WO2021235517A1 - Novel t-cell activator - Google Patents

Novel t-cell activator Download PDF

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WO2021235517A1
WO2021235517A1 PCT/JP2021/019156 JP2021019156W WO2021235517A1 WO 2021235517 A1 WO2021235517 A1 WO 2021235517A1 JP 2021019156 W JP2021019156 W JP 2021019156W WO 2021235517 A1 WO2021235517 A1 WO 2021235517A1
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cells
gaba
cell
cancer
inhibitor
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French (fr)
Japanese (ja)
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ファガラサン・シドニア
倫生 宮島
ヒャクコウ ショウ
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国立研究開発法人理化学研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
    • A61K31/5517—1,4-Benzodiazepines, e.g. diazepam or clozapine condensed with five-membered rings having nitrogen as a ring hetero atom, e.g. imidazobenzodiazepines, triazolam
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a novel T cell activator, specifically, a novel T cell activator that inhibits or reduces the action of B cells that suppress the killing activity of T cells on cancer cells and infected cells. ..
  • Non-Patent Document 1 cytotoxic CD8-positive T cells that accumulate in the liver developing non-alcoholic fatty liver and prevent the development of hepatocellular carcinoma in the liver.
  • Non-Patent Document 3 It has also been reported that the progression of hepatocellular carcinoma is promoted by suppressing the disease.
  • Non-Patent Documents 4, 5, 6 and 7 there are some reports on the relationship between gamma-aminobutyric acid (GABA) and tumor cells.
  • GABA gamma-aminobutyric acid
  • Patent Documents 4, 5, 6 and 7 the relationship between immune response and GABA is known.
  • An object of the present invention is to develop a novel T cell activator targeting B cells and immunotherapy for cancer and infectious diseases by understanding the function of regulatory B cells such as IgA-positive B cells.
  • the inventors were able to examine the results of metabolome analysis of immune cells in detail and identify small molecule metabolites characteristic of activated B cells, including IgA-positive B cells. Therefore, a novel T cell that inhibits or reduces the action of B cells that suppress the killing activity of cytotoxic T cells against cancer cells and infected cells by modulating the metabolic regulation and signal transduction of the small molecule metabolite. It is also an object of the present invention to develop a cell activator and a method for treating cancer and infectious diseases.
  • IgM-deficient homo-knockout mice in which B cells activated by antigen stimulation produce gamma aminobutyric acid (GABA), and B cells and cells belonging to their cell lineage hardly differentiate.
  • GABA gamma aminobutyric acid
  • MMT gamma aminobutyric acid
  • B cells and cells belonging to their cell lineage hardly differentiate.
  • GABA inhibits tumor growth and increases cytotoxic tumor-infiltrating CD8-positive cells, suppresses tumor growth in MMT, and increases cytotoxic tumor-infiltrating CD8-positive cells compared to wild-type mice.
  • Rukoto administration of inhibitors of GABA a receptors, the growth of the tumor is inhibited in wild-type mice, CD8 positive cells infiltrating the tumor wild-type mice treated with inhibitors of GABA a receptors in size , Found that the cell structure becomes complicated.
  • GABA A receptor inhibitors do not act directly on the tumor to suppress its growth, but rather suppress the growth of the tumor via immune cells (B cells and T cells). confirmed. Based on these findings, we have found a completely new therapeutic mechanism that inhibits or reduces the cytotoxic T cell inhibitory effect of B cells. By this therapeutic mechanism, tumors and infected cells can be eliminated (Fig. 17).
  • the present invention provides a T cell activator that inhibits or reduces the cytotoxic T cell inhibitory effect of B cells.
  • the B cells can also be B cells activated by antigen stimulation.
  • the T cell activator of the present invention can also contain an inhibitor of gamma-aminobutyric acid (GABA) -mediated signal transduction in T cells.
  • GABA gamma-aminobutyric acid
  • the inhibitor of GABA-mediated signal transduction in T cells can also be an inhibitor that suppresses or reduces the expression and / or function of GABA receptors expressed in human T cells. ..
  • the GABA receptor expressed in the T cell can be at least one of the GABA A receptor and the GABA- ⁇ receptor.
  • the GABA receptor expressed in the T cells can also consist of a subunit polypeptide expressed in human T cells.
  • the subunit polypeptide expressed in the human T cells may be at least one selected from the group consisting of human ⁇ 1, ⁇ 5, ⁇ 1, ⁇ and ⁇ 2.
  • the inhibitor of GABA-mediated signal transduction in the T cells is at least one selected from the group consisting of human ⁇ 1, ⁇ 5, ⁇ 1, ⁇ and ⁇ 2 expressed in the human T cells. It can include antisense nucleic acids, RNAi (RNA interference) inducible nucleic acids or ribozymes or expression vectors thereof, which suppress or reduce the expression of a type of subunit polypeptide.
  • RNAi RNA interference
  • the inhibitor of GABA-mediated signal transduction in the T cells is at least one selected from the group consisting of human ⁇ 1, ⁇ 5, ⁇ 1, ⁇ and ⁇ 2 expressed in the human T cells. At least one selected from the group consisting of antibodies, specific binding partners or fragments thereof that specifically bind to a subunit polypeptide of a type of GABA receptor and suppress or reduce the function of the GABA receptor. You can also do it.
  • the GABA A receptor inhibitor is derived from flumazenil, Ro15-4513, sarmazenil, cicutoxin, enanthotoxin, pentylenetetrazol, picrotoxin, thujone, linden, bicuculline, gabazine and derivatives thereof. It may be at least one type selected from the group.
  • the GABA- ⁇ receptor inhibitor comprises a group consisting of (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid (TPMPA) and its derivatives. It can also be at least one selected.
  • TPMPA (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid
  • the T cell activator of the present invention comprises an inhibitor of GABA biosynthesis in B cells, a promoter of GABA degradation in B cells, an inhibitor of GABA secretion in B cells, and / or a capture agent for free GABA. You can also.
  • the inhibitor of GABA biosynthesis in the B cell can also be an inhibitor of the expression and / or enzyme activity of glutamate decarboxylase and aldehyde dehydrogenase 9 family member A1, and the B cell.
  • the GABA degradation promoter in GABA can also be a promoter of expression and / or enzyme activity of 4-aminobutyric acid aminotransferase
  • the free GABA capture agent is a protein that specifically binds to free GABA, free. It can also include at least one selected from the group consisting of antibodies that specifically bind GABA, specific binding partners or fragments thereof.
  • the T cell activator of the present invention can also contain an antibody that specifically binds to GABA, a specific binding partner and / or a fragment thereof.
  • the T cell activator of the present invention can also contain antibodies, specific binding partners or fragments thereof, and / or B cell scavengers, which are cytotoxic to B cells.
  • the present invention provides a method for treating cancer, which comprises inhibiting or reducing the cytotoxic T cell inhibitory effect of B cells in a patient in need of treatment.
  • the present invention also provides a method for preventing and / or treating an infectious disease, which comprises inhibiting or reducing the cytotoxic T cell inhibitory effect of B cells in a patient in need of treatment.
  • the B cells can also be B cells activated by antigen stimulation.
  • the method for treating cancer and the method for preventing and / or treating an infectious disease according to the present invention can also include administering an inhibitor of GABA-mediated signal transduction in T cells to a patient in need of treatment.
  • the GABA-mediated signal transduction inhibitor in T cells suppresses the expression and / or function of GABA receptors expressed in human T cells.
  • it can be an inhibitor that reduces.
  • the GABA receptor can be at least one of GABA A receptor and GABA- ⁇ receptor.
  • the GABA receptor expressed in T cells can also be composed of a subunit polypeptide expressed in human T cells.
  • the subunit polypeptide expressed in the human T cells is selected from the group consisting of human ⁇ 1, ⁇ 5, ⁇ 1, ⁇ and ⁇ 2. It may be at least one type.
  • the GABA-mediated signal transduction inhibitor in the T cells is expressed in the human T cells as human ⁇ 1, ⁇ 5, ⁇ 1, ⁇ and It can include antisense nucleic acids, RNAi-inducible nucleic acids or ribozymes or expression vectors thereof that suppress or reduce the expression of at least one subunit polypeptide selected from the group consisting of ⁇ 2.
  • the GABA-mediated signal transduction inhibitor in the T cells is expressed in the human T cells as human ⁇ 1, ⁇ 5, ⁇ 1, ⁇ and It consists of an antibody, a specific binding partner or a fragment thereof that specifically binds to a subunit polypeptide of at least one GABA receptor selected from the group consisting of ⁇ 2 and suppresses or reduces the function of the GABA receptor. It can also be at least one selected from the group.
  • the GABA A receptor inhibitor is flumazenil, Ro15-4513, salmazenil, cicutoxin, enanthotoxin, pentylenetetrazol, picrotoxin, tujon, and the like. It can also be at least one selected from the group consisting of linden, bicuculline, gabazine and derivatives thereof.
  • the inhibitor of the GABA- ⁇ receptor is (1,2,5,6-tetrahydropyridine-4-yl) methyl phosphinic acid. It can also be at least one selected from the group consisting of (TPMPA) and its derivatives.
  • the methods for treating cancer and preventing and / or treating infectious diseases of the present invention include an inhibitor of GABA biosynthesis in B cells, a promoter of GABA degradation in B cells, an inhibitor of GABA secretion in B cells, and /.
  • a capture agent for free GABA may be administered to a patient in need of treatment.
  • the inhibitor of GABA biosynthesis in B cells is an inhibitor of the expression or enzyme activity of glutamate decarboxylase and aldehyde dehydrogenase 9 family member A1.
  • the GABA degradation promoter in B cells can also be a promoter of expression or enzyme activity of 4-aminobutyric acid aminotransferase, and the free GABA capture agent is specific for free GABA. It can also include at least one selected from the group consisting of proteins that bind to free GABA, antibodies that specifically bind to free GABA, specific binding partners or fragments thereof.
  • the methods of treating cancer and preventing and / or treating infectious diseases of the present invention administer an antibody that specifically binds to GABA, a specific binding partner and / or a fragment thereof to a patient in need of treatment. Can also be included.
  • the methods of treating cancer and preventing and / or treating infectious diseases of the present invention use antibodies, specific binding partners or fragments thereof, and / or B cell scavengers having cytotoxicity against B cells. It can also include administration to patients in need of treatment.
  • the T cell activator of the present invention kills cancer cells or infected cells, suppresses the growth of cancer cells or infected cells, or induces apoptosis of cancer cells or infected cells. It can also be used in combination with drugs.
  • the method for treating cancer and the method for preventing and / or treating an infectious disease according to the present invention kills cancer cells or infected cells, suppresses the growth of cancer cells or infected cells, or causes cancer cells or It can also be used in combination with other therapeutic methods that induce apoptosis of infected cells.
  • GABA is synthesized by glutamic acid decarboxylase (GAD1) from glutamic acid derived from alpha-ketoglutaric acid (a-KG) generated in the TCA cycle, or from the urea cycle or 4-aminobutanal derived from spermidine / spermine tamine to the aldehyde dehydrogenase 9 family. Synthesized by member A1 (ALDH9A1). GABA is decomposed into succinic semialdehyde by 4-aminobutyric acid transaminase, and further oxidized to enter the TCA cycle as succinic acid.
  • GAD1 glutamic acid decarboxylase
  • the vertical axis of the graph shows small intestinal IgA plasma cells (SI IgA PC), germinal centers (GC) or non-GC (nonGC) T cells and B cells of the Pier plate (PP), and B cells (B220) of lymph nodes (LN).
  • Dendritic cells DC
  • CD4 or CD8 positive effector memory EM
  • CM central memory
  • CD4 or CD8 positive or negative naive T cells naive
  • CD4 or CD8 positive and CD44 positive cells Represents each cell of (CD44).
  • the horizontal axis of the graph represents a relative value in which the GABA content of each cell is multiplied by the GABA content of naive CD4T cells.
  • Immunity to wild-type mice WT
  • CD3-deficient homo-knockout mice CD3 -/-
  • IgM-deficient homo-knockout mice mMT
  • RAG-1-deficient homo-knockout mice RAG-1 ⁇ / ⁇
  • a bar graph showing the GABA content of lymph node tissue ipsilaterally (ipsi) and contralaterally (contra) to the applied hindlimb footpad. Error bars represent the standard deviation of GABA content between samples of each lymph node tissue.
  • the vertical axis of the graph represents a relative value in which the GABA content of each tissue is multiplied by the GABA content of the lymph node opposite to the wild-type mouse.
  • the horizontal axis represents the lymph node from which each tissue was derived.
  • the p value of the difference was 0.0011.
  • “*****” in FIG. 3, that is, the p-value of the significant difference in the GABA content of the lymph node tissues of the ipsilateral (ipsi) and contra (contra) wild-type mice of the immunized hindlimb is 0. It was less than .0001.
  • “Ns” indicates that no statistically significant difference was observed.
  • CD4, CD8 and B220-positive cells (CD4, CD8 and B220, respectively) sorted by flow cytometry from lymph node tissue ipsilaterally (ipsi) and contralateral (contra) to the hindlimb footpad of immunized wild mice.
  • B220 a bar graph showing the GABA content of contralateral total lymph nodes (total control LN), ipsilateral total lymph nodes (total ipsi LN) and ipsilateral macrophages / dendritic cells (ipsi Mf / DC). Error bars represent the standard deviation of GABA content between samples of each sorted cell.
  • the vertical axis of the GABA content represents a relative value in which the GABA content of each selected cell is multiplied by the GABA content of the contralateral CD4 positive cell.
  • the horizontal axis represents the type of each selected cell.
  • the p value of the significant difference in GABA content of CD4) was 0.0232.
  • ** in FIG.
  • the error bars represent the standard deviation of the individual mouse tumor volume on each measurement day.
  • the vertical axis represents the tumor volume (mm 3 ) between the IgM-deficient homozygous knockout mouse (mMT) and the wild-type mouse (WT).
  • the horizontal axis represents the measurement date of the tumor volume.
  • the error bars represent the standard deviation of the tumor volume on each measurement day.
  • the vertical axis represents the tumor volume (mm 3 ) between the IgM-deficient homozygous knockout mouse (mMT) and the wild-type mouse (WT).
  • the horizontal axis represents the measurement date of the tumor volume.
  • the error bars represent the standard deviation of the tumor volume on each measurement day.
  • the vertical axis represents the tumor volume (mm 3 ) between IgM-deficient homozygous knockout mice (mMT (+ placebo) or mMT (+ GABA)) treated with placebo pellets or GABA sustained-release pellets and wild-type mice (WT). ..
  • the horizontal axis represents the measurement date of the tumor volume.
  • mice 8 are wild-type mice treated with placebo pellets (WT (+ histogram)), and the center and right are IgM-deficient homoknockout mice treated with placebo pellets or GABA sustained-release pellets (mMT (+ histogram) or mMT (mMT (+ histogram)). + GABA)) 2-parameter histogram of gating CD8-positive cells by flow cytometry.
  • the vertical axis is the fluorescence intensity of CD98
  • the horizontal axis is the fluorescence intensity of TCRb.
  • the vertical axis is the fluorescence intensity of Sca-1
  • the horizontal axis is the fluorescence intensity of TCRb.
  • the number on the left of the box represents the percentage of cells with fluorescence intensity in the box among the CD8-positive cells under each condition.
  • the upper and lower left of FIG. 9 are wild-type mice treated with placebo pellets (WT (+ histogram)), and the center and right are IgM-deficient homoknockout mice treated with placebo pellets or GABA sustained-release pellets (mMT (+ histogram) or mMT (mMT (+ histogram)). + GABA)) 2-parameter histogram of gating CD8-positive cells by flow cytometry.
  • the vertical axis is the fluorescence intensity of perforin
  • the horizontal axis is the fluorescence intensity of TCRb.
  • the vertical axis is the fluorescence intensity of GrzB (Granzyme B)
  • the horizontal axis is the fluorescence intensity of TCRb.
  • the number on the left of the box represents the percentage of cells with fluorescence intensity in the box among the CD8-positive cells under each condition.
  • the vertical axis is the perforin-positive cells of wild-type mice treated with placebo pellets (WT + placebo), IgM-deficient homozygous knockout mice treated with placebo pellets or GABA sustained-release pellets (muMT ⁇ / ⁇ + placebo or muMT ⁇ / ⁇ + GABAp). Represents a percentage. “**” in FIG. 10A, ie, IgM-deficient homozygous knockout mice treated with placebo pellets (muMT ⁇ / ⁇ + placebo) and IgM-deficient homozygous knockout mice treated with GABA sustained-release pellets (muMT ⁇ / ⁇ + GABAp). The significance p value of the percentage of perforin-positive cells with was 0.006.
  • the vertical axis represents the tumor volume (mm 3 ) of a tiagabine (GAT (transporter) inhibitor) -administered animal (Tiagabine), a PBS-administered animal (PBS) or a picrotoxin-administered animal (Picrotoxin).
  • GAT transporter
  • the horizontal axis represents the measurement date of the tumor volume.
  • the curve with the largest tumor volume is the tumor volume of thiagabin (GAT (transporter) inhibitor) -administered animals
  • the curve with the second largest tumor volume is the PBS-administered animal
  • the curve with the smallest tumor volume is the tumor volume of picrotoxin-administered animals. Shows the change over time.
  • the error bars represent the standard deviation of the tumor volume on each measurement day. “***” in FIG.
  • the vertical axis is the tumor volume (mm 3 ) of a flumazenil (GABA A receptor inhibitor) -administered animal or a PBS-administered animal.
  • the horizontal axis is the date of measurement of tumor volume.
  • the curve with the smallest tumor volume on the final measurement day shows the change over time in the tumor volume of the flumazenil-administered animal (Flumazenil), and the curve with the largest tumor volume shows the change over time in the tumor volume of the DMSO-administered animal (DMSO).
  • the error bars represent the standard deviation of the tumor volume on each measurement day.
  • the lower right is a two-parameter histogram of cells infiltrated into a tumor derived from MC38 cells inoculated into picrotoxin-treated wild-type mice.
  • the upper right is a two-parameter histogram of cells infiltrated into a tumor derived from MC38 cells inoculated into wild-type mice in a control experiment.
  • the intensity of the laterally scattered light is represented by the vertical axis
  • the intensity of the forward scattered light is represented by the horizontal axis.
  • the numbers near the gating range represent the percentage of all measured cells within the gating range.
  • the vertical axis is the percentage of T cells with high intensity of laterally scattered light and forward scattered light.
  • the left shows the percentage of tumor-infiltrated cells in the control mouse (Ctr)
  • the right shows the percentage of tumor-infiltrated cells in the picrotoxin-treated mouse (Pic)
  • the error bar shows the standard deviation of the cell number in each cell population.
  • the p value of the percentage of tumor-infiltrated CD8-positive cells between picrotoxin-treated mice (Pic) and mice in the control experiment (Ctr) was 0.0156.
  • a bar graph showing that GABA or picrotoxin does not affect the proliferation and viability of MC38 cells in culture experiments.
  • the vertical axis represents the number of cells per well and the viability (ratio of viable cells to the total number of cells), respectively.
  • the horizontal axis represents control (PBS) or GABA (dissolved in PBS, final concentration 1 mM or 5 mM) added to RMPI culture medium (containing 10% fetal bovine serum).
  • the vertical axis represents cell viability, and the horizontal axis is control (DMSO) added to RMPI culture medium (containing 10% fetal bovine serum) or GABA A receptor inhibitor-picrotoxin (dissolved in DMSO). , Final concentration 100 ⁇ M, 200 ⁇ M or 300 ⁇ M).
  • Error bars represent the standard deviation of the number of cells or viability between wells. “Ns” indicates that no statistically significant difference was observed.
  • a line graph showing the effect of a GABA A receptor inhibitor (picrotoxin) on the growth of tumors derived from MC38 cells inoculated into rag1 gene-deficient mice. The vertical axis represents the tumor volume (mm 3 ) of rag1 gene-deficient mice treated with DMSO or picrotoxin. The horizontal axis represents the measurement date of the tumor volume after ingestion of cells. The error bars represent the standard deviation of the individual mouse tumor volume on each measurement day. “Ns” indicates that no statistically significant difference was observed.
  • a model of immune activation through suppression of GABA A receptor signals are examples of the number of cells or viability between wells. “Ns” indicates that no statistically significant difference was observed.
  • B cells synthesize and secrete GABA by decarboxylation of glutamate with glutamate decarboxylase (GAD).
  • GABA glutamate decarboxylase
  • B cell-derived GABA suppresses the function of cytotoxic T cells via GABA A receptor signals.
  • the GABA A receptor inhibitor (antagonist) releases the suppression of T cell cytotoxicity by GABA, resulting in suppression of cancer growth and elimination of infected cells.
  • Gln Glutamine; Glu, Glutamic Acid; GABA, ⁇ -Aminobutyric Acid; GLS, Glutaminase; GAD1, Glutamic Acid Decarboxylase 1.
  • B cells are used to include not only mature B cells but also terminally differentiated plasma cells and any B progenitor cells other than preB cells and their progenitor cells.
  • T cells are lymphocytes that differentiate and mature mainly in the thoracic gland, and when they mature, they express T cell receptors on the cell surface. It also includes any T progenitor cells capable of differentiation. Expression of cell surface markers is characterized by T cell receptors + , CD3 + , CD4 + , CD8 + and the like.
  • cytotoxic T cells are a type of T cells that recognize and destroy cells that are foreign to the host, such as virus-infected cells and cancer cells, and are characterized by the cell surface marker CD8 +.
  • the cytotoxic activity of cytotoxic T cells is characterized by the presence of cytotoxic substances such as perforin and granzyme.
  • an anticancer drug is an activity that suppresses, inhibits, or stops the growth of cancer cells, specifically kills cancer cells, or induces apoptosis or other self-destruction of cancer cells.
  • the anticancer agent of the present invention inhibits or reduces the action of B cells that suppress the cancer cell killing activity of cytotoxic T cells, that is, suppresses the cancer cell killing activity of cytotoxic T cells.
  • the method of treating cancer is any method that suppresses, inhibits, or stops the growth of cancer cells, specifically kills cancer cells, or induces cancer cells to undergo apoptosis or other self-destruction.
  • a method of performing treatment The method for treating cancer of the present invention inhibits or reduces the action of B cells that suppress the cancer cell killing activity of cytotoxic T cells, that is, suppresses the cancer cell killing activity of cytotoxic T cells. , Includes administration of a drug that inhibits or reduces the interaction between B cells and cytotoxic T cells.
  • the types of cancer targeted by the anticancer agent or the method for treating cancer of the present invention include carcinoma, squamous cell carcinoma (eg, cervical canal, eyelid, conjunctival, vaginal lung, oral cavity, skin, bladder, tongue, etc.). Squamous cell carcinoma of the throat and esophagus), adenocarcinoma (eg, adenocarcinoma of the prostate, small intestine, endometrial, cervical canal, colon, lung, pancreas, esophagus, rectal, uterus, stomach, breast and ovary). It also includes sarcomas (eg, myogenic sarcomas, osteosarcomas, uterine fibroids), leukemias, neuromas, melanomas and lymphomas.
  • carcinoma eg, cervical canal, eyelid, conjunctival, vaginal lung, oral cavity, skin, bladder, tongue, etc.
  • Squamous cell carcinoma of the throat and esophagus
  • the cancers targeted by the anticancer agent or the method for treating cancer of the present invention include, for example, lung cancer (for example, non-small cell lung cancer, small cell lung cancer, malignant mesoderma, etc.), breast cancer (for example, invasive milk). Tube cancer, non-invasive breast cancer, inflammatory breast cancer, etc.), prostate cancer (eg, hormone-dependent prostate cancer, hormone-independent prostate cancer, etc.), pancreatic cancer (eg, pancreatic duct cancer) Etc.), gastric cancer (eg papillary adenocarcinoma, mucinous adenocarcinoma, glandular squamous epithelial cancer, etc.), colon cancer (eg, gastrointestinal stromal tumor, etc.), rectal cancer (eg, gastrointestinal stromal tumor, etc.) Etc.), colon cancer (eg, familial colon cancer, hereditary non-polyposis colon cancer, gastrointestinal stromal tumor, etc.), esophageal cancer, duodenal cancer, tongue cancer
  • the term "preventive and / or therapeutic agent for infectious disease” refers to any method that suppresses, inhibits, or stops the growth of infected cells, specifically kills infected cells, or induces infected cells to undergo apoptosis or other self-destruction.
  • a drug having the activity of refers to a drug having the activity of.
  • the prophylactic and / or therapeutic agent for infectious diseases of the present invention inhibits or reduces the action of B cells that suppress the killing activity of cytotoxic T cells on infected cells, that is, kills cytotoxic T cells against infected cells.
  • a drug used to inhibit or reduce the interaction between B cells and cytotoxic T cells, which suppresses activity refers to any method that suppresses, inhibits, or stops the growth of infected cells, specifically kills infected cells, or induces infected cells to undergo apoptosis or other self-destruction.
  • the prophylactic and / or therapeutic agent for infectious diseases of the present invention inhibits or reduce
  • the method for preventing and / or treating an infectious disease is any method that suppresses, inhibits, or stops the growth of infected cells, specifically kills infected cells, or induces infected cells to undergo apoptosis or other self-destruction.
  • the method for preventing and / or treating an infectious disease of the present invention inhibits or reduces the action of B cells that suppress the killing activity of cytotoxic T cells on infected cells, that is, kills cytotoxic T cells against infected cells. It involves administering a drug that suppresses activity and inhibits or reduces the interaction of B cells with cytotoxic T cells.
  • infectious disease subject to the preventive and / or therapeutic agent for the infectious disease of the present invention or the preventive and / or therapeutic method for the infectious disease is, for example, a human hepatitis virus (hepatitis B, hepatitis C, hepatitis A or type E).
  • Virus infections such as Ebola virus, Western Nile virus, and other pathogens include, for example, pathogenic protozoa (eg, malaria protozoa, tripanosoma, toxoplasma), bacteria (eg, Escherichia coli, staphylococcus, tuberculosis). ), Infections caused by fungi (eg, Aspergillus, Blast Mrs., Candida), etc., and are not limited thereto.
  • pathogenic protozoa eg, malaria protozoa, tripanosoma, toxoplasma
  • bacteria eg, Escherichia coli, staphylococcus, tuberculosis
  • Infections caused by fungi eg, Aspergillus, Blast Mrs., Candida
  • the GABA receptor refers to a receptor that specifically binds to gamma-aminobutyric acid (GABA).
  • GABA gamma-aminobutyric acid
  • GABA A receptor is an ion channel type receptor that allows chlorine ions to permeate into cells when bound to GABA
  • GABA B receptor is a G protein-bound type receptor.
  • the GABA- ⁇ receptor was once classified as an independent class as a GABA C receptor.
  • GABA A receptor is a pentamer ion channel type receptor consisting of five subunit polypeptides
  • ⁇ subunit is similar to other GABA A receptor subunits in terms of gene sequence, structure, and function.
  • GABA- ⁇ receptors are now classified as a subclass of GABA A receptors because they have the above-mentioned properties and differ only in that they consist only of ⁇ subunits.
  • the T cell activator of the present invention is an inhibitor of GABA A receptor
  • its active ingredients include antagonists such as bicuculline and gabazine, and negative allosteric regulators such as flumazenil, Ro15-4513, salmazenil and zinc.
  • Non-competitive channel blockers such as sictoxin, enanthotoxin, pentylenetetrazole, picrotoxin, tujon, linden, their derivatives, and human ⁇ 1, ⁇ 5, ⁇ 1, ⁇ and among the subunits of the receptor expressed in human T cells. It includes, but is not limited to, antisense nucleic acids, RNAi-inducible nucleic acids or ribozymes or expression vectors thereof that suppress or reduce the expression of at least one subunit selected from the group consisting of ⁇ 2.
  • the active ingredients thereof are (1,2,5,6-tetrahydropyridine-4-yl) methyl phosphinic acid (TPMPA) and Including, but not limited to, derivatives thereof.
  • TPMPA (1,2,5,6-tetrahydropyridine-4-yl) methyl phosphinic acid
  • GABA glutamic acid decarboxylase
  • a-KG alpha-ketoglutaric acid
  • a-KG alpha-ketoglutaric acid
  • 4-aminobutanal derived from spermidine / spermine tamine to the aldehyde dehydrogenase 9 family.
  • A1 aldehyde dehydrogenase 9
  • GABA is decomposed into succinic semialdehyde by 4-aminobutyric acid transaminase, and further oxidized to enter the tricarboxylic acid (TCA) cycle as succinic acid.
  • TCA tricarboxylic acid
  • Glutamine consumption in both T and B cells is increased by antigen receptor-mediated activation.
  • 80% of the intracellular glutamate pool 24 hours after antigen stimulation is derived from glutamine.
  • the labeled glutamine contributes to nucleotide biosynthesis in activated B and T cells via precursors of purines and pyrimidines.
  • B cells after 72 hours of culture almost 90% of glutathione is derived from glutamine.
  • B cells a significant portion of the ⁇ -ketoglutaric acid that is oxidized by the TCA cycle is derived from glutamine.
  • GABA neurotransmitter gamma-aminobutyric acid
  • RNA transcription analysis of the major enzymes of the glutamine degradation pathway also confirms that the expression of enzymes involved in GABA synthetic degradation differs between B cells and CD4T cells.
  • B cells Compared to CD4T cells, B cells have higher expression of the enzyme GAD1 that converts glutamate to GABA and the genes that encode the glutamate transporters Slc38a1, Slc38a2 and Slc38a5. Conversely, B cells have less expression of the gene encoding the enzyme ABAT that catabolizes GABA.
  • the expression of the enzyme GAD1 which also converts human B cells to GABA, is higher than that of T cells.
  • the expression of GAD1 in cancer cells is low in intestinal cancer, renal cancer and liver cancer.
  • the expression of GAD1 is low in thyroid cancer, lung cancer, head and neck cancer, gastric cancer, pancreatic cancer, urinary tract epithelial cancer, prostate cancer, testis cancer, breast cancer, ovarian cancer, melanoma and the like.
  • the prognosis is good when the expression of GAD1 is high in glioma, cervical cancer and endometrial cancer, and the expression of GAD1 is high in cervical cancer and endometrial cancer.
  • the T cell activator of the present invention can suppress the growth of cancer against a cancer having a low expression of GAD1, and can also suppress the growth of a cancer against a cancer having a high expression level of GAD1.
  • the active ingredient thereof is an antisense nucleic acid, RNAi-inducible nucleic acid or ribozyme against glutamate decarboxylase or aldehyde dehydrogenase 9 family member A1.
  • the active ingredient thereof is an antisense nucleic acid, RNAi-inducible nucleic acid or ribozyme against glutamate decarboxylase or aldehyde dehydrogenase 9 family member A1.
  • Glutamate at least one subunit selected herein from the group consisting of human ⁇ 1, ⁇ 5, ⁇ 1, ⁇ and ⁇ 2, whose expression is specifically suppressed by antisense nucleic acids, RNAi-inducible nucleic acids or ribozymes.
  • Decarboxylase and aldehyde dehydrogenase 9 family member A1 are hereinafter referred to as target genes.
  • an antisense nucleic acid, an RNAi-inducible nucleic acid or a ribozyme against the target gene is referred to as a target gene-inhibiting nucleic acid.
  • the antisense nucleic acid for the target gene consists of a base sequence capable of hybridizing with the transcript under the physiological conditions of cells expressing the transcript (mRNA or initial transcript) of the target gene, and is in a hybridized state.
  • antisense nucleic acids may be a chemically modified nucleotide such as -O-methyl type.
  • Other important factors in the design of antisense nucleic acids include enhancing water solubility and cell membrane permeability, which can be overcome by devising dosage forms such as the use of liposomes and microspheres.
  • the length of the antisense nucleic acid is not particularly limited as long as it can specifically hybridize with the transcript of the target gene, and includes a short sequence of about 6 bases and a long sequence complementary to the entire sequence of the transcript. It may be an arrangement like this.
  • examples thereof include oligonucleotides consisting of about 6 bases or more, preferably about 15 to about 40 bases, and more preferably about 15 to about 30 bases.
  • the antisense nucleic acid not only hybridizes with the transcription product of the target gene and inhibits translation, but also binds to double-stranded DNA to form a triplet and inhibits transcription into mRNA. It may be possible.
  • complementarity between sequence A and sequence B means the identity between the complementary sequence of sequence A and sequence B.
  • the complementarity of the antisense nucleic acid with the target gene of the antisense nucleic acid does not necessarily have to be 100%, and it hybridizes to the extent that it can complementarily bind to the DNA or RNA of the target gene in a living cell. , About 70% or more, about 80% or more, about 90% or more, or about 95% or more, provided that transcription and / or translation into mRNA can be inhibited.
  • RNAi-inducible nucleic acid refers to a polynucleotide capable of inducing RNA interference (RNAi) by being introduced into a cell, and is preferably RNA or a chimeric molecule of RNA and DNA.
  • RNA interference refers to the effect that double-stranded RNA containing the same base sequence (or a partial sequence thereof) as mRNA suppresses the expression of the mRNA. In order to obtain this RNAi effect, for example, it is preferable to use double-stranded RNA having the same base sequence (or a partial sequence thereof) as at least 19 consecutive target mRNAs.
  • the double-stranded structure may be composed of different strands of the sense strand and the antisense strand, or may be a double strand (SHRNA) provided by the stem-loop structure of one RNA.
  • SHRNA double strand
  • RNAi-inducible nucleic acids include siRNA and miRNA. The miRNA recognizes the 3'UTR of the target gene, destabilizes the mRNA of the target gene, and suppresses translation to suppress the expression of the target gene.
  • the RNAi-inducible nucleic acid is preferably siRNA from the viewpoint of strong transcriptional repressive activity.
  • the siRNA for the target gene can target any portion of the mRNA of the target gene.
  • the siRNA molecule for the target gene is not particularly limited as long as it can induce the RNAi effect, but is, for example, 18 to 27 base bases long, preferably 21 to 25 bases long.
  • the siRNA for the target gene is a double strand containing a sense strand and an antisense strand.
  • the siRNA for the target gene may have an overhang at the 5'end or 3'end of one or both of the sense strand and the antisense strand.
  • Overhangs are formed by the addition of one to several (eg, 1, 2 or 3) bases at the ends of the sense and / or antisense strands.
  • Methods for designing siRNA are known to those of skill in the art, and various siRNA design software or algorithms can be used to select an appropriate siRNA base sequence from the above base sequences.
  • ribozyme refers to RNA having an enzyme activity for cleaving nucleic acid, but recently, it has been clarified that oligo DNA having a base sequence of the enzyme activity site also has nucleic acid cleaving activity. In the specification, it is used as a concept including DNA as long as it has sequence-specific nucleic acid cleavage activity.
  • the ribozyme can specifically cleave the mRNA or early transcript encoding the target gene inside the coding region (including the intron moiety in the case of the early transcript).
  • the most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and hammer head type and hairpin type are known.
  • the hammer head type exerts enzymatic activity at about 40 bases, and several bases at both ends adjacent to the part having the hammer head structure (about 10 bases in total) are arranged in a sequence complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. Furthermore, when the ribozyme is used in the form of an expression vector containing the DNA encoding it, it should be a hybrid ribozyme in which tRNA-modified sequences are further linked in order to promote the transfer of the transcript to the cytoplasm. It can also be done (Nucleic Acids Res., 29 (13): 2780-2788 (2001)).
  • the T cell activator of the present invention can also be provided as an expression vector containing a polynucleotide encoding the target gene-inhibiting nucleic acid and a promoter operably linked to the polynucleotide.
  • the promoter may be appropriately selected depending on the type of nucleic acid to be expressed under its control, and may be, for example, a polIII promoter (eg, tRNA promoter, U6 promoter, H1 promoter), a mammalian promoter (eg, CMV promoter, CAG). Promoter, SV40 promoter).
  • the expression vector of the present invention further contains a selectable marker gene (a gene that imparts resistance to a drug such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinoslicin, a gene that complements an auxotrophic mutation, etc.). You may.
  • the backbone of the expression vector of the present invention is not particularly limited as long as it can produce the target gene-inhibiting nucleic acid in mammalian cells such as humans, and examples thereof include a plasmid vector and a viral vector.
