WO2021085450A1 - Cellules du type mait chez les souris et souris riche en cellules mait - Google Patents

Cellules du type mait chez les souris et souris riche en cellules mait Download PDF

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WO2021085450A1
WO2021085450A1 PCT/JP2020/040361 JP2020040361W WO2021085450A1 WO 2021085450 A1 WO2021085450 A1 WO 2021085450A1 JP 2020040361 W JP2020040361 W JP 2020040361W WO 2021085450 A1 WO2021085450 A1 WO 2021085450A1
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cells
mait
mouse
cell
mice
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宏 若尾
智恵 杉本
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学校法人獨協学園獨協医科大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the present invention relates to mouse MAIT-like cells and mice rich in MAIT cells.
  • MAIT cells are a type of innate immune T cells that play a "bridge" between innate immunity and acquired immunity through the production of various cytokines and control the immune response of individuals.
  • MAIT cells are abundant in humans, for example, 20-50% in T cells in the liver, in intestinal lamina limba lymphocytes (LPL) and peripheral blood mononuclear cells (PBMC). While accounting for 1-10%, it is a rare cell in mice (Non-Patent Document 1: Dusseaux et al., 2011; Non-Patent Document 2: Le Bourhis et al., 2011).
  • MAIT cells are associated with autoimmune diseases such as multiple sclerosis, inflammatory diseases, and the onset and progression of cancer.
  • CD8 + / CD161 high T cells accumulate in inflammatory sites, such as liver or joints, it is a T cell population that is eye-onset factors of multiple sclerosis, is in human PBMC of CD8 + / CD161 high T cells
  • V ⁇ 7.2 + which is a MAIT cell-specific T cell receptor (TCR) ⁇ chain
  • Non-Patent Document 4 Illes et al., 2004; Non-Patent Document 5: Miyazaki et al. , 2011). Accumulation of MAIT cells has also been reported in renal cancer and brain tumors (Non-Patent Document 6: Peterfalvi et al., 2008) and chronic inflammatory demyelinating polyneuritis (Non-Patent Document 4: Illes et al., 2004). ing.
  • inflammatory bowel diseases such as ulcerative colitis and Crohn's disease
  • Reference 7 Xiao Ruijing et al., 2012).
  • MAIT cells are suggested to be involved in various diseases and pathological conditions, but detailed mechanisms such as immune control mechanism, especially role in immune response and its mechanism, factors and molecules contributing to it, and further At present, the study and analysis of the significance of the onset and progression of pathological conditions have not been sufficiently advanced.
  • One of the major reasons for this is the problem of cell / animal sources that can be used for in vitro and in vivo tests.
  • MAIT cells are a very rare cell population in mice that are frequently used as experimental animals, and it is difficult to analyze their functions using the animals.
  • MAIT cells are abundant in humans as compared with mice, there is a limit to preparing large amounts of MAIT cells from human biological samples such as peripheral blood. Further, in such a method, there is a high possibility that the number and properties of the obtained MAIT cells fluctuate greatly, and there is a difficulty in the stability and reproducibility of the test using the cells.
  • MAIT cells are usually in a state of having little cell proliferation ability, and it is difficult to amplify them under in vitro conditions because the factors and stimuli that induce their proliferation have not been identified ().
  • MAIT cells As a cell source used for so-called cell transplantation therapy, in which MAIT cells or artificially modified MAIT cells are transferred and treated in patients suffering from various infectious diseases, autoimmune diseases, and cancers. Can be used.
  • it is essential to establish a method for preparing a large amount of MAIT cells, which also has stable quality. Since MAIT cells do not respond to any previously known T cell proliferation stimulus, it has been difficult to prepare a large amount of MAIT cells required for functional analysis.
  • MAIT cells are a very rare cell population in mice that are frequently used as laboratory animals, and there is a limit to the progress of research and development using conventional laboratory mice.
  • mice expressing MAIT cells as frequently as humans have been sought.
  • mouse MAIT cell-derived iPS cells are known (Patent Document 1: Patent No. 6275646), but there is no specific description of a method for concentrating and purifying mouse MAIT cells to convert them into iPS cells.
