent cell by a normal cell fusion method, and monoclonal antibody-producing cells are screened by a normal screening method.

[0041] The aptamer used in the present invention may be a nucleic acid aptamer or a peptide aptamer. In the case of a nucleic acid aptamer, the nucleic acid may be DNA, RNA, or a DNA / RNA chimera. Further, it may be a nucleic acid or peptide modified with a ribose, a phosphate backbone, a nucleobase, an amino acid residue, or both terminal portions. The nucleic acid aptamer may be double-stranded or single-stranded, but is preferably single-stranded. The aptamer of the present invention can be selected using methods well known to those skilled in the art. Without limitation, for example, it can be selected by the SELEX method (Systematic Evolution of Ligands by Exponential Enrichment) (Tuerk, C. and Gold, L., 1990, Science, 249: 505-510) or the yeast two-hybrid method.

[0042] The LILRB3 expression inhibitor used in the present invention may act at any stage such as the transcription level of the gene encoding LILRB3, the level of post-transcriptional regulation, the translation level into protein, and the level of post-translational modification. Therefore, examples of the LILRB3 expression inhibitor include nucleic acids (e.g., antigene) that inhibit the transcription of the gene encoding LILRB3, nucleic acids that inhibit the processing from the primary transcript to mRNA, nucleic acids that inhibit the translation from mRNA to protein (e.g., antisense nucleic acid, miRNA), or nucleic acids that degrade mRNA (e.g., siRNA, ribozyme, microRNA (miRNA)).

[0043] siRNA can be designed according to the rules proposed by, for example, Elbashir et al. (Genes Dev., 15, 188-200 (2001)) based on the cDNA sequence information of the LILRB3 gene. Further, a short hairpin RNA (shRNA), which is a precursor of siRNA, can be designed by appropriately selecting any linker sequence (e.g., about 5 to 25 bases) capable of forming a loop structure and linking the sense strand and the antisense strand of siRNA via the linker sequence.

[0044] The sequences of siRNA and / or shRNA can be searched using search software provided for free on various websites. Such websites include, for example, siDESIGN Center provided by Dharmacon (http: / / dharmacon.horizondiscovery.com / jp / design-center / ?rdr=true&LangType=1041&pageid=17179928204), siRNA Target Finder provided by GenScript (https: / / www.genscript.com / tools / sirna-target-finder), etc., but are not limited thereto.

[0045] miRNA can be searched using target prediction software provided for free on various websites. Such websites include, for example, TargetScan published by the Whitehead Institute of the United States (http: / / www.targetscan.org / vert_72 / ), DIANA-micro-T-CDS published by the Alexander Fleming Biomedical Sciences Research Center in Greece (http: / / diana.imis.athena-innovation.gr / DianaTools / index.php?r=microT_CDS / index), etc., but are not limited thereto. Alternatively, TarBase (http: / / carolina.imis.athena-innovation.gr / diana_tools / web / index.php?r=tarbasev8 / index), a database on miRNA that has been experimentally proven to act on target mRNA and is published by institutions such as the University of Chezalay and the Pasteur Institute, can also be used to search for miRNA that targets the mRNA encoding LILRB3.

[0046] siRNA can be prepared by synthesizing the sense strand and the antisense strand of the target sequence on mRNA using a DNA / RNA automatic synthesizer respectively, denaturing them at about 90 to about 95 °C for about 1 minute in an appropriate annealing buffer, and then annealing them at about 30 to about 70 °C for about 1 to about 8 hours. Also, shRNA, which is a precursor of siRNA, can be synthesized and then cleaved using dicer to prepare siRNA. miRNA and pre-miRNA can be synthesized using a DNA / RNA automatic synthesizer based on their sequence information.

[0047] The antisense nucleic acid can be DNA, RNA, or a DNA / RNA chimera. When the antisense nucleic acid is DNA, the RNA:DNA hybrid formed by the target RNA and the antisense DNA can be recognized by endogenous RNase H and cause selective degradation of the target RNA. The target region of the antisense nucleic acid has no particular limitation on its length as long as the antisense nucleic acid hybridizes to inhibit translation into protein as a result, and it can be the entire sequence or a partial sequence of the mRNA encoding the protein, with short ones being about 10 bases long and long ones being the entire sequence of the mRNA or the primary transcript. Also, the antisense nucleic acid may not only hybridize to the target mRNA or primary transcript to inhibit translation into protein, but also bind to these genes that are double-stranded DNA to form a triple helix (triplex) and inhibit transcription into RNA (antigene).

[0048] The antisense nucleic acid can be prepared by determining the target sequence of the mRNA or primary transcript based on the cDNA sequence or genomic DNA sequence of the target gene and synthesizing a sequence complementary to it using a commercially available DNA / RNA automatic synthesizer.

[0049] The agent of the present invention is prepared and used as a parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous, intradermal, intraperitoneal, intravaginal, intramuscular, intranasal, rectal, intraoral, intraocular, intraauricular, sublingual, etc.) preparation or an oral preparation. When administered orally, it can be taken before, after, or between meals. The agent of the present invention is prepared and used as a preparation diluted to a predetermined concentration and dosage with a solvent such as physiological saline and added with pharmaceutically acceptable additives. The agent of the present invention may be prepared as a pharmaceutical composition.

[0050] As the dosage form of the agent of the present invention, for example, a solution, dispersion, or emulsion of a protein containing an effective amount of the α3 domain of MHC class Ia or a substance that inhibits the binding of MHC class Ia and LILRB3 in a diluent or dispersion medium such as water or physiological saline, such as an injection, cream, ointment, beverage, aerosol, skin gel, eye drop, nasal drop, etc.; solid preparations such as tablets, capsules, powders, powder preparations, granules, tablets, sustained-release preparations, suppositories, etc. can be used, and these may be in the form of an encapsulated body in which the active ingredient is encapsulated in liposomes or sustained-release materials, or a carrier supported on a carrier. The dosage form of the agent of the present invention may be a preparation in a unit dosage form or a preparation in a multiple dosage form.