  • Suitable vectors for administration to mammals include virus vectors such as retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, Sindobis virus, and Sendai virus.
  • virus vectors such as retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, Sindobis virus, and Sendai virus.
  • viral vectors derived from retrovirus, adenovirus, adeno-associated virus, and vaccinia virus are preferable.
  • the active ingredient includes, but is limited to, an antibody that specifically binds to GABA, a specific binding partner and / or a fragment thereof. Not done.
  • the antibody refers to a natural antibody such as a polyclonal antibody or a monoclonal antibody, a chimeric antibody that can be produced by using a gene recombination technique, a humanized antibody or a single-stranded antibody, a human antibody-producing transgenic animal, or the like.
  • a natural antibody such as a polyclonal antibody or a monoclonal antibody
  • a chimeric antibody that can be produced by using a gene recombination technique
  • a humanized antibody or a single-stranded antibody a human antibody-producing transgenic animal, or the like.
  • human antibodies that can be produced using, antibodies produced by phage display.
  • a specific binding partner refers to a member of a specific binding pair.
  • Specific binding pairs include two different molecules that specifically bind to each other by chemical or physical means.
  • other specific binding pairs include biotin and avidin (or streptavidin), carbohydrates and lectins, complementary nucleotide sequences, effectors and. Examples include, but are not limited to, acceptor molecules, cofactors and enzymes, enzymes and enzyme inhibitors.
  • the specific binding pair a member that is an analog of the original specific binding member, for example, an analyte analog can be mentioned.
  • Immune response-specific binding members whether isolated or recombinantly produced, include antigens and fragments thereof, antibodies including monoclonal and polyclonal antibodies, and complexes and fragments thereof. Can be mentioned.
  • the class of antibody in the present specification is not particularly limited, and includes an antibody having any isotype such as IgG, IgM, IgA, IgD or IgE. It is preferably IgG or IgM, and more preferably IgG in consideration of easiness of purification and the like.
  • the polyclonal antibody, monoclonal antibody, chimeric antibody, humanized antibody and fragments thereof in the present specification can be produced by a known general production method.
  • the antibodies described herein are, for example, Greenfield, E. et al. A. Hen, Antibodies: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, 2014).
  • the T cell activator of the present invention can be used as a pharmaceutical (immunotherapeutic agent) for the treatment of cancer, the prevention and / or the treatment of infectious diseases.
  • the present invention includes the use of T cell activators for the treatment of cancer or the prevention and / or treatment of infectious diseases, which inhibit or reduce the cytotoxic T cell inhibitory effect of B cells.
  • the present invention also comprises the use of a T cell activator that inhibits or reduces the cytotoxic T cell inhibitory effect of B cells to produce an anticancer agent or a prophylactic and / or therapeutic agent for an infectious disease. Also includes the above-mentioned uses for.
  • the T cell activator of the present invention When used as an anticancer agent or a prophylactic and / or therapeutic agent for infectious diseases, it can be orally or parenterally administered to a patient, and as an administration form.
  • examples include oral administration, local administration, intravenous administration, transdermal administration, and the like, and if necessary, the drug is formulated into a dosage form suitable for administration together with a pharmaceutically acceptable additive.
  • Dosage forms suitable for oral administration include, for example, tablets, capsules, granules, powders, etc.
  • dosage forms suitable for parenteral administration include, for example, injections, ointments, lotions, creams, etc. Examples include patches. These can be prepared using conventional techniques commonly used in the art.
  • the anticancer agent and the preventive and / or therapeutic agent for an infectious disease of the present invention suppress or reduce the growth of cancer or infected cells in a patient, or suppress, shrink or eliminate the growth of cancer or infected tissue, etc.
  • the route of administration and dosage form are not particularly limited as long as they have a therapeutic effect on cancer, but the preferred route of administration is topical administration, and the dosage form is an injection, an ointment, a lotion, a cream or a patch. ..
  • the anticancer agent and the preventive and / or therapeutic agent for infectious diseases of the present invention shall be DDS (drug delivery system) -ized preparations such as intra-organ implant preparations and microspheres. You can also.
  • intramuscular topical administration in order to bring the anticancer agent and the preventive and / or therapeutic agent of the present invention to the desired cancer tissue or infected tissue (for example, primary lesion or metastatic cancer tissue), intramuscular topical administration , Subcutaneous local administration, direct application to the skin, local administration such as application, and systemic administration such as intravenous injection (drip) and subcutaneous administration may be used.
  • the anticancer agent and the preventive and / or therapeutic agent for infectious diseases of the present invention may contain a pharmaceutically acceptable carrier depending on the type of active ingredient and the route of administration thereof.
  • a pharmaceutically acceptable carrier include, for example, excipients such as sucrose, starch, mannitt, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate; cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin.
  • Arabium gum polyethylene glycol, sucrose, starch and other binders; starch, carboxymethyl cellulose, hydroxypropyl starch, sodium-glycol-starch, sodium hydrogen carbonate, calcium phosphate, calcium citrate and other disintegrants; magnesium stearate, aerodil , Lubricants such as talc, sodium lauryl sulfate; Preservatives such as sodium benzoate, sodium hydrogen sulfite, methylparaben, propylparaben; pH adjusters such as citrate, sodium citrate, acetic acid; methylcellulose, polyvinylpyrrolidone, aluminum stearate, etc.
  • Suspension agent such as surfactant
  • Dispersant such as surfactant
  • Dissolving agent such as water, physiological saline, ethanol, propylene glycol
  • Isotonic agent such as glucose, sodium chloride, potassium chloride
  • Cacao fat polyethylene glycol, white kerosene Examples include, but are not limited to, base waxes such as. Further, these carriers are not limited to a single action, and can be used for the purpose of exerting a plurality of actions.
  • an ointment for example, sodium hydrogen sulfite, sodium thiosulfate, etc.
  • a stabilizer for example, sodium hydrogen sulfite, sodium thiosulfate, etc.
  • sodium edetate sodium citrate, ascorbic acid, dibutylhydroxytoluene, etc.
  • solubilizers eg, glycerin, propylene glycol, macrogol, polyoxyethylene hydrogenated castor oil, etc.
  • suspending agents eg, polyvinylpyrrolidone, etc.) Hydroxypropylmethylcellulose, hydroxymethylcellulose, sodium carboxymethylcellulose, etc.
  • emulsifiers eg, polyvinylpyrrolidone, soybean lecithin, egg yolk lecithin, polyoxyethylene hydrogenated castor oil, polysorbate 80, etc.
  • buffers eg, phosphate buffer, acetate buffer, etc.
  • Derivatives sodium chondroitin sulfate, sodium hyaluronate, carboxyvinyl polymer, polyvinyl alcohol, polyvinylpyrrolidone, macrogol, etc., preservatives (eg, benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, chlorobutanol, benzyl alcohol, dehydroacetic acid) Sodium, paraoxybenzoic acid esters, sodium edetate, boric acid, etc.), isotonic agents (eg, sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, glucose, propylene glycol, etc.), pH adjusters (eg, sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, glucose, propylene glycol, etc.) For example, hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid
  • the anticancer agent and the preventive and / or therapeutic agent for an infectious disease of the present invention are operably linked to the target gene-inhibiting nucleic acid, the polynucleotide encoding the target gene-inhibiting nucleic acid, and the polynucleotide.
  • Expression vectors containing promoters can also be formulated using the lipofection method.
  • liposomes usually composed of phosphatidylserine are used. Since phosphatidylserine has a negative charge, it is easier to make more stable liposomes as a substitute for phosphatidylserine.
  • DOTMA N-triethylammonium chloride
  • positively charged liposomes as a whole can be adsorbed on the surface of negatively charged cells and fused with the cell membrane.
  • Nucleic acid can be introduced into cells.
  • the skin, peritoneum, thoracic membrane, etc. may be incised to expose the cancer lesion as in incision surgery or recovery surgery, or the cancer lesion may be accessed percutaneously by endoscopy.
  • An expression vector comprising the target gene-inhibiting nucleic acid, or a polynucleotide encoding the target gene-inhibiting nucleic acid, and a promoter operably linked to the polynucleotide can also be injected directly into the lesion.
  • Direct topical administration to such cancer lesions comprises expression of said target gene-inhibiting nucleic acid, or a polynucleotide encoding the target gene-inhibiting nucleic acid, and a promoter operably linked to the polynucleotide.
  • Side effects such as off-target effects may be reduced compared to systemic administration of the vector.
  • the ratio of the active ingredient contained in the anticancer agent and the preventive and / or therapeutic agent for infectious diseases of the present invention can be appropriately set within a range in which the desired effect can be obtained, but is usually 0.01. It is about 100% by weight, preferably 0.1 to 99.9% by weight, and more preferably 0.5 to 99.5% by weight.
  • the T cell activator of the present invention is an inhibitor of signal transduction mediated by GABA, an inhibitor of GABA biosynthesis in B cells, a promoter of GABA degradation in B cells, an inhibitor of GABA secretion in B cells, and GABA in B cells.
  • GABA-specific binding antibodies, specific binding partners and / or fragments thereof they act on the central or peripheral nerves and the psyche. Or it may cause side effects that alter nerve function.
  • the T cell activator of the invention has cytotoxicity against B cells, humoral immune function in the body. May cause weakening side effects.
  • a therapeutically or prophylactically effective amount of the T cell activator of the present invention is associated with cytotoxic T cells rather than side effects such as altering mental or neurological function or weakening humoral immune function.
  • the dose should be greater than the effect of inhibiting or reducing the action of B cells that suppress cytotoxic activity.
  • the dose to be administered must be carefully adjusted to take into account the age, weight and condition of the individual being treated or prevented, the route of administration, the form and method of administration, and the exact dose will be determined by the physician. Must be decided.
  • the actual dosage is within the discretion of the physician and may vary by setting the dose for the particular circumstances of the invention in order to obtain the desired therapeutic effect.
  • the dose of the T cell activator of the present invention is not unconditionally defined depending on the type of active ingredient, the body weight and age of the subject to be administered, symptoms, etc., but starts from 0.0001 mg per 1 kg of body weight at a time. It is possible to select in the range of 1000 mg.
  • the number of administrations of the therapeutic agent of the present invention is not particularly limited, but is usually about 1 to 5 times per day.
  • the administration period may be a short-term administration of several days to one week or a long-term administration of several weeks to several months. If the disease recurs at considerable intervals, the therapeutic and / or prophylactic agent of the present invention can be re-administered.
  • the number of administrations of the T cell activator of the present invention is not particularly limited, but is usually about 1 to 5 times per day.
  • the administration period may be a short-term administration of several days to one week or a long-term administration of several weeks to several months. If the cancer or infectious disease recurs at considerable intervals, the T cell activator of the present invention can be administered again.
  • specifically killing cancer cells means that the killing effect on cancer cells is usually 1.2 times or more, preferably 1.2 times or more, as compared with the killing effect on cells other than cancer cells. 1.3 times or more, 1.5 times or more, 1.7 times or more, 1.9 times or more, 2 times or more, 5 times or more, 10 times or more, 20 times or more, 50 times or more, 100 times or more, 200 It means that it is more than double, 500 times or more, 1000 times or more, 2000 times or more, 5000 times or more, and 10000 times or more higher. The same applies to "specifically killing infected cells".
  • target gene-inhibiting nucleic acid of the present invention “specifically suppressed expression” means that the expression of the target gene when the target gene-inhibiting nucleic acid is added is the target gene when the target gene-inhibiting nucleic acid is not added.
  • 1.2 times or more preferably 1.3 times or more, 1.5 times or more, 1.7 times or more, 1.9 times or more, 2 times or more, 5 times or more, 10 times or more, as compared with the expression of It means that it is 20 times or more, 50 times or more, 100 times or more, 200 times or more, 500 times or more, 1000 times or more, 2000 times or more, 5000 times or more, and 10000 times or more lower.
  • the term "specifically binding" to substance B means that the substance A and the substance B have a considerably high binding affinity, and the cross-reaction between the substance A and the substance other than the substance B. Refers to not showing sex. Significantly high binding affinities are those with a dissociation constant K d of 1 ⁇ 10 -7 M or less, 1 ⁇ 10 -8 M or less, and more particularly 1 ⁇ 10 -9 M or less or even more 1 ⁇ 10 -10 M or less. Refers to a bond.
  • the binding affinity of substance A and substance B in the technical field of the present invention is measured by a measuring device using a surface plasmon resonance method (for example, Biacore system (GE Healthcare Japan Co., Ltd.)) or a reflection type interference spectroscopy. Measurement can be performed by a measuring device including, but not limited to, a device (for example, forte-BIO series (Nippon Pole Co., Ltd.)). Further, for substance A and substance B, "cross-reactivity with substances other than substance A and substance B" can be determined using, for example, a competitive binding assay (eg, ELISA).
  • a competitive binding assay eg, ELISA
  • Treatment means that the anti-cancer agent of the present invention and the treatment method of cancer are effective, and the complete response to the complete disappearance of the cancer.
  • complete remission (CR) and partial response or partial remission (PR) the size of solid cancers decreases, the number of cancer cells decreases in blood cancers), and the size of the tumor does not change.
  • the anticancer agent and the therapeutic method for cancer of the present invention act to suppress the cancer cell killing activity of cytotoxic T cells, and to inhibit or reduce the interaction between B cells and cytotoxic T cells. Because of the mechanism, the mechanism of action differs from that of existing anticancer agents such as chemotherapeutic agents, immunotherapeutic agents, and irradiation therapy, or cancer treatment methods. Therefore, the anti-cancer agent and the method for treating cancer of the present invention can be used in combination with any existing anti-cancer agent or method for treating cancer.
  • Treatment means that the infectious disease prevention and / or therapeutic agent and the infectious disease prevention and / or treatment method of the present invention are effective. It refers to the effect, including the disappearance and alleviation of symptoms due to infectious diseases, the elimination of pathogens in the body, and the production of neutralizing antibodies against pathogens. "Prevention” includes preventing infection with a pathogen, and even if a pathogen causing an infectious disease invades the body, the symptoms do not appear or the symptoms are alleviated.
  • infectious disease prevention and / or therapeutic agent and the infectious disease prevention and / or treatment method of the present invention suppress the killing activity of cytotoxic T cells against infected cells, and the interaction between B cells and cytotoxic T cells. Since the mechanism of action is inhibition or reduction, the mechanism of action differs from the existing methods of infectious disease prevention and / or treatment with therapeutic agents and vaccines. Therefore, the infectious disease prevention and / or therapeutic agent and the infectious disease prevention and / or treatment method of the present invention can be used in combination with the existing infectious disease prevention and / or treatment method using a therapeutic agent or vaccine.
  • an antibody, a specific binding partner or a fragment thereof, which is cytotoxic to B cells has the ability to kill B cells alone or in a complex with complement or the like.
  • an antibody having, a specific binding partner, or a fragment thereof refers to an antibody having, a specific binding partner, or a fragment thereof.
  • the B cell removing agent refers to any drug having an activity of removing B cells that suppresses the killing activity of cytotoxic T cells against cancer cells and infected cells.
  • B cell removers herein include anti-CD20 monoclonal antibodies that specifically remove B cells that express CD20 on the cell surface, and proteins derived from the monoclonal antibodies, such as rituximab and its biosimilars. In addition, it contains an antibody against intestinal bacteria that eliminates intestinal-specific CD11b-positive IgA-producing cells.
  • the anti-cancer agent of the present invention and / or other pharmaceutical agents or therapeutic methods used in combination with the method for treating cancer of the present invention include, but are not limited to, the following. ⁇ Surgery to physically remove cancer cells, Chemotherapeutic and chemotherapeutic agents that administer chemicals, including but not limited to alkylating agents, platinum compounds, metabolic antagonists, topoisomerase inhibitors, microtube inhibitors, antibiotics, and cancer cells Chemotherapy and chemotherapeutic agents that are attacked by immune cells as non-self, and radiation that inhibits and kills cancer cells by irradiation with radiation, including but not limited to X-rays, electron beams, gamma rays, and neutron rays. Therapy.
  • Chemotherapeutic agents for the treatment of cancer include 6-O- (N-chloroacetylcarbamoyl) fumaguilol, bleomycin, methotrexate, actinomycin D, mitomycin C, doxorubicin, adriamycin, neocultinostatin, cytosine arabinoside.
  • the other pharmaceuticals may also contain immune checkpoint inhibitors.
  • the other treatment method may also include surgical removal of the cancerous tissue and / or irradiation with radiation that kills the cancer cells.
  • immune checkpoint inhibitors include, but are not limited to, anti-PD-1 antibody, anti-CTLA-4 antibody, and anti-PD-L1 antibody.
  • infectious disease preventive and / or therapeutic agent of the present invention and other pharmaceutical or therapeutic methods used in combination with the infectious disease preventive and / or therapeutic method include vaccination, antibacterial agent, antiviral agent, insecticide and the like. Drug administration, but is not limited to these.
  • the other pharmaceuticals may also include immune checkpoint inhibitors. Examples of immune checkpoint inhibitors include, but are not limited to, anti-PD-1 antibody, anti-CTLA-4 antibody, and anti-PD-L1 antibody.
  • the adnominal adjective "about” that modifies a numerical value means that the numerical value is in the numerical range of 90% or more and 110% or less of the numerical value.
  • “about 40 bases” refers to a base in a numerical range of 36 bases or more and 44 bases or less.
  • Example 1 (1) Materials and methods (1.1) Mouse-mouse mutants muMt ⁇ / ⁇ , Cd3e ⁇ / ⁇ and rag1 ⁇ / ⁇ (all backgrounds are C57BL / 6J strains) and wild-type mice are RIKEN. It was propagated and maintained under SPF conditions at the Center for Biomedical Sciences (IMS RIKEN). Sterile (GF) wild-type mice were delivered and maintained in a vinyl isolator at the RIKEN Center for Integrative Medical Sciences. C57BL / 6N or C57BL / 6J wild-type mice were purchased from Claire Japan. For the analysis, littermates or mice with appropriate age / sex matching were used. All animal studies were performed according to a protocol approved by the institution's Animal Care and Use Committee.
  • Intracellular staining was performed using Fixation / Permeabilization Solution Kit (Becton Dickinson, Inc., Japan). The data were analyzed using FlowJo software (FlowJo, LLC, Becton Dickinson, Inc., Japan).
  • CD4 or CD8T cells from wild type mice using (CD11c -, CD11b -, B220 -, CD4 + or CD8 + , CD44 int / -, and CD62L +), central memory (CM) CD4 or CD8T cells (CD11c -, CD11b -, B220 -, CD4 + or CD8 +, CD44 high, and CD62L +), effector memory (EM) CD4 or CD8T cells (CD11c -, CD11b -, B220 -, CD4 + or CD8 +, CD44 high, CD62L - ) and, B cells (CD11c -, CD11b -, CD4 -, CD8 -, B220 +) and, CD11b / c cells (B220 -, CD11c + and / or CD11b +
  • Germinal center (GC) or non-GCT cells TCR- ⁇ +, CD4 + , PD-1 - or PD-1 +, CXCR5 - or CXCR5 +), non-GCB cells (B220 +, IgD high) or GCB cells (B220 +, IgD -, it was sorted from FAS +) and Peyer's patches (PP).
  • IgA plasma cells (PC) were sorted from the small intestine lamina intestinal (SILP). Selected cells were washed with PBS and rapidly frozen in liquid nitrogen for analysis of metabolites.
  • Liquid Chromatography-mass spectrometry was used for analysis of metabolites.
  • Liquid Chromatography Mass Spectrometry is performed by UltiMate 3000-High Performance Liquid Chromatography (Thermo Fisher Scientific Co., Ltd.) linked with Q Executive-Orbitrap Hybrid Mass Spectrometer (MS) (Thermo Fisher Scientific Co., Ltd.). ), Miyajima, M. et al. Et al. (Nat Immunol. 18: 1342 (2017)).
  • T cells or B cells were selected from the lymph nodes and 10% (v / v) FBS, 1 ⁇ MEM.
  • the cells were cultured in RPMI1640 medium (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with NEAA, 10 mM HEPES, 50 ⁇ M 2-mercaptoethanol, 1 mM sodium pyruvate, 100 U / mL penicillin, and 100 U / mL streptomycin for 24 hours or 72 hours.
  • Anti-CD3e (2.5 ⁇ g / mL, 145- 2C11, Nippon Becton Dickinson Co., Ltd.) stimulated T cells using an immobilized 96-well plate.
  • Anti-IgM (8 ⁇ g / mL, Jackson Immuno Research, Fujifilm Wako Junyaku Co., Ltd.) alone, anti-IgM (8 ⁇ g / mL) and anti-CD40 (1 ⁇ g / mL, Nippon Becton Dickinson Co., Ltd.), LPS (100 ng / B cells were stimulated with mL, Sigma, Sigma-Aldrich Japan LLC).
  • PBMCs Human peripheral blood mononuclear cells
  • PBMCs Human peripheral blood mononuclear cells
  • PBMCs were stained with biotin-labeled anti-CD20 (Biolegend, clone 2H7) and biotin-labeled anti-CD19 (Biolegend, clone HIB19), and fractions binding to anti-biotin MicroBeads (Miltenyi Biotec, Inc.) were selected as human B cells. .. Fractions that did not bind were sorted as human T cells using a human pan T cell negative isolation kit (Miltenny Biotec, Inc.).
  • FIG. 2 is a bar graph of GABA content of various lymphocytes excised from wild-type mice and isolated by flow cytometry. Error bars represent the standard error of the sample mean between samples of each lymphocyte type. The standard error of the sample mean is the standard deviation of all combinations, which indicates how much the sample mean varies depending on the combination selected when a certain number of samples are selected from the population.
  • the vertical axis of the graph shows small intestinal IgA plasma cells (SI IgA PC), germinal center (GC) or non-GC (nonGC) T cells and B cells of the Pier plate (PP), and B cells (B220) of lymph nodes (LN).
  • Dendritic cells DC
  • CD4 or CD8 positive or negative naive T cells naive
  • CD4 or CD8 positive and CD44 positive cells Represents each cell of (CD44).
  • the horizontal axis of the graph represents a relative value in which the GABA content of each cell is multiplied by the GABA content of naive CD4T cells.
  • the GABA content of small intestinal IgA plasma cells is remarkably high, which is more than twice the GABA content of the next highest Peyer's patch nongerminal center B cells.
  • FIG. 1 A bar graph comparing the GABA content of lymph node tissue is shown in FIG.
  • Error bars represent the standard deviation of GABA content between samples of each lymph node tissue.
  • the vertical axis of the graph represents a relative value in which the GABA content of each tissue is multiplied by the GABA content of the lymph node opposite to the wild-type mouse.
  • the horizontal axis represents the lymph node from which each tissue was derived.
  • the p value of the difference was 0.0011. “*****” in FIG.
  • the vertical axis of the GABA content represents a relative value in which the GABA content of each selected cell is multiplied by the GABA content of the contralateral CD4 positive cell.
  • the horizontal axis represents the type of each selected cell. “*” In FIG. 4, that is, CD4 positive cells (ipsi CD4) selected from all lymph node tissues ipsilateral to the immunized hindlimb footpad and CD4 positive cells (contra) selected from lymph node tissue on the opposite side.
  • the p value of the significant difference in GABA content of CD4) was 0.0232.
  • FIG. 6 shows the effect of insufficiency of B cell differentiation bred under sterile conditions in mice. It is a broken line graph showing the influence on the growth (tumor lesion volume) of the tumor derived from MC38 cells. The error bars represent the standard deviation of the tumor volume on each measurement day.
  • the vertical axis represents the tumor volume (mm 3 ) between the IgM-deficient homozygous knockout mouse (mMT) and the wild-type mouse (WT).
  • the horizontal axis represents the measurement date of the tumor volume.
  • IgM-deficient homozygous knockout mice suppressed tumor growth more than wild-type mice.
  • the horizontal axis represents the measurement date of the tumor volume.
  • tumor growth was suppressed in IgM-deficient homozygous knockout mice (mMT (+ placebo)) treated with placebo pellets as compared to wild-type mice (WT).
  • IgM-deficient homozygous knockout mice (mMT (+ GABA)) treated with GABA sustained-release pellets promoted tumor growth compared to IgM-deficient homozygous knockout mice (mMT (+ placebo)) treated with placebo pellets. .. That is, GABA sustained-release pellets inhibited the tumor growth-suppressing effect in IgM-deficient homozygous knockout mice.
  • FIG. 8 shows that GABA sustained-release pellet treatment on B-cell differentiation-deficient mice was performed on tumor-infiltrating CD8-positive cells. It is a combination diagram of a two-parameter histogram showing the effect on activation. The upper and lower left of FIG. 8 are wild-type mice treated with placebo pellets (WT (+ histogram)), and the center and right are IgM-deficient homoknockout mice treated with placebo pellets or GABA sustained-release pellets (mMT (+ histogram) or mMT (mMT (+ histogram)).
  • + GABA is a two-parameter histogram obtained by gating CD8-positive cells by flow cytometry.
  • the vertical axis represents the fluorescence intensity of CD98
  • the horizontal axis represents the fluorescence intensity of TCRb.
  • the vertical axis represents the fluorescence intensity of Sca-1
  • the horizontal axis represents the fluorescence intensity of TCRb.
  • the numerical value in the vicinity of the box represents the percentage of the cells having the fluorescence intensity in the box among the CD8-positive cells under each condition. As shown in FIG.
  • IgM-deficient homozygous knockout mice (mMT (+ placebo)) treated with placebo pellets were CD98 or Sca1 positive and compared to wild-type mice treated with placebo pellets (WT (+ placebo)).
  • the number of TCRb-positive tumor-infiltrating CD8-positive cells increased.
  • IgM-deficient homozygous knockout mice (mMT (+ GABA)) treated with GABA sustained-release pellets were CD98 or Sca1 positive, compared to IgM-deficient homozygous knockout mice (mMT (+ placebo)) treated with placebo pellets.
  • the number of TCRb-positive tumor-infiltrating CD8-positive cells was reduced compared to wild-type mice treated with placebo pellets (WT (+ placebo)). That is, GABA sustained-release pellets inhibited the increase of tumor-infiltrated CD8-positive cells by IgM-deficient homozygous knockout mice.
  • FIG. 9 shows that GABA sustained-release pellet treatment on B-cell differentiation-deficient mice is tumor-infiltrating CD8-positive cells. It is a combination diagram of a two-parameter histogram showing the influence on the cell killing activity of.
  • the upper and lower left of FIG. 9 are wild-type mice treated with placebo pellets (WT (+ histogram)), and the center and right are IgM-deficient homoknockout mice treated with placebo pellets or GABA sustained-release pellets (mMT (+ histogram) or mMT (mMT (+ histogram)).
  • + GABA is a two-parameter histogram obtained by gating CD8-positive cells by flow cytometry.
  • the vertical axis represents the fluorescence intensity of perforin
  • the horizontal axis represents the fluorescence intensity of TCRb.
  • the vertical axis represents the fluorescence intensity of GrzB (Granzyme B)
  • the horizontal axis represents the fluorescence intensity of TCRb.
  • the numerical value in the vicinity of the box represents the percentage of the cells having the fluorescence intensity in the box among the CD8-positive cells under each condition.
  • FIG. 10A is a bar graph showing the effect of GABA sustained release pellet treatment on B cell differentiation-deficient mice on the proportion of tumor-infiltrating CD8-positive cells to perforin-positive cells, based on the two-parameter histogram of FIG.
  • the vertical axis represents the percentage of perforin-positive cells in wild-type mice treated with placebo pellets (WT + placebo), IgM-deficient homozygous knockout mice treated with placebo pellets or sustained-release GABA pellets (mMT + placebo or mMT + GABAp). “**” in FIG.
  • FIG. 10B is a bar graph showing the effect of GABA sustained release pellet treatment on B cell differentiation-deficient mice on the proportion of granzyme-positive cells of tumor-infiltrating CD8-positive cells, based on the two-parameter histogram of FIG.
  • the vertical axis is the percentage of granzyme-positive cells in wild-type mice treated with placebo pellets (WT + placebo), IgM-deficient homozygous knockout mice treated with placebo pellets or sustained-release GABA pellets (muMT ⁇ / ⁇ + placebo or muMT ⁇ / ⁇ + GABAp). Represents. “*” In FIG.
  • IgM-deficient homoknockout mice muMT ⁇ / ⁇ + placebo
  • placebo pellets WT + placebo
  • the number of positive and TCRb-positive cytotoxic tumor-infiltrating CD8-positive cells increased.
  • IgM-deficient homoknockout mice muMT ⁇ / ⁇ + GABAp
  • GABA sustained-release pellets were perforin or granzyme compared to IgM-deficient homoknockout mice (muMT ⁇ / ⁇ + placebo) treated with placebo pellets.
  • the number of positive and TCRb-positive cytotoxic tumor-infiltrating CD8-positive cells decreased to the same level as in wild-type mice (WT + placebo) treated with placebo pellets. That is, GABA sustained-release pellets inhibited the increase of cytotoxic tumor-infiltrating CD8-positive cells by IgM-deficient homozygous knockout mice.
  • FIG. 11 shows the GABA A receptor on the growth of MC38 cell-derived tumors inoculated into wild-type mice. It is a broken line graph showing the influence of the inhibitor of.
  • the vertical axis represents the tumor volume (mm 3 ) of tiagabine (GAT (transporter) inhibitor) -administered animal (Tiagabine), PBS-administered animal (PBS) and picrotoxin-administered animal (Picrotoxin).
  • the horizontal axis represents the measurement date of the tumor volume.
  • the curve with the largest tumor volume on the final measurement day shows the time course of the tumor volume of the animals treated with thiagabin (GAT (transporter) inhibitor), and the curve with the second largest tumor volume shows the time course of the tumor volume of the animals treated with PBS.
  • the curve with the smallest tumor volume shows the change over time in the tumor volume of the picrotoxin-administered animal.
  • the error bars represent the standard deviation of the tumor volume on each measurement day. “***” in FIG. 11, that is, the p value of the significant difference in tumor volume between the PBS-administered animal (PBS) and the picrotoxin-administered animal (Picrotoxin) measured on the 23rd day after inoculation with MC38 cells was 0.0003. rice field.
  • FIG. 12 shows the GABA A receptor on the growth of MC38 cell-derived tumors inoculated into wild-type mice. It is a broken line graph showing the influence of the inhibitor of.
  • the vertical axis represents the tumor volume (mm 3 ) of a flumazenil (GABA A receptor inhibitor) -administered animal or a PBS-administered animal.
  • the horizontal axis represents the measurement date of the tumor volume.
  • the curve with the smallest tumor volume on the final measurement day shows the change over time in the tumor volume of the flumazenil-administered animal (Flumazenil), and the curve with the largest tumor volume shows the change over time in the tumor volume of the DMSO-administered animal (DMSO).
  • the error bars represent the standard deviation of the tumor volume on each measurement day.
  • flumazenil suppressed the increase in tumor volume.
  • the p-value of "**" in FIG. 12, that is, the significant difference in tumor volume between the DMSO-administered animal (DMSO) and the flumazenil-administered animal (Flumazenil) measured on the final measurement day was 0.0092.
  • FIG. 13 shows the effects of CD8-positive cells infiltrating tumors derived from MC38 cells inoculated into wild-type mice. It is a combination diagram of a two-parameter histogram showing the effect of picrotoxin on the composition. On the left is a two-parameter histogram of cells infiltrated into a tumor derived from MC38 cells inoculated into wild-type mice, with the fluorescence intensity of CD8 on the horizontal axis and the fluorescence intensity of CD45-2 on the vertical axis.
  • the lower right is a two-parameter histogram of cells infiltrated into a tumor derived from MC38 cells inoculated into picrotoxin-treated wild-type mice.
  • the upper right is a two-parameter histogram of cells invaded into a tumor derived from MC38 cells inoculated into wild-type mice in a control experiment.
  • the intensity of the laterally scattered light is represented by the vertical axis
  • the intensity of the forward scattered light is represented by the horizontal axis.
  • the numbers near the gating range represent the percentage of all measured cells within the gating range. As shown in FIG.
  • FIG. 14 is a bar graph showing the percentage of tumor-infiltrating T cells that are CD8 positive and have high intensities of laterally scattered light and forward scattered light calculated from the two-parameter histogram of FIG.
  • the vertical axis represents the percentage of T cells that are CD8 positive and have high intensity of lateral and forward scattered light.
  • the left shows the percentage of tumor infiltrating cells in the control experiment mouse (Ctr), and the right shows the percentage of tumor infiltrating cells in the picrotoxin-treated mouse (Pic). Error bars represent the standard deviation of the number of cells in each cell population.
  • FIGS. 13 and 14 suggest that the suppression of tumor volume increase by pictrotoxin treatment shown in FIG. 11 is associated with the enlargement of CD8-positive cells infiltrating the tumor and the complication of the cell structure.
  • Example 2 The GABA effect on cancer was investigated.
  • Culture experiment Control (DMSO) or picrotoxin dissolved in DMSO, 40 ⁇ g per mouse was administered intraperitoneally to rag1 gene-deficient mice from 1 day before inoculation with MC38 cells, and repeated every 2 days. The change in tumor volume over time was measured. The results are shown in FIG.
  • the vertical axis in FIG. 15 represents the tumor volume (mm 3 ) of rag1 gene-deficient mice treated with DMSO or picrotoxin.
  • the horizontal axis represents the measurement date of the tumor volume after ingestion of cells.
  • the error bars represent the standard deviation of the individual mouse tumor volume on each measurement day. “Ns” indicates that no statistically significant difference was observed.
  • GABA or picrotoxin does not affect the proliferation and viability of MC38 cultured cells.
  • Picrotoxin also suppresses the growth of MC38 cell-derived tumors in wild-type mice, but does not affect the growth of MC38 cell-derived tumors in rag1 gene-deficient mice (T and B cell-deficient mice), and thus GABA.
  • picrotoxins have been shown to affect the growth of MC38 cell-derived tumors via immune cells (T and B cells) rather than directly affecting MC38 cells.
  • the T cell activator of the present invention and the treatment of cancer and the prevention and / or treatment method of infectious diseases are based on a completely new therapeutic mechanism of inhibiting or reducing the cytotoxic T cell inhibitory effect of B cells. It can be used in combination with all conventional anti-cancer agents and methods of treating cancer, or preventive and / or therapeutic agents of infectious diseases and methods of prevention and / or treatment of infectious diseases.
  • a GABA inhibitor which has been widely studied as a neurotransmitter, is used, it is excellent in that there is little risk of side effects.

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Abstract

Provided are: a novel T-cell activator that targets B cells; and an immunotherapy for cancer or infectious diseases. A T-cell activator according to the present invention comprises a substance, such as flumazenil, which inhibits signal transduction via a GABA receptor, in particular, a GABAA receptor. An immunotherapy for cancer or infectious diseases, according to the present invention, comprises administrating the substance, such as flumazenil, which inhibits signal transduction via the GABA receptor, in particular, the GABAA receptor.

Description

新規Ｔ細胞活性化剤New T cell activator
　本発明は新規のＴ細胞活性化剤に関し、具体的には、Ｔ細胞のがん細胞や感染細胞に対する殺傷活性を抑制するＢ細胞の作用を阻害または低減する、新規のＴ細胞活性化剤に関する。 The present invention relates to a novel T cell activator, specifically, a novel T cell activator that inhibits or reduces the action of B cells that suppress the killing activity of T cells on cancer cells and infected cells. ..