  • the patent does not state that MAIT-like cells are effective for cell therapy only for bacterial infections and for cancer.
  • mice rich in MAIT cells that enable functional elucidation in disease models of MAIT cells have not been described.
  • the present inventor has succeeded in reprogramming mouse MAIT cells to produce MAIT cell-derived induced pluripotent stem cells (iPS cells).
  • iPS cells MAIT cell-derived induced pluripotent stem cells
  • by transferring the obtained MAIT-like cells into a mouse we succeeded in producing a mouse rich in MAIT-like cells.
  • Mice and V ⁇ 8 mice were obtained to complete the present invention.
  • the present invention includes the following embodiments.
  • a MAIT cell-enriched mouse obtained by transferring MAIT-like cells obtained by inducing differentiation of induced pluripotent stem cells in which mouse-derived MAIT cells have been reprogrammed.
  • the mouse according to (1) wherein the transfer of MAIT-like cells is into the abdominal cavity or via the tail vein.
  • the mouse having an allelic gene.
  • the mouse according to (3) which has an allele in which the V ⁇ 19 gene and the J ⁇ 33 gene are rearranged so as to be adjacent to each other.
  • the mouse according to (3) which has an allele in which the V ⁇ gene, the D gene and the J gene are rearranged so as to be adjacent to each other.
  • a model mouse for pathological analysis or drug screening which comprises the mouse according to any one of (1) to (7).
  • the mouse according to (8) which has a pathological condition of cancer.
  • Production of MAIT cell-enriched mouse which comprises isolating MAIT cells from the mouse according to any one of (1) to (9) and transferring the isolated MAIT cells into a wild-type mouse.
  • Method. (11) A method for screening a cancer metastasis inhibitor, which comprises contacting a mouse with the candidate substance according to any one of (1) to (9).
  • a therapeutic and / or prophylactic agent for cancer which comprises MAIT-like cells obtained by inducing differentiation of induced pluripotent stem cells in which MAIT cells have been reprogrammed.
  • artificial pluripotent stem cells can be prepared from mouse MAIT cells
  • mouse MAIT-like cells can be prepared from artificial pluripotent stem cells.
  • the mice into which the MAIT-like cells have been transferred are rich in MAIT-like cells, and also show suppression of cancer metastasis and prolongation of survival time.
  • inhibition of metastasis and prolongation of survival of transplanted cancer were also observed in V ⁇ 8 mice rich in MAIT cells derived from induced pluripotent stem cells from mouse MAIT cells.
  • m-reMAIT cell MAIT-like cell
  • MAIT-iPS cells were seeded on OP9 / dlk-1, which are feeder cells, and differentiated into m-re MAIT cells via mesoderm and lymphocyte immature cells.
  • m-reMAIT cells are defined as TCR ⁇ + mMR1Tet + cells.
  • the m-reMAIT cell purity after differentiation was 92% or more (left figure), and most of them were so-called double positives of CD4 + CD8 + (center figure).
  • the TCR ⁇ chain repatova of m-reMAIT cells derived from this iPS cell was V ⁇ 8 (right figure). It is a figure which shows the suppression of melanoma lung metastasis by m-reMAIT cell transfer.
  • Intraperitoneal administration of m-reMAIT cells (m-reMAIT cell transfer group) or PBS (control group) was performed, and 5 days later, B16F10 melanoma was transplanted via the tail vein. Eighteen days after melanoma transplantation, the lungs were removed and the number of cancer nodules was measured.
  • A Representative lungs of each group. Many black cancer nodules were found in the control group.
  • Lung cancer cell (LLC) transplanted mouse (dashed line)
  • m-reMAIT cell transfer + LLC transplanted mouse (solid line)
  • P value is based on Log rank test. It is a figure which shows the amount (percentage) of MAIT cell in the blood of a novel mouse via a chimeric mouse derived from MAIT-iPS cell.