[0051] Examples of oral preparations include tablets, powders, powder preparations, granules, fine granules, pills, capsules, troches, chewable tablets, solutions, emulsions, microcapsules, suspensions, elixirs, syrups, sustained-release preparations, etc., and these may be in the form of an encapsulated body in which the active ingredient is encapsulated in liposomes or sustained-release materials, or a carrier supported on a carrier.

[0052] Examples of parenteral preparations include injections, infusions, creams, ointments, aerosols, skin gels, eye drops, nasal drops, etc., which are solutions, dispersions, or emulsions of an effective amount of adrenomedullin or a modified form thereof, or a derivative thereof, or a salt thereof in a diluent or dispersion medium such as water or physiological saline. Alternatively, it may be in the form of a powder preparation, transdermal patch, lotion, ointment, poultice, or suppository.

[0053] The pharmaceutically acceptable pharmaceutical additives can be prepared by adding additives such as stabilizers, antioxidants, pH adjusters, buffers, suspending agents, emulsifiers, surfactants, etc., which are known to those skilled in the art. The types of these pharmaceutical additives and their usage and dosage are described in the Pharmaceutical Additive Dictionary 2007 (edited by the Japan Pharmaceutical Additive Association, Yakujitsu Shimbunsha, July 2007), etc., and can be prepared and used according to these descriptions.

[0054] Specifically, as stabilizers, for example, organic acids such as tartaric acid, citric acid, succinic acid, fumaric acid, etc. can be used; as antioxidants, for example, ascorbic acid, dibutylhydroxytoluene or propyl gallate, etc. can be used; as pH adjusters, for example, dilute hydrochloric acid or aqueous sodium hydroxide solution, etc. can be used; as buffers, for example, citric acid, succinic acid, fumaric acid, tartaric acid or ascorbic acid or their salts, glutamic acid, glutamine, glycine, aspartic acid, alanine or arginine or their salts, magnesium oxide, zinc oxide, magnesium hydroxide, phosphoric acid or boric acid or their salts can be used; as suspending agents or emulsifiers, for example, lecithin, sucrose fatty acid ester, polyglycerin fatty acid ester, polyoxyethylene hydrogenated castor oil, polysorbate or polyoxyethylene-polyoxypropylene copolymer, etc. can be used; as surfactants, for example, polysorbate 80, sodium lauryl sulfate or polyoxyethylene hydrogenated castor oil, etc. can be used, but are not limited thereto.

[0055] The administration targets of the agent of the present invention include humans, mammals other than humans (for example, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, etc.). When adapting to mammals other than humans, the dosage of the agent of the present invention may be appropriately adjusted according to the weight and size of the animal.

[0056] Regarding the agent of the present invention, the dosage of the protein containing the α3 domain of MHC class Ia, which is the active ingredient per day for adults, or the agonist antibody against LILRB3, or the substance that inhibits the binding between MHC class Ia and LILRB3 can be appropriately adjusted according to gender, age, body weight, symptoms, administration route, dosage form, etc. For example, when the active ingredient is a protein, it can usually be selected in the range of about 0.0001 mg / kg to about 1,000 mg / kg. Alternatively, per subject (patient), for example, it can be selected in the range of about 0.001 mg / body to about 1,000,000 mg / body. However, the agent of the present invention is not limited to the above dosage.

[0057] The agent of the present invention may administer the above daily dosage once or divided into multiple times. Also, the timing of administration may be before meals, after meals, or between meals. Also, the administration interval is not particularly limited, and it may be daily, every other day, or intermittent administration. The administration period is not particularly limited, but long-term administration is possible.

[0058] When the agent for controlling cell competition of the present invention promotes cell competition, the cells eliminated by the cell competition (e.g., mutant cells, etc.) are caspase-positive cells as shown in the examples described later, and cell death (e.g., apoptosis) can be induced. Therefore, the agent is suitable for the prevention and treatment of cancer (including pre-cancerous conditions) alone. Also, even after cancer treatment (e.g., surgical treatment), the cells involved in cancer metastasis and recurrence can be eliminated with the agent, and the eliminated cells are caspase-positive cells as described above, and cell death (e.g., apoptosis) can be induced. Therefore, the agent is also suitable for the prevention and treatment of cancer metastasis and recurrence alone. Furthermore, as described above, the cells eliminated by the agent for controlling cell competition of the present invention are likely to have cell death (e.g., apoptosis) induced. For example, even in cancers that are resistant to immune checkpoint inhibitors (e.g., anti-PD-1 antibody, etc.), by combining the agent for controlling cell competition of the present invention with a drug as described later, a more excellent cancer prevention or treatment effect can be expected. When the agent for controlling cell competition of the present invention suppresses cell competition, as can be understood from the examples described later, for example, it is suitable for maintaining transplanted cells at a desired location. Further, for example, in order to further enhance the efficiency of cell transplantation, it may be used in combination with a drug as described later.

[0059] As described above, the agent of the present invention may be used in combination (combination agent), that is, used in combination with other drugs and / or other treatment methods. When used in combination, the agent of the present invention and other drugs may be provided in the form of a single medicine, or may be provided in the form of a pharmaceutical combination or kit containing a plurality of separately formulated preparations. The administration timing of the drug to be combined with the agent of the present invention is not limited, and the drug to be combined with the agent of the present invention may be administered simultaneously to the administration subject, or may be administered separately (eg, continuously, with a time interval, etc.). The dosage of the drug to be combined may conform to the dosage clinically used, and can be appropriately selected according to the administration subject, administration route, disease, combination, etc.

[0060] When the agent of the present invention is an accelerator of cell competition, the other drug may be, for example, an anticancer agent or an antiviral agent. When the agent of the present invention is an inhibitor of cell competition, the other drug may be an immunosuppressant, an inhibitor of rejection of transplanted organs, and / or an agent for promoting engraftment after transplantation.