　Ｂ細胞はＴ細胞のうちヘルパーＴ細胞に刺激されて形質細胞に分化し大量の抗体を産生する。逆にＢ細胞がＴ細胞の機能を調節する現象は制御性Ｂ細胞によるＩＬ－１０等の抗炎症性サイトカイン分泌が知られている（非特許文献１）。前立腺がんのマウスモデルで、ＩｇＡ産生形質細胞が、免疫原性がある腫瘍細胞の細胞死による細胞傷害性リンパ球の活性化を阻害し、オキサリプラチン抵抗性の発生を促進することが報告された（非特許文献２）。また、ＰＤ－Ｌ１を発現するＩｇＡ陽性Ｂ細胞は、非アルコール性脂肪肝を発症している肝臓に蓄積して、肝臓で肝細胞がんの発症を防止している細胞傷害性ＣＤ８陽性Ｔ細胞を抑制することにより、肝細胞がんの進行を促進することも報告されている（非特許文献３）。
　一方、ガンマアミノ酪酸（ＧＡＢＡ）と腫瘍細胞の関係について、いくつかの報告がある（非特許文献４、５、６及び７）。また、免疫応答とＧＡＢＡの関係が知られている（特許文献１） Among T cells, B cells are stimulated by helper T cells to differentiate into plasma cells and produce a large amount of antibody. On the contrary, the phenomenon that B cells regulate the function of T cells is known to secrete anti-inflammatory cytokines such as IL-10 by regulatory B cells (Non-Patent Document 1). In a mouse model of prostate cancer, IgA-producing plasma cells have been reported to inhibit the activation of cytotoxic lymphocytes by cell death of immunogenic tumor cells and promote the development of oxaliplatin resistance. (Non-Patent Document 2). In addition, IgA-positive B cells expressing PD-L1 are cytotoxic CD8-positive T cells that accumulate in the liver developing non-alcoholic fatty liver and prevent the development of hepatocellular carcinoma in the liver. It has also been reported that the progression of hepatocellular carcinoma is promoted by suppressing the disease (Non-Patent Document 3).
On the other hand, there are some reports on the relationship between gamma-aminobutyric acid (GABA) and tumor cells ( Non-Patent Documents 4, 5, 6 and 7). In addition, the relationship between immune response and GABA is known (Patent Document 1).
特表２００２０－５２４７０２Special table 20020-524702
　本発明の課題は、ＩｇＡ陽性Ｂ細胞のような制御性Ｂ細胞の機能を理解することでＢ細胞を標的とする新規のＴ細胞活性化剤およびがんや感染症の免疫療法を開発することである。発明者らは、免疫細胞のメタボローム解析結果を詳細に検討し、ＩｇＡ陽性Ｂ細胞をはじめとする活性化Ｂ細胞に特徴的な低分子代謝物を突き止めることができた。そこで、当該低分子代謝物の代謝調節やシグナル伝達をモジュレーションすることにより、細胞傷害性Ｔ細胞のがん細胞や感染細胞に対する殺傷活性を抑制するＢ細胞の作用を阻害または低減する、新規のＴ細胞活性化剤およびがんや感染症の治療方法を開発することも本発明の課題である。 An object of the present invention is to develop a novel T cell activator targeting B cells and immunotherapy for cancer and infectious diseases by understanding the function of regulatory B cells such as IgA-positive B cells. Is. The inventors were able to examine the results of metabolome analysis of immune cells in detail and identify small molecule metabolites characteristic of activated B cells, including IgA-positive B cells. Therefore, a novel T cell that inhibits or reduces the action of B cells that suppress the killing activity of cytotoxic T cells against cancer cells and infected cells by modulating the metabolic regulation and signal transduction of the small molecule metabolite. It is also an object of the present invention to develop a cell activator and a method for treating cancer and infectious diseases.
　本発明者らは、抗原刺激により活性化されたＢ細胞がガンマアミノ酪酸（ＧＡＢＡ）を産生すること、Ｂ細胞およびその細胞系譜に属する細胞がほとんど分化しない、ＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ）では、野生型マウスよりも腫瘍の成長が抑制され、かつ細胞傷害性腫瘍浸潤ＣＤ８陽性細胞が増加すること、ｍＭＴにおける腫瘍成長の抑制と細胞傷害性腫瘍浸潤ＣＤ８陽性細胞の増加がＧＡＢＡにより阻害されること、ＧＡＢＡＡレセプターの阻害薬の投与により、野生型マウスにおける腫瘍の成長が抑制されること、ＧＡＢＡＡレセプターの阻害薬を投与した野生型マウスの腫瘍に浸潤したＣＤ８陽性細胞は大型化して、細胞構造が複雑化することを見出した。さらに、本発明者らは、ＧＡＢＡＡレセプターの阻害薬は腫瘍に直接作用してその成長を抑制するのではなく、免疫細胞（Ｂ細胞とＴ細胞）を介して腫瘍の成長を抑制することも確認した。これらの知見に基づき、Ｂ細胞による細胞傷害性Ｔ細胞抑制作用を阻害または低減するという全く新しい治療機序を見出した。この治療機序により、腫瘍や感染細胞を排除することができる（図１７）。 We found IgM-deficient homo-knockout mice (mMT) in which B cells activated by antigen stimulation produce gamma aminobutyric acid (GABA), and B cells and cells belonging to their cell lineage hardly differentiate. In, GABA inhibits tumor growth and increases cytotoxic tumor-infiltrating CD8-positive cells, suppresses tumor growth in MMT, and increases cytotoxic tumor-infiltrating CD8-positive cells compared to wild-type mice. Rukoto, administration of inhibitors of GABA a receptors, the growth of the tumor is inhibited in wild-type mice, CD8 positive cells infiltrating the tumor wild-type mice treated with inhibitors of GABA a receptors in size , Found that the cell structure becomes complicated. Furthermore, we also indicate that GABA A receptor inhibitors do not act directly on the tumor to suppress its growth, but rather suppress the growth of the tumor via immune cells (B cells and T cells). confirmed. Based on these findings, we have found a completely new therapeutic mechanism that inhibits or reduces the cytotoxic T cell inhibitory effect of B cells. By this therapeutic mechanism, tumors and infected cells can be eliminated (Fig. 17).
　本発明は、Ｂ細胞による細胞傷害性Ｔ細胞抑制作用を阻害または低減する、Ｔ細胞活性化剤を提供する。 The present invention provides a T cell activator that inhibits or reduces the cytotoxic T cell inhibitory effect of B cells.
　本発明のＴ細胞活性化剤において、前記Ｂ細胞は抗原刺激により活性化されたＢ細胞とすることもできる。 In the T cell activator of the present invention, the B cells can also be B cells activated by antigen stimulation.
　本発明のＴ細胞活性化剤は、Ｔ細胞におけるガンマアミノ酪酸（ＧＡＢＡ）を介するシグナル伝達の阻害薬を含むこともできる。 The T cell activator of the present invention can also contain an inhibitor of gamma-aminobutyric acid (GABA) -mediated signal transduction in T cells.
　本発明のＴ細胞活性化剤において、前記Ｔ細胞におけるＧＡＢＡを介するシグナル伝達の阻害薬は、ヒトＴ細胞で発現するＧＡＢＡレセプターの発現および／または機能を抑制または低減する阻害薬とすることもできる。 In the T cell activator of the present invention, the inhibitor of GABA-mediated signal transduction in T cells can also be an inhibitor that suppresses or reduces the expression and / or function of GABA receptors expressed in human T cells. ..
　本発明のＴ細胞活性化剤において、前記Ｔ細胞で発現するＧＡＢＡレセプターは、ＧＡＢＡＡレセプター、および、ＧＡＢＡ－ρレセプターのうちの少なくとも１つとすることができる。 In the T cell activator of the present invention, the GABA receptor expressed in the T cell can be at least one of the GABA A receptor and the GABA-ρ receptor.
　本発明のＴ細胞活性化剤において、前記Ｔ細胞で発現するＧＡＢＡレセプターは、ヒトＴ細胞で発現するサブユニットポリペプチドからなることもできる。 In the T cell activator of the present invention, the GABA receptor expressed in the T cells can also consist of a subunit polypeptide expressed in human T cells.
　本発明のＴ細胞活性化剤において、前記ヒトＴ細胞で発現するサブユニットポリペプチドは、ヒトα１、α５、β１、πおよびρ２からなる群から選択される少なくとも１種類であってもよい。 In the T cell activator of the present invention, the subunit polypeptide expressed in the human T cells may be at least one selected from the group consisting of human α1, α5, β1, π and ρ2.
　本発明のＴ細胞活性化剤において、前記Ｔ細胞におけるＧＡＢＡを介するシグナル伝達の阻害薬は、前記ヒトＴ細胞で発現するヒトα１、α５、β１、πおよびρ２からなる群から選択される少なくとも１種類のサブユニットポリペプチドの発現を抑制または低減する、アンチセンス核酸、ＲＮＡｉ（ＲＮＡ干渉）誘導性核酸もしくはリボザイムまたはそれらの発現ベクターを含むことができる。 In the T cell activator of the present invention, the inhibitor of GABA-mediated signal transduction in the T cells is at least one selected from the group consisting of human α1, α5, β1, π and ρ2 expressed in the human T cells. It can include antisense nucleic acids, RNAi (RNA interference) inducible nucleic acids or ribozymes or expression vectors thereof, which suppress or reduce the expression of a type of subunit polypeptide.
　本発明のＴ細胞活性化剤において、前記Ｔ細胞におけるＧＡＢＡを介するシグナル伝達の阻害薬は、前記ヒトＴ細胞で発現するヒトα１、α５、β１、πおよびρ２からなる群から選択される少なくとも１種類のＧＡＢＡレセプターのサブユニットポリペプチドと特異的に結合して、該ＧＡＢＡレセプターの機能を抑制または低減する、抗体、特異的結合パートナーまたはそれらの断片からなる群から選択される少なくとも１種類とすることもできる。 In the T cell activator of the present invention, the inhibitor of GABA-mediated signal transduction in the T cells is at least one selected from the group consisting of human α1, α5, β1, π and ρ2 expressed in the human T cells. At least one selected from the group consisting of antibodies, specific binding partners or fragments thereof that specifically bind to a subunit polypeptide of a type of GABA receptor and suppress or reduce the function of the GABA receptor. You can also do it.
　本発明のＴ細胞活性化剤において、前記ＧＡＢＡＡレセプターの阻害薬は、フルマゼニル、Ｒｏ１５－４５１３、サルマゼニル、シクトキシン、エナントトキシン、ペンチレンテトラゾール、ピクロトキシン、ツジョン、リンデン、ビククリン、ガバジンおよびこれらの誘導体からなる群から選択される、少なくとも１種類とすることもできる。 In the T cell activator of the present invention, the GABA A receptor inhibitor is derived from flumazenil, Ro15-4513, sarmazenil, cicutoxin, enanthotoxin, pentylenetetrazol, picrotoxin, thujone, linden, bicuculline, gabazine and derivatives thereof. It may be at least one type selected from the group.
　本発明のＴ細胞活性化剤において、前記ＧＡＢＡ－ρレセプターの阻害薬は、（１，２，５，６－テトラヒドロピリジン－４－イル）メチル・ホスフィン酸（ＴＰＭＰＡ）およびその誘導体からなる群から選択される少なくとも１種類とすることもできる。 In the T cell activator of the present invention, the GABA-ρ receptor inhibitor comprises a group consisting of (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid (TPMPA) and its derivatives. It can also be at least one selected.
　本発明のＴ細胞活性化剤は、Ｂ細胞におけるＧＡＢＡ生合成の阻害薬、Ｂ細胞におけるＧＡＢＡ分解の促進薬、Ｂ細胞におけるＧＡＢＡ分泌の阻害薬、および／または、遊離ＧＡＢＡの捕捉薬を含むこともできる。 The T cell activator of the present invention comprises an inhibitor of GABA biosynthesis in B cells, a promoter of GABA degradation in B cells, an inhibitor of GABA secretion in B cells, and / or a capture agent for free GABA. You can also.
　本発明のＴ細胞活性化剤において、前記Ｂ細胞におけるＧＡＢＡ生合成の阻害薬はグルタミン酸デカルボキシラーゼおよびアルデヒドデヒドロゲナーゼ９ファミリーメンバーＡ１の発現および／または酵素活性の阻害剤とすることもでき、前記Ｂ細胞におけるＧＡＢＡ分解の促進薬は４－アミノ酪酸アミノ基転移酵素の発現および／または酵素活性の促進剤とすることもでき、前記遊離ＧＡＢＡの捕捉薬は、遊離ＧＡＢＡに特異的に結合するタンパク質、遊離ＧＡＢＡと特異的に結合する抗体、特異的結合パートナーまたはそれらの断片からなる群から選択される少なくとも１種類を含むこともできる。 In the T cell activator of the present invention, the inhibitor of GABA biosynthesis in the B cell can also be an inhibitor of the expression and / or enzyme activity of glutamate decarboxylase and aldehyde dehydrogenase 9 family member A1, and the B cell. The GABA degradation promoter in GABA can also be a promoter of expression and / or enzyme activity of 4-aminobutyric acid aminotransferase, and the free GABA capture agent is a protein that specifically binds to free GABA, free. It can also include at least one selected from the group consisting of antibodies that specifically bind GABA, specific binding partners or fragments thereof.
　本発明のＴ細胞活性化剤は、ＧＡＢＡと特異的に結合する抗体、特異的結合パートナーおよび／またはそれらの断片を含むこともできる。 The T cell activator of the present invention can also contain an antibody that specifically binds to GABA, a specific binding partner and / or a fragment thereof.
　本発明のＴ細胞活性化剤は、Ｂ細胞に対する細胞傷害性を有する、抗体、特異的結合パートナーまたはそれらの断片、および／または、Ｂ細胞の除去剤を含むこともできる。 The T cell activator of the present invention can also contain antibodies, specific binding partners or fragments thereof, and / or B cell scavengers, which are cytotoxic to B cells.
　本発明は、治療を必要とする患者におけるＢ細胞による細胞傷害性Ｔ細胞抑制作用を阻害または低減することを含む、がんの治療方法を提供する。また、本発明は、治療を必要とする患者におけるＢ細胞による細胞傷害性Ｔ細胞抑制作用を阻害または低減することを含む、感染症の予防及び/又は治療方法を提供する。 The present invention provides a method for treating cancer, which comprises inhibiting or reducing the cytotoxic T cell inhibitory effect of B cells in a patient in need of treatment. The present invention also provides a method for preventing and / or treating an infectious disease, which comprises inhibiting or reducing the cytotoxic T cell inhibitory effect of B cells in a patient in need of treatment.
　本発明のがんの治療方法並びに感染症の予防及び/又は治療方法において、前記Ｂ細胞は抗原刺激により活性化されたＢ細胞とすることもできる。 In the method for treating cancer and the method for preventing and / or treating infectious diseases of the present invention, the B cells can also be B cells activated by antigen stimulation.
　本発明のがんの治療方法並びに感染症の予防及び/又は治療方法は、治療を必要とする患者にＴ細胞におけるＧＡＢＡを介するシグナル伝達の阻害薬を投与することを含むこともできる。 The method for treating cancer and the method for preventing and / or treating an infectious disease according to the present invention can also include administering an inhibitor of GABA-mediated signal transduction in T cells to a patient in need of treatment.
　本発明のがんの治療方法並びに感染症の予防及び/又は治療方法において、前記Ｔ細胞におけるＧＡＢＡを介するシグナル伝達の阻害薬は、ヒトＴ細胞で発現するＧＡＢＡレセプターの発現および／または機能を抑制または低減する阻害薬とすることもできる。 In the method for treating cancer and the method for preventing and / or treating infectious diseases of the present invention, the GABA-mediated signal transduction inhibitor in T cells suppresses the expression and / or function of GABA receptors expressed in human T cells. Alternatively, it can be an inhibitor that reduces.
　本発明のがんの治療方法並びに感染症の予防及び/又は治療方法において、前記ＧＡＢＡレセプターは、ＧＡＢＡＡレセプター、および、ＧＡＢＡ－ρレセプターのうちの少なくとも１つとすることができる。 In the method for treating cancer and the method for preventing and / or treating infectious diseases of the present invention, the GABA receptor can be at least one of GABA A receptor and GABA-ρ receptor.
　本発明のがんの治療方法並びに感染症の予防及び/又は治療方法において、前記Ｔ細胞で発現するＧＡＢＡレセプターは、ヒトＴ細胞で発現するサブユニットポリペプチドからなることもできる。 In the method for treating cancer and the method for preventing and / or treating infectious diseases of the present invention, the GABA receptor expressed in T cells can also be composed of a subunit polypeptide expressed in human T cells.
　本発明のがんの治療方法並びに感染症の予防及び/又は治療方法において、前記ヒトＴ細胞で発現するサブユニットポリペプチドは、ヒトα１、α５、β１、πおよびρ２からなる群から選択される少なくとも１種類であってもよい。 In the method for treating cancer and the method for preventing and / or treating infectious diseases of the present invention, the subunit polypeptide expressed in the human T cells is selected from the group consisting of human α1, α5, β1, π and ρ2. It may be at least one type.
　本発明のがんの治療方法並びに感染症の予防及び/又は治療方法において、前記Ｔ細胞におけるＧＡＢＡを介するシグナル伝達の阻害薬は、前記ヒトＴ細胞で発現するヒトα１、α５、β１、πおよびρ２からなる群から選択される少なくとも１種類のサブユニットポリペプチドの発現を抑制または低減する、アンチセンス核酸、ＲＮＡｉ誘導性核酸もしくはリボザイムまたはそれらの発現ベクターを含むことができる。 In the method for treating cancer and the method for preventing and / or treating infectious diseases of the present invention, the GABA-mediated signal transduction inhibitor in the T cells is expressed in the human T cells as human α1, α5, β1, π and It can include antisense nucleic acids, RNAi-inducible nucleic acids or ribozymes or expression vectors thereof that suppress or reduce the expression of at least one subunit polypeptide selected from the group consisting of ρ2.
　本発明のがんの治療方法並びに感染症の予防及び/又は治療方法において、前記Ｔ細胞におけるＧＡＢＡを介するシグナル伝達の阻害薬は、前記ヒトＴ細胞で発現するヒトα１、α５、β１、πおよびρ２からなる群から選択される少なくとも１種類のＧＡＢＡレセプターのサブユニットポリペプチドと特異的に結合して、該ＧＡＢＡレセプターの機能を抑制または低減する、抗体、特異的結合パートナーまたはそれらの断片からなる群から選択される少なくとも１種類とすることもできる。 In the method for treating cancer and the method for preventing and / or treating infectious diseases of the present invention, the GABA-mediated signal transduction inhibitor in the T cells is expressed in the human T cells as human α1, α5, β1, π and It consists of an antibody, a specific binding partner or a fragment thereof that specifically binds to a subunit polypeptide of at least one GABA receptor selected from the group consisting of ρ2 and suppresses or reduces the function of the GABA receptor. It can also be at least one selected from the group.
　本発明のがんの治療方法並びに感染症の予防及び/又は治療方法において、前記ＧＡＢＡＡレセプターの阻害薬は、フルマゼニル、Ｒｏ１５－４５１３、サルマゼニル、シクトキシン、エナントトキシン、ペンチレンテトラゾール、ピクロトキシン、ツジョン、リンデン、ビククリン、ガバジンおよびこれらの誘導体からなる群から選択される、少なくとも１種類とすることもできる。 In the method for treating cancer and the method for preventing and / or treating infectious diseases of the present invention, the GABA A receptor inhibitor is flumazenil, Ro15-4513, salmazenil, cicutoxin, enanthotoxin, pentylenetetrazol, picrotoxin, tujon, and the like. It can also be at least one selected from the group consisting of linden, bicuculline, gabazine and derivatives thereof.
　本発明のがんの治療方法並びに感染症の予防及び/又は治療方法において、前記ＧＡＢＡ－ρレセプターの阻害薬は、（１，２，５，６－テトラヒドロピリジン－４－イル）メチル・ホスフィン酸（ＴＰＭＰＡ）およびその誘導体からなる群から選択される少なくとも１種類とすることもできる。 In the method for treating cancer and the method for preventing and / or treating infectious diseases of the present invention, the inhibitor of the GABA-ρ receptor is (1,2,5,6-tetrahydropyridine-4-yl) methyl phosphinic acid. It can also be at least one selected from the group consisting of (TPMPA) and its derivatives.
　本発明のがんの治療方法並びに感染症の予防及び/又は治療方法は、Ｂ細胞におけるＧＡＢＡ生合成の阻害薬、Ｂ細胞におけるＧＡＢＡ分解の促進薬、Ｂ細胞におけるＧＡＢＡ分泌の阻害薬、および／または、遊離ＧＡＢＡの捕捉薬を、治療を必要とする患者に投与することを含むこともできる。 The methods for treating cancer and preventing and / or treating infectious diseases of the present invention include an inhibitor of GABA biosynthesis in B cells, a promoter of GABA degradation in B cells, an inhibitor of GABA secretion in B cells, and /. Alternatively, a capture agent for free GABA may be administered to a patient in need of treatment.
　本発明のがんの治療方法並びに感染症の予防及び/又は治療方法において、前記Ｂ細胞におけるＧＡＢＡ生合成の阻害薬はグルタミン酸デカルボキシラーゼおよびアルデヒドデヒドロゲナーゼ９ファミリーメンバーＡ１の発現または酵素活性の阻害剤とすることもでき、前記Ｂ細胞におけるＧＡＢＡ分解の促進薬は４－アミノ酪酸アミノ基転移酵素の発現または酵素活性の促進剤とすることもでき、前記遊離ＧＡＢＡの捕捉薬は、遊離ＧＡＢＡに特異的に結合するタンパク質、遊離ＧＡＢＡと特異的に結合する抗体、特異的結合パートナーまたはそれらの断片からなる群から選択される少なくとも１種類を含むこともできる。 In the method for treating cancer and the method for preventing and / or treating infectious diseases of the present invention, the inhibitor of GABA biosynthesis in B cells is an inhibitor of the expression or enzyme activity of glutamate decarboxylase and aldehyde dehydrogenase 9 family member A1. The GABA degradation promoter in B cells can also be a promoter of expression or enzyme activity of 4-aminobutyric acid aminotransferase, and the free GABA capture agent is specific for free GABA. It can also include at least one selected from the group consisting of proteins that bind to free GABA, antibodies that specifically bind to free GABA, specific binding partners or fragments thereof.
　本発明のがんの治療方法並びに感染症の予防及び/又は治療方法は、ＧＡＢＡと特異的に結合する抗体、特異的結合パートナーおよび／またはそれらの断片を、治療を必要とする患者に投与することを含むこともできる。 The methods of treating cancer and preventing and / or treating infectious diseases of the present invention administer an antibody that specifically binds to GABA, a specific binding partner and / or a fragment thereof to a patient in need of treatment. Can also be included.
　本発明のがんの治療方法並びに感染症の予防及び/又は治療方法は、Ｂ細胞に対する細胞傷害性を有する、抗体、特異的結合パートナーまたはそれらの断片、および／または、Ｂ細胞の除去剤を、治療を必要とする患者に投与することを含むこともできる。 The methods of treating cancer and preventing and / or treating infectious diseases of the present invention use antibodies, specific binding partners or fragments thereof, and / or B cell scavengers having cytotoxicity against B cells. It can also include administration to patients in need of treatment.
　本発明のＴ細胞活性化剤は、がん細胞又は感染細胞を殺傷するか、がん細胞又は感染細胞の増殖を抑制するか、あるいは、がん細胞又は感染細胞のアポトーシスを誘導する、他の医薬を併用することもできる。本発明のがんの治療方法並びに感染症の予防及び/又は治療方法は、がん細胞又は感染細胞を殺傷するか、がん細胞又は感染細胞の増殖を抑制するか、あるいは、がん細胞又は感染細胞のアポトーシスを誘導する、他の治療方法と併用することもできる。 The T cell activator of the present invention kills cancer cells or infected cells, suppresses the growth of cancer cells or infected cells, or induces apoptosis of cancer cells or infected cells. It can also be used in combination with drugs. The method for treating cancer and the method for preventing and / or treating an infectious disease according to the present invention kills cancer cells or infected cells, suppresses the growth of cancer cells or infected cells, or causes cancer cells or It can also be used in combination with other therapeutic methods that induce apoptosis of infected cells.
　本明細書は、本願の優先権の基礎である日本国特許出願、特願2020‐88948の明細書および／または図面に記載される内容を包含する。 This specification includes the contents described in the Japanese patent application, Japanese Patent Application No. 2020-88948, and / or the drawings, which are the basis of the priority of the present application.
ＧＡＢＡおよび関連代謝物の合成および分解経路とこれらに関与する酵素を示す代謝マップ。ＧＡＢＡは、ＴＣＡ回路で生じるアルファケトグルタル酸（ａ－ＫＧ）由来のグルタミン酸からグルタミン酸デカルボキシラーゼ（ＧＡＤ１）により合成されるか、尿素回路またはスペルミジン／スペルミングルタミン由来の４－アミノブタナールからアルデヒドデヒドロゲナーゼ９ファミリーメンバーＡ１（ＡＬＤＨ９Ａ１）により合成される。ＧＡＢＡは４－アミノ酪酸アミノ基転移酵素によりコハク酸セミアルデヒドに分解され、さらに酸化されてコハク酸としてＴＣＡ回路に入る。A metabolic map showing the synthetic and degradative pathways of GABA and related metabolites and the enzymes involved in them. GABA is synthesized by glutamic acid decarboxylase (GAD1) from glutamic acid derived from alpha-ketoglutaric acid (a-KG) generated in the TCA cycle, or from the urea cycle or 4-aminobutanal derived from spermidine / spermine tamine to the aldehyde dehydrogenase 9 family. Synthesized by member A1 (ALDH9A1). GABA is decomposed into succinic semialdehyde by 4-aminobutyric acid transaminase, and further oxidized to enter the TCA cycle as succinic acid. 野生型マウスから摘出され、フロー・サイトメトリー法で単離された各種リンパ球のＧＡＢＡ含量の棒グラフ。誤差棒は各リンパ球タイプの試料間の標本平均の標準誤差を表す。標本平均の標準誤差とは、母集団からある数の標本を選ぶとき、選ぶ組み合わせによって標本平均がどの程度ばらつくかを、全ての組み合わせについての標準偏差で表したものをいう。グラフ縦軸は、小腸ＩｇＡ形質細胞（ＳＩ　ＩｇＡ　ＰＣ）、パイエル板（ＰＰ）の胚中心（ＧＣ）または非ＧＣ（ｎｏｎＧＣ）Ｔ細胞およびＢ細胞、リンパ節（ＬＮ）のＢ細胞（Ｂ２２０）、樹状細胞（ＤＣ）、ＣＤ４またはＣＤ８陽性エフェクターメモリー（ＥＭ）またはセントラルメモリー（ＣＭ）Ｔ細胞、ＣＤ４またはＣＤ８が陽性または陰性のナイーブＴ細胞（ｎａｉｖｅ）、ＣＤ４またはＣＤ８が陽性でＣＤ４４陽性の細胞（ＣＤ４４）の各細胞を表す。グラフ横軸は各細胞のＧＡＢＡ含量をナイーブなＣＤ４Ｔ細胞のＧＡＢＡ含量を１倍とする相対値を表す。Bar graph of GABA content of various lymphocytes removed from wild-type mice and isolated by flow cytometry. Error bars represent the standard error of the sample mean between samples of each lymphocyte type. The standard error of the sample mean is the standard deviation of all combinations, which indicates how much the sample mean varies depending on the combination selected when a certain number of samples are selected from the population. The vertical axis of the graph shows small intestinal IgA plasma cells (SI IgA PC), germinal centers (GC) or non-GC (nonGC) T cells and B cells of the Pier plate (PP), and B cells (B220) of lymph nodes (LN). Dendritic cells (DC), CD4 or CD8 positive effector memory (EM) or central memory (CM) T cells, CD4 or CD8 positive or negative naive T cells (naive), CD4 or CD8 positive and CD44 positive cells Represents each cell of (CD44). The horizontal axis of the graph represents a relative value in which the GABA content of each cell is multiplied by the GABA content of naive CD4T cells. 野生型マウス（ＷＴ）、ＣＤ３欠失ホモノックアウトマウス（ＣＤ３－／－）、ＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ）、ＲＡＧ－１欠失ホモノックアウトマウス（ＲＡＧ－１－／－）のそれぞれに免疫付与した後肢足蹠と同側（ｉｐｓｉ）および反対側（ｃｏｎｔｒａ）のリンパ節組織のＧＡＢＡ含量を示す棒グラフ。誤差棒は各リンパ節組織の試料間のＧＡＢＡ含量の標準偏差を表す。グラフの縦軸は、各組織のＧＡＢＡ含量を野生型マウス反対側リンパ節のＧＡＢＡ含量を１倍とする相対値を表す。横軸は、各組織が由来したリンパ節を表す。図３の「＊＊」、すなわち、免疫付与した後肢足蹠と同側（ｉｐｓｉ）および反対側（ｃｏｎｔｒａ）のＣＤ３欠失ホモノックアウトマウス（ＣＤ３－／－）のリンパ節組織のＧＡＢＡ含量の有意差のｐ値は、０．００１１であった。図３の「＊＊＊＊」、すなわち、免疫付与した後肢足蹠と同側（ｉｐｓｉ）および反対側（ｃｏｎｔｒａ）の野生型マウスのリンパ節組織のＧＡＢＡ含量の有意差のｐ値は、０．０００１未満であった。「ｎｓ」は統計的な有意差は認められなかったことを示す。Immunity to wild-type mice (WT), CD3-deficient homo-knockout mice (CD3 -/- ), IgM-deficient homo-knockout mice (mMT), and RAG-1-deficient homo-knockout mice (RAG-1 − / − ) A bar graph showing the GABA content of lymph node tissue ipsilaterally (ipsi) and contralaterally (contra) to the applied hindlimb footpad. Error bars represent the standard deviation of GABA content between samples of each lymph node tissue. The vertical axis of the graph represents a relative value in which the GABA content of each tissue is multiplied by the GABA content of the lymph node opposite to the wild-type mouse. The horizontal axis represents the lymph node from which each tissue was derived. Significant GABA content in the lymph node tissue of the "**" in FIG. 3, i.e., CD3-deficient homo-knockout mice (CD3 − / −) on the ipsilateral (ipsi) and contra (contra) immunized hindlimb footpads. The p value of the difference was 0.0011. “*****” in FIG. 3, that is, the p-value of the significant difference in the GABA content of the lymph node tissues of the ipsilateral (ipsi) and contra (contra) wild-type mice of the immunized hindlimb is 0. It was less than .0001. “Ns” indicates that no statistically significant difference was observed. 免疫付与した野生型マウスの後肢足蹠と同側（ｉｐｓｉ）および反対側（ｃｏｎｔｒａ）のリンパ節組織からフロー・サイトメトリー法で選別されたＣＤ４、ＣＤ８およびＢ２２０陽性細胞（それぞれ、ＣＤ４、ＣＤ８およびＢ２２０）と、反対側全リンパ節（ｔｏｔａｌ　ｃｏｎｔｒａ　ＬＮ）、同側全リンパ節（ｔｏｔａｌ　ｉｐｓｉ　ＬＮ）および同側マクロファージ/樹状細胞（ｉｐｓｉ　Ｍｆ／ＤＣ）とのＧＡＢＡ含量を示す棒グラフ。誤差棒は各選別細胞の試料間のＧＡＢＡ含量の標準偏差を表す。ＧＡＢＡ含量の縦軸は、各選別細胞のＧＡＢＡ含量を反対側ＣＤ４陽性細胞のＧＡＢＡ含量を１倍とする相対値を表す。横軸は、各選別細胞の種類を表す。図４の「＊」、すなわち、免疫付与した後肢足蹠と同側の全リンパ節組織から選別されたＣＤ４陽性細胞（ｉｐｓｉ　ＣＤ４）および反対側のリンパ節組織から選別されたＣＤ４陽性細胞（ｃｏｎｔｒａ　ＣＤ４）のＧＡＢＡ含量の有意差のｐ値は、０．０２３２であった。図４の「＊＊」、すなわち、同側の全リンパ節組織から選別されたＢ２２０陽性細胞（ｉｐｓｉ　Ｂ２２０）および反対側のリンパ節組織から選別されたＢ２２０陽性細胞（ｃｏｎｔｒａ　Ｂ２２０）のＧＡＢＡ含量の有意差のｐ値は、０．００１５であった。図４の「＊＊＊」、すなわち、同側の全リンパ節組織から選別されたＣＤ８陽性細胞（ｉｐｓｉ　ＣＤ８）および反対側のリンパ節組織から選別されたＣＤ８陽性細胞（ｃｏｎｔｒａ　ＣＤ８）のＧＡＢＡ含量の有意差のｐ値は、０．００１０であった。図４の「＊＊＊＊」、すなわち、反対側全リンパ節（ｔｏｔａｌ　ｃｏｎｔｒａ　ＬＮ）と同側全リンパ節（ｔｏｔａｌ　ｉｐｓｉ　ＬＮ）とのＧＡＢＡ含量の有意差のｐ値は、０．００６２であった。CD4, CD8 and B220-positive cells (CD4, CD8 and B220, respectively) sorted by flow cytometry from lymph node tissue ipsilaterally (ipsi) and contralateral (contra) to the hindlimb footpad of immunized wild mice. B220), a bar graph showing the GABA content of contralateral total lymph nodes (total control LN), ipsilateral total lymph nodes (total ipsi LN) and ipsilateral macrophages / dendritic cells (ipsi Mf / DC). Error bars represent the standard deviation of GABA content between samples of each sorted cell. The vertical axis of the GABA content represents a relative value in which the GABA content of each selected cell is multiplied by the GABA content of the contralateral CD4 positive cell. The horizontal axis represents the type of each selected cell. "*" In FIG. 4, that is, CD4 positive cells (ipsi CD4) selected from all lymph node tissues on the same side as the immunized hindlimb footpad and CD4 positive cells (contra) selected from the lymph node tissue on the opposite side. The p value of the significant difference in GABA content of CD4) was 0.0232. “**” in FIG. 4, that is, the GABA content of B220-positive cells (ipsi B220) selected from all lymph node tissues on the same side and B220-positive cells (contra B220) selected from lymph node tissues on the opposite side. The p value of the significant difference was 0.0015. “***” in FIG. 4, that is, the GABA content of CD8-positive cells (ipsi CD8) selected from all lymph node tissues on the same side and CD8-positive cells (contra CD8) selected from lymph node tissues on the opposite side. The p-value of the significant difference was 0.0010. The p value of "*****" in FIG. 4, that is, the significant difference in GABA content between the ipsilateral total lymph node (total control LN) and the ipsilateral total lymph node (total ipsi LN) was 0.0062. rice field. Ｂ細胞分化不全がＳＰＦ環境下のマウスに接種されたＭＣ３８細胞由来の腫瘍の成長に及ぼす影響を示す折れ線グラフ。誤差棒は各測定日のマウス個体腫瘍体積の標準偏差を表す。縦軸は、ＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ）と、野生型マウス（ＷＴ）との腫瘍の体積（ｍｍ３）を表す。横軸は、腫瘍体積の測定日を表す。A line graph showing the effect of B cell differentiation deficiency on the growth of tumors derived from MC38 cells inoculated into mice in an SPF environment. The error bars represent the standard deviation of the individual mouse tumor volume on each measurement day. The vertical axis represents the tumor volume (mm 3 ) between the IgM-deficient homozygous knockout mouse (mMT) and the wild-type mouse (WT). The horizontal axis represents the measurement date of the tumor volume. 無菌条件下で飼育されたＢ細胞分化不全マウスに接種されたＭＣ３８細胞由来の腫瘍の成長に及ぼす影響を示す折れ線グラフ。誤差棒は各測定日の腫瘍体積の標準偏差を表す。縦軸は、ＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ）と、野生型マウス（ＷＴ）との腫瘍の体積（ｍｍ３）を表す。横軸は、腫瘍体積の測定日を表す。