  • Wild-type mice MAIT cell mass in (C57BL / 6) (percentage of MMR1Tet + cells numbers circled TCR [beta] + cells) (left), Vbeta8 mouse MAIT cell-specific gene reconstructed V ⁇ chain (Vbeta8 .2-D1-J1.2) the MAIT cell mass in mice peripheral blood with the allele of one side (the ratio of MMR1Tet + cells numbers circled TCR [beta] + cells) (middle), Varufa19 mouse MAIT cell specificity MAIT cell mass in mice peripheral blood with genetic reconstructed V ⁇ chain (V ⁇ 19-J ⁇ 33) alleles of one side (the ratio of MMR1Tet + cells numbers circled TCR [beta] + cells) (right).
  • V ⁇ 8 mouse a mouse which is genetically rich in MAIT cells. LLC was transplanted into wild-type (C57BL / 6) mice (broken line) and V ⁇ 8 mice (solid line), and the survival time was measured. The P value depends on the Log rank test. It is a figure which shows the TCR ⁇ and TCR ⁇ gene arrangement at the TCR locus in MAIT cell-derived iPS cell, V ⁇ 19 mouse, and V ⁇ 8 mouse, and the TCR ⁇ and TCR ⁇ gene arrangement in a wild-type mouse.
  • MAIT cell-derived iPS cells and V ⁇ 19 mice have gene-rearranged arrangements of MAIT cell-specific TCR ⁇ V ⁇ 19 and ⁇ J33, but this rearrangement is not observed in wild-type mice.
  • TCR ⁇ s V ⁇ 8 or V ⁇ 6 and D, J which are often found in MAIT cells, have a gene-rearranged arrangement, but this rearrangement is not observed in wild-type mice.
  • Figure 8 shows Ly5.2m-reMAIT cells (1.0 x 10 6 ) divided into a wild-type mouse (C57BL / 6) intraperitoneal adoptive group (also referred to as “L7-1”) and a non-transfer group.
  • the vertical axis represents the survival rate of mice, and the horizontal axis represents the number of days of survival. * Indicates that there is a significant difference due to the Log Rank test (correction of multiple comparisons by the Bonferroni method).
  • Figure 9 shows the group in which Ly5.2 m-reMAIT cells (1.0 x 10 6 ) were adopted into the abdominal cavity of wild-type mice (C57BL / 6) (also referred to as “L7-1”) and the non-transfer group.
  • C57BL / 6 wild-type mice
  • L7-1 wild-type mice
  • the vertical axis represents the survival rate of mice
  • the horizontal axis represents the number of days of survival. * Indicates that there is a significant difference due to the Log Rank test (correction of multiple comparisons by the Bonferroni method).
  • the present invention relates to a mouse rich in MAIT cells, in which mouse MAIT cells are reprogrammed (reprogrammed) to obtain iPS cells, and MAIT-like cells (m-reMAIT) induced to differentiate from the iPS cells are transferred.
  • the present invention also relates to MAIT cell-rich V ⁇ 19 mice and V ⁇ 8 mice established via chimeric mice produced from chimeric embryos into which the MAIT cell-derived iPS cells have been transferred.
  • the present invention also relates to a method for producing these mice. When cancer is transplanted after adopting m-reMAIT cells into wild-type mice, or when cancer is transplanted into V ⁇ 8 mice rich in MAIT cells, the transplanted mice are compared to wild-type mice. The effect of suppressing lung metastasis and significantly prolonging the survival time of mice was obtained.
  • MAIT cells are T cells with the highest abundance ratio in humans and are considered to be involved in pathological control of cancer, infectious diseases, autoimmune diseases, asthma, lifestyle-related diseases and skin diseases.
  • MAIT cells are extremely small in mice, which are frequently used in disease models, functional analysis in mice is extremely difficult.
  • the present inventor reprograms (iPS) mouse MAIT cells, induces differentiation of a large amount of mouse MAIT-like cells (hereinafter, also referred to as "m-reMAIT cells”) from the reprogrammed iPS cells, and chimeric mice.
  • m-reMAIT cells mouse MAIT-like cells
  • MHC that binds adaptive immune T cells such as CAR-T cells exhibits diversity
  • MR1 which is a molecule that binds MAIT cells, exhibits unity. Therefore, m-produced by the present invention. Immune cell therapy using reMAIT cells can be expected to have a uniform effect among individuals.