[0061] In the accelerator of cell competition of the present invention, the anticancer agent as the other drug includes, but is not limited to, alkylating agents, cytotoxic antibiotics, platinum preparations, antimetabolites, kinase inhibitors, angiogenesis inhibitors, hormonal agents, DNA modifying enzyme inhibitors, proteasome inhibitors, alkaloid agents, type I and type II topoisomerase inhibitors, histone deacetylase inhibitors, cytokine preparations, hormonal agents, immune checkpoint inhibitors, natural killer cell activators, indoleamine 2,3-dioxygenase (IDO) inhibitors, monoclonal antibodies, and other molecular target therapeutic agents.

[0062] The anti-cancer agent is, more specifically, doxorubicin, daunorubicin, cisplatin, oxaliplatin, carboplatin, paclitaxel, irinotecan, SN-38, actinomycin D, vincristine, vinblastine, methotrexate, azathioprine, fluorouracil, mitomycin C, docetaxel, cyclophosphamide, capecitabine, epirubicin, gemcitabine, mitoxantrone, leucovorin, vinorelbine, trastuzumab, etoposide, estramustine, prednisone, interferon α, interleukin-2, bleomycin, ifosfamide, mesna, altretamine, topotecan, cytarabine, methylprednisolone, dexamethasone, mercaptopurine, thioguanine, fludarabine, gemtuzumab, idarubicin, mitoxantrone, tretinoin, alemtuzumab, chlorambucil, cladribine, imatinib, epirubicin, dacarbazine, procarbazine, mechlorethamine, rituximab, denileukin diftitox, trimethoprim / sulfamethoxazole, allopurinol, carmustine, tamoxifen, filgrastim, temozolomide, melphalan, vinorelbine, azacitidine, thalidomide, mitomycin, regorafenib, cetuximab, panitumumab, ramucirumab, gefitinib, erlotinib, afatinib, crizotinib, alectinib, ceritinib, lenvatinib, lapatinib, pertuzumab, sunitinib, sorafenib, axitinib, pazopanib, nivolumab, pembrolizumab, ipilimumab, vemurafenib, everolimus, temsirolimus, bevacizumab, geldanamycin, etc., but not limited thereto.

[0063] The promoter of cell competition of the present invention, the antiviral agent as the other drug, includes interferon, nucleoside and nucleotide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, fusion inhibitors, maturation inhibitors, guanosine analogs, purine analogs, pyrimidine analogs, and other "unclassified" antiviral agents recognized in the art that are not included in any of the above classes (for example, foscarnet and miltefosine), but not limited thereto.

[0064] More specifically, the antiviral agent includes, but is not limited to, abacavir, acyclovir, adefovir, amantadine, amdoxovir, amprenavir, apricitabine, aplaviroc, albendazole, atazanavir, bevirimat, BMS-488043, boceprevir, brequinar, cidofovir, DCM205, docosanol, delavirdine, didanosine, darunavir, efavirenz, elvitegravir, erbucitabine, emtricitabine, enfuvirtide, epigallocatechin gallate, etravirine, famciclovir, fosamprenavir, ganciclovir, globoidnan A, griffithsin, ibalizumab, idoxuridine, indinavir, lamivudine, lopinavir, loviride, maraviroc, nelfinavir, nevirapine, oseltamivir, pegylated interferon α-2a, pegylated interferon α-2b, penciclovir, peramivir, prexasertib, PRO 140, racivir, raltegravir, ritonavir, ribavirin, rimantadine, rilpivirine, saquinavir, stampidine, stubidine, tenofovir, tipranavir, TNX-355, trifluridine, tromantadine, valacyclovir, valganciclovir, vicriviroc, vidarabine, viramidine, vivecon, zalcitabine, zanamivir, and zidovudine, etc.

[0065] In the inhibitor for suppressing cell competition of the present invention, the immunosuppressant as the other agent includes, but is not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide, methylprednisolone, prednisone, azathioprine, tacrolimus hydrate, 15-deoxyspergualin, natalizumab, rapamycin, and etanercept.

[0066] In the present invention, the dosage of the above combination agent can be set to any amount as long as side effects are not a problem. The daily dosage of the combined drugs varies depending on the degree of symptoms, age, gender, weight, sensitivity difference of the administration subject, administration time, interval, nature of the pharmaceutical preparation, formulation, type, type of active ingredient, etc., and is not particularly limited.

[0067] (2) Control method of the present invention, etc. Another embodiment of the present invention is a method for controlling cell competition, which comprises administering a therapeutically effective amount of an agonist antibody against a protein containing the α3 domain of MHC class Ia or LILRB3, or a substance that inhibits the binding of MHC class Ia and LILRB3 (more specifically, the binding of the α3 domain of MHC class Ia and the D1 and D2 domains of LILRB3) to a subject in need thereof. Further, in the method for controlling cell competition, when an agonist antibody against a protein containing the α3 domain of MHC class Ia or LILRB3 is administered, the control method may be for the prevention or treatment of cancer or viral infection. Furthermore, in the method for controlling cell competition, when a substance that inhibits the binding of MHC class Ia and LILRB3 is administered, the control method may be for improving the engraftment failure of organ transplantation, tissue transplantation or cell transplantation, or for suppressing the progression of symptoms of neurodegenerative diseases, myodegenerative diseases and other various degenerative diseases. Another further embodiment of the present invention is the use of an agonist antibody against a protein containing the α3 domain of MHC class Ia or LILRB3, or a substance that inhibits the binding of MHC class Ia and LILRB3, in the manufacture of a medicament for controlling cell competition. Regarding the control method, etc. of the present invention described above, all the contents of the control agent of the present invention (1) above can be incorporated by reference.

[0068] All documents mentioned in this specification are hereby incorporated by reference in their entirety. The detailed description and examples of this specification are illustrative of the embodiments of the present invention and should not be construed as limiting the scope of the present invention.

Examples

[0069] In the examples of this specification, cloning, genome editing, cell culture, microscopic observation, protein expression, induction, and purification were carried out according to the manufacturer's instructions using commercially available vectors, reagents, and kits, etc.