A line graph showing the effect on the growth of tumors derived from MC38 cells inoculated into B cell differentiation-deficient mice bred under sterile conditions. The error bars represent the standard deviation of the tumor volume on each measurement day. The vertical axis represents the tumor volume (mm 3 ) between the IgM-deficient homozygous knockout mouse (mMT) and the wild-type mouse (WT). The horizontal axis represents the measurement date of the tumor volume. 無菌条件下で飼育されたＳＰＦ環境下のＢ細胞分化不全マウスに接種されたＭＣ３８細胞由来の腫瘍の成長に及ぼすＧＡＢＡ徐放ペレット処置の影響を示す折れ線グラフ。誤差棒は各測定日の腫瘍体積の標準偏差を表す。縦軸は、プラセボペレットまたはＧＡＢＡ徐放ペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ（＋ｐｌａｃｅｂｏ）またはｍＭＴ（＋ＧＡＢＡ））と、野生型マウス（ＷＴ）との腫瘍の体積（ｍｍ３）を表す。横軸は、腫瘍体積の測定日を表す。A line graph showing the effect of GABA sustained release pellet treatment on the growth of tumors derived from MC38 cells inoculated into B cell differentiation-deficient mice in an SPF environment bred under sterile conditions. The error bars represent the standard deviation of the tumor volume on each measurement day. The vertical axis represents the tumor volume (mm 3 ) between IgM-deficient homozygous knockout mice (mMT (+ placebo) or mMT (+ GABA)) treated with placebo pellets or GABA sustained-release pellets and wild-type mice (WT). .. The horizontal axis represents the measurement date of the tumor volume. Ｂ細胞分化不全マウスへのＧＡＢＡ徐放ペレット処置が腫瘍浸潤ＣＤ８陽性細胞の活性化に及ぼす影響を示す２パラメーターヒストグラムの組み合わせ図。図８の左の上下はプラセボペレットを処置した野生型マウス（ＷＴ（＋ｐｌａｃｅｂｏ））、中央および右はプラセボペレットまたはＧＡＢＡ徐放ペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ（＋ｐｌａｃｅｂｏ）またはｍＭＴ（＋ＧＡＢＡ））のフロー・サイトメトリーでＣＤ８陽性細胞をゲーティングした２パラメーターヒストグラム。上段は、縦軸がＣＤ９８の蛍光強度、横軸がＴＣＲｂの蛍光強度。下段は、縦軸がＳｃａ－１の蛍光強度、横軸がＴＣＲｂの蛍光強度。ボックスの左の数値は、各条件のＣＤ８陽性細胞のうちボックス内の蛍光強度の細胞の百分率を表す。A combination diagram of a two-parameter histogram showing the effect of GABA sustained-release pellet treatment on B cell differentiation-deficient mice on the activation of tumor-infiltrating CD8-positive cells. The upper and lower left of FIG. 8 are wild-type mice treated with placebo pellets (WT (+ histogram)), and the center and right are IgM-deficient homoknockout mice treated with placebo pellets or GABA sustained-release pellets (mMT (+ histogram) or mMT (mMT (+ histogram)). + GABA)) 2-parameter histogram of gating CD8-positive cells by flow cytometry. In the upper row, the vertical axis is the fluorescence intensity of CD98, and the horizontal axis is the fluorescence intensity of TCRb. In the lower row, the vertical axis is the fluorescence intensity of Sca-1, and the horizontal axis is the fluorescence intensity of TCRb. The number on the left of the box represents the percentage of cells with fluorescence intensity in the box among the CD8-positive cells under each condition. Ｂ細胞分化不全マウスへのＧＡＢＡ徐放ペレット処置が腫瘍浸潤ＣＤ８陽性細胞の細胞殺傷活性に及ぼす影響を示す２パラメーターヒストグラムの組み合わせ図。図９の左の上下はプラセボペレットを処置した野生型マウス（ＷＴ（＋ｐｌａｃｅｂｏ））、中央および右はプラセボペレットまたはＧＡＢＡ徐放ペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ（＋ｐｌａｃｅｂｏ）またはｍＭＴ（＋ＧＡＢＡ））のフロー・サイトメトリーでＣＤ８陽性細胞をゲーティングした２パラメーターヒストグラム。上段は、縦軸がパーフォリンの蛍光強度、横軸がＴＣＲｂの蛍光強度。下段は、縦軸がＧｒｚＢ（グランザイムＢ）の蛍光強度、横軸がＴＣＲｂの蛍光強度。ボックスの左の数値は、各条件のＣＤ８陽性細胞のうちボックス内の蛍光強度の細胞の百分率を表す。A combination diagram of a two-parameter histogram showing the effect of GABA sustained-release pellet treatment on B cell differentiation-deficient mice on the cytokilling activity of tumor-infiltrating CD8-positive cells. The upper and lower left of FIG. 9 are wild-type mice treated with placebo pellets (WT (+ histogram)), and the center and right are IgM-deficient homoknockout mice treated with placebo pellets or GABA sustained-release pellets (mMT (+ histogram) or mMT (mMT (+ histogram)). + GABA)) 2-parameter histogram of gating CD8-positive cells by flow cytometry. In the upper row, the vertical axis is the fluorescence intensity of perforin, and the horizontal axis is the fluorescence intensity of TCRb. In the lower row, the vertical axis is the fluorescence intensity of GrzB (Granzyme B), and the horizontal axis is the fluorescence intensity of TCRb. The number on the left of the box represents the percentage of cells with fluorescence intensity in the box among the CD8-positive cells under each condition. 図９の２パラメーターヒストグラムにもとづいて、Ｂ細胞分化不全マウスへのＧＡＢＡ徐放ペレット処置が腫瘍浸潤ＣＤ８陽性細胞のパーフォリン陽性細胞の割合に及ぼす影響を示す棒グラフ。縦軸は、プラセボペレットを処置した野生型マウス（ＷＴ＋ｐｌａｃｅｂｏ）、プラセボペレットまたはＧＡＢＡ徐放ペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍｕＭＴ－／－＋ｐｌａｃｅｂｏまたはｍｕＭＴ－／－＋ＧＡＢＡｐ）のパーフォリン陽性細胞の百分率を表す。図１０Ａの「＊＊」、すなわち、プラセボペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍｕＭＴ－／－＋ｐｌａｃｅｂｏ）と、ＧＡＢＡ徐放ペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍｕＭＴ－／－＋ＧＡＢＡｐ）とのパーフォリン陽性細胞の割合の百分率の有意性のｐ値は０．００６であった。図１０Ａの「＊＊＊」、すなわち、プラセボペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍｕＭＴ－／－＋ｐｌａｃｅｂｏ）と、プラセボペレットを処置した野生型マウス（ＷＴ＋ｐｌａｃｅｂｏ）とのパーフォリン陽性細胞の割合の百分率の有意性のｐ値は０．００１２であった。A bar graph showing the effect of GABA sustained release pellet treatment on B cell differentiation-deficient mice on the proportion of tumor-infiltrating CD8-positive cells to perforin-positive cells based on the two-parameter histogram of FIG. The vertical axis is the perforin-positive cells of wild-type mice treated with placebo pellets (WT + placebo), IgM-deficient homozygous knockout mice treated with placebo pellets or GABA sustained-release pellets (muMT − / − + placebo or muMT − / − + GABAp). Represents a percentage. “**” in FIG. 10A, ie, IgM-deficient homozygous knockout mice treated with placebo pellets (muMT − / − + placebo) and IgM-deficient homozygous knockout mice treated with GABA sustained-release pellets (muMT − / − + GABAp). The significance p value of the percentage of perforin-positive cells with was 0.006. “***” in FIG. 10A, that is, the proportion of perforin-positive cells between IgM-deficient homo-knockout mice treated with placebo pellets (muMT − / − + placebo) and wild-type mice treated with placebo pellets (WT + placebo). The p-value for the significance of the percentage was 0.0012. 図９の２パラメーターヒストグラムにもとづいて、Ｂ細胞分化不全マウスへのＧＡＢＡ徐放ペレット処置が腫瘍浸潤ＣＤ８陽性細胞のグランザイム陽性細胞の割合に及ぼす影響を示す棒グラフ。プラセボペレットを処置した野生型マウス（ＷＴ＋ｐｌａｃｅｂｏ）、プラセボペレットまたはＧＡＢＡ徐放ペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍｕＭＴ－／－＋ｐｌａｃｅｂｏまたはｍｕＭＴ－／－＋ＧＡＢＡｐ）のグランザイム陽性細胞の百分率を表す。図１０Ｂの「＊」、すなわち、プラセボペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍｕＭＴ－／－＋ｐｌａｃｅｂｏ）と、ＧＡＢＡ徐放ペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍｕＭＴ－／－＋ＧＡＢＡｐ）とのグランザイム陽性細胞の割合の百分率の有意性のｐ値は０．０２２であった。図１０Ｂの「＊＊」、すなわち、プラセボペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍｕＭＴ－／－＋ｐｌａｃｅｂｏ）と、プラセボペレットを処置した野生型マウス（ＷＴ＋ｐｌａｃｅｂｏ）とのグランザイム陽性細胞の割合の百分率の有意性のｐ値は０．００１２であった。A bar graph showing the effect of GABA sustained release pellet treatment on B cell differentiation-deficient mice on the proportion of granzyme-positive cells of tumor-infiltrating CD8-positive cells based on the two-parameter histogram of FIG. Represents the percentage of granzyme-positive cells in wild-type mice treated with placebo pellets (WT + placebo), IgM-deficient homozygous knockout mice treated with placebo pellets or sustained-release GABA pellets (muMT − / − + placebo or muMT − / − + GABAp). “*” In FIG. 10B, that is, IgM-deficient homozygous knockout mice treated with placebo pellets (muMT − / − + placebo) and IgM-deficient homozygous knockout mice treated with GABA sustained-release pellets (muMT − / − + GABAp). The significance p value of the percentage of granzyme-positive cells was 0.022. “**” in FIG. 10B, ie, the percentage of granzyme-positive cells between IgM-deficient homo-knockout mice treated with placebo pellets (muMT − / − + placebo) and wild-type mice treated with placebo pellets (WT + placebo). The significance p value of was 0.0012. 野生型マウスに接種されたＭＣ３８細胞由来の腫瘍の成長に及ぼすＧＡＢＡＡレセプターの阻害薬の影響を示す折れ線グラフ。縦軸はチアガビン（ＧＡＴ（トランスポーター）阻害剤）投与動物（Ｔｉａｇａｂｉｎ）、ＰＢＳ投与動物（ＰＢＳ）またはピクロトキシン投与動物（Ｐｉｃｒｏｔｏｘｉｎ）の腫瘍の体積（ｍｍ３）を表す。横軸は、腫瘍体積の測定日を表す。最終測定日に最も腫瘍体積の大きい曲線はチアガビン（ＧＡＴ（トランスポーター）阻害剤）投与動物、２番目に腫瘍体積の大きい曲線はＰＢＳ投与動物、最も腫瘍体積の小さい曲線はピクロトキシン投与動物の腫瘍体積の経時的変化を示す。誤差棒は各測定日の腫瘍体積の標準偏差を表す。図１１の「＊＊＊」、すなわち、ＭＣ３８細胞接種後２３日に測定したＰＢＳ投与動物（ＰＢＳ）とピクロトキシン投与動物（Ｐｉｃｒｏｔｏｘｉｎ）との腫瘍の体積の有意差のｐ値は０．０００３であった。図１１の「＊＊＊＊」、すなわち、ＭＣ３８細胞接種後２５日および２７日に測定したＰＢＳ投与動物（ＰＢＳ）とピクロトキシン投与動物（Ｐｉｃｒｏｔｏｘｉｎ）との腫瘍の体積の有意差のｐ値は、いずれも、０．０００１未満であった。 A line graph showing the effect of GABA A receptor inhibitors on the growth of tumors derived from MC38 cells inoculated into wild-type mice. The vertical axis represents the tumor volume (mm 3 ) of a tiagabine (GAT (transporter) inhibitor) -administered animal (Tiagabine), a PBS-administered animal (PBS) or a picrotoxin-administered animal (Picrotoxin). The horizontal axis represents the measurement date of the tumor volume. On the final measurement day, the curve with the largest tumor volume is the tumor volume of thiagabin (GAT (transporter) inhibitor) -administered animals, the curve with the second largest tumor volume is the PBS-administered animal, and the curve with the smallest tumor volume is the tumor volume of picrotoxin-administered animals. Shows the change over time. The error bars represent the standard deviation of the tumor volume on each measurement day. “***” in FIG. 11, that is, the p value of the significant difference in tumor volume between the PBS-administered animal (PBS) and the picrotoxin-administered animal (Picrotoxin) measured on the 23rd day after inoculation with MC38 cells was 0.0003. rice field. “*****” in FIG. 11, that is, the p-value of the significant difference in tumor volume between the PBS-administered animal (PBS) and the picrotoxin-administered animal (Picrotoxin) measured 25 days and 27 days after MC38 cell inoculation. Both were less than 0.0001. 野生型マウスに接種されたＭＣ３８細胞由来の腫瘍の成長に及ぼすＧＡＢＡＡレセプターの阻害薬の影響を示す折れ線グラフ。縦軸はフルマゼニル（ＧＡＢＡＡレセプター阻害剤）投与動物またはＰＢＳ投与動物の腫瘍の体積（ｍｍ３）。横軸は、腫瘍体積の測定日。最終測定日に最も腫瘍体積の小さい曲線はフルマゼニル投与動物（Ｆｌｕｍａｚｅｎｉｌ）の腫瘍体積の経時的変化を示し、最も腫瘍体積の大きい曲線はＤＭＳＯ投与動物（ＤＭＳＯ）の腫瘍体積の経時的変化を示す。誤差棒は各測定日の腫瘍体積の標準偏差を表す。図１２の「＊＊」、すなわち、最終測定日に測定したＤＭＳＯ投与動物（ＤＭＳＯ）とフルマゼニル投与動物（Ｆｌｕｍａｚｅｎｉｌ）との腫瘍の体積の有意差のｐ値は、０．００９２であった。 A line graph showing the effect of GABA A receptor inhibitors on the growth of tumors derived from MC38 cells inoculated into wild-type mice. The vertical axis is the tumor volume (mm 3 ) of a flumazenil (GABA A receptor inhibitor) -administered animal or a PBS-administered animal. The horizontal axis is the date of measurement of tumor volume. The curve with the smallest tumor volume on the final measurement day shows the change over time in the tumor volume of the flumazenil-administered animal (Flumazenil), and the curve with the largest tumor volume shows the change over time in the tumor volume of the DMSO-administered animal (DMSO). The error bars represent the standard deviation of the tumor volume on each measurement day. The p-value of "**" in FIG. 12, that is, the significant difference in tumor volume between the DMSO-administered animal (DMSO) and the flumazenil-administered animal (Flumazenil) measured on the final measurement day was 0.0092. 野生型マウスに接種されたＭＣ３８細胞由来の腫瘍に浸潤するＣＤ８陽性細胞の組成に対するピクロトキシンの影響を示す２パラメーターヒストグラムの組み合わせ図。左はＣＤ８の蛍光強度を横軸に、ＣＤ４５－２の蛍光強度を縦軸に表す野生型マウスに接種したＭＣ３８細胞由来の腫瘍に浸潤した細胞の２パラメーターヒストグラム。右下は、ピクロトキシン処理した野生型マウスに接種したＭＣ３８細胞由来の腫瘍に浸潤した細胞の２パラメーターヒストグラム。右上は対照実験の野生型マウスに接種したＭＣ３８細胞由来の腫瘍に浸潤した細胞の２パラメーターヒストグラム。右上および右下は、ともに、側方散乱光の強度を縦軸に、前方散乱光の強度を横軸に表す。ゲーティング範囲の近傍の数字は測定された細胞全てのうちゲーティング範囲内の細胞の百分率を表す。A combination diagram of a two-parameter histogram showing the effect of picrotoxin on the composition of CD8-positive cells infiltrating tumors derived from MC38 cells inoculated into wild-type mice. On the left is a two-parameter histogram of cells infiltrated into a tumor derived from MC38 cells inoculated into wild-type mice, with the fluorescence intensity of CD8 on the horizontal axis and the fluorescence intensity of CD45-2 on the vertical axis. The lower right is a two-parameter histogram of cells infiltrated into a tumor derived from MC38 cells inoculated into picrotoxin-treated wild-type mice. The upper right is a two-parameter histogram of cells infiltrated into a tumor derived from MC38 cells inoculated into wild-type mice in a control experiment. In both the upper right and the lower right, the intensity of the laterally scattered light is represented by the vertical axis, and the intensity of the forward scattered light is represented by the horizontal axis. The numbers near the gating range represent the percentage of all measured cells within the gating range. 図１３の２パラメーターヒストグラムから算出した腫瘍浸潤ＣＤ８陽性で、かつ、側方散乱光および前方散乱光の強度も高いＴ細胞の百分率を示す棒グラフ。縦軸は側方散乱光および前方散乱光の強度も高いＴ細胞の百分率。左は対照実験のマウス（Ｃｔｒ）の腫瘍浸潤細胞の百分率、右はピクロトキシン処理マウス（Ｐｉｃ）の腫瘍浸潤細胞の百分率、誤差棒は各細胞集団の細胞数の標準偏差を表す。ピクロトキシン処理マウス（Ｐｉｃ）と対照実験のマウス（Ｃｔｒ）との腫瘍浸潤ＣＤ８陽性細胞の百分率のｐ値は０．０１５６であった。A bar graph showing the percentage of T cells positive for tumor infiltration CD8 calculated from the two-parameter histogram of FIG. 13 and having high intensity of laterally scattered light and forward scattered light. The vertical axis is the percentage of T cells with high intensity of laterally scattered light and forward scattered light. The left shows the percentage of tumor-infiltrated cells in the control mouse (Ctr), the right shows the percentage of tumor-infiltrated cells in the picrotoxin-treated mouse (Pic), and the error bar shows the standard deviation of the cell number in each cell population. The p value of the percentage of tumor-infiltrated CD8-positive cells between picrotoxin-treated mice (Pic) and mice in the control experiment (Ctr) was 0.0156. 培養実験において、GABAあるいはピクロトキシンはＭＣ３８細胞の増殖と生存率に影響を与えないことを示す棒グラフ。左と中間の図は、それぞれ、縦軸が１ウェルあたりの細胞の数と生存率（全細胞数における生存細胞の割合）を表す。横軸は、RMPI培養液（１０％ウシ胎児血清入り）に添加したコントロール（PBS)あるいはGABA（PBSに溶解し、最終濃度1mMまたは5mM)を表す。右の図は、縦軸が細胞の生存率を表わし、横軸がRMPI培養液（１０％ウシ胎児血清入り）に添加したコントロール（DMSO)あるいはＧＡＢＡＡレセプターの阻害薬－ピクロトキシン（DMSOに溶解し、最終濃度１００μM、 ２００μMまたは３００μM)を表す。誤差棒はウェル間の細胞の数あるいは生存率の標準偏差を表す。「ｎｓ」は統計的な有意差は認められなかったことを示す。A bar graph showing that GABA or picrotoxin does not affect the proliferation and viability of MC38 cells in culture experiments. In the figures on the left and in the middle, the vertical axis represents the number of cells per well and the viability (ratio of viable cells to the total number of cells), respectively. The horizontal axis represents control (PBS) or GABA (dissolved in PBS, final concentration 1 mM or 5 mM) added to RMPI culture medium (containing 10% fetal bovine serum). In the figure on the right, the vertical axis represents cell viability, and the horizontal axis is control (DMSO) added to RMPI culture medium (containing 10% fetal bovine serum) or GABA A receptor inhibitor-picrotoxin (dissolved in DMSO). , Final concentration 100 μM, 200 μM or 300 μM). Error bars represent the standard deviation of the number of cells or viability between wells. “Ns” indicates that no statistically significant difference was observed. rag1遺伝子欠損マウスに接種されたＭＣ３８細胞由来の腫瘍の成長に及ぼすＧＡＢＡＡレセプターの阻害薬（ピクロトキシン）の影響を示す折れ線グラフ。縦軸はDMSOあるいはピクロトキシンを投与したrag1遺伝子欠損マウスの腫瘍の体積（ｍｍ３）を表す。横軸は、細胞摂取後の腫瘍体積の測定日を表す。誤差棒は各測定日のマウス個体腫瘍体積の標準偏差を表す。「ｎｓ」は統計的な有意差は認められなかったことを示す。 A line graph showing the effect of a GABA A receptor inhibitor (picrotoxin) on the growth of tumors derived from MC38 cells inoculated into rag1 gene-deficient mice. The vertical axis represents the tumor volume (mm 3 ) of rag1 gene-deficient mice treated with DMSO or picrotoxin. The horizontal axis represents the measurement date of the tumor volume after ingestion of cells. The error bars represent the standard deviation of the individual mouse tumor volume on each measurement day. “Ns” indicates that no statistically significant difference was observed. ＧＡＢＡＡレセプターシグナルの抑制を介した免疫活性化のモデル。B細胞はグルタミン酸デカルボキシラーゼ（GAD）によるグルタミン酸の脱炭酸反応によってGABAを合成し、分泌する。B細胞由来のGABAは、ＧＡＢＡＡレセプターシグナルを介して、細胞傷害性Ｔ細胞の機能を抑制する。ＧＡＢＡＡレセプターの阻害薬（アンタゴニスト）により、GABAによるＴ細胞の細胞傷害性の抑制が解除され、その結果、癌の増殖が抑制されたり、感染細胞が排除される。Gln, グルタミン; Glu, グルタミン酸; GABA, γ-アミノ酪酸; GLS, グルタミナーゼ; GAD1, グルタミン酸デカルボキシラーゼ1。 A model of immune activation through suppression of GABA A receptor signals. B cells synthesize and secrete GABA by decarboxylation of glutamate with glutamate decarboxylase (GAD). B cell-derived GABA suppresses the function of cytotoxic T cells via GABA A receptor signals. The GABA A receptor inhibitor (antagonist) releases the suppression of T cell cytotoxicity by GABA, resulting in suppression of cancer growth and elimination of infected cells. Gln, Glutamine; Glu, Glutamic Acid; GABA, γ-Aminobutyric Acid; GLS, Glutaminase; GAD1, Glutamic Acid Decarboxylase 1.
　本明細書においてＢ細胞とは、成熟Ｂ細胞だけでなく、最終分化した形質細胞や、ｐｒｅＢ細胞およびその前駆細胞を除く任意のＢ前駆細胞をも含む意味で用いられる。細胞表面マーカーの発現としては、ＩｇＭ＋、ＩｇＤ＋、ＩｇＧ＋、ＣＤ１９＋、Ｂ２２０＋、ＣＤ２４＋、ＣＤ４３－、ＣＤ２５－、ｃ－ｋｉｔ－、ＩＬ－７Ｒ－などにより特徴付けられる。 As used herein, B cells are used to include not only mature B cells but also terminally differentiated plasma cells and any B progenitor cells other than preB cells and their progenitor cells. The expression of cell surface markers, IgM +, IgD +, IgG +, CD19 +, B220 +, CD24 +, CD43 -, CD25 -, c-kit -, IL-7R - characterized by such.
　本明細書においてＴ細胞とは、主に胸腺内で分化、成熟するリンパ球で、成熟するとＴ細胞レセプターを細胞表面に発現するリンパ球であって、成熟したＴ細胞のほか、Ｔ細胞への分化能を有する任意のＴ前駆細胞をも含む。細胞表面マーカーの発現としては、Ｔ細胞レセプター＋、ＣＤ３＋、ＣＤ４＋、ＣＤ８＋などにより特徴付けられる。 As used herein, T cells are lymphocytes that differentiate and mature mainly in the thoracic gland, and when they mature, they express T cell receptors on the cell surface. It also includes any T progenitor cells capable of differentiation. Expression of cell surface markers is characterized by T cell receptors + , CD3 + , CD4 + , CD8 + and the like.
　本明細書において細胞傷害性Ｔ細胞とは、ウイルス感染細胞やがん細胞など宿主にとって異物となる細胞を認識して破壊するＴ細胞の一種で、細胞表面マーカーＣＤ８＋で特徴づけられる。細胞傷害性Ｔ細胞の細胞傷害活性は、細胞傷害物質であるパーフォリン、グランザイム等の存在で特徴付けられる。 As used herein, cytotoxic T cells are a type of T cells that recognize and destroy cells that are foreign to the host, such as virus-infected cells and cancer cells, and are characterized by the cell surface marker CD8 +. The cytotoxic activity of cytotoxic T cells is characterized by the presence of cytotoxic substances such as perforin and granzyme.
　本明細書において抗がん剤とは、がん細胞の増殖を抑制、阻害または停止させるか、がん細胞を特異的に殺傷するか、がん細胞にアポトーシスその他自ら死滅を誘導させる任意の活性を有する医薬を指す。本発明の抗がん剤は、細胞傷害性Ｔ細胞のがん細胞殺傷活性を抑制するＢ細胞の作用を阻害または低減する、すなわち、細胞傷害性Ｔ細胞のがん細胞殺傷活性を抑制する、Ｂ細胞と細胞傷害性Ｔ細胞との相互作用を阻害または低減するために用いる医薬を指す。 As used herein, an anticancer drug is an activity that suppresses, inhibits, or stops the growth of cancer cells, specifically kills cancer cells, or induces apoptosis or other self-destruction of cancer cells. Refers to a drug having. The anticancer agent of the present invention inhibits or reduces the action of B cells that suppress the cancer cell killing activity of cytotoxic T cells, that is, suppresses the cancer cell killing activity of cytotoxic T cells. Refers to a drug used to inhibit or reduce the interaction between B cells and cytotoxic T cells.
　本明細書においてがんの治療方法とは、がん細胞の増殖を抑制、阻害または停止させるか、がん細胞を特異的に殺傷するか、がん細胞にアポトーシスその他自ら死滅を誘導させる任意の処置を行う方法をいう。本発明のがんの治療方法は、細胞傷害性Ｔ細胞のがん細胞殺傷活性を抑制するＢ細胞の作用を阻害または低減する、すなわち、細胞傷害性Ｔ細胞のがん細胞殺傷活性を抑制する、Ｂ細胞と細胞傷害性Ｔ細胞との相互作用を阻害または低減する医薬を投与することを含む。 As used herein, the method of treating cancer is any method that suppresses, inhibits, or stops the growth of cancer cells, specifically kills cancer cells, or induces cancer cells to undergo apoptosis or other self-destruction. A method of performing treatment. The method for treating cancer of the present invention inhibits or reduces the action of B cells that suppress the cancer cell killing activity of cytotoxic T cells, that is, suppresses the cancer cell killing activity of cytotoxic T cells. , Includes administration of a drug that inhibits or reduces the interaction between B cells and cytotoxic T cells.
　本発明の抗がん剤またはがんの治療方法の対象となるがんの種類は、癌腫、扁平上皮癌（例えば、子宮頚管、瞼、結膜、膣肺、口腔、皮膚、膀胱、舌、喉頭および食道の扁平上皮癌）、腺癌（例えば、前立腺、小腸、子宮内膜、子宮頚管、大腸、肺、膵、食道、直腸、子宮、胃、乳房および卵巣の腺癌）を含む。さらに、肉腫（例えば、筋原性肉腫、骨肉腫、子宮筋腫）、白血病、神経腫、メラノーマおよびリンパ腫も含む。 The types of cancer targeted by the anticancer agent or the method for treating cancer of the present invention include carcinoma, squamous cell carcinoma (eg, cervical canal, eyelid, conjunctival, vaginal lung, oral cavity, skin, bladder, tongue, etc.). Squamous cell carcinoma of the throat and esophagus), adenocarcinoma (eg, adenocarcinoma of the prostate, small intestine, endometrial, cervical canal, colon, lung, pancreas, esophagus, rectal, uterus, stomach, breast and ovary). It also includes sarcomas (eg, myogenic sarcomas, osteosarcomas, uterine fibroids), leukemias, neuromas, melanomas and lymphomas.
　本発明の抗がん剤またはがんの治療方法の対象となるがんは、例えば、肺がん（例えば、非小細胞肺がん、小細胞肺がん、悪性中皮腫等）、乳がん（例えば、浸潤性乳管がん、非浸潤性乳管がん、炎症性乳がん等）、前立腺がん（例えば、ホルモン依存性前立腺がん、ホルモン非依存性前立腺がん等）、膵がん（例えば、膵管がん等）、胃がん（例えば、乳頭腺がん、粘液性腺がん、腺扁平上皮がん等）、結腸がん（例えば、消化管間質腫瘍等）、直腸がん（例えば、消化管間質腫瘍等）、大腸がん（例えば、家族性大腸がん、遺伝性非ポリポーシス大腸がん、消化管間質腫瘍等）、食道がん、十二指腸がん、舌がん、咽頭がん（例えば、上咽頭がん、中咽頭がん、下咽頭がん等）、頭頚部がん、唾液腺がん、脳腫瘍（例えば、松果体星細胞腫瘍、毛様細胞性星細胞腫、びまん性星細胞腫、退形成性星細胞腫等）、肝臓がん（例えば、原発性肝がん、肝外胆管がん等）、腎臓がん（例えば、腎細胞がん、腎盂と尿管の移行上皮がん等）、胆嚢がん、胆管がん、膵臓がん、子宮内膜がん、子宮頸がん、卵巣がん（例、上皮性卵巣がん、性腺外胚細胞腫瘍、卵巣性胚細胞腫瘍、卵巣低悪性度腫瘍等）、膀胱がん、尿道がん、皮膚がん（例えば、眼内（眼）黒色腫、メルケル細胞がん等）、血管腫、悪性リンパ腫（例えば、非ホジキンリンパ腫、ホジキン病等）、メラノーマ（悪性黒色腫）、甲状腺がん（例えば、甲状腺髄様がん等）、副甲状腺がん、鼻腔がん、副鼻腔がん、陰茎がん、精巣腫瘍、小児固形がん（例えば、ウィルムス腫瘍、小児腎腫瘍等）、上顎洞腫瘍、骨腫瘍等が挙げられ、これらに限定されない。 The cancers targeted by the anticancer agent or the method for treating cancer of the present invention include, for example, lung cancer (for example, non-small cell lung cancer, small cell lung cancer, malignant mesoderma, etc.), breast cancer (for example, invasive milk). Tube cancer, non-invasive breast cancer, inflammatory breast cancer, etc.), prostate cancer (eg, hormone-dependent prostate cancer, hormone-independent prostate cancer, etc.), pancreatic cancer (eg, pancreatic duct cancer) Etc.), gastric cancer (eg papillary adenocarcinoma, mucinous adenocarcinoma, glandular squamous epithelial cancer, etc.), colon cancer (eg, gastrointestinal stromal tumor, etc.), rectal cancer (eg, gastrointestinal stromal tumor, etc.) Etc.), colon cancer (eg, familial colon cancer, hereditary non-polyposis colon cancer, gastrointestinal stromal tumor, etc.), esophageal cancer, duodenal cancer, tongue cancer, pharyngeal cancer (eg, top) Pharyngeal cancer, mesopharyngeal cancer, hypopharyngeal cancer, etc.), head and neck cancer, salivary adenocarcinoma, brain tumor (eg, pineapple stellate cell tumor, hairy cell stellate cell tumor, diffuse stellate cell tumor, etc.) Degenerative stellate cell tumor, etc.), Liver cancer (eg, primary liver cancer, extrahepatic bile duct cancer, etc.), Kidney cancer (eg, renal cell cancer, transitional epithelial cancer of renal pelvis and urinary tract, etc.) ), Biliary sac cancer, bile duct cancer, pancreatic cancer, endometrial cancer, cervical cancer, ovarian cancer (eg, epithelial ovarian cancer, extragonal embryonic cell tumor, ovarian embryonic cell tumor, ovary) Low-grade tumors, etc.), bladder cancer, urinary tract cancer, skin cancer (eg, intraocular (eye) melanoma, Mercel cell cancer, etc.), hemangiomas, malignant lymphomas (eg, non-Hodgkin lymphoma, Hodgkin's disease, etc.) Etc.), melanoma (malignant melanoma), thyroid cancer (eg, thyroid medullary cancer, etc.), parathyroid cancer, nasal cavity cancer, sinus cavity cancer, penis cancer, testis tumor, pediatric solid cancer (eg, pediatric solid cancer) For example, Wilms tumor, pediatric kidney tumor, etc.), maxillary sinus tumor, bone tumor, etc., but are not limited thereto.
　本明細書において感染症の予防及び/又は治療剤とは、感染細胞の増殖を抑制、阻害または停止させるか、感染細胞を特異的に殺傷するか、感染細胞にアポトーシスその他自ら死滅を誘導させる任意の活性を有する医薬を指す。本発明の感染症の予防及び/又は治療剤は、細胞傷害性Ｔ細胞の感染細胞に対する殺傷活性を抑制するＢ細胞の作用を阻害または低減する、すなわち、細胞傷害性Ｔ細胞の感染細胞に対する殺傷活性を抑制する、Ｂ細胞と細胞傷害性Ｔ細胞との相互作用を阻害または低減するために用いる医薬を指す。 As used herein, the term "preventive and / or therapeutic agent for infectious disease" refers to any method that suppresses, inhibits, or stops the growth of infected cells, specifically kills infected cells, or induces infected cells to undergo apoptosis or other self-destruction. Refers to a drug having the activity of. The prophylactic and / or therapeutic agent for infectious diseases of the present invention inhibits or reduces the action of B cells that suppress the killing activity of cytotoxic T cells on infected cells, that is, kills cytotoxic T cells against infected cells. Refers to a drug used to inhibit or reduce the interaction between B cells and cytotoxic T cells, which suppresses activity.
　本明細書において感染症の予防及び/又は治療方法とは、感染細胞の増殖を抑制、阻害または停止させるか、感染細胞を特異的に殺傷するか、感染細胞にアポトーシスその他自ら死滅を誘導させる任意の処置を行う方法をいう。本発明の感染症の予防及び/又は治療方法は、細胞傷害性Ｔ細胞の感染細胞に対する殺傷活性を抑制するＢ細胞の作用を阻害または低減する、すなわち、細胞傷害性Ｔ細胞の感染細胞に対する殺傷活性を抑制する、Ｂ細胞と細胞傷害性Ｔ細胞との相互作用を阻害または低減する医薬を投与することを含む。 As used herein, the method for preventing and / or treating an infectious disease is any method that suppresses, inhibits, or stops the growth of infected cells, specifically kills infected cells, or induces infected cells to undergo apoptosis or other self-destruction. Refers to the method of performing the treatment of. The method for preventing and / or treating an infectious disease of the present invention inhibits or reduces the action of B cells that suppress the killing activity of cytotoxic T cells on infected cells, that is, kills cytotoxic T cells against infected cells. It involves administering a drug that suppresses activity and inhibits or reduces the interaction of B cells with cytotoxic T cells.
　本発明の感染症の予防及び/又は治療剤または感染症の予防及び/又は治療方法の対象となる感染症は、例えば、ヒト肝炎ウイルス（Ｂ型肝炎、Ｃ型肝炎、Ａ型肝炎またはＥ型肝炎）、ヒトレトロウイルス、ヒト免疫不全ウイルス（ＨＩＶ１、ＨＩＶ２）、ヒトＴ細胞白血病ウイルス、ヒトＴリンパ向性ウイルス（ＨＴＬＶ１、ＨＴＬＶ２）、単純ヘルペスウイルス１型および２型、エプスタイン・バーウイルス、サイトメガロウイルス、水痘－帯状疱疹ウイルス、ヒトヘルペスウイルス、ポリオウイルス、麻疹ウイルス、風疹ウイルス、日本脳炎ウイルス、おたふくウイルス、インフルエンザウイルス、アデノウイルス、エンテロウイルス、ライノウイルス、重症急性呼吸器症候群（ＳＡＲＳ）を発症するウイルス、エボラウイルス、西ナイルウイルスなどのウイルスの感染症、その他の病原体として、例えば、病原性原生動物（例えば、マラリア原虫、トリパノソーマ、トキソプラズマ）、細菌（例えば、大腸菌、黄色ブドウ球菌、結核菌）、真菌（例えば、アスペルギルス、ブラストミセス、カンジダ）による感染症等が挙げられ、これらに限定されない。 The infectious disease subject to the preventive and / or therapeutic agent for the infectious disease of the present invention or the preventive and / or therapeutic method for the infectious disease is, for example, a human hepatitis virus (hepatitis B, hepatitis C, hepatitis A or type E). Hepatitis), human retrovirus, human immunodeficiency virus (HIV1, HIV2), human T cell leukemia virus, human T lymphotrophic virus (HTLV1, HTLV2), simple herpesvirus types 1 and 2, Epstein bar virus, site Megalovirus, varicella-belt virus, human herpesvirus, poliovirus, measles virus, ruin virus, Japanese encephalitis virus, mumps virus, influenza virus, adenovirus, enterovirus, rhinovirus, severe acute respiratory syndrome (SARS) Virus infections such as Ebola virus, Western Nile virus, and other pathogens include, for example, pathogenic protozoa (eg, malaria protozoa, tripanosoma, toxoplasma), bacteria (eg, Escherichia coli, staphylococcus, tuberculosis). ), Infections caused by fungi (eg, Aspergillus, Blast Mrs., Candida), etc., and are not limited thereto.