  • iPS cells are artificially differentiated pluripotency and self-renewal ability by introducing a reprogramming factor (nuclear reprogramming factor) into somatic cells and expressing it. Is a cell that has acquired traits and has similar traits to ES cells.
  • reprogramming factor nuclear reprogramming factor
  • Potency is defined as a cell capable of differentiating into cells of all lineages under appropriate conditions, but in the practice of the present invention, the ability to differentiate into cells of all lineages is not necessarily the case. It is not necessary to have MAIT cells, and it is sufficient that they have the ability to differentiate into MAIT cells and their stem cells or progenitor cells, and have the ability to differentiate into one or more other cell lines.
  • the traits similar to ES cells are the presence of surface marker molecules specific to ES cells, cell biological properties specific to ES cells such as teratoma forming ability, expression of genes specific to ES cells, or target cells. It can be defined by the degree of similarity in the expression patterns of a large number of gene groups in.
  • the pluripotency of the MAIT cell-derived induced pluripotent stem cells (iPS cells) obtained in the present invention is guaranteed by the ability to produce chimeric mice.
  • the "MAIT cell” is a T cell in which the TCR ⁇ chain gene is uniquely and uniformly composed of V ⁇ -J ⁇ (V ⁇ 19-J ⁇ 33 in mouse), and more preferably expresses CD26 or IL-18R ⁇ . It can be defined as a cell.
  • Another characteristic of MAIT cells is that their TCR is constrained by a single MR1.
  • the TCR ⁇ chain gene was biased to a specific combination, and one iPS cell obtained in the present invention showed gene-reconstituted V ⁇ 8.2, whereas another iPS cell showed V ⁇ 6 and V ⁇ 5. Met.
  • the MAIT cells used for reprogramming can be derived from mice.
  • Examples of the mouse used here include, but are not limited to, C57BL / 6, Balb / c, C3H, and DBA strains. Since MAIT cells in vivo have very poor proliferative capacity and techniques for in vitro proliferation have not been established, MAIT cells need to be collected from in vivo.
  • the collection site is not particularly limited, and examples thereof include umbilical cord blood, peripheral blood, lung, liver, thymus, spleen, bone marrow, and intestinal tract (lamina basement, Peyer's patch), but lung is preferable. ..
  • MAIT-iPS cells When MAIT cells are collected from tissues such as lung, the efficiency of inducing differentiation of iPS cells (MAIT-iPS cells) obtained by reprogramming MAIT cells into MAIT-like cells is higher than when MAIT cells are collected from umbilical cord blood. May be higher.
  • MAIT cells can be obtained, for example, by the following methods. For example, tissue is removed from wild-type mice, then shredded and crushed to become unicellular. Mononuclear cells are collected from these cells and washed. Recovery of MAIT cell fractions can be performed using, for example, APC-labeled 5-OP-RU-loaded murine MR1 tetramer reagents, anti-APC beads and MS columns. If the mononuclear cell fraction recovered from the tissue is extremely rich in other immune cells, the negative cell population is treated with one or more of biotin-labeled CD19, CD62L, Gr1, CD11b and TCR ⁇ antibodies, for example.
  • the recovered MAIT cell fraction is treated with a CD44 antibody, a B220 antibody, a mouse F4 / 80 antibody, and a TCR ⁇ antibody.
  • Each antibody may be labeled, for example, the CD44 antibody may be FITC labeled, the B220 antibody and mouse F4 / 80 antibody may be PE labeled, and the TCR ⁇ antibody may be PE-Cy7 labeled.
  • Cells were then washed with MACS buffer and the like, mMR1-Tet + TCR ⁇ + B220 - Gr1 - CD44 high (MAIT cells) purified by cell sorter.
  • a known reprogramming factor for reprogramming MAIT cells
  • a proteinaceous factor or a nucleic acid encoding the same (a form incorporated in a vector)
  • a proteinaceous factor or a nucleic acid encoding the same (a form incorporated in a vector)
  • a proteinaceous factor or a nucleic acid encoding the same (a form incorporated in a vector)
  • Or may be composed of any substance such as a low molecular weight compound.