[0070] <Suppression of the ability of normal MDCK cells to eliminate abnormal cells by suppressing antigen presentation in RasV12-expressing cells> 1. Experimental method Establishment of RasV12-expressing cells The pTRE3G-GFP-RasV12 and Hyper PiggyBac transposase expression vectors were introduced into MDCK cells, which are cell lines derived from canine renal tubular epithelial cells, by Nucleofection. The transfected MDCK cells were selected with Blasticidin, and monoclonal pTRE3G-GFP-RasV12-expressing MDCK stable cell lines were obtained by isolating viable cell colonies. Furthermore, 24 hours after adding doxycycline (200 ng / mL), RasV12-expressing MDCK cells in which GFP-RasV12 was expressed more uniformly in all cells were finally obtained as usable stable cell lines using the presence or absence of GFP fluorescence as an indicator under a fluorescence microscope. In the following examples, the above-mentioned RasV12-expressing MDCK cells were used as abnormal cells to examine cell competition with normal MDCK cells.

[0071] Establishment of RasV12-expressing cells deficient in the TAPl gene The pCDH-QC-sgRNA (Maruyama et al., Nat. Biotechnol. 2015) into which the guide sequence (target sequence (gRNA)) of SEQ ID NO: 3 was inserted was introduced into the tetracycline-inducible Cas9 stable MDCK cell line (pCW-Cas9-MDCK).

[0072] After gene introduction, Cas9 was induced with doxycycline (200 ng / mL). Furthermore, only the cells with pCDH-QC-sgRNA were selected by culturing for 7 days in the presence of hygromycin (400 μg / mL). The selected cells were serially diluted to a concentration of 0.5 cells per well to obtain single clones. Subsequently, clones with gene deficiency were identified by sequence analysis and used as KO cells of the TAPl gene. To eliminate clonal effects, two clones of cells (TAP-KO#1 and TAP-KO#2) were selected and TAPl gene-deficient RasV12-expressing MDCK cells were established using the PB-pTRE3G-RasV12 vector by the same procedure as the establishment of the above RasV12-expressing cell line.

[0073] Evaluation of non-autonomous cell effects between normal and abnormal cells Type I-A Collagen, 5X DMEM solution, and pH-adjusting buffer solution were mixed on ice at a ratio of 7:2:1 to prepare a collagen gel solution. A 15 mm coverslip was placed in a 12-well plate, and 500 μL of the collagen gel solution was added thereon and allowed to stand at 37°C for 30 minutes to solidify.

[0074] MDCK normal cells and RasV12-expressing MDCK cells (TAPl gene wild-type cells (TAP-WT-RasV12) or TAPl gene-deficient cells (TAP-KO#1-RasV12 or TAP-KO#2-RasV12)) were mixed at a ratio of 50:1, at 1×10 6 cells and 2×10 4 cells, respectively, seeded on the collagen gel, cultured at 37°C for 8 - 12 hours until a monolayer was formed, and then treated with doxycycline (200 ng / mL) for 24 hours.

[0075] After performing doxycycline treatment for 24 hours, 4% Paraformaldehyde / PBS was added, and the cells were fixed by incubating at room temperature for 15 minutes while shielding from light. After fixation, 0.1% Triton X-100 / PBS was added, and the cells were permeabilized by incubating at room temperature for 15 minutes while shielding from light. Blocking was performed by allowing the cells to stand at room temperature for 1 hour while shielding from light with 1% BSA / PBS. Subsequently, the cells were reacted with Alexa-Fluor-568-conjugated phalloidin diluted 200-fold with 1% BSA / PBS at room temperature for 1 hour. After further washing with PBS three times for 5 minutes each, the cells were reacted with Hoechst 33342 diluted 4,000-fold with PBS at room temperature for 10 minutes. Finally, the cells were mounted on glass slides using Mowiol.

[0076] The accumulation of Filamin was statistically processed using open-source ImageJ (Figure 1B). The exclusion efficiency of RasV12-expressing cells (TAP-WT-RasV12, TAP-KO#1-RasV12, and TAP-KO#2-RasV12) resulting from the accumulation of Filamin was determined under a confocal microscope by searching for colonies of GFP-fluorescent positive cells consisting of 2 to 8 cells, counting the number of cells that escaped to the apical side among them, and finally expressing it as the ratio of the number of escaped cells to the total number of RasV12-expressing cells.

[0077] To examine whether antigen presentation by RasV12-expressing MDCK cells promotes the elimination ability of normal MDCK cells, the elimination efficiency of wild-type TAP1 gene RasV12-expressing cells (TAP-WT-RasV12) by normal MDCK cells was compared with the elimination efficiency of TAP1 gene-deficient RasV12-expressing cells (TAP-KO#1-RasV12 and TAP-KO#2-RasV12) by normal MDCK cells.

[0078] 2. Experimental Results When co - culturing RasV12 - expressing cells (TAP - WT - RasV12, TAP - KO#1 or TAP - KO#2) with MDCK normal cells, accumulation of Filamin on the side of MDCK normal cells was confirmed due to the non - autonomous effect between normal and abnormal cells (Figure 1A). On the other hand, the accumulation of Filamin in MDCK normal cells surrounding TAPl - gene - deficient RasV12 - expressing cells (TAP - KO#1 and TAP - KO#2) was statistically significantly suppressed compared to the accumulation of Filamin in MDCK normal cells surrounding TAPl - gene - wild - type RasV12 - expressing cells (TAP - WT - RasV12) (Figure 1B). Also, when examining the elimination efficiency of RasV12 - expressing cells caused by the accumulation of Filamin, suppression of the elimination efficiency was confirmed in TAPl - gene - deficient RasV12 - expressing cells (TAP - KO#1 - RasV12 and TAP - KO#2 - RasV12) compared to the elimination efficiency in RasV12 - expressing cells (TAP - WT - RasV12) (Figures 1C, 1D).