　本明細書において、ＧＡＢＡレセプターとは、ガンマアミノ酪酸（ＧＡＢＡ）と特異的に結合するレセプターを指す。哺乳類および霊長類では、ＧＡＢＡレセプターは、ＧＡＢＡＡレセプターとＧＡＢＡＢレセプターとに分類される。ＧＡＢＡＡレセプターは、ＧＡＢＡと結合すると塩素イオンを細胞内に透過させるイオンチャンネル型受容体で、ＧＡＢＡＢレセプターはＧタンパク質結合型受容体である。ＧＡＢＡ－ρレセプターは、かつてはＧＡＢＡＣレセプターとして独立のクラスに分類されていたこともあった。しかし、サブユニットポリペプチド５本からなる５量体イオンチャンネル型受容体である点でＧＡＢＡＡレセプターと共通で、遺伝子配列や構造、機能についてもρサブユニットは他のＧＡＢＡＡレセプターサブユニットと類似の性質を持ち、ρサブユニットのみからなる点のみが異なることから、今ではＧＡＢＡ－ρレセプターはＧＡＢＡＡレセプターのサブクラスに分類される。 As used herein, the GABA receptor refers to a receptor that specifically binds to gamma-aminobutyric acid (GABA). In mammals and primates, GABA receptors are classified into GABA A receptors and GABA B receptors. The GABA A receptor is an ion channel type receptor that allows chlorine ions to permeate into cells when bound to GABA, and the GABA B receptor is a G protein-bound type receptor. The GABA-ρ receptor was once classified as an independent class as a GABA C receptor. However, it is common with the GABA A receptor in that it is a pentamer ion channel type receptor consisting of five subunit polypeptides , and the ρ subunit is similar to other GABA A receptor subunits in terms of gene sequence, structure, and function. GABA-ρ receptors are now classified as a subclass of GABA A receptors because they have the above-mentioned properties and differ only in that they consist only of ρ subunits.
　本発明のＴ細胞活性化剤がＧＡＢＡＡレセプターの阻害薬の場合には、その有効成分は、ビククリン、ガバジンなどのアンタゴニストと、フルマゼニル、Ｒｏ１５－４５１３、サルマゼニル、亜鉛などのネガティブアロステリック調節因子と、シクトキシン、エナントトキシン、ペンチレンテトラゾール、ピクロトキシン、ツジョン、リンデンなどの非競合的チャネルブロッカーと、これらの誘導体と、前記レセプターのサブユニットのうちヒトＴ細胞で発現するヒトα１、α５、β１、πおよびρ２からなる群から選択される少なくとも１種類のサブユニットの発現を抑制または低減する、アンチセンス核酸、ＲＮＡｉ誘導性核酸もしくはリボザイムまたはそれらの発現ベクターとを含むが、これらに限定されない。 When the T cell activator of the present invention is an inhibitor of GABA A receptor, its active ingredients include antagonists such as bicuculline and gabazine, and negative allosteric regulators such as flumazenil, Ro15-4513, salmazenil and zinc. Non-competitive channel blockers such as sictoxin, enanthotoxin, pentylenetetrazole, picrotoxin, tujon, linden, their derivatives, and human α1, α5, β1, π and among the subunits of the receptor expressed in human T cells. It includes, but is not limited to, antisense nucleic acids, RNAi-inducible nucleic acids or ribozymes or expression vectors thereof that suppress or reduce the expression of at least one subunit selected from the group consisting of ρ2.
　本発明のＴ細胞活性化剤がＧＡＢＡ－ρレセプターの阻害薬の場合には、その有効成分は、（１，２，５，６－テトラヒドロピリジン－４－イル）メチル・ホスフィン酸（ＴＰＭＰＡ）およびその誘導体を含むが、これらに限定されない。 When the T cell activator of the present invention is an inhibitor of GABA-ρ receptor, the active ingredients thereof are (1,2,5,6-tetrahydropyridine-4-yl) methyl phosphinic acid (TPMPA) and Including, but not limited to, derivatives thereof.
　Ｂ細胞におけるＧＡＢＡ生合成経路を、図１のＧＡＢＡおよび関連代謝物の合成および分解経路とこれらに関与する酵素を示す代謝マップを用いて説明する。ＧＡＢＡは、ＴＣＡ回路で生じるアルファケトグルタル酸（ａ－ＫＧ）由来のグルタミン酸からグルタミン酸デカルボキシラーゼ（ＧＡＤ１）により合成されるか、尿素回路またはスペルミジン／スペルミングルタミン由来の４－アミノブタナールからアルデヒドデヒドロゲナーゼ９ファミリーメンバーＡ１（ＡＬＤＨ９Ａ１）により合成される。ＧＡＢＡは４－アミノ酪酸アミノ基転移酵素によりコハク酸セミアルデヒドに分解され、さらに酸化されてコハク酸としてトリカルボン酸（ＴＣＡ）回路に入る。Ｔ細胞およびＢ細胞におけるグルタミンの消費はいずれも抗原レセプターを介する活性化により増加する。Ｔ細胞もＢ細胞も、抗原刺激の２４時間後の細胞内グルタミン酸プールの８０％はグルタミン由来である。前記標識グルタミンは、プリンおよびピリミジンの前駆体を経て、活性化Ｂ細胞およびＴ細胞におけるヌクレオチド生合成に寄与する。また培養７２時間後のＢ細胞ではグルタチオンのほぼ９０％がグルタミン由来である。そして、グルタミン由来のアミノ酸のうち、グルタミン酸、または、ＴＣＡ回路経由で流れ込むグルタミンのいずれかからのアスパラギン酸合成はＢ細胞のほうが顕著である。Ｂ細胞ではＴＣＡ回路で酸化されるα－ケトグルタル酸のかなりの部分はグルタミン由来である。Ｂ細胞とＴ細胞とで明らかに異なるグルタミン由来代謝物のひとつが、神経伝達物質のγアミノ酪酸（ＧＡＢＡ）である。抗原レセプターまたはｔｏｌｌ様レセプターを介する刺激の７２時間後、Ｂ細胞はＧＡＢＡを合成し細胞外に輸送するが、Ｔ細胞はＧＡＢＡ合成も細胞外輸送もしない。グルタミン分解経路の主要な酵素のＲＮＡ転写解析も、ＧＡＢＡ合成分解に関与する酵素の発現がＢ細胞とＣＤ４Ｔ細胞とで異なることを裏付ける。ＣＤ４Ｔ細胞と比較すると、グルタミン酸をＧＡＢＡに変換する酵素ＧＡＤ１と、グルタミントランスポーターのＳｌｃ３８ａ１、Ｓｌｃ３８ａ２およびＳｌｃ３８ａ５をコード化する遺伝子の発現はＢ細胞のほうがより多い。逆に、ＧＡＢＡを異化する酵素ＡＢＡＴをコード化する遺伝子の発現はＢ細胞のほうがより少ない。ヒトのＢ細胞もＧＡＢＡに変換する酵素ＧＡＤ１の発現はＴ細胞よりも高い。 The GABA biosynthesis pathway in B cells will be described using the synthesis and degradation pathways of GABA and related metabolites in FIG. 1 and a metabolic map showing enzymes involved in these. GABA is synthesized by glutamic acid decarboxylase (GAD1) from glutamic acid derived from alpha-ketoglutaric acid (a-KG) generated in the TCA cycle, or from the urea cycle or 4-aminobutanal derived from spermidine / spermine tamine to the aldehyde dehydrogenase 9 family. Synthesized by member A1 (ALDH9A1). GABA is decomposed into succinic semialdehyde by 4-aminobutyric acid transaminase, and further oxidized to enter the tricarboxylic acid (TCA) cycle as succinic acid. Glutamine consumption in both T and B cells is increased by antigen receptor-mediated activation. In both T and B cells, 80% of the intracellular glutamate pool 24 hours after antigen stimulation is derived from glutamine. The labeled glutamine contributes to nucleotide biosynthesis in activated B and T cells via precursors of purines and pyrimidines. In B cells after 72 hours of culture, almost 90% of glutathione is derived from glutamine. And, among the amino acids derived from glutamine, the synthesis of aspartic acid from either glutamic acid or glutamine flowing through the TCA cycle is more remarkable in B cells. In B cells, a significant portion of the α-ketoglutaric acid that is oxidized by the TCA cycle is derived from glutamine. One of the glutamine-derived metabolites that is clearly different between B cells and T cells is the neurotransmitter gamma-aminobutyric acid (GABA). After 72 hours of stimulation via antigen receptors or toll-like receptors, B cells synthesize GABA and transport it extracellularly, whereas T cells do not synthesize GABA or transport extracellularly. RNA transcription analysis of the major enzymes of the glutamine degradation pathway also confirms that the expression of enzymes involved in GABA synthetic degradation differs between B cells and CD4T cells. Compared to CD4T cells, B cells have higher expression of the enzyme GAD1 that converts glutamate to GABA and the genes that encode the glutamate transporters Slc38a1, Slc38a2 and Slc38a5. Conversely, B cells have less expression of the gene encoding the enzyme ABAT that catabolizes GABA. The expression of the enzyme GAD1, which also converts human B cells to GABA, is higher than that of T cells.
　癌細胞におけるＧＡＤ１の発現については、腸癌、腎癌及び肝癌で発現が低い。その他、甲状腺癌、肺癌、頭頸部癌、胃癌、膵癌、尿路上皮がん、前立腺癌、精巣癌、乳癌、卵巣癌、黒色腫などでＧＡＤ１の発現が低い。一方、神経膠腫、子宮頸がん及び子宮内膜がんではＧＡＤ１の発現が高く、子宮頸がん及び子宮内膜がんではＧＡＤ１の発現が高いと予後が良い。本発明のＴ細胞活性化剤は、ＧＡＤ１の発現が低い癌に対して、癌の増殖を抑制しうるし、ＧＡＤ１の発現量が高い癌に対しても、癌の増殖を抑制しうる。 Regarding the expression of GAD1 in cancer cells, the expression is low in intestinal cancer, renal cancer and liver cancer. In addition, the expression of GAD1 is low in thyroid cancer, lung cancer, head and neck cancer, gastric cancer, pancreatic cancer, urinary tract epithelial cancer, prostate cancer, testis cancer, breast cancer, ovarian cancer, melanoma and the like. On the other hand, the prognosis is good when the expression of GAD1 is high in glioma, cervical cancer and endometrial cancer, and the expression of GAD1 is high in cervical cancer and endometrial cancer. The T cell activator of the present invention can suppress the growth of cancer against a cancer having a low expression of GAD1, and can also suppress the growth of a cancer against a cancer having a high expression level of GAD1.
　本発明のＴ細胞活性化剤が前記Ｂ細胞におけるＧＡＢＡ生合成の阻害薬の場合には、その有効成分は、グルタミン酸デカルボキシラーゼまたはアルデヒドデヒドロゲナーゼ９ファミリーメンバーＡ１に対するアンチセンス核酸、ＲＮＡｉ誘導性核酸もしくはリボザイムまたはそれらの発現ベクターを含むが、これらに限定されない。 When the T cell activator of the present invention is an inhibitor of GABA biosynthesis in B cells, the active ingredient thereof is an antisense nucleic acid, RNAi-inducible nucleic acid or ribozyme against glutamate decarboxylase or aldehyde dehydrogenase 9 family member A1. Or include, but is not limited to, their expression vectors.
　本明細書において、アンチセンス核酸、ＲＮＡｉ誘導性核酸またはリボザイムによって特異的に発現が抑制される、ヒトα１、α５、β１、πおよびρ２からなる群から選択される少なくとも１種類のサブユニット、グルタミン酸デカルボキシラーゼおよびアルデヒドデヒドロゲナーゼ９ファミリーメンバーＡ１を以下では標的遺伝子という。また該標的遺伝子に対するアンチセンス核酸、ＲＮＡｉ誘導性核酸またはリボザイムを標的遺伝子阻害核酸という。前記標的遺伝子に対するアンチセンス核酸は、前記標的遺伝子の転写産物（ｍＲＮＡまたは初期転写産物）を発現する細胞の生理的条件下で前記転写産物とハイブリダイズし得る塩基配列からなり、且つハイブリダイズした状態で前記転写産物にコードされるポリペプチドの翻訳を阻害し得るポリヌクレオチドをいう。アンチセンス核酸の種類はＤＮＡであってもＲＮＡであってもよいし、あるいはＤＮＡ／ＲＮＡキメラであってもよい。アンチセンス核酸は、非修飾（天然型）のリン酸ジエステル結合を有するものであっても、分解酵素に安定なチオリン酸型（リン酸結合のＰ＝ＯをＰ＝Ｓに置換）や２’－Ｏ－メチル型等の化学修飾されたヌクレオチドであってもよい。アンチセンス核酸の設計に重要な他の要素として、水溶性および細胞膜透過性を高めること等が挙げられるが、これらはリポソームやマイクロスフェアを使用するなどの剤形の工夫によっても克服できる。アンチセンス核酸の長さは、前記標的遺伝子の転写産物と特異的にハイブリダイズし得る限り特に制限はなく、短いもので約６塩基程度、長いもので転写産物の全配列に相補的な配列を含むような配列であってもよい。合成の容易さや抗原性の問題等から、例えば約６塩基以上、好ましくは約１５～約４０塩基、より好ましくは約１５塩基～約３０塩基からなるオリゴヌクレオチドが例示される。さらに、アンチセンス核酸は、前記標的遺伝子の転写産物とハイブリダイズして翻訳を阻害するだけでなく、二本鎖ＤＮＡと結合して三重鎖（トリプレックス）を形成し、ｍＲＮＡへの転写を阻害し得るものであってもよい。 Glutamate, at least one subunit selected herein from the group consisting of human α1, α5, β1, π and ρ2, whose expression is specifically suppressed by antisense nucleic acids, RNAi-inducible nucleic acids or ribozymes. Decarboxylase and aldehyde dehydrogenase 9 family member A1 are hereinafter referred to as target genes. Further, an antisense nucleic acid, an RNAi-inducible nucleic acid or a ribozyme against the target gene is referred to as a target gene-inhibiting nucleic acid. The antisense nucleic acid for the target gene consists of a base sequence capable of hybridizing with the transcript under the physiological conditions of cells expressing the transcript (mRNA or initial transcript) of the target gene, and is in a hybridized state. Refers to a polynucleotide capable of inhibiting translation of a polypeptide encoded by the transcript. The type of antisense nucleic acid may be DNA, RNA, or DNA / RNA chimera. Even if the antisense nucleic acid has an unmodified (natural type) phosphodiester bond, it has a thiophosphate type stable to a degrading enzyme (P = O of the phosphate bond is replaced with P = S) or 2'. It may be a chemically modified nucleotide such as -O-methyl type. Other important factors in the design of antisense nucleic acids include enhancing water solubility and cell membrane permeability, which can be overcome by devising dosage forms such as the use of liposomes and microspheres. The length of the antisense nucleic acid is not particularly limited as long as it can specifically hybridize with the transcript of the target gene, and includes a short sequence of about 6 bases and a long sequence complementary to the entire sequence of the transcript. It may be an arrangement like this. From the viewpoint of ease of synthesis, antigenicity, and the like, examples thereof include oligonucleotides consisting of about 6 bases or more, preferably about 15 to about 40 bases, and more preferably about 15 to about 30 bases. Furthermore, the antisense nucleic acid not only hybridizes with the transcription product of the target gene and inhibits translation, but also binds to double-stranded DNA to form a triplet and inhibits transcription into mRNA. It may be possible.
　本明細書において、配列Ａと配列Ｂとの「相補性」とは、配列Ａの相補配列と配列Ｂとの同一性をいう。アンチセンス核酸と、該アンチセンス核酸の標的遺伝子との相補性は必ずしも１００％である必要はなく、生体細胞内で前記標的遺伝子のＤＮＡまたはＲＮＡと相補的に結合しうる程度でハイブリダイズして、ｍＲＮＡへの転写および／または翻訳を阻害できることを条件として、約７０％以上、約８０％以上、約９０％以上、または、約９５％以上であってもかまわない。 As used herein, the term "complementarity" between sequence A and sequence B means the identity between the complementary sequence of sequence A and sequence B. The complementarity of the antisense nucleic acid with the target gene of the antisense nucleic acid does not necessarily have to be 100%, and it hybridizes to the extent that it can complementarily bind to the DNA or RNA of the target gene in a living cell. , About 70% or more, about 80% or more, about 90% or more, or about 95% or more, provided that transcription and / or translation into mRNA can be inhibited.
　前記ＲＮＡｉ誘導性核酸とは、細胞内に導入されることにより、ＲＮＡ干渉（ＲＮＡｉ）を誘導し得るポリヌクレオチドをいい、好ましくはＲＮＡまたはＲＮＡとＤＮＡのキメラ分子である。ＲＮＡ干渉とは、ｍＲＮＡと同一の塩基配列（またはその部分配列）を含む２本鎖構造のＲＮＡが、当該ｍＲＮＡの発現を抑制する効果をいう。このＲＮＡｉ効果を得るには、例えば、少なくとも１９の連続する標的ｍＲＮＡと同一の塩基配列（またはその部分配列）を有する２本鎖構造のＲＮＡを用いることが好ましい。ただし、前記標的遺伝子の発現阻害作用を有していれば数塩基置換されているものであってもよく、１９塩基長よりも短いＲＮＡであってもよい。２本鎖構造は、センス鎖とアンチセンス鎖の異なるストランドで構成されていてもよいし、一つのＲＮＡのステムループ構造によって与えられる２本鎖（ｓｈＲＮＡ）であってもよい。ＲＮＡｉ誘導性核酸としては、例えばｓｉＲＮＡ、ｍｉＲＮＡなどが挙げられる。ｍｉＲＮＡは、前記標的遺伝子の３’ＵＴＲを認識して、該標的遺伝子のｍＲＮＡを不安定化するとともに翻訳抑制を行うことで前記標的遺伝子の発現を抑制する。 The RNAi-inducible nucleic acid refers to a polynucleotide capable of inducing RNA interference (RNAi) by being introduced into a cell, and is preferably RNA or a chimeric molecule of RNA and DNA. RNA interference refers to the effect that double-stranded RNA containing the same base sequence (or a partial sequence thereof) as mRNA suppresses the expression of the mRNA. In order to obtain this RNAi effect, for example, it is preferable to use double-stranded RNA having the same base sequence (or a partial sequence thereof) as at least 19 consecutive target mRNAs. However, as long as it has an effect of inhibiting the expression of the target gene, it may be substituted with several bases, or it may be an RNA shorter than 19 bases in length. The double-stranded structure may be composed of different strands of the sense strand and the antisense strand, or may be a double strand (SHRNA) provided by the stem-loop structure of one RNA. Examples of RNAi-inducible nucleic acids include siRNA and miRNA. The miRNA recognizes the 3'UTR of the target gene, destabilizes the mRNA of the target gene, and suppresses translation to suppress the expression of the target gene.
　ＲＮＡｉ誘導性核酸は、転写抑制活性が強いという観点から、ｓｉＲＮＡが好ましい。前記標的遺伝子に対するｓｉＲＮＡは、該標的遺伝子のｍＲＮＡの任意の部分を標的とすることができる。前記標的遺伝子に対するｓｉＲＮＡ分子は、ＲＮＡｉ効果を誘導できる限り特に制限されないが、例えば１８～２７塩基長、好ましくは２１～２５塩基長である。前記標的遺伝子に対するｓｉＲＮＡは、センス鎖およびアンチセンス鎖を含む二重鎖である。前記標的遺伝子に対するｓｉＲＮＡは、センス鎖、アンチセンス鎖の一方または双方の５’末端または３’末端においてオーバーハングを有していてもよい。オーバーハングは、センス鎖および／またはアンチセンス鎖の末端における１～数個（例、１、２または３個）の塩基の付加により形成されるものである。ｓｉＲＮＡの設計方法は、当業者に公知であり、ｓｉＲＮＡの様々な設計ソフトウェアまたはアルゴリズムを用いて、上記塩基配列から適切なｓｉＲＮＡの塩基配列を選択することができる。 The RNAi-inducible nucleic acid is preferably siRNA from the viewpoint of strong transcriptional repressive activity. The siRNA for the target gene can target any portion of the mRNA of the target gene. The siRNA molecule for the target gene is not particularly limited as long as it can induce the RNAi effect, but is, for example, 18 to 27 base bases long, preferably 21 to 25 bases long. The siRNA for the target gene is a double strand containing a sense strand and an antisense strand. The siRNA for the target gene may have an overhang at the 5'end or 3'end of one or both of the sense strand and the antisense strand. Overhangs are formed by the addition of one to several (eg, 1, 2 or 3) bases at the ends of the sense and / or antisense strands. Methods for designing siRNA are known to those of skill in the art, and various siRNA design software or algorithms can be used to select an appropriate siRNA base sequence from the above base sequences.
　前記「リボザイム」とは核酸を切断する酵素活性を有するＲＮＡをいうが、最近では当該酵素活性部位の塩基配列を有するオリゴＤＮＡも同様に核酸切断活性を有することが明らかになっているので、本明細書では配列特異的な核酸切断活性を有する限りＤＮＡをも包含する概念として用いる。具体的には、リボザイムは、前記標的遺伝子をコードするｍＲＮＡまたは初期転写産物を、コード領域の内部（初期転写産物の場合はイントロン部分を含む）で特異的に切断し得る。リボザイムとして最も汎用性の高いものとしては、ウイロイドやウイルソイド等の感染性ＲＮＡに見られるセルフスプライシングＲＮＡがあり、ハンマーヘッド型やヘアピン型等が知られている。ハンマーヘッド型は約４０塩基程度で酵素活性を発揮し、ハンマーヘッド構造をとる部分に隣接する両端の数塩基ずつ（合わせて約１０塩基程度）をｍＲＮＡの所望の切断部位と相補的な配列にすることにより、標的ｍＲＮＡのみを特異的に切断することが可能である。さらに、リボザイムを、それをコードするＤＮＡを含む発現ベクターの形態で使用する場合には、転写産物の細胞質への移行を促進するために、ｔＲＮＡを改変した配列をさらに連結したハイブリッドリボザイムとすることもできる（Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓ．，　２９（１３）：　２７８０－２７８８　（２００１））。 The above-mentioned "ribozyme" refers to RNA having an enzyme activity for cleaving nucleic acid, but recently, it has been clarified that oligo DNA having a base sequence of the enzyme activity site also has nucleic acid cleaving activity. In the specification, it is used as a concept including DNA as long as it has sequence-specific nucleic acid cleavage activity. Specifically, the ribozyme can specifically cleave the mRNA or early transcript encoding the target gene inside the coding region (including the intron moiety in the case of the early transcript). The most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and hammer head type and hairpin type are known. The hammer head type exerts enzymatic activity at about 40 bases, and several bases at both ends adjacent to the part having the hammer head structure (about 10 bases in total) are arranged in a sequence complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. Furthermore, when the ribozyme is used in the form of an expression vector containing the DNA encoding it, it should be a hybrid ribozyme in which tRNA-modified sequences are further linked in order to promote the transfer of the transcript to the cytoplasm. It can also be done (Nucleic Acids Res., 29 (13): 2780-2788 (2001)).
　本発明のＴ細胞活性化剤は、前記標的遺伝子阻害核酸をコードするポリヌクレオチド、および当該ポリヌクレオチドに機能可能に連結されたプロモーターを含む、発現ベクターとして提供することもできる。前記プロモーターは、その制御下にある発現対象の核酸の種類により適宜選択され得るが、例えば、ｐｏｌＩＩＩプロモーター（例、ｔＲＮＡプロモーター、Ｕ６プロモーター、Ｈ１プロモーター）、哺乳動物用プロモーター（例、ＣＭＶプロモーター、ＣＡＧプロモーター、ＳＶ４０プロモーター）が挙げられる。本発明の発現ベクターはさらに、選択マーカー遺伝子（テトラサイクリン、アンピシリン、カナマイシン、ハイグロマイシン、ホスフィノスリシン等の薬剤に対する抵抗性を付与する遺伝子、栄養要求性変異を相補する遺伝子等）をさらに含んでいてもよい。本発明の発現ベクターのバックボーン（ｂａｃｋｂｏｎｅ）としては、ヒト等の哺乳動物細胞中で前記標的遺伝子阻害核酸を産生できるものであれば特に制限されないが、例えば、プラスミドベクター、ウイルスベクターが挙げられる。哺乳動物への投与に好適なベクターとしては、レトロウイルス、アデノウイルス、アデノ随伴ウイルス、ヘルペスウイルス、ワクシニアウイルス、ポックスウイルス、ポリオウイルス、シンドビスウイルス、センダイウイルス等のウイルスベクターが挙げられる。なかでも、レトロウイルス、アデノウイルス、アデノ随伴ウイルス、ワクシニアウイルス由来のウイルスベクターが好ましい。 The T cell activator of the present invention can also be provided as an expression vector containing a polynucleotide encoding the target gene-inhibiting nucleic acid and a promoter operably linked to the polynucleotide. The promoter may be appropriately selected depending on the type of nucleic acid to be expressed under its control, and may be, for example, a polIII promoter (eg, tRNA promoter, U6 promoter, H1 promoter), a mammalian promoter (eg, CMV promoter, CAG). Promoter, SV40 promoter). The expression vector of the present invention further contains a selectable marker gene (a gene that imparts resistance to a drug such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinoslicin, a gene that complements an auxotrophic mutation, etc.). You may. The backbone of the expression vector of the present invention is not particularly limited as long as it can produce the target gene-inhibiting nucleic acid in mammalian cells such as humans, and examples thereof include a plasmid vector and a viral vector. Suitable vectors for administration to mammals include virus vectors such as retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, sindobis virus, and Sendai virus. Of these, viral vectors derived from retrovirus, adenovirus, adeno-associated virus, and vaccinia virus are preferable.
　本発明のＴ細胞活性化剤が遊離ＧＡＢＡの捕捉薬の場合には、その有効成分は、ＧＡＢＡと特異的に結合する抗体、特異的結合パートナーおよび／またはそれらの断片を含むが、これらに限定されない。 When the T cell activator of the present invention is a capture agent for free GABA, the active ingredient includes, but is limited to, an antibody that specifically binds to GABA, a specific binding partner and / or a fragment thereof. Not done.
　本明細書において、抗体とは、ポリクローナル抗体、モノクローナル抗体等の天然型抗体、遺伝子組換技術を用いて製造され得るキメラ抗体、ヒト化抗体や一本鎖抗体、ヒト抗体産生トランスジェニック動物等を用いて製造され得るヒト抗体、ファージディスプレイによって作製された抗体を指す。 In the present specification, the antibody refers to a natural antibody such as a polyclonal antibody or a monoclonal antibody, a chimeric antibody that can be produced by using a gene recombination technique, a humanized antibody or a single-stranded antibody, a human antibody-producing transgenic animal, or the like. Refers to human antibodies that can be produced using, antibodies produced by phage display.
　本明細書において、特異的結合パートナーとは、特異的結合対のメンバーを指す。特異的結合対は、化学的または物理的手段によって互いに特異的に結合する２つの異なる分子を含む。従って、一般的な免疫反応の抗原と抗体との特異的結合対に加えて、他の特異的結合対としては、ビオチンおよびアビジン（またはストレプトアビジン）、炭水化物およびレクチン、相補的ヌクレオチド配列、エフェクターおよび受容体分子、補助因子および酵素、酵素および酵素阻害剤などを挙げることができるが、これらに限られない。さらに、特異的結合対としては、元の特異的結合メンバーの類似体であるメンバー、例えば、分析物類似体を挙げることができる。免疫反応特異的結合メンバーとしては、単離されているかまたは組換え的に生産されたかにかかわらず、抗原およびその断片と、モノクローナル抗体およびポリクローナル抗体を含む抗体と、これらの複合体およびその断片を挙げることができる。 As used herein, a specific binding partner refers to a member of a specific binding pair. Specific binding pairs include two different molecules that specifically bind to each other by chemical or physical means. Thus, in addition to the general immune response antigen-antibody specific binding pairs, other specific binding pairs include biotin and avidin (or streptavidin), carbohydrates and lectins, complementary nucleotide sequences, effectors and. Examples include, but are not limited to, acceptor molecules, cofactors and enzymes, enzymes and enzyme inhibitors. Further, as the specific binding pair, a member that is an analog of the original specific binding member, for example, an analyte analog can be mentioned. Immune response-specific binding members, whether isolated or recombinantly produced, include antigens and fragments thereof, antibodies including monoclonal and polyclonal antibodies, and complexes and fragments thereof. Can be mentioned.
　本明細書における「これらの断片」または「その断片」という文言は、抗体の断片を指す場合には、本発明の「これらの断片」または「その断片」は抗体の一部分の領域を意味する。具体的には、例えばＦ（ａｂ’）２、Ｆａｂ’、Ｆａｂ、Ｆｖ（ｖａｒｉａｂｌｅ　ｆｒａｇｍｅｎｔ　ｏｆ　ａｎｔｉｂｏｄｙ）、ｓＦｖ、ｄｓＦｖ（ｄｉｓｕｌｐｈｉｄｅ　ｓｔａｂｉｌｉｚｅｄ　Ｆｖ）、ｄＡｂ（ｓｉｎｇｌｅ　ｄｏｍａｉｎ　ａｎｔｉｂｏｄｙ）を指すが、これらに限られない。本発明の「これらの断片」または「その断片」という文言が、抗体以外の特異的結合パートナーの断片を指す場合には、本発明の「これらの断片」または「その断片」は当該特異的結合パートナー分子の断片を意味する。 When the terms "these fragments" or "the fragments thereof" in the present specification refer to fragments of an antibody, "these fragments" or "the fragments thereof" of the present invention mean a region of a part of the antibody. Specifically, for example, F (ab') 2, Fab', Fab, Fv (variable fragment of antibody), sFv, dsFv (disulphid stapled Fv), dAb (single domain antibodies) are not limited to these. .. When the words "these fragments" or "fragments thereof" of the present invention refer to fragments of specific binding partners other than antibodies, "these fragments" or "fragments thereof" of the present invention refer to the specific binding. Means a fragment of a partner molecule.
　本明細書における抗体のクラスは、特に限定されず、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＤまたはＩｇＥ等のいずれのアイソタイプを有する抗体をも包含する。好ましくは、ＩｇＧまたはＩｇＭであり、精製の容易性等を考慮するとより好ましくはＩｇＧである。 The class of antibody in the present specification is not particularly limited, and includes an antibody having any isotype such as IgG, IgM, IgA, IgD or IgE. It is preferably IgG or IgM, and more preferably IgG in consideration of easiness of purification and the like.
　本明細書におけるポリクローナル抗体、モノクローナル抗体、キメラ抗体、ヒト化抗体およびこれらの断片は、既知の一般的な製造方法によって製造することができる。本明細書に記載の抗体は、例えばＧｒｅｅｎｆｉｅｌｄ，　Ｅ．　Ａ．編、Ａｎｔｉｂｏｄｉｅｓ：　Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ、第２版（Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ、２０１４）に詳しく説明されている。 The polyclonal antibody, monoclonal antibody, chimeric antibody, humanized antibody and fragments thereof in the present specification can be produced by a known general production method. The antibodies described herein are, for example, Greenfield, E. et al. A. Hen, Antibodies: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, 2014).
　本発明のＴ細胞活性化剤は、医薬（免疫療法剤）として、がんの治療、感染症の予防及び/又は治療などに利用することができる。本発明は、Ｂ細胞による細胞傷害性Ｔ細胞抑制作用を阻害または低減する、Ｔ細胞活性化剤のがんの治療あるいは感染症の予防及び/又は治療のための使用を包含する。また、本発明は、Ｂ細胞による細胞傷害性Ｔ細胞抑制作用を阻害または低減する、Ｔ細胞活性化剤の使用であって、抗がん剤あるいは感染症の予防及び/又は治療剤を製造するための前記使用も包含する。 The T cell activator of the present invention can be used as a pharmaceutical (immunotherapeutic agent) for the treatment of cancer, the prevention and / or the treatment of infectious diseases. The present invention includes the use of T cell activators for the treatment of cancer or the prevention and / or treatment of infectious diseases, which inhibit or reduce the cytotoxic T cell inhibitory effect of B cells. The present invention also comprises the use of a T cell activator that inhibits or reduces the cytotoxic T cell inhibitory effect of B cells to produce an anticancer agent or a prophylactic and / or therapeutic agent for an infectious disease. Also includes the above-mentioned uses for.
　本発明のＴ細胞活性化剤を抗がん剤あるいは感染症の予防及び/又は治療剤として使用する場合には、患者に対して経口的または非経口的に投与することができ、投与形態としては、経口投与、局所投与、静脈内投与、経皮投与などが挙げられ、必要に応じて、製薬学的に許容され得る添加剤と共に、投与に適した剤形に製剤化される。経口投与に適した剤形としては、例えば、錠剤、カプセル剤、顆粒剤、散剤などが挙げられ、非経口投与に適した剤形としては、例えば、注射剤、軟膏、ローション剤、クリーム剤、貼付剤などが挙げられる。これらは当該分野で汎用されている通常の技術を用い、調製することができる。本発明の抗がん剤並びに感染症の予防及び/又は治療剤は、患者のがんや感染細胞の増殖を抑制または軽減し、あるいは、がんや感染組織の成長抑制、縮小または消滅する等のがんに対する治療効果を奏する限り、その投与経路および剤形は特に限定されないが、好ましい投与経路は局所投与であり、その剤形は注射剤、軟膏、ローション剤、クリーム剤または貼付剤である。また、本発明の抗がん剤並びに感染症の予防及び/又は治療剤は、これらの製剤の他に臓器内インプラント用製剤やマイクロスフェア等のＤＤＳ（ドラッグデリバリーシステム）化された製剤にすることもできる。さらに、本発明の抗がん剤や感染症の予防及び/又は治療剤を所望のがん組織や感染組織（例えば、原発巣または転移がん組織）に到達させるためには、筋肉内局所投与、皮下局所投与、皮膚への直接塗布、貼付等の局所投与に限らず、静脈内注射（点滴）、皮下投与等の全身性投与であってもよい。 When the T cell activator of the present invention is used as an anticancer agent or a prophylactic and / or therapeutic agent for infectious diseases, it can be orally or parenterally administered to a patient, and as an administration form. Examples include oral administration, local administration, intravenous administration, transdermal administration, and the like, and if necessary, the drug is formulated into a dosage form suitable for administration together with a pharmaceutically acceptable additive. Dosage forms suitable for oral administration include, for example, tablets, capsules, granules, powders, etc., and dosage forms suitable for parenteral administration include, for example, injections, ointments, lotions, creams, etc. Examples include patches. These can be prepared using conventional techniques commonly used in the art. The anticancer agent and the preventive and / or therapeutic agent for an infectious disease of the present invention suppress or reduce the growth of cancer or infected cells in a patient, or suppress, shrink or eliminate the growth of cancer or infected tissue, etc. The route of administration and dosage form are not particularly limited as long as they have a therapeutic effect on cancer, but the preferred route of administration is topical administration, and the dosage form is an injection, an ointment, a lotion, a cream or a patch. .. In addition to these preparations, the anticancer agent and the preventive and / or therapeutic agent for infectious diseases of the present invention shall be DDS (drug delivery system) -ized preparations such as intra-organ implant preparations and microspheres. You can also. Furthermore, in order to bring the anticancer agent and the preventive and / or therapeutic agent of the present invention to the desired cancer tissue or infected tissue (for example, primary lesion or metastatic cancer tissue), intramuscular topical administration , Subcutaneous local administration, direct application to the skin, local administration such as application, and systemic administration such as intravenous injection (drip) and subcutaneous administration may be used.