  • four factors can be used: Oct3 / 4 gene product known as Yamanaka factor, Klf family gene product such as Klf4, Myc family gene product such as c-Myc, and Sox family gene product such as Sox2 (Takahashi K). , Yamanaka S. (2006). “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors”.
  • iPS cells can also be obtained by introducing three factors, Oct3 / 4 gene product, Klf family gene product, and Sox family gene product, and then culturing in the presence of basic fibroblast growth factor (bFGF) or the like. (See WO2007 / 69666).
  • bFGF basic fibroblast growth factor
  • the method of introducing the above factor as a protein is also adopted, or it is used in the form of a nucleic acid (DNA, RNA, DNA / RNA chimera) encoding the protein. You can also do it.
  • the nucleic acid (preferably cDNA) is inserted into a plasmid vector or viral vector capable of functioning in a host MAIT cell to construct an expression vector, and is subjected to a nuclear reprogramming step.
  • Sendai virus vector is a virus having a single-stranded non-segmented negative-strand RNA as a genome, and has been widely used in the field of cell biology.
  • the Sendai virus vector has the advantage that genes can be introduced into cells and tissues of many mammals, and the vector genome remains in the cytoplasm in the RNA state, so that the host chromosome is not affected. Kit products for constructing Sendai virus vectors are commercially available and can be appropriately obtained by those skilled in the art.
  • the iPS cells obtained by reprogramming MAIT cells oppose the TCR ⁇ chain gene whose gene rearrangement has been completed specifically for MAIT cells and / or the TCR ⁇ chain gene that selectively binds to this TCR ⁇ chain. It differs from the conventional pluripotent stem cells such as ES cells and iPS cells in that it has a gene.
  • a pluripotent stem cell having a TCR ⁇ chain gene whose gene rearrangement has been completed specifically for MAIT cells has characteristics similar to those of MAIT cells by being placed under conditions capable of inducing differentiation into T cells. It can be obtained to selectively produce cells.
  • the MAIT-iPS cells can be recovered in a high purity and in a large amount by a cell recovery, separation, purification method or the like by a known method.
  • MAIT-like cells are obtained by inducing differentiation of MAIT-iPS cells.
  • the cells induced to differentiate in this way have characteristics similar to those of MAIT cells, and since MAIT cells are derived from mice, they are referred to as "m-re MAIT cells" in the present invention.
  • m-reMAIT cells are mMR1-tetramer + (recognized by the MR1 tetramer molecule presented with 5-OP-RU) and TCR ⁇ + , but are characterized by very weak expression of the activation marker CD44. ..
  • MAIT cell-enriched mice V ⁇ 8 and V ⁇ 19 mice
  • MAIT cells are mMR1-tetramer + (recognized by the MR1 tetramer molecule presented with 5-OP-RU) and TCR ⁇ + , as well as CD44 is highly expressed, and the expression of the naive marker CD62L is very low.
  • the culture method for obtaining m-reMAIT-like cells by inducing differentiation is not particularly limited, and for example, a co-culture method with feeder cells, a suspension culture method, a hanging drop culture method, and a swirling culture method. , Soft agar culture method, microcarrier culture method and the like.
  • co-culture is preferable in order to induce differentiation of iPS cells to obtain m-reMAIT cells.
  • stromal cells such as OP9 cells are co-cultured as feeder cells, and further.
  • M-re MAIT cells can be efficiently obtained from MAIT-iPS cells.
  • the m-reMAIT cells thus obtained can be recovered, separated and purified by a known method.
  • Any known method for purifying m-reMAIT cells can be used as long as it is a known cell separation and purification method, but it is based on an antigen-antibody reaction such as a flow cytometer, magnetic beads, or a panning method. Examples thereof include a method and a cell fractionation method by density gradient centrifugation using a carrier such as sucrose or Percoll.
  • the m-reMAIT cells obtained by the present invention are cells exhibiting morphological, physiological and / or immunological characteristics almost equivalent to those of MAIT cells in vivo.