Example

[0079] <Positive regulation of promoting the elimination of abnormal cells by DLA88 of the DLA family> 1. Experimental method Establishment of DLA88 - KO cells, DLA79 - KO cells, DLA64 - KO cells and DLA12 - KO cells Attention was paid to DLA12, DLA64, DLA79 and DLA88 of the DLA family, which are orthologs of MHC class I proteins in dogs, as proteins involved in antigen presentation. pCDH - QC - sgRNA (Maruyama et al., Nat. Biotechnol. 2015) into which the guide sequences (target sequences (gRNA)) of SEQ ID NOs: 4 - 7 (DLA88, DLA79, DLA64, and DLA12, respectively) were inserted was transfected into a tetracycline - inducible Cas9 - stable MDCK cell line (pCW - Cas9 - MDCK).

[0080] After gene introduction, Cas9 was induced with doxycycline (200 ng / mL). Furthermore, only the cells with pCDH-QC-sgRNA were selected by culturing for 7 days in the presence of hygromycin (400 μg / mL). The selected cells were serially diluted to a concentration of 0.5 cells per well to obtain single clones. Thereafter, clones with gene deficiency were used as KO cells for each gene by sequence analysis. Hereinafter, DLA88 gene-deficient RasV12-expressing cells, DLA79 gene-deficient RasV12-expressing cells, DLA64 gene-deficient RasV12-expressing cells, and DLA12 gene-deficient RasV12-expressing cells are referred to as DLA88-KO-RasV12, DLA79-KO-RasV12, DLA64-KO-RasV12, and DLA12-KO-RasV12, respectively.

[0081] To rule out clonal effects, two clones of cells were selected for each gene-deficient RasV12-expressing cell, and gene-deficient RasV12-expressing cells were established using the PB-pTRE3G-RasV12 vector by the same procedure as the above RasV12-expressing cell establishment.

[0082] Similar to Example 1, the accumulation of filamin was measured, and the elimination efficiency of abnormal cells (DLA88-KO-RasV12, DLA79-KO-RasV12, DLA64-KO-RasV12, and DLA12-KO-RasV12) caused by the accumulation of filamin was calculated. Also, similar to Example 1, the cells were fixed and then observed under a fluorescence microscope.

[0083] 2. Experimental results As a result of gene deletion of DLA12, DLA64, DLA79, and DLA88 using the CRISPR / Cas9 system, the elimination efficiency by MDCK normal cells was suppressed only in gene-deleted RasV12-expressing cells of DLA88 (DLA88-KO-RasV12) (Figure 2A). From this, it was shown that DLA88 positively controls the elimination of RasV12-expressing cells. Furthermore, when the behavior of abnormal cells (DLA88-KO-RasV12, DLA79-KO-RasV12, DLA64-KO-RasV12, and DLA12-KO-RasV12) was analyzed by confocal microscopy, gene-deleted RasV12-expressing cells of DLA88 (DLA88-KO-RasV12) showed basal protrusion that dives basally instead of apical extrusion seen in RasV12-expressing cells lacking the DLA88 gene (DLA-WT-RasV12, DLA79-KO-RasV12, DLA64-KO-RasV12, and DLA12-KO-RasV12) (Figure 2B).

Example

[0084] <Promotion of elimination of abnormal cells by extracellular domain protein of DLA88> 1. Experimental method Preparation of recombinant protein DLA88 α(alpha)3 The extracellular domain of DLA88 is composed of three α domains (Figure 3A). Therefore, a recombinant protein of α1 and α2 domains (DLA88-alpha1 / 2) and a recombinant protein of α3 domain (DLA88-alpha3) were prepared. To prepare the recombinant protein of DLA88-alpha3 (SEQ ID NO: 1) and the recombinant protein of DLA88-alpha1 / 2, a DNA fragment encoding DLA88-alpha3 (SEQ ID NO: 2) and a DNA fragment encoding DLA88-alpha1 / 2 were inserted into the EcoRI / XhoI restriction enzyme sites of pGEX-6p-1 to prepare expression plasmids pGEX-6p-1-DLA88-alpha3 and pGEX-6p-1-DLA88-alpha1 / 2.

[0085] IPTG (final concentration 0.1 mM) was added to the culture of BL21 transformed with pGEX-6p-1-DLA88-alpha3 or DLA88-alpha1 / 2, and the expression of the recombinant protein was induced at 25°C for 2 hours. The collected Escherichia coli was solubilized, and the recombinant proteins of DLA88-alpha3 (SEQ ID NO: 1) and DLA88-alpha1 / 2 were purified from the extract using Gluthatione Sepharose 4B.

[0086] Evaluation of the non-autonomous cell effects between normal cells and abnormal cells The non-autonomous cell effects were evaluated in the same manner as in Example 1, except that in addition to the doxorubicin treatment, a recombinant protein treatment (30 nM or 300 nM) was performed.

[0087] 2. Experimental results The extracellular domain of DLA88 is composed of three α domains. Therefore, a recombinant protein of the α3 domain was prepared. Also, after mixing at a cell ratio of normal cells:mutant cells = 50:1 to form a single cell layer, the GFP-RasV12 protein was expressed with doxorubicin. When the recombinant protein DLA88-α3 was treated simultaneously with doxorubicin, the elimination of mutant cells was promoted in a concentration-dependent manner (Figures 3A and 3B). Also, even under the condition of normal cells:DLA88-deficient mutant cells = 50:1, the presence of the same recombinant protein promoted the elimination of abnormal cells. From this, it was shown that DLA88 promotes the elimination of abnormal cells, and the recombinant protein of the extracellular domain promotes the elimination of abnormal cells (Figures 3C, 3D).

Example

[0088] Knockout of LILRB3 1. Experimental method To knockout the canine LILRB3 (ENSCAFG00000028453, hereinafter also referred to as "28453") gene, genome editing was performed using the guide sequence (target sequence) of LILRB3 described in SEQ ID NO: 10 in the same manner as in Example 2.