　本発明の抗がん剤並びに感染症の予防及び/又は治療剤は、有効成分の種類とその投与経路に応じて、薬学的に許容される担体を含んでいてもよい。当業者であればかかる状況に適切な担体を適宜選択することができる。選択可能な担体としては、例えば、ショ糖、デンプン、マンニット、ソルビット、乳糖、グルコース、セルロース、タルク、リン酸カルシウム、炭酸カルシウム等の賦形剤；セルロース、メチルセルロース、ヒドロキシプロピルセルロース、ポリプロピルピロリドン、ゼラチン、アラビアゴム、ポリエチレングリコール、ショ糖、デンプン等の結合剤；デンプン、カルボキシメチルセルロース、ヒドロキシプロピルスターチ、ナトリウム－グリコール－スターチ、炭酸水素ナトリウム、リン酸カルシウム、クエン酸カルシウム等の崩壊剤；ステアリン酸マグネシウム、エアロジル、タルク、ラウリル硫酸ナトリウム等の滑剤；安息香酸ナトリウム、亜硫酸水素ナトリウム、メチルパラベン、プロピルパラベン等の保存剤；クエン酸、クエン酸ナトリウム、酢酸等のｐＨ調節剤；メチルセルロース、ポリビニルピロリドン、ステアリン酸アルミニウム等の懸濁剤；界面活性剤等の分散剤；水、生理食塩水、エタノール、プロピレングリコール等の溶解剤；グルコース、塩化ナトリウム、塩化カリウム等の等張化剤；カカオ脂、ポリエチレングリコール、白灯油等のベースワックスなどがあげられるが、それらに限定されるものではない。また、これらの担体は単独の作用に限定されず、複数の作用を発揮する目的で使用することができる。 The anticancer agent and the preventive and / or therapeutic agent for infectious diseases of the present invention may contain a pharmaceutically acceptable carrier depending on the type of active ingredient and the route of administration thereof. A person skilled in the art can appropriately select a carrier suitable for such a situation. Selectable carriers include, for example, excipients such as sucrose, starch, mannitt, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate; cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin. , Arabium gum, polyethylene glycol, sucrose, starch and other binders; starch, carboxymethyl cellulose, hydroxypropyl starch, sodium-glycol-starch, sodium hydrogen carbonate, calcium phosphate, calcium citrate and other disintegrants; magnesium stearate, aerodil , Lubricants such as talc, sodium lauryl sulfate; Preservatives such as sodium benzoate, sodium hydrogen sulfite, methylparaben, propylparaben; pH adjusters such as citrate, sodium citrate, acetic acid; methylcellulose, polyvinylpyrrolidone, aluminum stearate, etc. Suspension agent; Dispersant such as surfactant; Dissolving agent such as water, physiological saline, ethanol, propylene glycol; Isotonic agent such as glucose, sodium chloride, potassium chloride; Cacao fat, polyethylene glycol, white kerosene Examples include, but are not limited to, base waxes such as. Further, these carriers are not limited to a single action, and can be used for the purpose of exerting a plurality of actions.
　例えば、本発明の抗がん剤並びに感染症の予防及び/又は治療剤を注射剤、軟膏、ローション剤、クリーム剤または貼付剤として用いる場合、安定剤（例えば、亜硫酸水素ナトリウム、チオ硫酸ナトリウム、エデト酸ナトリウム、クエン酸ナトリウム、アスコルビン酸、ジブチルヒドロキシトルエンなど）、溶解補助剤（例えば、グリセリン、プロピレングリコール、マクロゴール、ポリオキシエチレン硬化ヒマシ油など）、懸濁化剤（例えば、ポリビニルピロリドン、ヒドロキシプロピルメチルセルロース、ヒドロキシメチルセルロース、カルボキシメチルセルロースナトリウムなど）、乳化剤（例えば、ポリビニルピロリドン、大豆レシチン、卵黄レシチン、ポリオキシエチレン硬化ヒマシ油、ポリソルベート８０など）、緩衝剤（例えば、リン酸緩衝液、酢酸緩衝液、ホウ酸緩衝液、炭酸緩衝液、クエン酸緩衝液、トリス緩衝液、グルタミン酸、イプシロンアミノカプロン酸など）、粘稠剤（例えば、メチルセルロース、ヒドロキシエチルセルロース、ヒドロキシプロピルメチルセルロース、カルボキシメチルセルロースなどの水溶性セルロース誘導体、コンドロイチン硫酸ナトリウム、ヒアルロン酸ナトリウム、カルボキシビニルポリマー、ポリビニルアルコール、ポリビニルピロリドン、マクロゴールなど）、保存剤（例えば、塩化ベンザルコニウム、塩化ベンゼトニウム、グルコン酸クロルヘキシジン、クロロブタノール、ベンジルアルコール、デヒドロ酢酸ナトリウム、パラオキシ安息香酸エステル類、エデト酸ナトリウム、ホウ酸など）、等張化剤（例えば、塩化ナトリウム、塩化カリウム、グリセリン、マンニトール、ソルビトール、ホウ酸、ブドウ糖、プロピレングリコールなど）、ｐＨ調整剤（例えば、塩酸、水酸化ナトリウム、リン酸、酢酸など）、清涼化剤（例えば、ｌ－メントール、ｄ－カンフル、ｄ－ボルネオール、ハッカ油など）、軟膏基剤（例えば、白色ワセリン、精製ラノリン、流動パラフィン、植物油（オリーブ油、椿油、落花生油など）など）などを添加剤として加えることができる。これら添加剤の添加量は、添加する添加剤の種類、用途などによって異なるが、添加剤の目的を達成し得る濃度を添加すればよい。 For example, when the anticancer agent and the preventive and / or therapeutic agent of the present invention are used as an injection, an ointment, a lotion, a cream or a patch, a stabilizer (for example, sodium hydrogen sulfite, sodium thiosulfate, etc.) Sodium edetate, sodium citrate, ascorbic acid, dibutylhydroxytoluene, etc.), solubilizers (eg, glycerin, propylene glycol, macrogol, polyoxyethylene hydrogenated castor oil, etc.), suspending agents (eg, polyvinylpyrrolidone, etc.) Hydroxypropylmethylcellulose, hydroxymethylcellulose, sodium carboxymethylcellulose, etc.), emulsifiers (eg, polyvinylpyrrolidone, soybean lecithin, egg yolk lecithin, polyoxyethylene hydrogenated castor oil, polysorbate 80, etc.), buffers (eg, phosphate buffer, acetate buffer, etc.) Liquids, borate buffers, carbonate buffers, citrate buffers, Tris buffers, glutamic acid, epsilon aminocaproic acid, etc.), viscous agents (eg, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose and other water-soluble celluloses. Derivatives, sodium chondroitin sulfate, sodium hyaluronate, carboxyvinyl polymer, polyvinyl alcohol, polyvinylpyrrolidone, macrogol, etc., preservatives (eg, benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, chlorobutanol, benzyl alcohol, dehydroacetic acid) Sodium, paraoxybenzoic acid esters, sodium edetate, boric acid, etc.), isotonic agents (eg, sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, glucose, propylene glycol, etc.), pH adjusters (eg, sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, glucose, propylene glycol, etc.) For example, hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, etc.), refreshing agents (eg, l-menthol, d-camfur, d-bornol, peppermint oil, etc.), ointment bases (eg, white vaseline, purified lanolin, etc.) Liquid paraffin, vegetable oil (olive oil, camellia oil, peanut oil, etc.), etc. can be added as additives. The amount of these additives added varies depending on the type of the additive to be added, the intended use, and the like, but a concentration that can achieve the purpose of the additive may be added.
　本発明の抗がん剤並びに感染症の予防及び/又は治療剤は、前記標的遺伝子阻害核酸、または、該記標的遺伝子阻害核酸をコードするポリヌクレオチド、および当該ポリヌクレオチドに機能可能に連結されたプロモーターを含む、発現ベクターを、リポフェクション法を用いて製剤化することもできる。リポフェクション法には、通常ホスファチジルセリンからなるリポソームが用いられる。ホスファチジルセリンは陰電荷を有するため、ホスファチジルセリンの代用として、より安定したリポソームを作りやすいＮ－［１－（２，３－ジオレイルオキシ）プロピル］－Ｎ，Ｎ，Ｎ－トリエチルアンモニウムクロライド（ＤＯＴＭＡ）という陽イオン性脂質（商品名：トランスフェクタム、リポフェクトアミン）を用いることが好ましい。これらの陽イオン性脂質と陰電荷を持つ核酸との複合体を形成させると、全体として正に荷電しているリポソームが、負に荷電している細胞の表面に吸着し、細胞膜と融合できることで核酸を細胞内に導入することができる。切開手術または回復手術のように皮膚、腹膜、胸膜等を切開してがんの病巣を露出するか、あるいは、内視鏡による経皮経管的にがんの病巣にアクセスするかして、前記標的遺伝子阻害核酸、または、該記標的遺伝子阻害核酸をコードするポリヌクレオチド、および当該ポリヌクレオチドに機能可能に連結されたプロモーターを含む、発現ベクターを、病巣に直接注射することもできる。このようながんの病巣への直接局所投与は、前記標的遺伝子阻害核酸、または、該記標的遺伝子阻害核酸をコードするポリヌクレオチド、および当該ポリヌクレオチドに機能可能に連結されたプロモーターを含む、発現ベクターを全身投与するのに比べて、オフターゲット効果のような副作用を軽減できることがある。 The anticancer agent and the preventive and / or therapeutic agent for an infectious disease of the present invention are operably linked to the target gene-inhibiting nucleic acid, the polynucleotide encoding the target gene-inhibiting nucleic acid, and the polynucleotide. Expression vectors containing promoters can also be formulated using the lipofection method. For the lipofection method, liposomes usually composed of phosphatidylserine are used. Since phosphatidylserine has a negative charge, it is easier to make more stable liposomes as a substitute for phosphatidylserine. N- [1- (2,3-dioreyloxy) propyl] -N, N, N-triethylammonium chloride (DOTMA) ), Which is a cationic lipid (trade name: liposome, lipofectamine) is preferably used. When a complex of these cationic lipids and negatively charged nucleic acids is formed, positively charged liposomes as a whole can be adsorbed on the surface of negatively charged cells and fused with the cell membrane. Nucleic acid can be introduced into cells. The skin, peritoneum, thoracic membrane, etc. may be incised to expose the cancer lesion as in incision surgery or recovery surgery, or the cancer lesion may be accessed percutaneously by endoscopy. An expression vector comprising the target gene-inhibiting nucleic acid, or a polynucleotide encoding the target gene-inhibiting nucleic acid, and a promoter operably linked to the polynucleotide can also be injected directly into the lesion. Direct topical administration to such cancer lesions comprises expression of said target gene-inhibiting nucleic acid, or a polynucleotide encoding the target gene-inhibiting nucleic acid, and a promoter operably linked to the polynucleotide. Side effects such as off-target effects may be reduced compared to systemic administration of the vector.
　本発明の抗がん剤並びに感染症の予防及び/又は治療剤に含まれる前記有効成分の割合は、所望の効果を奏することができる範囲で適宜設定することができるが、通常、０．０１～１００重量％であり、好ましくは０．１～９９．９重量％、より好ましくは０．５～９９．５重量％である。 The ratio of the active ingredient contained in the anticancer agent and the preventive and / or therapeutic agent for infectious diseases of the present invention can be appropriately set within a range in which the desired effect can be obtained, but is usually 0.01. It is about 100% by weight, preferably 0.1 to 99.9% by weight, and more preferably 0.5 to 99.5% by weight.
　本発明のＴ細胞活性化剤がＧＡＢＡを介するシグナル伝達の阻害薬、Ｂ細胞におけるＧＡＢＡ生合成の阻害薬、Ｂ細胞におけるＧＡＢＡ分解の促進薬、Ｂ細胞におけるＧＡＢＡ分泌の阻害薬、Ｂ細胞におけるＧＡＢＡ再吸収の促進薬、および／または、遊離ＧＡＢＡの捕捉薬、ＧＡＢＡと特異的に結合する抗体、特異的結合パートナーおよび／またはそれらの断片の場合には、中枢神経または末梢神経に作用して精神または神経機能を変容させる副作用を惹起するおそれがある。本発明のＴ細胞活性化剤がＢ細胞に対する細胞傷害性を有する、抗体、特異的結合パートナーまたはそれらの断片、および／または、Ｂ細胞の除去剤の場合には、体内の液性免疫機能を弱体化させる副作用を惹起するおそれがある。そこで、本発明のＴ細胞活性化剤の治療又は予防上有効な量は、精神または神経機能を変容させたり、液性免疫機能を弱体化させたりする副作用よりも、細胞傷害性Ｔ細胞のがん細胞殺傷活性を抑制するＢ細胞の作用を阻害または低減する効果が上回る用量であるとよい。 The T cell activator of the present invention is an inhibitor of signal transduction mediated by GABA, an inhibitor of GABA biosynthesis in B cells, a promoter of GABA degradation in B cells, an inhibitor of GABA secretion in B cells, and GABA in B cells. In the case of reabsorption promoters and / or free GABA capture agents, GABA-specific binding antibodies, specific binding partners and / or fragments thereof, they act on the central or peripheral nerves and the psyche. Or it may cause side effects that alter nerve function. In the case of antibodies, specific binding partners or fragments thereof, and / or B cell scavengers, the T cell activator of the invention has cytotoxicity against B cells, humoral immune function in the body. May cause weakening side effects. Therefore, a therapeutically or prophylactically effective amount of the T cell activator of the present invention is associated with cytotoxic T cells rather than side effects such as altering mental or neurological function or weakening humoral immune function. The dose should be greater than the effect of inhibiting or reducing the action of B cells that suppress cytotoxic activity.
　投与される用量は、治療又は予防の対象となる個人の年齢、体重および状態と、投与経路、投与形態および方法とを考慮して注意深く調整されなければならず、そして正確な投与量は医師によって決定されなければならない。実際の投与量は医師の裁量の範囲内であり、望ましい治療効果を得るために本発明の特別な状況に対して用量を設定することにより変動することがある。しかし本発明のＴ細胞活性化剤の投与量としては、有効成分の種類、投与対象の体重や年齢、症状などにより一概に規定されるものではないが、１回につき体重１ｋｇあたり０．０００１ｍｇから１０００ｍｇの範囲で選ぶことが可能である。本発明の治療薬の投与回数は、特に限定されるものではないが、通常、１日当たり１～５回程度である。また、投与期間は、数日～１週間程度の短期服用であっても、数週間～数ヶ月程度の長期服用であってもよい。なお、相当程度の間隔を置いて前記疾患が再発した場合、本発明の治療及び/又は予防薬の再度の投与が可能である。 The dose to be administered must be carefully adjusted to take into account the age, weight and condition of the individual being treated or prevented, the route of administration, the form and method of administration, and the exact dose will be determined by the physician. Must be decided. The actual dosage is within the discretion of the physician and may vary by setting the dose for the particular circumstances of the invention in order to obtain the desired therapeutic effect. However, the dose of the T cell activator of the present invention is not unconditionally defined depending on the type of active ingredient, the body weight and age of the subject to be administered, symptoms, etc., but starts from 0.0001 mg per 1 kg of body weight at a time. It is possible to select in the range of 1000 mg. The number of administrations of the therapeutic agent of the present invention is not particularly limited, but is usually about 1 to 5 times per day. The administration period may be a short-term administration of several days to one week or a long-term administration of several weeks to several months. If the disease recurs at considerable intervals, the therapeutic and / or prophylactic agent of the present invention can be re-administered.
　本発明のＴ細胞活性化剤の投与回数は、特に限定されるものではないが、通常、１日当たり１～５回程度である。また、投与期間は、数日～１週間程度の短期服用であっても、数週間～数ヶ月程度の長期服用であってもよい。なお、相当程度の間隔を置いてがんや感染症が再発した場合、本発明のＴ細胞活性化剤を再度投与することもできる。 The number of administrations of the T cell activator of the present invention is not particularly limited, but is usually about 1 to 5 times per day. The administration period may be a short-term administration of several days to one week or a long-term administration of several weeks to several months. If the cancer or infectious disease recurs at considerable intervals, the T cell activator of the present invention can be administered again.
　本明細書において、「がん細胞を特異的に殺傷する」とは、がん細胞への殺傷効果ががん細胞以外の細胞への殺傷効果と比較して通常１.２倍以上、好ましくは、１.３倍以上、１.５倍以上、１.７倍以上、１.９倍以上、２倍以上、５倍以上、１０倍以上、２０倍以上、５０倍以上、１００倍以上、２００倍以上、５００倍以上、１０００倍以上、２０００倍以上、５０００倍以上、１００００倍以上高いことをいう。「感染細胞を特異的に殺傷する」についても同様である。また、本発明の標的遺伝子阻害核酸について「特異的に発現が抑制される」とは、標的遺伝子阻害核酸を添加した場合の標的遺伝子の発現が標的遺伝子阻害核酸を添加しなかった場合の標的遺伝子の発現と比較して通常１.２倍以上、好ましくは、１.３倍以上、１.５倍以上、１.７倍以上、１.９倍以上、２倍以上、５倍以上、１０倍以上、２０倍以上、５０倍以上、１００倍以上、２００倍以上、５００倍以上、１０００倍以上、２０００倍以上、５０００倍以上、１００００倍以上低いことをいう。 In the present specification, "specifically killing cancer cells" means that the killing effect on cancer cells is usually 1.2 times or more, preferably 1.2 times or more, as compared with the killing effect on cells other than cancer cells. 1.3 times or more, 1.5 times or more, 1.7 times or more, 1.9 times or more, 2 times or more, 5 times or more, 10 times or more, 20 times or more, 50 times or more, 100 times or more, 200 It means that it is more than double, 500 times or more, 1000 times or more, 2000 times or more, 5000 times or more, and 10000 times or more higher. The same applies to "specifically killing infected cells". Further, regarding the target gene-inhibiting nucleic acid of the present invention, "specifically suppressed expression" means that the expression of the target gene when the target gene-inhibiting nucleic acid is added is the target gene when the target gene-inhibiting nucleic acid is not added. Usually 1.2 times or more, preferably 1.3 times or more, 1.5 times or more, 1.7 times or more, 1.9 times or more, 2 times or more, 5 times or more, 10 times or more, as compared with the expression of It means that it is 20 times or more, 50 times or more, 100 times or more, 200 times or more, 500 times or more, 1000 times or more, 2000 times or more, 5000 times or more, and 10000 times or more lower.
　本明細書において、物質Ａが物質Ｂと「特異的に結合する」とは、物質Ａと物質Ｂとがかなり高い結合親和性を示し、かつ、物質Ａおよび物質Ｂ以外の物質との交差反応性を示さないことを指す。かなり高い結合親和性とは、１×１０－７Ｍ以下、１×１０－８Ｍ以下、さらに特に１×１０－9Ｍ以下またはよりさらに１×１０－10Ｍ以下の解離定数Ｋｄでの結合を指す。本発明の技術分野における物質Ａおよび物質Ｂの結合親和性は、表面プラズモン共鳴法を利用する測定装置（例えばＢｉａｃｏｒｅシステム（ＧＥヘルスケア・ジャパン株式会社））、反射型干渉分光法を利用する測定装置（例えばｆｏｒｔｅ－ＢＩＯシリーズ（日本ポール株式会社））を含むが、これらに限られない測定装置により測定することができる。また、物質Ａおよび物質Ｂについて「物質Ａおよび物質Ｂ以外の物質との交差反応性」は、例えば、競合結合アッセイ（例えばＥＬＩＳＡ）を用いて決定することができる。 As used herein, the term "specifically binding" to substance B means that the substance A and the substance B have a considerably high binding affinity, and the cross-reaction between the substance A and the substance other than the substance B. Refers to not showing sex. Significantly high binding affinities are those with a dissociation constant K d of 1 × 10 -7 M or less, 1 × 10 -8 M or less, and more particularly 1 × 10 -9 M or less or even more 1 × 10 -10 M or less. Refers to a bond. The binding affinity of substance A and substance B in the technical field of the present invention is measured by a measuring device using a surface plasmon resonance method (for example, Biacore system (GE Healthcare Japan Co., Ltd.)) or a reflection type interference spectroscopy. Measurement can be performed by a measuring device including, but not limited to, a device (for example, forte-BIO series (Nippon Pole Co., Ltd.)). Further, for substance A and substance B, "cross-reactivity with substances other than substance A and substance B" can be determined using, for example, a competitive binding assay (eg, ELISA).
　本発明の抗がん剤およびがんの治療方法について「治療」とは、本発明の抗がん剤およびがんの治療方法が効果を奏することを指し、がんが完全に消失した完全奏効または完全寛解（ＣＲ）と、部分奏効または部分寛解（ＰＲ、固形がんについてそのサイズが減少すること、血液がんではがん細胞の数が減少すること）と、腫瘍のサイズが変わらない「安定（ＳＤ）」状態とを含む。 About the anti-cancer agent of the present invention and the treatment method of cancer "Treatment" means that the anti-cancer agent of the present invention and the treatment method of cancer are effective, and the complete response to the complete disappearance of the cancer. Or complete remission (CR) and partial response or partial remission (PR, the size of solid cancers decreases, the number of cancer cells decreases in blood cancers), and the size of the tumor does not change. Includes "stable (SD)" state.
　本発明の抗がん剤およびがんの治療方法は、細胞傷害性Ｔ細胞のがん細胞殺傷活性を抑制する、Ｂ細胞と細胞傷害性Ｔ細胞との相互作用を阻害または低減することを作用機序とするため、化学療法剤、免疫療法剤、放射線照射療法等の既存の抗がん剤またはがんの治療方法と作用機序が異なる。したがって、本発明の抗がん剤およびがんの治療方法は、既存の任意の抗がん剤またはがんの治療方法と併用することができる。 The anticancer agent and the therapeutic method for cancer of the present invention act to suppress the cancer cell killing activity of cytotoxic T cells, and to inhibit or reduce the interaction between B cells and cytotoxic T cells. Because of the mechanism, the mechanism of action differs from that of existing anticancer agents such as chemotherapeutic agents, immunotherapeutic agents, and irradiation therapy, or cancer treatment methods. Therefore, the anti-cancer agent and the method for treating cancer of the present invention can be used in combination with any existing anti-cancer agent or method for treating cancer.
　本発明の感染症予防及び/又は治療剤並びに感染症予防及び/又は治療方法について「治療」とは、本発明の感染症予防及び/又は治療剤並びに感染症予防及び/又は治療方法が効果を奏することを指し、感染症による症状の消失や緩和がもたらされること、体内から病原体が検出されなくなること、病原体に対する中和抗体が産生されることなどを含む。「予防」とは、病原体への感染を防ぐことの他、感染症の原因となる病原体がからだに侵入しても、症状が現れない、または症状が軽くなることを含む。 About the infectious disease prevention and / or therapeutic agent and the infectious disease prevention and / or treatment method of the present invention "Treatment" means that the infectious disease prevention and / or therapeutic agent and the infectious disease prevention and / or treatment method of the present invention are effective. It refers to the effect, including the disappearance and alleviation of symptoms due to infectious diseases, the elimination of pathogens in the body, and the production of neutralizing antibodies against pathogens. "Prevention" includes preventing infection with a pathogen, and even if a pathogen causing an infectious disease invades the body, the symptoms do not appear or the symptoms are alleviated.
　本発明の感染症予防及び/又は治療剤並びに感染症予防及び/又は治療方法は、細胞傷害性Ｔ細胞の感染細胞に対する殺傷活性を抑制する、Ｂ細胞と細胞傷害性Ｔ細胞との相互作用を阻害または低減することを作用機序とするため、既存の感染症予防及び/又は治療剤やワクチンによる治療方法と作用機序が異なる。したがって、本発明の感染症予防及び/又は治療剤並びに感染症予防及び/又は治療方法は、既存の感染症予防及び/又は治療剤やワクチンによる治療方法と併用することができる。 The infectious disease prevention and / or therapeutic agent and the infectious disease prevention and / or treatment method of the present invention suppress the killing activity of cytotoxic T cells against infected cells, and the interaction between B cells and cytotoxic T cells. Since the mechanism of action is inhibition or reduction, the mechanism of action differs from the existing methods of infectious disease prevention and / or treatment with therapeutic agents and vaccines. Therefore, the infectious disease prevention and / or therapeutic agent and the infectious disease prevention and / or treatment method of the present invention can be used in combination with the existing infectious disease prevention and / or treatment method using a therapeutic agent or vaccine.
　本明細書において、Ｂ細胞に対する細胞傷害性を有する、抗体、特異的結合パートナーまたはそれらの断片とは、単独で、あるいは、補体その他と複合体を形成して、Ｂ細胞を殺傷する能力を有する抗体か、特異的結合パートナーか、これらの断片を指す。 As used herein, an antibody, a specific binding partner or a fragment thereof, which is cytotoxic to B cells, has the ability to kill B cells alone or in a complex with complement or the like. Refers to an antibody having, a specific binding partner, or a fragment thereof.
　本明細書において、Ｂ細胞の除去剤とは、細胞傷害性Ｔ細胞のがん細胞や感染細胞に対する殺傷活性を抑制するＢ細胞を除去する活性を有する任意の薬剤を指す。本明細書のＢ細胞の除去剤には、ＣＤ２０を細胞表面に発現するＢ細胞を特異的に除去する抗ＣＤ２０モノクローナル抗体や、該モノクローナル抗体に由来するタンパク質、例えば、リツキシマブおよびそのバイオシミラーの他、腸管特異的なＣＤ１１ｂ陽性ＩｇＡ産生細胞を除去する腸内細菌に対する抗生物質を含む。 In the present specification, the B cell removing agent refers to any drug having an activity of removing B cells that suppresses the killing activity of cytotoxic T cells against cancer cells and infected cells. B cell removers herein include anti-CD20 monoclonal antibodies that specifically remove B cells that express CD20 on the cell surface, and proteins derived from the monoclonal antibodies, such as rituximab and its biosimilars. In addition, it contains an antibody against intestinal bacteria that eliminates intestinal-specific CD11b-positive IgA-producing cells.
　本明細書において、本発明の抗がん剤および／または本発明のがんの治療方法と併用する他の医薬または治療方法には、以下が含まれるが、これらに限られない。
・がん細胞を物理的に摘出除去する外科手術療法、
・アルキル化薬、白金化合物、代謝拮抗薬、トポイソメラーゼ阻害薬、微小管阻害薬、抗生物質を含むが、これらに限られない、化学物質を投与する化学療法および化学療法剤、・がん細胞を非自己として免疫細胞が攻撃する免疫療法および免疫療法剤、および
・エックス線、電子線、ガンマ線、中性子線を含むがこれらに限られない放射線の照射によりがん細胞の増殖を阻害し、死滅させる放射線療法。 As used herein, the anti-cancer agent of the present invention and / or other pharmaceutical agents or therapeutic methods used in combination with the method for treating cancer of the present invention include, but are not limited to, the following.
・ Surgery to physically remove cancer cells,
Chemotherapeutic and chemotherapeutic agents that administer chemicals, including but not limited to alkylating agents, platinum compounds, metabolic antagonists, topoisomerase inhibitors, microtube inhibitors, antibiotics, and cancer cells Chemotherapy and chemotherapeutic agents that are attacked by immune cells as non-self, and radiation that inhibits and kills cancer cells by irradiation with radiation, including but not limited to X-rays, electron beams, gamma rays, and neutron rays. Therapy.
　がんを治療するための化学療法薬には、６－Ｏ－（Ｎ－クロロアセチルカルバモイル）フマギロール、ブレオマイシン、メトトレキサート、アクチノマイシンＤ、マイトマイシンＣ、ダウノルビシン、アドリアマイシン、ネオカルチノスタチン、シトシンアラビノシド、フルオロウラシル、テトラヒドロフリル－５－フルオロウラシル、ピシバニール、レンチナン、レバミゾール、ベスタチン、アジメキソン、グリチルリチン、塩酸ドキソルビシン、塩酸アクラルビシン、塩酸ブレオマイシン、硫酸ヘプロマイシン、硫酸ビンクリスチン、硫酸ビンブラスチン、塩酸イリノテカン、シクロフォスファミド、メルファラン、ブスルファン、チオテパ、塩酸プロカルバジン、シスプラチン、アザチオプリン、メルカプトプリン、テガフール、カルモフール、シタラビン、メチルテストステロン、プロピオン酸テストステロン、エナント酸テストステロン、メピチオスタン、ホスフェストロール、酢酸クロルマジノン、酢酸リュープロレリン、酢酸ブセレリン等を含む。 Chemotherapeutic agents for the treatment of cancer include 6-O- (N-chloroacetylcarbamoyl) fumaguilol, bleomycin, methotrexate, actinomycin D, mitomycin C, doxorubicin, adriamycin, neocultinostatin, cytosine arabinoside. , Fluorouracil, Tetrahydrofuryl-5-Fluorouracil, Pisibanil, Lentinan, Revamizol, Bestatin, Azimexone, Glycyrrhizin, Doxorubicin Hydrochloride, Acralvisin Hydrochloride, Bleomycin Hydrochloride, Hepromycin Sulfate, Bincristin Sulfate, Binblastin Sulfate, Irinotecan Hydrochloride, Cyclophosphamide , Busulfan, thiotepa, procarbazine hydrochloride, cisplatin, azathiopurine, mercaptopurine, tegafur, carmofur, citarabin, methyltestosterone, propionate teststerone, enanthate testosterone, mepitiostane, phosfestol, chlormaginone acetate, leuprorelin acetate, bleomycin acetate, etc. include.
　前記他の医薬は、免疫チェックポイント阻害剤を含むこともできる。前記他の治療方法は、がん組織を外科的手術によって摘出除去すること、および／または、がん細胞を殺傷する放射線を照射することを含むこともできる。免疫チェックポイント阻害剤としては、抗PD-1抗体、抗CTLA-4抗体、抗PD-L1抗体を挙げることができるが、これらに限定されない。 The other pharmaceuticals may also contain immune checkpoint inhibitors. The other treatment method may also include surgical removal of the cancerous tissue and / or irradiation with radiation that kills the cancer cells. Examples of immune checkpoint inhibitors include, but are not limited to, anti-PD-1 antibody, anti-CTLA-4 antibody, and anti-PD-L1 antibody.
　本明細書において、本発明の感染症予防及び/又は治療剤並びに感染症予防及び/又は治療方法と併用する他の医薬または治療方法には、ワクチン接種、抗菌剤、抗ウイルス剤、駆虫剤などの薬物投与が含まれるが、これらに限られない。前記他の医薬は、免疫チェックポイント阻害剤を含むこともできる。免疫チェックポイント阻害剤としては、抗PD-1抗体、抗CTLA-4抗体、抗PD-L1抗体を挙げることができるが、これらに限定されない。 In the present specification, the infectious disease preventive and / or therapeutic agent of the present invention and other pharmaceutical or therapeutic methods used in combination with the infectious disease preventive and / or therapeutic method include vaccination, antibacterial agent, antiviral agent, insecticide and the like. Drug administration, but is not limited to these. The other pharmaceuticals may also include immune checkpoint inhibitors. Examples of immune checkpoint inhibitors include, but are not limited to, anti-PD-1 antibody, anti-CTLA-4 antibody, and anti-PD-L1 antibody.
　本明細書において数値について修飾する連体詞「約」は、当該数値の９０％以上、かつ、１１０％以内の数値範囲であることを意味する。例えば、「約４０塩基」とは、３６塩基以上４４塩基以内の数値範囲の塩基を指す。 In the present specification, the adnominal adjective "about" that modifies a numerical value means that the numerical value is in the numerical range of 90% or more and 110% or less of the numerical value. For example, "about 40 bases" refers to a base in a numerical range of 36 bases or more and 44 bases or less.
　本明細書において言及される全ての文献はその全体が引用により本明細書に取り込まれる。 All documents referred to herein are incorporated herein by reference in their entirety.
　以下に説明する本発明の実施例は例示のみを目的とし、本発明の技術的範囲を限定するものではない。本発明の技術的範囲は特許請求の範囲の記載によってのみ限定される。本発明の趣旨を逸脱しないことを条件として、本発明の変更、例えば、本発明の構成要件の追加、削除および置換を行うことができる。 The examples of the present invention described below are for illustration purposes only and do not limit the technical scope of the present invention. The technical scope of the invention is limited only by the description of the claims. Modifications of the present invention, for example, addition, deletion and replacement of the constituent elements of the present invention may be made on condition that the gist of the present invention is not deviated.
〔実施例１〕
　（１）　材料と方法
　（1.1）マウス
　マウス突然変異体のｍｕＭｔ－／－、Ｃｄ３ｅ－／－およびｒａｇ１－／－（いずれもバックグラウンドはＣ５７ＢＬ／６Ｊ系統）と、野生型マウスとは、理化学研究所生命医科学研究センター（ＩＭＳ　ＲＩＫＥＮ）においてＳＰＦ条件下で繁殖・維持された。無菌（ＧＦ）野生型マウスは理化学研究所生命医科学研究センターにおいてビニールアイソレータ内で出産維持された。Ｃ５７ＢＬ／６ＮまたはＣ５７ＢＬ／６Ｊ野生型マウスは日本クレアから購入した。解析には、同腹仔か、週齢／性別が適切にマッチしたマウスかを用いた。全ての動物実験は施設内の動物実験委員会から承認されたプロトコールに従って実施された。 [Example 1]
(1) Materials and methods (1.1) Mouse-mouse mutants muMt − / − , Cd3e − / − and rag1 − / − (all backgrounds are C57BL / 6J strains) and wild-type mice are RIKEN. It was propagated and maintained under SPF conditions at the Center for Biomedical Sciences (IMS RIKEN). Sterile (GF) wild-type mice were delivered and maintained in a vinyl isolator at the RIKEN Center for Integrative Medical Sciences. C57BL / 6N or C57BL / 6J wild-type mice were purchased from Claire Japan. For the analysis, littermates or mice with appropriate age / sex matching were used. All animal studies were performed according to a protocol approved by the institution's Animal Care and Use Committee.
　（1.2）フロー・サイトメトリー
　細胞は、以下の抗体で染色され、フロー・サイトメトリーはＢＤ　ＦＡＣＳ　Ａｒｉａ
　ＩＩフロー・サイトメトリーシステム（日本ベクトン・ディッキンソン株式会社）で実施された。抗ＣＤ８ａ（Ｂｉｏｌｅｇｅｎｄ（ＢｉｏＬｅｇｅｎｄ　Ｊａｐａｎ株式会社）、クローン５３－６．７）、抗ＴＣＲ－β（Ｂｉｏｌｅｇｅｎｄ、クローンＨ５７－５９７）、抗ＣＤ４（Ｂｉｏｌｅｇｅｎｄ、クローンＲＭ４－５）、抗ＣＤ６２Ｌ（Ｂｉｏｌｅｇｅｎｄ、クローンＭＥＬ－１４）、抗ＣＤ１１ｃ（Ｂｉｏｌｅｇｅｎｄ、クローンＮ４１８）、抗ＣＤ１１ｂ（Ｂｉｏｌｅｇｅｎｄ、クローンＭ１／７０）、抗ＣＤ３（Ｂｉｏｌｅｇｅｎｄ、クローン１４５－２Ｃ１１）、抗ＣＤ４５．２（Ｂｉｏｌｅｇｅｎｄ、クローン１０４）、抗Ｇｒａｎｚｙｍｅ　Ｂ（Ｂｉｏｌｅｇｅｎｄ、クローンＱＡ１６Ａ０２）、抗Ｐｅｒｆｏｒｉｎ（Ｂｉｏｌｅｇｅｎｄ、クローンＳ１６００９Ｂ）、抗ＣＤ９８（Ｂｉｏｌｅｇｅｎｄ、クローンＲＬ３８８）、抗ＩＦＮ－γ（ｅＢｉｏｓｃｉｅｎｃｅ（サーモフィッシャーサイエンティフィック株式会社）、クローンＸＭＧ１．２）、抗ＣＤ４４（ｅＢｉｏｓｃｉｅｎｃｅ、クローンＩＭ７）、抗Ｂ２２０（ｅＢｉｏｓｃｉｅｎｃｅ、クローンＲＡ３－６Ｂ２）、抗ＴＮＦ－α（ｅＢｉｏｓｃｉｅｎｃｅ、クローンＭＰ６－ＸＴ２２）、抗ＩｇＤ（ｅｂｉｏｓｃｉｅｎｃｅ、クローン１１－２６ｃ）、抗ＴＣＲ－β（ＢＤ（サーモフィッシャーサイエンティフィック株式会社）、クローンＨ５７－５９７）、抗ＩＬ－２（ＢＤ、クローンＪＥＳ６－５Ｈ４）、抗ＰＤ－１（ＢＤ、クローンＪ４３）、抗ＣＸＣＲ５（ＢＤ、２Ｇ８）、抗ＦＡＳ（ＢＤ、クローンＪｏ２）および抗ＩｇＡ（ＳｏｕｔｈｅｒｎＢｉｏｔｅｃｈ（コスモ・バイオ株式会社）、ポリクローナル抗体）。細胞内サイトカイン産生を測定するために、ＧｏｌｇｉＳｔｏｐ（商標、ＢＤ（日本ベクトン・ディッキンソン株式会社））存在下ＰＭＡおよびイオノマイシン（シグマ、シグマ　アルドリッチ　ジャパン合同会社）で細胞を4時間刺激した。細胞
内染色はＦｉｘａｔｉｏｎ／Ｐｅｒｍｅａｂｉｌｉｚａｔｉｏｎ　Ｓｏｌｕｔｉｏｎ　Ｋｉｔ（日本ベクトン・ディッキンソン株式会社）を用いて実施した。データはＦｌｏｗＪｏソフトウェア（ＦｌｏｗＪｏ，ＬＬＣ、日本ベクトン・ディッキンソン株式会社）を用いて解析した。 (1.2) Flow cytometry The cells are stained with the following antibody, and the flow cytometry is BD FACS Maria.