  • Identification of a MAIT-like cell can be made by confirming the expression of one or more markers specific to the MAIT cell. Marker expression should be confirmed by known cell tissue biological methods such as immunostaining using antibodies, reverse transcriptase-mediated polymerase chain reaction (RT-PCR), hybridization analysis, or molecular biological methods. Can be done.
  • the present invention is obtained by transferring MAIT-like cells obtained by inducing differentiation of induced pluripotent stem cells in which mouse-derived MAIT cells have been reprogrammed. , MAIT cell-enriched mouse, or a method for producing the MAIT cell-enriched mouse.
  • the m-reMAIT cells established by the present invention have almost the same cell surface antigen, gene expression, and cytokine production ability as in vivo MAIT cells (below), and by transferring into mice, the intestinal tract, spleen, and liver. Localized in tissues such as.
  • 1.0 x10 6 to 1.0 x10 7 cells may be suspended in a suitable buffer or Hanks balanced salt solution (HBSS) and injected intraperitoneally or via the tail vein.
  • HBSS Hanks balanced salt solution
  • Cell surface antigen mouse MR1 tetramer reagent (obtained from NIH) and anti-mouse TCR ⁇ antibody presenting 5-OP-RU
  • a chimeric mouse can be performed by a standard method.
  • the type of mouse used in the present invention is not particularly limited.
  • Balb / c system, ICR, etc. are used.
  • an ICR that is easy to handle and reproduce is preferable.
  • a chimeric embryo is prepared by transferring MAIT-iPS cells, which is the purpose of lineage establishment, into the embryo. Chimeric mice are produced by transplanting this chimeric embryo into the uterus of a pseudopregnant foster mother and giving birth.
  • the "embryo" to which m-reMAIT cells are transferred means an embryo in the stage from fertilization to birth in ontogeny, a 2-cell stage embryo, a 4-cell stage embryo, an 8-cell stage embryo, and a mulberry. Includes stage embryos, blastocysts, etc.
  • a known method such as a microinjection method or an agglutination method can be used.
  • a known method such as a microinjection method or an agglutination method can be used.
  • To prepare a chimeric embryo first, a female mouse that has been overovulated with a hormonal agent is mated with a male mouse. Then, for example, early developmental embryos are collected from the oviduct or uterus 2-2.5 days after fertilization when using 8-cell stage embryos and 3.5-4 days after fertilization when using blastocysts. .. M-reMAIT cells are injected into the collected embryos to prepare chimeric embryos.
  • MAIT-iPS cells are injected into the collected embryos with an injector.
  • MAIT-iPS cells may be mixed with normal embryos from which the zona pellucida has been removed and aggregated.
  • a pseudopregnant female mouse to be a foster parent can be obtained by mating a female mouse with a normal cycle with a male mouse castrated by vas deferens ligation or the like.
  • a chimeric mouse can be produced by intrauterine transplantation of a chimeric embryo prepared by the above-mentioned method into the created pseudopregnant mouse and then giving birth.
  • mice derived from MAIT-iPS cells are selected. After the male chimeric mouse with a high chimeric rate has matured, this mouse is mated with a female mouse of a pure mouse lineage. Then, it is possible that the gene-reconstructed TCR locus derived from MAIT cells was introduced into the germline of chimeric mice by the appearance of the coat color of mice derived from MAIT-iPS cells in the born offspring mice. I can judge.
  • mice From the mice thus obtained, offspring having a TCR ⁇ chain or TCR ⁇ chain locus for which MAIT cell-specific gene rearrangement has been completed for the allele is selected by the PCR method.
  • TCR ⁇ chain examples include V ⁇ 19 and J ⁇ 33
  • examples of the TCR ⁇ chain include the V ⁇ , D and J genes.
  • V ⁇ genes include V ⁇ 8.2, V ⁇ 8.1, V ⁇ 8.3, V ⁇ 6, V ⁇ 5.1, etc.
  • D genes include D1, D2, etc.
  • J genes include 12 types such as J1.2. J fragments (J1.1-J1.6 and J2.1-J2.6) of.
  • V ⁇ 19-J ⁇ 33 V ⁇ fragment, D fragment, and V (x) -D (y) -J (z) (y, z are the fragments described above, in which the V ⁇ 19 fragment and the J ⁇ 33 fragment are bound.