[0089] 2. Experimental results By knocking out LILRB3 (ENSCAFG00000028453, hereinafter also referred to as "28453"), the elimination efficiency of abnormal cells (LILRB3 gene-deficient RasV12-expressing MDCK cells, 28453-KO-RasV12) by normal cells (MDCK cells) decreased significantly statistically (Figure 4A).

Example

[0090] Production of extracellular domain D1-D2 recombinant protein of 28453 1. Experimental method 28453 D1-D2 To prepare the recombinant protein of 28453 D1-D2 (SEQ ID NO: 8), a DNA fragment (SEQ ID NO: 9) encoding 28453 D1-D2 was inserted into the EcoRI / XhoI restriction enzyme sites of pGEX-6p-1 to prepare the expression plasmid pGEX-6p-1-28453 D1-D2 (Figure 4B).

[0091] IPTG (final concentration 0.1 mM) was added to the culture solution of BL21 transformed with pGEX-6p-1-DLA88-alpha3, and the expression of the recombinant protein was induced at 25 °C for 2 hours. The collected Escherichia coli was solubilized, and the recombinant protein of 28453 D1-D2 (SEQ ID NO: 8) was purified from the extract using Gluthatione Sepharose 4B.

[0092] 2. Experimental results The target recombinant protein of "28453-D1 / 2" (SEQ ID NO: 8) was obtained.

Example

[0093] Evaluation of the binding property between 28453-D1 / 2 and DLA88-α3 1. Experimental method GST-28453-D1 / 2-HA and GST-DLA88-α3-Flag were mixed at a 1:1 ratio in PBS containing 0.1% TritonX-100 (0.1% TritonX-100-PBS). Then, the mixture was allowed to stand at 37°C for 2 hours, and beads carrying anti-Flag antibody (hereinafter referred to as "Flag beads") were added, followed by inversion mixing at 4°C for 30 minutes. After washing the Flag beads three times with 0.1% TritonX-100-PBS, they were separated by SDS-PAGE. The immunoprecipitated GST-DLA88-α3-Flag was detected with anti-Flag antibody, and the co-precipitated GST-28453-D1 / 2-HA was detected with anti-HA antibody.

[0094] 2. Experimental Results After mixing 28453-D1 / 2 labeled with HA and DLA88-α3 labeled with FLAG and performing immunoprecipitation, both HA and FLAG labels were observed. From this result, it was shown that 28453-D1 / 2 and DLA88-α3 have binding activity (Figure 4C).

Example

[0095] Evaluation of the non-autonomous effect of cells between normal cells and mutant cells 1. Experimental Method In addition to doxycycline treatment, the non-autonomous effect of cells was evaluated in the same manner as in Examples 1 and 3, except that recombinant protein treatment (300 nM or 1,000 nM) was performed.

[0096] 2. Experimental Results When 300 nM and 1,000 nM of 28453-D1 / 2 were added to the mixed culture of normal cells (MDCK cells) and abnormal cells (RasV12-expressing cells), a statistically significant decrease in the elimination rate of mutant cells was observed in the 1,000 nM addition group (Figure 4D).

Example

[0097] Chemical kinetic analysis of the binding between a recombinant protein containing only the α3 domain of DLA88 and a recombinant protein of the D1 / D2 domain of LILRB3 1. Experimental Method BLItz TM (PRIMETECH) was used to analyze the dynamics between DLA88-α3 and LILRB3-D1 / D2. A GST sensor and an anti-GST antibody-immobilized sensor (PRIMETECH) were used. GST-DLA88-α3 and GST-LILRB3-D1 / D2 were individually incubated at 37 °C for 2 hours. GST-DLA88-α3 was immobilized as a ligand molecule on the GST sensor for 5 minutes, completely covering the GST sensor. After washing with PBS-T (5% Tween-PBS) for 30 seconds, GST-LILRB3 D1 / D2 was loaded as a sample while vibrating for 2 minutes. In addition to the association kinetics, the dissociation kinetics was evaluated in PBS-T buffer for 2 minutes. Recombinant protein GST-GFP was used as a control.

[0098] 2. Experimental Results As a result of the above measurement, the binding constant Kd was determined to be 6.54 ± 0.28 μM.

Example

[0099] Cell Competition Experiment in Human Cells 1. Experimental Method In the previous examples, MDCK cells, a cell line derived from canine kidney tubular epithelial cells, were used as a model experimental system for the competition phenomenon of epithelial cells. In this example, considering the application to humans, it was examined using HaCaT cells, a cell line of human epidermal keratinocytes.

[0100] Similar to the example with MDCK cells, RasV12-expressing HaCaT cells (LILRB3 WT -RasV12) expressing GFP-RasV12 were created by gene transfection. Normal HaCaT cells and RasV12-expressing HaCaT cells (LILRB3 WT-RasV12) were mixed at a ratio of 30:1, and the efficiency with which RasV12-expressing HaCaT cells were excluded as abnormal cells to the apical side by cell competition was measured every 2 hours after adding doxycycline 20 hours after seeding as the ratio of the number of cells that escaped to the total number of RasV12-expressing cells in the same manner as in Example 1.

[0101] 2. Experimental results The results are shown in Fig. 6. Doxycycline only When added and treated (LILRB3 WT ) ), the exclusion efficiency of RasV12-expressing HaCat cells by normal HaCat cells increased monotonically after adding doxycycline and reached 40% 44 hours after seeding. In contrast, when doxycycline and simultaneously with the recombinant protein 28453-Dl / 2 was added (LILRB3 WT +28453-D1 / 2 ), the exclusion efficiency of RasV12-expressing HaCat cells by normal HaCat cells only increased slightly. From this result, it was shown that the recombinant protein of the D1 / D2 domain of canine LILRB3 competes cells in human HaCat cells. inhibit Also, in LILRB3-KO cells, the exclusion efficiency was not rescued by α3 treatment.

Example

[0102] Tumor formation of human HaCat cells and RasV12-expressing HaCat cells transplanted into nude mice 1. Experimental method Pellets of cells obtained by mixing human HaCat normal cells (HaCat):RasV12-expressing human HaCat cells (RasV12) at a ratio of 3:1, 10:1 or 30:1 in the presence or absence of DLA88-α3 in Matrigel were transplanted into the abdomen of nude mice, and the total volume and weight of the tumor masses formed 2 weeks later were measured.