It was carried out by II Flow Cytometry System (Becton Dickinson Japan Co., Ltd.). Anti-CD8a (Biolegend (BioLegend Japan Co., Ltd.), clone 53-6.7), anti-TCR-β (Biolegend, clone H57-597), anti-CD4 (Biolegend, clone RM4-5), anti-CD62L (Biolegend, clone MEL) -14), anti-CD11c (Biolegend, clone N418), anti-CD11b (Biolegend, clone M1 / 70), anti-CD3 (Biolegend, clone 145-2C11), anti-CD45.2 (Biolegend, clone 104), anti-Granzyme B (Biolegend, clone 104). Biolegend, clone QA16A02), anti-Perforin (Biolegend, clone S16009B), anti-CD98 (Biolegend, clone RL388), anti-IFN-γ (eBioscience (Thermofisher Scientific Co., Ltd.), clone XMG1.2), anti-CD44. , Clone IM7), anti-B220 (eBioscience, clone RA3-6B2), anti-TNF-α (eBioscience, clone MP6-XT22), anti-IgD (ebioscience, clone 11-26c), anti-TCR-β (BD (thermofisher scientist)). Tiffic Co., Ltd.), clone H57-597), anti-IL-2 (BD, clone JES6-5H4), anti-PD-1 (BD, clone J43), anti-CXCR5 (BD, 2G8), anti-FAS (BD, clone) Jo2) and anti-IgA (SouthernBiotech (Cosmo Bio Co., Ltd.), polyclonal antibody). To measure intracellular cytokine production, cells were stimulated with PMA and ionomycin (Sigma, Sigma-Aldrich Japan GK) in the presence of GolgiStop (trademark, BD (Japan Becton Dickinson, Inc.)) for 4 hours. Intracellular staining was performed using Fixation / Permeabilization Solution Kit (Becton Dickinson, Inc., Japan). The data were analyzed using FlowJo software (FlowJo, LLC, Becton Dickinson, Inc., Japan).
　（1.3）セルソーティング
　ＢＤ　ＦＡＣＳ　Ａｒｉａ　ＩＩフロー・サイトメトリーシステム（日本ベクトン・ディッキンソン株式会社）を用いて野生型マウスからナイーブなＣＤ4またはＣＤ8Ｔ細胞（ＣＤ１１ｃ－、ＣＤ１１ｂ－、Ｂ２２０－、ＣＤ４＋またはＣＤ８＋、ＣＤ４４ｉｎｔ／－、ＣＤ６２Ｌ＋）と、セントラルメモリー（ＣＭ）ＣＤ４またはＣＤ８Ｔ細胞（ＣＤ１１ｃ－、ＣＤ１１ｂ－、Ｂ２２０－、ＣＤ４＋またはＣＤ８＋、ＣＤ４４ｈｉｇｈ、ＣＤ６２Ｌ＋）と、エフェクターメモリー（ＥＭ）ＣＤ４またはＣＤ８Ｔ細胞（ＣＤ１１ｃ－、ＣＤ１１ｂ－、Ｂ２２０－、ＣＤ４＋またはＣＤ８＋、ＣＤ４４ｈｉｇｈ、ＣＤ６２Ｌ－）と、Ｂ細胞（ＣＤ１１ｃ－、ＣＤ１１ｂ－、ＣＤ４－、ＣＤ８－、Ｂ２２０＋）と、ＣＤ１１ｂ／ｃ細胞（Ｂ２２０－、ＣＤ１１ｃ＋および／またはＣＤ１１ｂ＋）を、（腋窩、上腕および鼠径）リンパ節から選別した。胚中心（ＧＣ）または非ＧＣＴ細胞（ＴＣＲ－β＋、ＣＤ４＋、ＰＤ－１－またはＰＤ－１＋、ＣＸＣＲ５－またはＣＸＣＲ５＋）、非ＧＣＢ細胞（Ｂ２２０＋、ＩｇＤｈｉｇｈ）またはＧＣＢ細胞（Ｂ２２０＋、ＩｇＤ－、ＦＡＳ＋）をパイエル板（ＰＰ）から選別した。ＩｇＡ形質細胞（ＰＣ）を小腸固有層（ＳＩＬＰ）から選別した。選別された細胞は、代謝物の解析のために、ＰＢＳで洗浄し迅速に液体窒素で凍結した。 (1.3) cell sorting BD FACS Aria II flow cytometry system (Nippon Becton Dickinson Co., Ltd.) naive CD4 or CD8T cells from wild type mice using (CD11c -, CD11b -, B220 -, CD4 + or CD8 + , CD44 int / -, and CD62L +), central memory (CM) CD4 or CD8T cells (CD11c -, CD11b -, B220 -, CD4 + or CD8 +, CD44 high, and CD62L +), effector memory (EM) CD4 or CD8T cells (CD11c -, CD11b -, B220 -, CD4 + or CD8 +, CD44 high, CD62L - ) and, B cells (CD11c -, CD11b -, CD4 -, CD8 -, B220 +) and, CD11b / c cells (B220 -, CD11c + and / or CD11b +) were sorted from (axillary, brachial and inguinal) lymph nodes. Germinal center (GC) or non-GCT cells (TCR-β +, CD4 + , PD-1 - or PD-1 +, CXCR5 - or CXCR5 +), non-GCB cells (B220 +, IgD high) or GCB cells (B220 +, IgD -, it was sorted from FAS +) and Peyer's patches (PP). IgA plasma cells (PC) were sorted from the small intestine lamina propria (SILP). Selected cells were washed with PBS and rapidly frozen in liquid nitrogen for analysis of metabolites.
　（1.4）代謝物の解析
　代謝物の解析は、Miyajima, M.ら、（Ｎａｔ　Ｉｍｍｕｎｏｌ．　１８：１３４２　（２０１７））に記載のとおり実施された。簡潔には、凍結された組織または細胞は破砕され超純水に溶解した。ろ過後、溶媒は真空濃縮器（ＳｐｅｅｄＶａｃ、サーモフィッシャーサイエンティフィック株式会社）を用いて除去した。濃縮されたろ液を超純水に溶解して、代謝物の解析に供した。 (1.4) Analysis of metabolites Analysis of metabolites was carried out as described in Miyajima, M. et al. (Nat Immunol. 18: 1342 (2017)). Briefly, frozen tissue or cells were crushed and dissolved in ultrapure water. After filtration, the solvent was removed using a vacuum concentrator (SpeedVac, Thermo Fisher Scientific, Inc.). The concentrated filtrate was dissolved in ultrapure water and used for analysis of metabolites.
　（1.5）１３Ｃ５－標識および／または１５Ｃ２－標識グルタミントレーサー法
　１３Ｃ標識および／または１５Ｃ２－標識グルタミントレーサー法には、グルタミン不含培地に１３Ｃ５－標識および／または１５Ｃ２－標識Ｌ－グルタミン（２ｍＭ、大陽日酸株式会社）を添加した。 (1.5) 13 C 5 -labeled and / or 15 C 2 -labeled glutamine tracer method 13 C-labeled and / or 15 C 2 -labeled glutamine tracer method for 13 C 5 -labeled and / or 15 on glutamine-free medium. C 2 - labeled L- glutamine (2mM, Sanso Co., Ltd.) was added.
　（1.6）代謝物の解析
　代謝物の解析には液体クロマトグラフィー質量分析を用いた。液体クロマトグラフィー質量分析は、Ｑ　Ｅｘａｃｔｉｖｅ－四重極（Ｏｒｂｉｔｒａｐ）ハイブリッド質量分析計（ＭＳ）（サーモフィッシャーサイエンティフィック株式会社）と連結したＵｌｔｉＭａｔｅ　３０００－高速液体クロマトグラフィー（サーモフィッシャーサイエンティフィック株式会社）を用いて、Ｍｉｙａｊｉｍａ，Ｍ．ら、（Ｎａｔ　Ｉｍｍｕｎｏｌ．１８：１３４２（２０１７））に記載のとおり実施した。 (1.6) Analysis of metabolites Liquid chromatography-mass spectrometry was used for analysis of metabolites. Liquid Chromatography Mass Spectrometry is performed by UltiMate 3000-High Performance Liquid Chromatography (Thermo Fisher Scientific Co., Ltd.) linked with Q Executive-Orbitrap Hybrid Mass Spectrometer (MS) (Thermo Fisher Scientific Co., Ltd.). ), Miyajima, M. et al. Et al. (Nat Immunol. 18: 1342 (2017)).
　（1.7）培養下での活性化
　リンパ節から全Ｔ細胞またはＢ細胞を選別して、１０％（ｖ／ｖ）ＦＢＳ、１×ＭＥＭ
　ＮＥＡＡ、１０ｍＭ　ＨＥＰＥＳ、５０μＭ　２－メルカプトエタノール、１ｍＭ　ピルビン酸ナトリウム、１００Ｕ／ｍＬ　ペニシリン、１００Ｕ／ｍＬ　ストレプトマイシンを添加したＲＰＭＩ１６４０培地（富士フイルム和光純薬株式会社）中で２４時間または７２時間培養した。抗ＣＤ２８（２μｇ／ｍＬ、３７．５１、日本ベクトン・ディッキンソン株式会社）およびＩＬ－２（２０ｎｇ／ｍＬ、Ｒ＆Ｄシステムズ、フナコシ株式会社）の存在下で、抗ＣＤ３ｅ（２．５μｇ／ｍＬ、１４５－２Ｃ１１、日本ベクトン・ディッキンソン株式会社）が固定化された９６穴プレートを用いてＴ細胞を刺激した。抗ＩｇＭ（８μｇ／ｍＬ、Ｊａｃｋｓｏｎ　Ｉｍｍｕｎｏ　Ｒｅｓｅｒｃｈ、富士フイルム和光純薬株式会社）単独か、抗ＩｇＭ（８μｇ／ｍＬ）および抗ＣＤ４０（１μｇ／ｍＬ、日本ベクトン・ディッキンソン株式会社）か、ＬＰＳ（１００ｎｇ／ｍＬ、シグマ、シグマ　アルドリッチ　ジャパン合同会社）かでＢ細胞を刺激した。 (1.7) Activation in culture All T cells or B cells were selected from the lymph nodes and 10% (v / v) FBS, 1 × MEM.
The cells were cultured in RPMI1640 medium (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with NEAA, 10 mM HEPES, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, 100 U / mL penicillin, and 100 U / mL streptomycin for 24 hours or 72 hours. Anti-CD3e (2.5 μg / mL, 145- 2C11, Nippon Becton Dickinson Co., Ltd.) stimulated T cells using an immobilized 96-well plate. Anti-IgM (8 μg / mL, Jackson Immuno Research, Fujifilm Wako Junyaku Co., Ltd.) alone, anti-IgM (8 μg / mL) and anti-CD40 (1 μg / mL, Nippon Becton Dickinson Co., Ltd.), LPS (100 ng / B cells were stimulated with mL, Sigma, Sigma-Aldrich Japan LLC).
　（1.8）足蹠免疫付与
　マウス左後肢足蹠に完全フロイントアジュバント（ＣＦＡ）とニワトリオバルブミンとの１：１混合乳化物（個体あたり２０μｇ）で７日間免疫付与して、同側および反対側のリンパ節を単離して代謝物の解析に供した。 (1.8) Immunization of the footpad Immunization of the left hindlimb footpad of the mouse with a 1: 1 mixed emulsion of complete Freund's adjuvant (CFA) and chicken triovalbumin (20 μg per individual) for 7 days was performed on the ipsilateral and contralateral sides. Lymph nodes were isolated and subjected to metabolite analysis.
　（1.9）ヒト細胞の単離
　ヒト末梢血単核細胞（ＰＢＭＣｓ）をＦｉｃｏｌｌ勾配遠心により単離した。ＰＢＭＣｓをビオチン標識抗ＣＤ２０（Ｂｉｏｌｅｇｅｎｄ、クローン２Ｈ７）およびビオチン標識抗ＣＤ１９（Ｂｉｏｌｅｇｅｎｄ、クローンＨＩＢ１９）で染色して、抗ビオチンＭｉｃｒｏＢｅａｄｓ（ミルテニーバイオテク株式会社）と結合する分画をヒトＢ細胞として選別した。結合しない分画をヒトｐａｎ　Ｔ　ｃｅｌｌ　ｎｅｇａｔｉｖｅ　ｉｓｏｌａｔｉｏｎ　ｋｉt（ミルテニーバイオテク株式会社）を用いてヒトＴ細胞として選別した
。 (1.9) Isolation of human cells Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient centrifugation. PBMCs were stained with biotin-labeled anti-CD20 (Biolegend, clone 2H7) and biotin-labeled anti-CD19 (Biolegend, clone HIB19), and fractions binding to anti-biotin MicroBeads (Miltenyi Biotec, Inc.) were selected as human B cells. .. Fractions that did not bind were sorted as human T cells using a human pan T cell negative isolation kit (Miltenny Biotec, Inc.).
　（1.10）腫瘍モデル
　ＰＢＳ、ＤＭＳＯ、チアガビン（マウス１匹あたり８００μｇ、東京化成工業株式会社）、フルマゼニル（マウス１匹あたり１４μｇ、東京化成工業株式会社）またはピクロトキシン（マウス１匹あたり４０μｇ、アブカム株式会社）をマウスに１日おきに腹腔内注射した。あるいは、プラセボペレットまたはＧＡＢＡペレット（ペレットあたり３１．５ｍｇ、２１日間徐放用、Ｉｎｎｏｖａｔｉｖｅ　Ｒｅｓｅａｒｃｈ　ｏｆ　Ａｍｅｒｉｃａ（ＩＲＡ））を挿入し、１日後に、ＭＣ３８がん細胞（５×１０５個）を右側横腹に皮下注射した。第７日に腫瘍組織を回収し、フロー・サイトメトリー解析用にコラーゲナーゼ（富士フイルム和光純薬株式会社）で消化した。腫瘍体積を表記の日に測定し、以下の式を用いて計算した。
　　　　　π　×　（長さ×幅×高さ）／６ (1.10) Tumor model PBS, DMSO, Thiagabin (800 μg per mouse, Tokyo Chemical Industry Co., Ltd.), Flumazenil (14 μg per mouse, Tokyo Chemical Industry Co., Ltd.) or Picrotoxin (40 μg per mouse, Abcam Co., Ltd.) ) Was intraperitoneally injected into mice every other day. Alternatively, placebo pellets or GABA pellets (31.5 mg per pellet, sustained release for 21 days, Innovative Research of America (IRA)) were inserted, and one day later, MC38 cancer cells (5 x 10 5 ) were placed on the right flank. Was injected subcutaneously. On the 7th day, the tumor tissue was collected and digested with coragenase (Fujifilm Wako Pure Chemical Industries, Ltd.) for flow cytometry analysis. Tumor volume was measured on the indicated day and calculated using the formula below.
π x (length x width x height) / 6
　（1.11）統計分析
　統計分析をＰＲＩＳＭ（Ｇｒａｐｈｐａｄ）で実行した。両側独立ステューデントｔ－検定または反復測定分散分析（ＮＯＶＡ）を用いて分析を行った。 (1.11) Statistical analysis Statistical analysis was performed by PRISM (Graphpad). Analysis was performed using two-sided independent student's t-test or repeated measures analysis of variance (NOVA).
　（２）結果
（2.1）野生型マウスにおける各種リンパ球のＧＡＢＡ含量
　図２は、野生型マウスから摘出され、フロー・サイトメトリー法で単離された各種リンパ球のＧＡＢＡ含量の棒グラフである。誤差棒は各リンパ球タイプの試料間の標本平均の標準誤差を表す。標本平均の標準誤差とは、母集団からある数の標本を選ぶとき、選ぶ組み合わせによって標本平均がどの程度ばらつくかを、全ての組み合わせについての標準偏差で表したものをいう。グラフ縦軸は、小腸ＩｇＡ形質細胞（ＳＩ　ＩｇＡ　ＰＣ）、パイエル板（ＰＰ）の胚中心（ＧＣ）または非ＧＣ（ｎｏｎＧＣ）Ｔ細胞およびＢ細胞、リンパ節（ＬＮ）のＢ細胞（Ｂ２２０）、樹状細胞（ＤＣ）、ＣＤ４またはＣＤ８陽性エフェクターメモリー（ＥＭ）またはセントラルメモリー（ＣＭ）Ｔ細胞、ＣＤ４またはＣＤ８が陽性または陰性のナイーブＴ細胞（ｎａｉｖｅ）、ＣＤ４またはＣＤ８が陽性でＣＤ４４陽性の細胞（ＣＤ４４）の各細胞を表す。グラフ横軸は各細胞のＧＡＢＡ含量をナイーブなＣＤ４Ｔ細胞のＧＡＢＡ含量を１倍とする相対値を表す。図２に示すとおり、小腸ＩｇＡ形質細胞のＧＡＢＡ含量は際立って多く、その次に多いパイエル板非胚中心Ｂ細胞のＧＡＢＡ含量の２倍を超える。 (2) Results (2.1) GABA content of various lymphocytes in wild-type mice FIG. 2 is a bar graph of GABA content of various lymphocytes excised from wild-type mice and isolated by flow cytometry. Error bars represent the standard error of the sample mean between samples of each lymphocyte type. The standard error of the sample mean is the standard deviation of all combinations, which indicates how much the sample mean varies depending on the combination selected when a certain number of samples are selected from the population. The vertical axis of the graph shows small intestinal IgA plasma cells (SI IgA PC), germinal center (GC) or non-GC (nonGC) T cells and B cells of the Pier plate (PP), and B cells (B220) of lymph nodes (LN). Dendritic cells (DC), CD4 or CD8 positive effector memory (EM) or central memory (CM) T cells, CD4 or CD8 positive or negative naive T cells (naive), CD4 or CD8 positive and CD44 positive cells Represents each cell of (CD44). The horizontal axis of the graph represents a relative value in which the GABA content of each cell is multiplied by the GABA content of naive CD4T cells. As shown in FIG. 2, the GABA content of small intestinal IgA plasma cells is remarkably high, which is more than twice the GABA content of the next highest Peyer's patch nongerminal center B cells.
　（2.2）活性化リンパ節のＧＡＢＡ含量に与えるＢ細胞およびＴ細胞の免疫不全突然変
異の影響
　末梢での抗原刺激に対するリンパ節での適応免疫応答とＢ細胞におけるＧＡＢＡ産生との関係を古典的な足蹠免疫プロトコールを用いて検討した。完全フロイントアジュバントに乳化したオバルブミンタンパク質を野生型Ｃ５７ＢＬ/６系統のマウス（ＷＴ）、ＣＤ３欠失ホモノックアウトマウス（ＣＤ３－／－）、ＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ）、および、ＲＡＧ－１欠失ホモノックアウトマウス（ＲＡＧ－１－／－）のそれぞれに接種して７日後に、活性化（免疫した足蹠と同側（ｉｐｓｉ））リンパ節および非活性化（反対側（ｃｏｎｔｒａ））リンパ節の組織のＧＡＢＡ含量を比較した棒グラフを図３に示す。誤差棒は各リンパ節組織の試料間のＧＡＢＡ含量の標準偏差を表す。グラフの縦軸は、各組織のＧＡＢＡ含量を野生型マウス反対側リンパ節のＧＡＢＡ含量を１倍とする相対値を表す。横軸は、各組織が由来したリンパ節を表す。図３の「＊＊」、すなわち、ＣＤ３欠失ホモノックアウトマウス（ＣＤ３－／－）の免疫付与した後肢足蹠と同側（ｉｐｓｉ）および反対側（ｃｏｎｔｒａ）のリンパ節組織のＧＡＢＡ含量の有意差のｐ値は、０．００１１であった。図３の「＊＊＊＊」、すなわち、野生型マウス（ＷＴ）の免疫付与した後肢足蹠と同側（ｉｐｓｉ）および反対側（ｃｏｎｔｒａ）のリンパ節組織のＧＡＢＡ含量の有意差のｐ値は、０．０００１未満であった。Ｔ細胞およびその細胞系譜に属する細胞がほとんど分化しないＣＤ３欠失ホモノックアウトマウス（ＣＤ３－／－）では野生型マウス（ＷＴ）に次ぐＧＡＢＡ含量が検出されたが、Ｂ細胞およびその細胞系譜に属する細胞がほとんど分化しない、ＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ）、および、ＲＡＧ－１欠失ホモノックアウトマウス（ＲＡＧ－１－／－）では、同側リンパ節でも反対側リンパ節でも、ほとんどＧＡＢＡが検出されなかった。したがって同側リンパ節でＧＡＢＡを含む細胞は活性化Ｂ細胞またはその細胞系譜に属する細胞であることが示唆された。 (2.2) Effect of immunodeficiency mutations on B and T cells on the GABA content of activated lymph nodes The relationship between adaptive immune responses in lymph nodes to peripheral antigen stimulation and GABA production in B cells is classical. The study was performed using the footpad immunoprotocol. Wild-type C57BL / 6 strain mice (WT), CD3-deficient homoknockout mice (CD3-/- ), IgM-deficient homoknockout mice (mMT), and RAG-1 emulsified with a complete Freund's adjuvant. Seven days after inoculation into each of the deleted homozygous knockout mice (RAG-1 − / − ), activated (immune and ipsi) lymph nodes and deactivated (contra). A bar graph comparing the GABA content of lymph node tissue is shown in FIG. Error bars represent the standard deviation of GABA content between samples of each lymph node tissue. The vertical axis of the graph represents a relative value in which the GABA content of each tissue is multiplied by the GABA content of the lymph node opposite to the wild-type mouse. The horizontal axis represents the lymph node from which each tissue was derived. Significant GABA content of "**" in FIG. 3, ie, lymph node tissue ipsilateral and contra (ipsi) and contra (contra) immunized hindlimb footpads of CD3-deficient homo-knockout mice (CD3 − / −). The p value of the difference was 0.0011. “*****” in FIG. 3, that is, the p-value of the significant difference in GABA content of the lymph node tissues ipsilateral and contra-immunized hindlimb footpads of wild-type mice (WT). Was less than 0.0001. In CD3-deleted homo-knockout mice (CD3 − / − ) in which T cells and cells belonging to the cell lineage hardly differentiated, GABA content was detected next to wild-type mice (WT), but it belongs to B cells and their cell lineages. In IgM-deficient homo-knockout mice (mMT) and RAG-1 -deficient homo-knockout mice (RAG-1 -/- ), in which cells hardly differentiate, almost GABA is present in both ipsilateral and contralateral lymph nodes. Not detected. Therefore, it was suggested that the cells containing GABA in the ipsilateral lymph node are activated B cells or cells belonging to the cell lineage thereof.
　（2.3）活性化リンパ節のさまざまな細胞タイプにおけるＧＡＢＡ含量
　免疫付与した野生型マウスの後肢足蹠と同側（ｉｐｓｉ）および反対側（ｃｏｎｔｒａ）のリンパ節組織からフロー・サイトメトリー法で選別されたＣＤ４、ＣＤ８およびＢ２２０陽性細胞（それぞれ、ＣＤ４、ＣＤ８およびＢ２２０）と、反対側全リンパ節（ｔｏｔａｌ　ｃｏｎｔｒａ　ＬＮ）、同側全リンパ節（ｔｏｔａｌ　ｉｐｓｉ　ＬＮ）および同側マクロファージ/樹状細胞（ｉｐｓｉ　Ｍｆ／ＤＣ）とのＧＡＢＡ含量を示す棒グラ
フを図４に示す。誤差棒は各選別細胞の試料間のＧＡＢＡ含量の標準偏差を表す。ＧＡＢＡ含量の縦軸は、各選別細胞のＧＡＢＡ含量を反対側ＣＤ４陽性細胞のＧＡＢＡ含量を１倍とする相対値を表す。横軸は、各選別細胞の種類を表す。図４の「＊」、すなわち、免疫付与した後肢足蹠と同側の全リンパ節組織から選別されたＣＤ４陽性細胞（ｉｐｓｉ　ＣＤ４）および反対側のリンパ節組織から選別されたＣＤ４陽性細胞（ｃｏｎｔｒａ　ＣＤ４）のＧＡＢＡ含量の有意差のｐ値は、０．０２３２であった。図４の「＊＊」、すなわち、同側の全リンパ節組織から選別されたＢ２２０陽性細胞（ｉｐｓｉ　Ｂ２２０）および反対側のリンパ節組織から選別されたＢ２２０陽性細胞（ｃｏｎｔｒａ　Ｂ２２０）のＧＡＢＡ含量の有意差のｐ値は、０．００１５であった。図４の「＊＊＊」、すなわち、同側の全リンパ節組織から選別されたＣＤ８陽性細胞（ｉｐｓｉ　ＣＤ８）および反対側のリンパ節組織から選別されたＣＤ８陽性細胞（ｃｏｎｔｒａ　ＣＤ８）のＧＡＢＡ含量の有意差のｐ値は、０．００１０であった。図４の「＊＊＊＊」、すなわち、反対側全リンパ節（ｔｏｔａｌ　ｃｏｎｔｒａ　ＬＮ）と同側全リンパ節（ｔｏｔａｌ　ｉｐｓｉ　ＬＮ）とのＧＡＢＡ含量の有意差のｐ値は、０．００６２であった。図４に示すとおり、活性化リンパ節の組織のさまざまな細胞タイプのうち、Ｂ２２０を発現する細胞、すなわち、Ｂ細胞およびその細胞系譜に属する細胞で最もＧＡＢＡ含量が多かった。そこで、活性化リンパ節組織のＧＡＢＡの主な供給源はＢ細胞およびその細胞系譜に属する細胞である。 (2.3) GABA content in various cell types of activated lymph nodes Selected by flow cytometry from lymph node tissue ipsilaterally and contralateral to the hindlimb footpads of immunized wild mice. CD4, CD8 and B220 positive cells (CD4, CD8 and B220, respectively) and contralateral total lymph nodes (total control LN), ipsilateral total lymph nodes (total ipsi LN) and ipsilateral macrophages / dendritic cells (ipsi). A bar graph showing the GABA content with Mf / DC) is shown in FIG. Error bars represent the standard deviation of GABA content between samples of each sorted cell. The vertical axis of the GABA content represents a relative value in which the GABA content of each selected cell is multiplied by the GABA content of the contralateral CD4 positive cell. The horizontal axis represents the type of each selected cell. “*” In FIG. 4, that is, CD4 positive cells (ipsi CD4) selected from all lymph node tissues ipsilateral to the immunized hindlimb footpad and CD4 positive cells (contra) selected from lymph node tissue on the opposite side. The p value of the significant difference in GABA content of CD4) was 0.0232. The GABA content of "**" in FIG. 4, i.e., B220-positive cells (ipsi B220) selected from all ipsilateral lymph node tissue and B220-positive cells (contra B220) selected from contralateral lymph node tissue. The p value of the significant difference was 0.0015. “***” in FIG. 4, i.e., GABA content of CD8-positive cells (ipsi CD8) selected from all ipsilateral lymph node tissue and CD8-positive cells (contra CD8) selected from contralateral lymph node tissue. The p-value of the significant difference was 0.0010. The p value of "*****" in FIG. 4, that is, the significant difference in GABA content between the ipsilateral total lymph node (total control LN) and the ipsilateral total lymph node (total ipsi LN) was 0.0062. rice field. As shown in FIG. 4, among the various cell types of activated lymph node tissue, cells expressing B220, ie B cells and cells belonging to their cell lineage, had the highest GABA content. Therefore, the main source of GABA in activated lymph node tissue is B cells and cells belonging to their genealogy.
　（2.4）Ｂ細胞免疫不全がＳＰＦ環境下のＩｇＭ欠失ホモノックアウトマウスに接種されたＭＣ３８細胞由来の腫瘍の成長に与える影響
　図５は、ＳＰＦ環境下のＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ）または野生型マウス（ＷＴ）に接種されたＭＣ３８細胞由来の腫瘍の成長（腫瘍病巣の体積）の経時的変化を調べた折れ線グラフである。誤差棒は各測定日のマウス個体腫瘍体積の標準偏差を表す。縦軸は、ＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ）と、野生型マウス（ＷＴ）との腫瘍の体積（ｍｍ３）を表す。横軸は、腫瘍体積の測定日を表す。図５に示すとおり、ＩｇＭ欠失ホモノックアウトマウスのほうが野生型マウスより腫瘍の成長は抑制された。 (2.4) Effect of B cell immunodeficiency on the growth of tumors derived from MC38 cells inoculated into IgM-deficient homo-knockout mice under SPF environment. It is a broken line graph which investigated the time-dependent change of the tumor growth (tumor lesion volume) derived from MC38 cells inoculated into wild-type mice (WT). The error bars represent the standard deviation of the individual mouse tumor volume on each measurement day. The vertical axis represents the tumor volume (mm 3 ) between the IgM-deficient homozygous knockout mouse (mMT) and the wild-type mouse (WT). The horizontal axis represents the measurement date of the tumor volume. As shown in FIG. 5, IgM-deficient homozygous knockout mice suppressed tumor growth more than wild-type mice.
　（2.5）無菌条件下で飼育されたＢ細胞分化不全がマウスに接種されたＭＣ３８細胞由来の腫瘍の成長に及ぼす影響
　図６は、無菌条件下で飼育されたＢ細胞分化不全がマウスに接種されたＭＣ３８細胞由来の腫瘍の成長（腫瘍病巣の体積）に及ぼす影響を示す折れ線グラフである。誤差棒は各測定日の腫瘍体積の標準偏差を表す。縦軸は、ＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ）と、野生型マウス（ＷＴ）との腫瘍の体積（ｍｍ３）を表す。横軸は、腫瘍体積の測定日を表す。図６に示すとおり、ＩｇＭ欠失ホモノックアウトマウスのほうが野生型マウスより腫瘍の成長を抑制した。 (2.5) Effect of insufficiency of B cell differentiation bred under sterile conditions on the growth of tumors derived from MC38 cells inoculated into mice Figure 6 shows the effect of insufficiency of B cell differentiation bred under sterile conditions in mice. It is a broken line graph showing the influence on the growth (tumor lesion volume) of the tumor derived from MC38 cells. The error bars represent the standard deviation of the tumor volume on each measurement day. The vertical axis represents the tumor volume (mm 3 ) between the IgM-deficient homozygous knockout mouse (mMT) and the wild-type mouse (WT). The horizontal axis represents the measurement date of the tumor volume. As shown in FIG. 6, IgM-deficient homozygous knockout mice suppressed tumor growth more than wild-type mice.
　（2.6）無菌条件下で飼育されたＳＰＦ環境下のＢ細胞分化不全マウスに接種されたＭＣ３８細胞由来の腫瘍の成長に及ぼすＧＡＢＡ徐放ペレット処置の影響
　図７は、無菌条件下で飼育されたＳＰＦ環境下のＢ細胞分化不全マウスに接種されたＭＣ３８細胞由来の腫瘍の成長に及ぼすＧＡＢＡ徐放ペレット処置の影響を示す折れ線グラフ。誤差棒は各測定日の腫瘍体積の標準偏差を表す。縦軸は、プラセボペレットまたはＧＡＢＡ徐放ペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ（＋ｐｌａｃｅｂｏ）またはｍＭＴ（＋ＧＡＢＡ））と、野生型マウス（ＷＴ）との腫瘍の体積（ｍｍ３）を表す。横軸は、腫瘍体積の測定日を表す。図７に示すとおり、野生型マウス（ＷＴ）と比較してプラセボペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ（＋ｐｌａｃｅｂｏ））では腫瘍の成長が抑制された。しかし、ＧＡＢＡ徐放ペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ（＋ＧＡＢＡ））では、プラセボペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ（＋ｐｌａｃｅｂｏ））と比較して腫瘍の成長を促進した。すなわち、ＩｇＭ欠失ホモノックアウトマウスでの腫瘍の成長抑制効果をＧＡＢＡ徐放ペレットが阻害した。 (2.6) Effect of GABA sustained-release pellet treatment on the growth of tumors derived from MC38 cells inoculated into B cell differentiation-deficient mice in an SPF environment bred under sterile conditions Figure 7 was bred under sterile conditions. A broken line graph showing the effect of GABA sustained release pellet treatment on the growth of tumors derived from MC38 cells inoculated into B cell differentiation-deficient mice under SPF environment. The error bars represent the standard deviation of the tumor volume on each measurement day. The vertical axis represents the tumor volume (mm 3 ) between IgM-deficient homozygous knockout mice (mMT (+ placebo) or mMT (+ GABA)) treated with placebo pellets or GABA sustained-release pellets and wild-type mice (WT). .. The horizontal axis represents the measurement date of the tumor volume. As shown in FIG. 7, tumor growth was suppressed in IgM-deficient homozygous knockout mice (mMT (+ placebo)) treated with placebo pellets as compared to wild-type mice (WT). However, IgM-deficient homozygous knockout mice (mMT (+ GABA)) treated with GABA sustained-release pellets promoted tumor growth compared to IgM-deficient homozygous knockout mice (mMT (+ placebo)) treated with placebo pellets. .. That is, GABA sustained-release pellets inhibited the tumor growth-suppressing effect in IgM-deficient homozygous knockout mice.
　（2.7）Ｂ細胞分化不全マウスへのＧＡＢＡ徐放ペレット処置が腫瘍浸潤ＣＤ８陽性細胞の活性化に及ぼす影響
　図８は、Ｂ細胞分化不全マウスへのＧＡＢＡ徐放ペレット処置が腫瘍浸潤ＣＤ８陽性細胞の活性化に及ぼす影響を示す２パラメーターヒストグラムの組み合わせ図である。図８の左の上下はプラセボペレットを処置した野生型マウス（ＷＴ（＋ｐｌａｃｅｂｏ））、中央および右はプラセボペレットまたはＧＡＢＡ徐放ペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ（＋ｐｌａｃｅｂｏ）またはｍＭＴ（＋ＧＡＢＡ））のフロー・サイトメトリーでＣＤ８陽性細胞をゲーティングした２パラメーターヒストグラムである。上段は、縦軸がＣＤ９８の蛍光強度、横軸がＴＣＲｂの蛍光強度を表す。下段は、縦軸がＳｃａ－１の蛍光強度、横軸がＴＣＲｂの蛍光強度を表す。ボックスの近傍の数値は、各条件のＣＤ８陽性細胞のうちボックス内の蛍光強度の細胞の百分率を表す。図８に示すとおり、プラセボペレットを処置した野生型マウス（ＷＴ（＋ｐｌａｃｅｂｏ））と比較してプラセボペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ（＋ｐｌａｃｅｂｏ））では、ＣＤ９８またはＳｃａ１陽性、かつ、ＴＣＲｂ陽性の腫瘍浸潤ＣＤ８陽性細胞が増加した。しかし、ＧＡＢＡ徐放ペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ（＋ＧＡＢＡ））では、プラセボペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ（＋ｐｌａｃｅｂｏ））と比較して、ＣＤ９８またはＳｃａ１陽性、かつ、ＴＣＲｂ陽性の腫瘍浸潤ＣＤ８陽性細胞は減少し、プラセボペレットを処置した野生型マウス（ＷＴ（＋ｐｌａｃｅｂｏ））よりも減少した。すなわち、ＩｇＭ欠失ホモノックアウトマウスによる腫瘍浸潤ＣＤ８陽性細胞の増加をＧＡＢＡ徐放ペレットが阻害した。 (2.7) Effect of GABA sustained-release pellet treatment on B cell differentiation-deficient mice on activation of tumor-infiltrating CD8-positive cells Figure 8 shows that GABA sustained-release pellet treatment on B-cell differentiation-deficient mice was performed on tumor-infiltrating CD8-positive cells. It is a combination diagram of a two-parameter histogram showing the effect on activation. The upper and lower left of FIG. 8 are wild-type mice treated with placebo pellets (WT (+ histogram)), and the center and right are IgM-deficient homoknockout mice treated with placebo pellets or GABA sustained-release pellets (mMT (+ histogram) or mMT (mMT (+ histogram)). + GABA)) is a two-parameter histogram obtained by gating CD8-positive cells by flow cytometry. In the upper row, the vertical axis represents the fluorescence intensity of CD98, and the horizontal axis represents the fluorescence intensity of TCRb. In the lower row, the vertical axis represents the fluorescence intensity of Sca-1, and the horizontal axis represents the fluorescence intensity of TCRb. The numerical value in the vicinity of the box represents the percentage of the cells having the fluorescence intensity in the box among the CD8-positive cells under each condition. As shown in FIG. 8, IgM-deficient homozygous knockout mice (mMT (+ placebo)) treated with placebo pellets were CD98 or Sca1 positive and compared to wild-type mice treated with placebo pellets (WT (+ placebo)). The number of TCRb-positive tumor-infiltrating CD8-positive cells increased. However, IgM-deficient homozygous knockout mice (mMT (+ GABA)) treated with GABA sustained-release pellets were CD98 or Sca1 positive, compared to IgM-deficient homozygous knockout mice (mMT (+ placebo)) treated with placebo pellets. Moreover, the number of TCRb-positive tumor-infiltrating CD8-positive cells was reduced compared to wild-type mice treated with placebo pellets (WT (+ placebo)). That is, GABA sustained-release pellets inhibited the increase of tumor-infiltrated CD8-positive cells by IgM-deficient homozygous knockout mice.