[0103] 2. Experimental results The results are shown in Fig. 7. The increase in the normal cell rate suppressed tumor growth, indicating that normal cells play an inhibitory role in tumor formation. Furthermore, co-injection of the α3 recombinant protein substantially and specifically suppressed tumor formation under mixed conditions. The inhibitory effect of α3 was not observed at a mixing ratio of 30:1, but was most clearly observed at a ratio of 10:1. At a ratio of 10:1, RasV12 cells mainly occupied the tumor region of the frozen sections, while normal cells predominantly occupied the mosaic-like regions following α3. The inhibitory effect of α3 on HLA-tKO was further tested. The tumor size of RasV12 HLA - tKO cells was slightly smaller than that of RasV12 WT cells, but RasV12 cells predominantly occupied the tumor region. α3 treatment showed statistically significant suppression. These results suggest that LILRB3 surrounding normal cells is stimulated by α3 to promote an eliminating force.

[0104] Induction of polar migration of adjacent normal cells surrounding normal cells by HLA-B / LILRB3 interaction 1. Experimental methods Live imaging and particle image velocimetry Normal HaCaT and HaCaT-RasV12 cells were mixed at a ratio of 30:1 on a bottom dish coated with collagen and cultured at 37 °C for 16 h until the cells formed a monolayer. The cells were incubated in the presence of doxycycline. Eight hours after incubation, JuLI TM Stage (NanoEntek) was used to acquire live imaging movies every 5 min for 48 h. For particle image velocimetry (PIV) analysis, movies per 2 h were analyzed by PIV of an ImageJ plugin. The PIV parameter set was as follows: normalized median test parameter noise of NMT (0.3) and threshold of NMT (0.8), dynamics mean test of DMT parameter C1 (0.5), parameter C2 (0.3).

[0105] Evaluation of Caspase-3 activity Normal HaCaT cells and HaCaT-RasV12 cells were mixed at a ratio of 50:1 and seeded onto a collagen gel. After a single phase was formed, tetracycline and α3 were added. After 16 hours, the cells were fixed, and the antibody recognizing cleaved Caspase-3 and Actin were stained with Pallodin. The samples were observed under a confocal microscope. The XZ images at that time are shown. (-): untreated, (+): α3-treated.

[0106] 2. Experimental Results The results are shown in Figure 8. Since RasV12 cells are mostly removed between 16 hours and 36 hours, the images from 16 hours to 36 hours were subjected to PIV analysis. Interestingly, a part of the normal cells moved towards RasV12 cells at 28 hours before apical extrusion at 32 hours, and 4 hours later, RasV12 cells were extruded from the epithelial monolayer. This polarized movement of normal cells near RasV12 cells was increased by α3 treatment, but the number of polarized normal cells almost disappeared due to LILRB3 knockout (left figure). These results suggest that the interaction between MHC-I and LILRB3 induces the polarized movement of adjacent normal cells to eliminate RasV12 cells. Also, it was demonstrated that apoptosis was induced in the cells eliminated by the addition of the α3 domain (right figure).

[0107] Apoptosis of mutant cells surrounded by normal cells when treated with α3 was detected with an antibody capable of detecting caspase-3 activation-dependent cleavage. Treatment with α3 increased the number of cells eliminated during co-culture and the number of caspase-3 positive cells cleaved in the eliminated cells (right figure in Figure 8).

Example

[0108] Human papillomavirus (HPV) is known to cause carcinogenesis by degrading Scribble, a tumor suppressor gene, after infecting cells. On the other hand, it is known that cells with suppressed Scribble expression are eliminated by surrounding normal cells while being induced to undergo apoptosis (left figure in Figure 9).

[0109] 1. Experimental method Elimination of Scribble knockdown cells via α3(ECA-1-ex3) ECAR α3(ECA-1-ex3) ECAR promotes the elimination of Scribble knockdown cells. Tetracycline-inducible Scribble knockdown MDCK cells (GFP-positive cells) were mixed and seeded at a ratio of normal cells or 28453(ECAR) to 1:10 (MDCK:SCRB KD = 10:1). After forming a single phase, tetracycline and α3 were added, and the ratio of GFP-positive cells (Scribble knockdown cells) to the number of cells in the microscopic field 24 hours later was calculated. ns.: not significant, **P < 0.01.

[0110] 2. Experimental results The results are shown in Figure 9. SCRB KD cells had a survival rate of about 35%, but after treatment with α3, the survival rate was about 20%. This is because the elimination of SCRBKD cells was promoted by the effect of α3. Also, in ECAR knockout cells, the survival rate increased regardless of the presence or absence of α3. From these facts, it was suggested that α3 promotes the elimination of SCRB KD cells via ECAR.

Example

[0111] 1. Experimental method The elimination of HPV full-genome stable MCF10A cells is promoted by α3 treatment. After MCF10A (HPV18) cells that stably retain the entire genome of HPV (HPV18 genome) were made distinguishable by CMFDA (green fluorescence staining) after co-culture, they were mixed with normal MCF10A at a ratio of 10:1 (Normal MCF10A:HPV18 = 10:1) and then seeded. The ratio of GFP-positive cells (HPV18 cells) to the total number of cells in the microscopic field 24 hours later was calculated. Cont: Residual rate of HPV18 in the epithelial cell layer, a3: Residual rate of HPV18 cells during α3 treatment. ns.: not significant, **P < 0.01.

[0112] 2. Experimental Results The results are shown in Figure 10. The residual rate of cells (HPV18) that stably retain the entire genome of HPV decreased upon treatment with α3. This means that cells with the entire genome of HPV are also promoted to be eliminated by α3.

Example

[0113] Identification of a Novel Cell Membrane Protein as a Cell Competition Regulatory Factor 1. Experimental Method Verification of the Induction Level of LILRB3 and the Cell Removal Efficiency LILRB3 is specifically induced during co-culture. Normal cells and RasV12 cells were co-cultured at a ratio of 1:1 on a silicone plate with a hardness of 0.5 kPa. After forming a single phase, they were treated with tetracycline for 8 hours. The cells were collected, and the induction level of LILRB3 was measured by quantitative PCR and shown as a graph. *P < 0.05, **P < 0.01, ****P < 0.001 by Student’s t-test (upper left figure in Figure 11).

[0114] LILR3 is induced at the protein level on the normal cell side under co-culture conditions. Only normal cells, only RasV12 cells, and a mixture of normal-mutant cells were seeded on a collagen gel, and when a single phase was formed, tetracycline was added. Then, after 24 hours, the cells were fixed and stained without membrane permeabilization treatment. Anti-LILRB3 antibody staining, HLA-B. Representative XY image (left figure in Fig. 11) Quantification of the protein induction level of LILRB3 (lower left figure in Fig. 11). Scale bars: 10 μm. *P < 0.05, **P < 0.01, ****P < 0.001 by Student’s t-test.

[0115] Deficiency of LILRB3 in MDCK suppresses the elimination of mutant cells. MDCK RasV12 cells were mixed with MDCK-WT, -AltR-KO or -AltR-KO-OE cells, respectively, and allowed to form a single phase on collagen, and then tetracycline was added. Then, the cell elimination efficiency after 24 hours was calculated. The cells were stained with Phalloidin and Hoechst. Representative XZ image (upper left figure in Fig. 12) and the result of quantification of the elimination efficiency (lower left figure in Fig. 12) are shown. Scale bars: 10 μm. *P < 0.05, **P < 0.01, ****P < 0.001 by Student’s t-test.

[0116] Knockout of LILRB3 in HaCaT normal cells suppresses the elimination of mutant cells. RasV12 HaCaT cells were normal HaCaT-WT, -LILRB3-KO (LILRB3 KO ) or -LILRB5-KO (LILRB5 KO) After mixing with cells and forming a single phase on collagen, tetracycline was added. Then, the cells were fixed in 16 hours and stained with Phalloidin and Hoechst. Representative XZ images are shown (upper right figure in Fig. 12). Also, quantitative data of the exclusion efficiency are shown as a graph (lower right figure in Fig. 12). Scale bars: 10 μm. *P < 0.05, **P < 0.01, ****P < 0.001 by Student’s t-test。

[0117] 2. Experimental results The results are shown in Figs. 11 and 12. Non-cell-autonomous induction of LILRB3 mRNA under mixed conditions and the most homologous LILRB3 protein levels in normal cells surrounding RasV12 cells were confirmed. LILRB3-KO in normal cells suppressed apical extrusion.

[0118] The present invention is based on Japanese Patent Application No. 2019-063594 (filing date: March 28, 2019) and Japanese Patent Application No. 2019-069777 (filing date: April 1, 2019), the contents of which are all incorporated herein.

Industrial applicability

[0119] The present invention can be used as a medicament for the prevention or treatment of cancer and viral infections. The present invention can also be used as a medicament for improving engraftment failure in organ transplantation, tissue transplantation or cell transplantation, or for suppressing the progression of symptoms of neurodegenerative diseases.

Claims

1. An agent for promoting cell competition, comprising a protein containing the α3 domain of MHC class Ia or an agonist antibody against LILRB3.

2. The agent according to claim 1, wherein the MHC class Ia is HLA-B or DLA-88.

3. The protein containing the α3 domain of the MHC class Ia is as follows: a) A protein having the amino acid sequence shown in SEQ ID NO: 1, b) A protein having an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, and specifically binds to a protein containing the D1 and D2 domains of LILRB3, c) A protein having an amino acid sequence having at least 90% similarity to the amino acid sequence shown in SEQ ID NO: 1, and specifically binds to a protein containing the D1 and D2 domains of LILRB3, and d) A protein containing the α3 domain of an ortholog of DLA-88 The agent according to claim 1 or 2, comprising at least one protein selected from the group consisting of.

4. The agent according to claim 3, wherein the protein containing the D1 and D2 domains of LILRB3 is a protein having the amino acid sequence shown in SEQ ID NO:

8.

5. The agent according to any one of claims 1 to 4, wherein the cell competition is the elimination of abnormal cells by non-immune normal cells.

6. The agent according to claim 5, wherein the abnormal cells are mutant cells, cancer cells, or virus-infected cells.

7. The agent according to claim 6, wherein the agent for promoting cell competition is a prophylactic or therapeutic agent for cancer or virus infection.

8. An inhibitor of cell competition, comprising a substance that inhibits the binding of MHC class Ia and LILRB3, wherein the substance that inhibits the binding of MHC class Ia and LILRB3 is at least one substance selected from the group consisting of a protein containing the D1 and D2 domains of LILRB3, an antibody against the D1 and D2 domains of LILRB3, siRNA and shRNA against the transcript of the LILRB3 gene, and an antisense nucleic acid against the LILRB3 gene or its transcript.

9. The protein containing the D1 and D2 domains of LILRB3 is as follows: e) A protein having the amino acid sequence shown in SEQ ID NO: 8, f) A protein having an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 8, and specifically binds to a protein containing the α3 domain of MHC class Ia. g) A protein having an amino acid sequence having at least 80% similarity to the amino acid sequence shown in SEQ ID NO: 8, and specifically binds to a protein containing the α3 domain of MHC class Ia. h) The agent according to claim 8, comprising at least one protein selected from proteins containing the D1 and D2 domains of an ortholog of ENSCAFG00000028453.

10. The agent according to claim 8 or 9, wherein the cell competition is the elimination of transplanted cells by non-immune normal cells.

11. The agent according to any one of claims 8 to 10, wherein the inhibition of cell competition is the improvement of engraftment failure in organ transplantation, tissue transplantation or cell transplantation, or the suppression of symptom progression of neurodegenerative diseases and myopathic diseases.

Images