　（2.8）Ｂ細胞分化不全マウスへのＧＡＢＡ徐放ペレット処置が腫瘍浸潤ＣＤ８陽性細胞の細胞殺傷活性に及ぼす影響
　図９は、Ｂ細胞分化不全マウスへのＧＡＢＡ徐放ペレット処置が腫瘍浸潤ＣＤ８陽性細胞の細胞殺傷活性に及ぼす影響を示す２パラメーターヒストグラムの組み合わせ図である。図９の左の上下はプラセボペレットを処置した野生型マウス（ＷＴ（＋ｐｌａｃｅｂｏ））、中央および右はプラセボペレットまたはＧＡＢＡ徐放ペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ（＋ｐｌａｃｅｂｏ）またはｍＭＴ（＋ＧＡＢＡ））のフロー・サイトメトリーでＣＤ８陽性細胞をゲーティングした２パラメーターヒストグラムである。上段は、縦軸がパーフォリンの蛍光強度、横軸がＴＣＲｂの蛍光強度を表す。下段は、縦軸がＧｒｚＢ（グランザイムＢ）の蛍光強度、横軸がＴＣＲｂの蛍光強度を表す。ボックスの近傍の数値は、各条件のＣＤ８陽性細胞のうちボックス内の蛍光強度の細胞の百分率を表す。 (2.8) Effect of GABA sustained-release pellet treatment on B cell differentiation-deficient mice on cell killing activity of tumor-infiltrating CD8-positive cells Figure 9 shows that GABA sustained-release pellet treatment on B-cell differentiation-deficient mice is tumor-infiltrating CD8-positive cells. It is a combination diagram of a two-parameter histogram showing the influence on the cell killing activity of. The upper and lower left of FIG. 9 are wild-type mice treated with placebo pellets (WT (+ histogram)), and the center and right are IgM-deficient homoknockout mice treated with placebo pellets or GABA sustained-release pellets (mMT (+ histogram) or mMT (mMT (+ histogram)). + GABA)) is a two-parameter histogram obtained by gating CD8-positive cells by flow cytometry. In the upper row, the vertical axis represents the fluorescence intensity of perforin, and the horizontal axis represents the fluorescence intensity of TCRb. In the lower row, the vertical axis represents the fluorescence intensity of GrzB (Granzyme B), and the horizontal axis represents the fluorescence intensity of TCRb. The numerical value in the vicinity of the box represents the percentage of the cells having the fluorescence intensity in the box among the CD8-positive cells under each condition.
　図１０Ａは、図９の２パラメーターヒストグラムにもとづいて、Ｂ細胞分化不全マウスへのＧＡＢＡ徐放ペレット処置が腫瘍浸潤ＣＤ８陽性細胞のパーフォリン陽性細胞の割合に及ぼす影響を示す棒グラフである。縦軸はプラセボペレットを処置した野生型マウス（ＷＴ＋ｐｌａｃｅｂｏ）、プラセボペレットまたはＧＡＢＡ徐放ペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍＭＴ＋ｐｌａｃｅｂｏまたはｍＭＴ＋ＧＡＢＡｐ）のパーフォリン陽性細胞の百分率を表す。図１０Ａの「＊＊」、すなわち、プラセボペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍｕＭＴ－／－＋ｐｌａｃｅｂｏ）と、ＧＡＢＡ徐放ペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍｕＭＴ－／－＋ＧＡＢＡｐ）とのパーフォリン陽性細胞の割合の百分率の有意性のｐ値は０．００６であった。図１０Ａの「＊＊＊」、すなわち、プラセボペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍｕＭＴ－／－＋ｐｌａｃｅｂｏ）と、プラセボペレットを処置した野生型マウス（ＷＴ＋ｐｌａｃｅｂｏ）とのパーフォリン陽性細胞の割合の百分率の有意性のｐ値は０．００１２であった。 FIG. 10A is a bar graph showing the effect of GABA sustained release pellet treatment on B cell differentiation-deficient mice on the proportion of tumor-infiltrating CD8-positive cells to perforin-positive cells, based on the two-parameter histogram of FIG. The vertical axis represents the percentage of perforin-positive cells in wild-type mice treated with placebo pellets (WT + placebo), IgM-deficient homozygous knockout mice treated with placebo pellets or sustained-release GABA pellets (mMT + placebo or mMT + GABAp). “**” in FIG. 10A, ie, IgM-deficient homozygous knockout mice treated with placebo pellets (muMT − / − + placebo) and IgM-deficient homozygous knockout mice treated with GABA sustained-release pellets (muMT − / − + GABAp). The significance p value of the percentage of perforin-positive cells with was 0.006. “***” in FIG. 10A, that is, the proportion of perforin-positive cells between IgM-deficient homo-knockout mice treated with placebo pellets (muMT − / − + placebo) and wild-type mice treated with placebo pellets (WT + placebo). The p-value for the significance of the percentage was 0.0012.
　図１０Ｂは、図９の２パラメーターヒストグラムにもとづいて、Ｂ細胞分化不全マウスへのＧＡＢＡ徐放ペレット処置が腫瘍浸潤ＣＤ８陽性細胞のグランザイム陽性細胞の割合に及ぼす影響を示す棒グラフである。縦軸はプラセボペレットを処置した野生型マウス（ＷＴ＋ｐｌａｃｅｂｏ）、プラセボペレットまたはＧＡＢＡ徐放ペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍｕＭＴ－／－＋ｐｌａｃｅｂｏまたはｍｕＭＴ－／－＋ＧＡＢＡｐ）のグランザイム陽性細胞の百分率を表す。図１０Ｂの「＊」、すなわち、プラセボペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍｕＭＴ－／－＋ｐｌａｃｅｂｏ）と、ＧＡＢＡ徐放ペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍｕＭＴ－／－＋ＧＡＢＡｐ）とのグランザイム陽性細胞の割合の百分率の有意性のｐ値は０．０２２であった。図１０Ｂの「＊＊」、すなわち、プラセボペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍｕＭＴ－／－＋ｐｌａｃｅｂｏ）と、プラセボペレットを処置した野生型マウス（ＷＴ＋ｐｌａｃｅｂｏ）とのグランザイム陽性細胞の割合の百分率の有意性のｐ値は０．００１２であった。 FIG. 10B is a bar graph showing the effect of GABA sustained release pellet treatment on B cell differentiation-deficient mice on the proportion of granzyme-positive cells of tumor-infiltrating CD8-positive cells, based on the two-parameter histogram of FIG. The vertical axis is the percentage of granzyme-positive cells in wild-type mice treated with placebo pellets (WT + placebo), IgM-deficient homozygous knockout mice treated with placebo pellets or sustained-release GABA pellets (muMT − / − + placebo or muMT − / − + GABAp). Represents. “*” In FIG. 10B, that is, IgM-deficient homozygous knockout mice treated with placebo pellets (muMT − / − + placebo) and IgM-deficient homozygous knockout mice treated with GABA sustained-release pellets (muMT − / − + GABAp). The significance p value of the percentage of granzyme-positive cells was 0.022. “**” in FIG. 10B, ie, the percentage of granzyme-positive cells between IgM-deficient homo-knockout mice treated with placebo pellets (muMT − / − + placebo) and wild-type mice treated with placebo pellets (WT + placebo). The significance p value of was 0.0012.
　図９、図１０Ａおよび図１０Ｂに示すとおり、プラセボペレットを処置した野生型マウス（ＷＴ＋ｐｌａｃｅｂｏ）と比較してプラセボペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍｕＭＴ－／－＋ｐｌａｃｅｂｏ）では、パーフォリンまたはグランザイム陽性、かつ、ＴＣＲｂ陽性の細胞傷害性腫瘍浸潤ＣＤ８陽性細胞が増加した。しかし、ＧＡＢＡ徐放ペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍｕＭＴ－／－＋ＧＡＢＡｐ）では、プラセボペレットを処置したＩｇＭ欠失ホモノックアウトマウス（ｍｕＭＴ－／－＋ｐｌａｃｅｂｏ）と比較して、パーフォリンまたはグランザイム陽性、かつ、ＴＣＲｂ陽性の細胞傷害性腫瘍浸潤ＣＤ８陽性細胞は減少し、プラセボペレットを処置した野生型マウス（ＷＴ＋ｐｌａｃｅｂｏ）と同程度まで減少した。すなわち、ＩｇＭ欠失ホモノックアウトマウスによる細胞傷害性腫瘍浸潤ＣＤ８陽性細胞の増加をＧＡＢＡ徐放ペレットが阻害した。 As shown in FIGS. 9, 10A and 10B, perforin or granzyme in IgM-deficient homoknockout mice (muMT − / − + placebo) treated with placebo pellets compared to wild-type mice treated with placebo pellets (WT + placebo). The number of positive and TCRb-positive cytotoxic tumor-infiltrating CD8-positive cells increased. However, IgM-deficient homoknockout mice (muMT − / − + GABAp) treated with GABA sustained-release pellets were perforin or granzyme compared to IgM-deficient homoknockout mice (muMT − / − + placebo) treated with placebo pellets. The number of positive and TCRb-positive cytotoxic tumor-infiltrating CD8-positive cells decreased to the same level as in wild-type mice (WT + placebo) treated with placebo pellets. That is, GABA sustained-release pellets inhibited the increase of cytotoxic tumor-infiltrating CD8-positive cells by IgM-deficient homozygous knockout mice.
　（2.9）野生型マウスに接種されたＭＣ３８細胞由来の腫瘍の成長に及ぼすＧＡＢＡＡレセプターの阻害薬の影響
　図１１は、野生型マウスに接種されたＭＣ３８細胞由来の腫瘍の成長に及ぼすＧＡＢＡＡレセプターの阻害薬の影響を示す折れ線グラフである。縦軸はチアガビン（ＧＡＴ（トランスポーター）阻害剤）投与動物（Ｔｉａｇａｂｉｎ）、ＰＢＳ投与動物（ＰＢＳ）およびピクロトキシン投与動物（Ｐｉｃｒｏｔｏｘｉｎ）の腫瘍の体積（ｍｍ３）を表す。横軸は、腫瘍体積の測定日を表す。最終測定日に最も腫瘍体積の大きい曲線はチアガビン（ＧＡＴ（トランスポーター）阻害剤）投与動物の腫瘍体積の経時的変化を表し、２番目に腫瘍体積の大きい曲線はＰＢＳ投与動物の腫瘍体積の経時的変化を表し、最も腫瘍体積の小さい曲線はピクロトキシン投与動物の腫瘍体積の経時的変化を示す。誤差棒は各測定日の腫瘍体積の標準偏差を表す。図１１の「＊＊＊」、すなわち、ＭＣ３８細胞接種後２３日に測定したＰＢＳ投与動物（ＰＢＳ）とピクロトキシン投与動物（Ｐｉｃｒｏｔｏｘｉｎ）との腫瘍の体積の有意差のｐ値は０．０００３であった。図１１の「＊＊＊＊」、すなわち、ＭＣ３８細胞接種後２５日および２７日に測定したＰＢＳ投与動物（ＰＢＳ）とピクロトキシン投与動物（Ｐｉｃｒｏｔｏｘｉｎ）との腫瘍の体積の有意差のｐ値は、いずれも、０．０００１未満であった。図１１に示すとおり、チアガビンは腫瘍体積の増加にほとんど影響がないのに比べて、ピクロトキシンは腫瘍体積の増加を有意に抑制した。 (2.9) Effect of GABA A receptor inhibitor on the growth of MC38 cell-derived tumors inoculated into wild-type mice FIG. 11 shows the GABA A receptor on the growth of MC38 cell-derived tumors inoculated into wild-type mice. It is a broken line graph showing the influence of the inhibitor of. The vertical axis represents the tumor volume (mm 3 ) of tiagabine (GAT (transporter) inhibitor) -administered animal (Tiagabine), PBS-administered animal (PBS) and picrotoxin-administered animal (Picrotoxin). The horizontal axis represents the measurement date of the tumor volume. The curve with the largest tumor volume on the final measurement day shows the time course of the tumor volume of the animals treated with thiagabin (GAT (transporter) inhibitor), and the curve with the second largest tumor volume shows the time course of the tumor volume of the animals treated with PBS. The curve with the smallest tumor volume shows the change over time in the tumor volume of the picrotoxin-administered animal. The error bars represent the standard deviation of the tumor volume on each measurement day. “***” in FIG. 11, that is, the p value of the significant difference in tumor volume between the PBS-administered animal (PBS) and the picrotoxin-administered animal (Picrotoxin) measured on the 23rd day after inoculation with MC38 cells was 0.0003. rice field. “*****” in FIG. 11, that is, the p-value of the significant difference in tumor volume between the PBS-administered animal (PBS) and the picrotoxin-administered animal (Picrotoxin) measured 25 days and 27 days after MC38 cell inoculation. Both were less than 0.0001. As shown in FIG. 11, tiagabine had little effect on the increase in tumor volume, whereas picrotoxin significantly suppressed the increase in tumor volume.
　（2.10）野生型マウスに接種されたＭＣ３８細胞由来の腫瘍の成長に及ぼすＧＡＢＡＡレセプターの阻害薬の影響
　図１２は、野生型マウスに接種されたＭＣ３８細胞由来の腫瘍の成長に及ぼすＧＡＢＡＡレセプターの阻害薬の影響を示す折れ線グラフである。縦軸はフルマゼニル（ＧＡＢＡＡレセプター阻害剤）投与動物またはＰＢＳ投与動物の腫瘍の体積（ｍｍ３）を表す。横軸は、腫瘍体積の測定日を表す。最終測定日に最も腫瘍体積の小さい曲線はフルマゼニル投与動物（Ｆｌｕｍａｚｅｎｉｌ）の腫瘍体積の経時的変化を示し、最も腫瘍体積の大きい曲線はＤＭＳＯ投与動物（ＤＭＳＯ）の腫瘍体積の経時的変化を示す。誤差棒は各測定日の腫瘍体積の標準偏差を表す。図１２に示すとおり、フルマゼニルは腫瘍体積の増加を抑制した。図１２の「＊＊」、すなわち、最終測定日に測定したＤＭＳＯ投与動物（ＤＭＳＯ）とフルマゼニル投与動物（Ｆｌｕｍａｚｅｎｉｌ）との腫瘍の体積の有意差のｐ値は、０．００９２であった。 (2.10) Effect of GABA A receptor inhibitor on the growth of MC38 cell-derived tumors inoculated into wild-type mice FIG. 12 shows the GABA A receptor on the growth of MC38 cell-derived tumors inoculated into wild-type mice. It is a broken line graph showing the influence of the inhibitor of. The vertical axis represents the tumor volume (mm 3 ) of a flumazenil (GABA A receptor inhibitor) -administered animal or a PBS-administered animal. The horizontal axis represents the measurement date of the tumor volume. The curve with the smallest tumor volume on the final measurement day shows the change over time in the tumor volume of the flumazenil-administered animal (Flumazenil), and the curve with the largest tumor volume shows the change over time in the tumor volume of the DMSO-administered animal (DMSO). The error bars represent the standard deviation of the tumor volume on each measurement day. As shown in FIG. 12, flumazenil suppressed the increase in tumor volume. The p-value of "**" in FIG. 12, that is, the significant difference in tumor volume between the DMSO-administered animal (DMSO) and the flumazenil-administered animal (Flumazenil) measured on the final measurement day was 0.0092.
　（2.11）野生型マウスに接種されたＭＣ３８細胞由来の腫瘍に浸潤するＣＤ８陽性細胞の組成に対するピクロトキシンの影響
　図１３は、野生型マウスに接種されたＭＣ３８細胞由来の腫瘍に浸潤するＣＤ８陽性細胞の組成に対するピクロトキシンの影響を示す２パラメーターヒストグラムの組み合わせ図である。左はＣＤ８の蛍光強度を横軸に、ＣＤ４５－２の蛍光強度を縦軸に表す野生型マウスに接種したＭＣ３８細胞由来の腫瘍に浸潤した細胞の２パラメーターヒストグラムである。右下は、ピクロトキシン処理した野生型マウスに接種したＭＣ３８細胞由来の腫瘍に浸潤した細胞の２パラメーターヒストグラムである。右上は対照実験の野生型マウスに接種したＭＣ３８細胞由来の腫瘍に浸潤した細胞の２パラメーターヒストグラムである。右上および右下は、ともに、側方散乱光の強度を縦軸に、前方散乱光の強度を横軸に表す。ゲーティング範囲の近傍の数字は測定された細胞全てのうちゲーティング範囲内の細胞の百分率を表す。図１３に示すとおり、ピクロトキシン処理した野生型マウスに接種したＭＣ３８細胞由来の腫瘍に浸潤した細胞は、対照実験の野生型マウスに接種したＭＣ３８細胞由来の腫瘍に浸潤した細胞に比べて、側方散乱光も前方散乱光も強い細胞の割合が多かった。これはピクトロトキシン処理したマウスの腫瘍に浸潤したＣＤ８陽性細胞が対照実験のマウスの腫瘍に浸潤したＣＤ８陽性細胞に比べて、細胞が大きくおよび細胞内部構造が複雑であることを示す。 (2.11) Effect of picrotoxin on the composition of CD8-positive cells infiltrating tumors derived from MC38 cells inoculated into wild-type mice FIG. 13 shows the effects of CD8-positive cells infiltrating tumors derived from MC38 cells inoculated into wild-type mice. It is a combination diagram of a two-parameter histogram showing the effect of picrotoxin on the composition. On the left is a two-parameter histogram of cells infiltrated into a tumor derived from MC38 cells inoculated into wild-type mice, with the fluorescence intensity of CD8 on the horizontal axis and the fluorescence intensity of CD45-2 on the vertical axis. The lower right is a two-parameter histogram of cells infiltrated into a tumor derived from MC38 cells inoculated into picrotoxin-treated wild-type mice. The upper right is a two-parameter histogram of cells invaded into a tumor derived from MC38 cells inoculated into wild-type mice in a control experiment. In both the upper right and the lower right, the intensity of the laterally scattered light is represented by the vertical axis, and the intensity of the forward scattered light is represented by the horizontal axis. The numbers near the gating range represent the percentage of all measured cells within the gating range. As shown in FIG. 13, cells infiltrated with MC38 cell-derived tumors inoculated into picrotoxin-treated wild-type mice were laterally compared with cells infiltrated with MC38 cell-derived tumors inoculated into wild-type mice in the control experiment. The proportion of cells with strong scattered light and forward scattered light was high. This indicates that the CD8-positive cells infiltrated into the tumor of the pictrotoxin-treated mouse are larger and the cell internal structure is more complicated than the CD8-positive cells infiltrated into the tumor of the control experiment mouse.
　図１４は、図１３の２パラメーターヒストグラムから算出したＣＤ８陽性で、かつ、側方散乱光および前方散乱光の強度も高い腫瘍浸潤Ｔ細胞の百分率を示す棒グラフである。縦軸は、ＣＤ８陽性で、かつ、側方散乱光および前方散乱光の強度も高いＴ細胞の百分率を表す。左は対照実験のマウス（Ｃｔｒ）の腫瘍浸潤細胞の百分率を、右はピクロトキシン処理マウス（Ｐｉｃ）の腫瘍浸潤細胞の百分率を表す。誤差棒は各細胞集団の細胞数の標準偏差を表す。図１３及び１４の結果から、図１１に示されるピクトロトキシン処理による腫瘍体積増加の抑制は、腫瘍に浸潤したＣＤ８陽性細胞の大型化及び細胞構造の複雑化と関連することを示唆する。 FIG. 14 is a bar graph showing the percentage of tumor-infiltrating T cells that are CD8 positive and have high intensities of laterally scattered light and forward scattered light calculated from the two-parameter histogram of FIG. The vertical axis represents the percentage of T cells that are CD8 positive and have high intensity of lateral and forward scattered light. The left shows the percentage of tumor infiltrating cells in the control experiment mouse (Ctr), and the right shows the percentage of tumor infiltrating cells in the picrotoxin-treated mouse (Pic). Error bars represent the standard deviation of the number of cells in each cell population. The results of FIGS. 13 and 14 suggest that the suppression of tumor volume increase by pictrotoxin treatment shown in FIG. 11 is associated with the enlargement of CD8-positive cells infiltrating the tumor and the complication of the cell structure.
〔実施例２〕
　癌におけるGABA効果を調べた。
（１）培養実験
　コントロール（DMSO) あるいはピクロトキシン（DMSOに溶解し、マウス１匹あたりに４０μg）をＭＣ３８細胞を接種する1日前からrag1遺伝子欠損マウスの腹腔内に投与し、2日ごとに繰り返し投与した時の腫瘍体積の経時的変化を測定した。結果を図１５に示す。図１５の縦軸はDMSOあるいはピクロトキシンを投与したrag1遺伝子欠損マウスの腫瘍の体積（ｍｍ３）を表す。横軸は、細胞摂取後の腫瘍体積の測定日を表す。誤差棒は各測定日のマウス個体腫瘍体積の標準偏差を表す。「ｎｓ」は統計的な有意差は認められなかったことを示す。 [Example 2]
The GABA effect on cancer was investigated.
(1) Culture experiment Control (DMSO) or picrotoxin (dissolved in DMSO, 40 μg per mouse) was administered intraperitoneally to rag1 gene-deficient mice from 1 day before inoculation with MC38 cells, and repeated every 2 days. The change in tumor volume over time was measured. The results are shown in FIG. The vertical axis in FIG. 15 represents the tumor volume (mm 3 ) of rag1 gene-deficient mice treated with DMSO or picrotoxin. The horizontal axis represents the measurement date of the tumor volume after ingestion of cells. The error bars represent the standard deviation of the individual mouse tumor volume on each measurement day. “Ns” indicates that no statistically significant difference was observed.
（２）rag1遺伝子欠損マウス（TとB細胞が欠損される）の実験
　コントロール（DMSO) あるいはピクロトキシン（DMSOに溶解し、マウス１匹あたりに４０μg）をＭＣ３８細胞を接種する1日前からrag1遺伝子欠損マウスの腹腔内に投与し、2日ごとに繰り返し投与した時の腫瘍体積の経時的変化を測定した。結果を図１６に示す。図１６の縦軸はDMSOあるいはピクロトキシンを投与したrag1遺伝子欠損マウスの腫瘍の体積（ｍｍ３）を表す。横軸は、細胞摂取後の腫瘍体積の測定日を表す。誤差棒は各測定日のマウス個体腫瘍体積の標準偏差を表す。「ｎｓ」は統計的な有意差は認められなかったことを示す。 (2) Experimental control (DMSO) or picrotoxin (dissolved in DMSO, 40 μg per mouse) of rag1 gene-deficient mice (T and B cells are deficient) is rag1 gene-deficient from 1 day before inoculation with MC38 cells. The changes over time in tumor volume were measured when the mice were administered intraperitoneally and repeatedly administered every two days. The results are shown in FIG. The vertical axis of FIG. 16 represents the tumor volume (mm 3 ) of rag1 gene-deficient mice treated with DMSO or picrotoxin. The horizontal axis represents the measurement date of the tumor volume after ingestion of cells. The error bars represent the standard deviation of the individual mouse tumor volume on each measurement day. “Ns” indicates that no statistically significant difference was observed.
考察
　GABAあるいはピクロトキシンはＭＣ３８培養細胞の増殖と生存率に影響を与えない。またピクロトキシンは野生型マウスにおけるＭＣ３８細胞由来の腫瘍の成長を抑制するが、 rag1遺伝子欠損マウス（TとB細胞を欠損したマウス）におけるＭＣ３８細胞由来の腫瘍の成長に影響を与えないことから、GABAあるいはピクロトキシンはＭＣ３８細胞に直接影響を及ぼすのではなく、免疫細胞（ TとB細胞）を介して、ＭＣ３８細胞由来の腫瘍の成長に影響を及ぼす事が示される。
Discussion GABA or picrotoxin does not affect the proliferation and viability of MC38 cultured cells. Picrotoxin also suppresses the growth of MC38 cell-derived tumors in wild-type mice, but does not affect the growth of MC38 cell-derived tumors in rag1 gene-deficient mice (T and B cell-deficient mice), and thus GABA. Alternatively, picrotoxins have been shown to affect the growth of MC38 cell-derived tumors via immune cells (T and B cells) rather than directly affecting MC38 cells.
　本発明のＴ細胞活性化剤およびがんの治療や感染症の予防及び/又は治療方法は、Ｂ細胞による細胞傷害性Ｔ細胞抑制作用を阻害または低減するという全く新しい治療機序に基づくため、従来のすべての抗がん剤およびがんの治療方法、あるいは感染症の予防及び/又は治療剤ならびに感染症の予防及び/又は治療方法と併用することができる。また、従来神経伝達物質として広範に研究されてきたＧＡＢＡの阻害薬を用いるため、副作用のおそれが少ない点で優れている。 Since the T cell activator of the present invention and the treatment of cancer and the prevention and / or treatment method of infectious diseases are based on a completely new therapeutic mechanism of inhibiting or reducing the cytotoxic T cell inhibitory effect of B cells. It can be used in combination with all conventional anti-cancer agents and methods of treating cancer, or preventive and / or therapeutic agents of infectious diseases and methods of prevention and / or treatment of infectious diseases. In addition, since a GABA inhibitor, which has been widely studied as a neurotransmitter, is used, it is excellent in that there is little risk of side effects.

Claims (17)

  1. 　Ｂ細胞による細胞傷害性Ｔ細胞抑制作用を阻害または低減する、Ｔ細胞活性化剤。 A T cell activator that inhibits or reduces the cytotoxic T cell inhibitory effect of B cells.
  2. 　前記Ｂ細胞は抗原刺激により活性化されたＢ細胞である、請求項１に記載のＴ細胞活性化剤。 The T cell activator according to claim 1, wherein the B cell is a B cell activated by antigen stimulation.
  3. 　Ｔ細胞におけるガンマアミノ酪酸（ＧＡＢＡ）を介するシグナル伝達の阻害薬を含む、請求項１または２に記載のＴ細胞活性化剤。 The T cell activator according to claim 1 or 2, which comprises an inhibitor of gamma-aminobutyric acid (GABA) -mediated signal transduction in T cells.
  4. 　前記Ｔ細胞におけるＧＡＢＡを介するシグナル伝達の阻害薬は、ヒトＴ細胞で発現するＧＡＢＡレセプターの発現および／または機能を抑制または低減する阻害薬である、請求項３に記載のＴ細胞活性化剤。 The T cell activator according to claim 3, wherein the GABA-mediated signal transduction inhibitor in T cells is an inhibitor that suppresses or reduces the expression and / or function of GABA receptors expressed in human T cells.
  5. 　前記Ｔ細胞で発現するＧＡＢＡレセプターは、ＧＡＢＡＡレセプター、および、ＧＡＢＡ－ρレセプターのうちの少なくとも１つである、請求項４に記載のＴ細胞活性化剤。 The T cell activator according to claim 4, wherein the GABA receptor expressed in the T cells is at least one of the GABA A receptor and the GABA-ρ receptor.
  6. 　前記Ｔ細胞で発現するＧＡＢＡレセプターは、ヒトＴ細胞で発現するサブユニットポリペプチドからなる、請求項４または５に記載のＴ細胞活性化剤。 The T cell activator according to claim 4 or 5, wherein the GABA receptor expressed in the T cells comprises a subunit polypeptide expressed in human T cells.
  7. 　前記ヒトＴ細胞で発現するサブユニットポリペプチドは、ヒトα１、α５、β１、πおよびρ２からなる群から選択される少なくとも１種類である、請求項６に記載のＴ細胞活性化剤。 The T cell activator according to claim 6, wherein the subunit polypeptide expressed in the human T cell is at least one selected from the group consisting of human α1, α5, β1, π and ρ2.
  8. 　前記Ｔ細胞におけるＧＡＢＡを介するシグナル伝達の阻害薬は、前記ヒトＴ細胞で発現するヒトα１、α５、β１、πおよびρ２からなる群から選択される少なくとも１種類のサブユニットポリペプチドの発現を抑制または低減する、アンチセンス核酸、ＲＮＡｉ誘導性核酸もしくはリボザイムまたはそれらの発現ベクターを含む、請求項７に記載のＴ細胞活性化剤。 The GABA-mediated signal transduction inhibitor in the T cells suppresses the expression of at least one subunit polypeptide selected from the group consisting of human α1, α5, β1, π and ρ2 expressed in the human T cells. The T cell activator according to claim 7, which comprises an antisense nucleic acid, an RNAi-inducible nucleic acid or a ribozyme, or an expression vector thereof, which reduces or reduces.
  9. 　前記Ｔ細胞におけるＧＡＢＡを介するシグナル伝達の阻害薬は、前記ヒトＴ細胞で発現するヒトα１、α５、β１、πおよびρ２からなる群から選択される少なくとも１種類のＧＡＢＡレセプターのサブユニットポリペプチドと特異的に結合して、該ＧＡＢＡレセプターの機能を抑制または低減する、抗体、特異的結合パートナーまたはそれらの断片からなる群から選択される少なくとも１種類を含む、請求項７に記載のＴ細胞活性化剤。 The GABA-mediated signal transduction inhibitor in the T cells is a subunit polypeptide of at least one GABA receptor selected from the group consisting of human α1, α5, β1, π and ρ2 expressed in the human T cells. The T cell activity of claim 7, comprising at least one selected from the group consisting of antibodies, specific binding partners or fragments thereof that specifically bind to suppress or reduce the function of the GABA receptor. Agent.
  10. 　前記ＧＡＢＡＡレセプターの阻害薬が、フルマゼニル、Ｒｏ１５－４５１３、サルマゼニル、シクトキシン、エナントトキシン、ペンチレンテトラゾール、ピクロトキシン、ツジョン、リンデン、ビククリン、ガバジンおよびこれらの誘導体からなる群から選択される、少なくとも１種類である、請求項５に記載のＴ細胞活性化剤。 The GABA A receptor inhibitor is selected from the group consisting of flumazenil, Ro15-4513, sarmazenil, cicutoxin, enanthotoxin, pentylenetetrazol, picrotoxin, thujone, linden, bicuculline, gabazine and derivatives thereof. The T cell activator according to claim 5.
  11. 　前記ＧＡＢＡ－ρレセプターの阻害薬が、（１，２，５，６－テトラヒドロピリジン－４－イル）メチル・ホスフィン酸（ＴＰＭＰＡ）およびその誘導体からなる群から選択される少なくとも１種類である、請求項５に記載のＴ細胞活性化剤。 Claimed that the inhibitor of the GABA-ρ receptor is at least one selected from the group consisting of (1,2,5,6-tetrahydropyridine-4-yl) methyl phosphinic acid (TPMPA) and its derivatives. Item 5. The T cell activator according to Item 5.
  12. 　Ｂ細胞におけるＧＡＢＡ生合成の阻害薬、Ｂ細胞におけるＧＡＢＡ分解の促進薬、Ｂ細胞におけるＧＡＢＡ分泌の阻害薬、および／または、遊離ＧＡＢＡの捕捉薬を含む、請求項１または２に記載のＴ細胞活性化剤。 The T cell according to claim 1 or 2, which comprises an inhibitor of GABA biosynthesis in B cells, a promoter of GABA degradation in B cells, an inhibitor of GABA secretion in B cells, and / or a capture agent for free GABA. Activator.
  13. 　前記Ｂ細胞におけるＧＡＢＡ生合成の阻害薬は、グルタミン酸デカルボキシラーゼおよびアルデヒドデヒドロゲナーゼ９ファミリーメンバーＡ１の発現および／または酵素活性の阻害剤であり、および／または、前記Ｂ細胞におけるＧＡＢＡ分解の促進薬は４－アミノ酪酸アミノ基転移酵素の発現および／または酵素活性の促進剤であり、および／または、前記遊離ＧＡＢＡの捕捉薬は、遊離ＧＡＢＡに特異的に結合するタンパク質、遊離ＧＡＢＡと特異的に結合する抗体、特異的結合パートナーまたはそれらの断片からなる群から選択される少なくとも１種類である、請求項９に記載のＴ細胞活性化剤。 The inhibitor of GABA biosynthesis in the B cells is an inhibitor of the expression and / or enzymatic activity of glutamate decarboxylase and aldehyde dehydrogenase 9 family member A1, and / or the promoter of GABA degradation in the B cells is 4 -Aminobutyric acid Aminobutyric acid is an agent for promoting the expression and / or enzyme activity of aminotransferase, and / or the capture agent for free GABA specifically binds to free GABA, a protein that specifically binds to free GABA. The T cell activator according to claim 9, which is at least one selected from the group consisting of an antibody, a specific binding partner or a fragment thereof.
  14. 　ＧＡＢＡと特異的に結合する抗体、特異的結合パートナーおよび／またはそれらの断片を含む、請求項１または２に記載のＴ細胞活性化剤。 The T cell activator according to claim 1 or 2, which comprises an antibody that specifically binds to GABA, a specific binding partner and / or a fragment thereof.
  15. 　Ｂ細胞に対する細胞傷害性を有する、抗体、特異的結合パートナーまたはそれらの断片、および／または、Ｂ細胞の除去剤を含む、請求項１または２に記載のＴ細胞活性化剤。 The T cell activator according to claim 1 or 2, which comprises an antibody, a specific binding partner or a fragment thereof, and / or a B cell removing agent having cytotoxicity against B cells.
  16. 　抗がん剤として使用される、請求項１～１５のいずれかに記載のＴ細胞活性化剤。 The T cell activator according to any one of claims 1 to 15, which is used as an anticancer agent.
  17. 　感染症予防及び/又は治療剤として使用される、請求項１～１５のいずれかに記載のＴ細胞活性化剤。 The T cell activator according to any one of claims 1 to 15, which is used as an infectious disease preventive and / or therapeutic agent.
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US20170198359A1 (en) * 2016-01-07 2017-07-13 The Wistar Institute Of Anatomy And Biology Methods and compositions for treatment or diagnosis of cancers related to gabra3
WO2018236955A1 (en) * 2017-06-23 2018-12-27 The Regents Of The University Of California Enhancing gaba's ability to modulate immune responses

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114990062A (en) * 2022-04-28 2022-09-02 中国人民解放军军事科学院军事医学研究院 Method for promoting plasma cell generation or promoting differentiation of naive B cells into plasma cells in vitro and application thereof

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