  • x reflects the genomic arrangement such as (preferably 8, 6 and 5).
  • V ⁇ 19 and J ⁇ 33 are adjacently arranged on one allele.
  • V ⁇ , D and J such as V ⁇ 8.2, D1 and J1-2, are adjacently arranged on one allele (Fig. 7).
  • the mice of the present invention are rich in MAIT cells and have more MAIT cells than wild-type mice.
  • the present invention provides a method for producing a MAIT cell-enriched mouse, which comprises isolating MAIT cells from the mice prepared as described above and transferring the isolated MAIT cells into a wild-type mouse. ..
  • a method for producing a MAIT cell-enriched mouse which comprises isolating MAIT cells from the mice prepared as described above and transferring the isolated MAIT cells into a wild-type mouse. ..
  • the same method as described in the above section "2. iPS conversion of mouse MAIT cells” can be adopted. After the MAIT cells are isolated, the MAIT cells are transferred to wild-type mice by intraperitoneal administration or via the tail vein.
  • the mouse of the present invention can be used as a model mouse for various disease studies. For example, since V ⁇ 8 mice show an anticancer effect, they can be used for screening for drugs that enhance the suppression of cancer metastasis by transplanting cancer cells into these mice and using the degree of metastasis as an index. In addition, since mice adopting m-reMAIT cells and V ⁇ 8 mice show prolongation of survival against transplanted cancers, they can be used to screen for drugs that prolong survival. Since the mouse of the present invention suppresses lung metastasis of melanoma and lung cancer cells, for example, a drug that further enhances this metastasis-suppressing activity can be screened as an anticancer agent. Therefore, the present invention provides a method for screening a cancer metastasis inhibitor, which comprises contacting a mouse prepared as described above with a candidate substance.
  • the present invention includes the following steps.
  • the candidate substance is not particularly limited, and may be an existing drug, or may be in any form such as a peptide, a low molecular weight compound, a high molecular weight compound, a salt or a precursor thereof.
  • "contact" is an embodiment in which a candidate substance is administered to a mouse. Administration may be oral or parenteral. That is, the administration route of the candidate substance is not particularly limited as long as it is a route generally adopted for administration of the drug, for example, oral, sublingual, nasal, transpulmonary, transdigestive tract, trans. Skin, eye drops, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, local injection, surgical transplantation are mentioned, and oral administration is preferable.
  • the item to be inspected in the step (b) is at least one of the following (i) to (vii).
  • (ii) Shrinkage of metastatic cancer cells iii) Mouse body weight (iv) Mouse feeding (v) Mouse survival
  • the candidate substance can be selected as a cancer metastasis inhibitor.
  • a candidate expected to have metastasis-suppressing activity may be administered to the mouse of the present invention, and whether or not the metastasis is suppressed may be evaluated.
  • MAIT cells are considered to be involved in pathological control in human infectious diseases, obesity / type II diabetes, allergies, asthma, and various autoimmune diseases. Therefore, by analyzing these disease models using the mice of the present invention (V ⁇ 19 mouse and V ⁇ 8 mouse), it is possible to obtain new findings on disease onset and pathological conditions that cannot be obtained by the conventional mouse model. For example, when the above disease model is created using V ⁇ 19 mice and V ⁇ 8 mice, it can be expected that the exacerbation of the pathological condition is suppressed or promoted as compared with the wild-type mouse, and in any case, the deterioration is expected. By elucidating the mechanism, new findings on the disease can be obtained.
  • the invention comprises MAIT-like cells obtained by inducing differentiation of induced pluripotent stem cells in which MAIT cells have been reprogrammed. And / or preventive agents.
  • the present invention comprises administering to a subject a MAIT-like cell obtained by inducing differentiation of induced pluripotent stem cells in which MAIT cells have been reprogrammed, or a therapeutic and / or prophylactic agent for cancer. Including cancer treatment and / or prevention methods.
  • the origin of MAIT cells is not limited, and examples thereof include mammals such as primates such as humans, experimental animals such as rats, mice, and rats, and domestic animals such as pigs, cows, horses, and sheep, and examples thereof include humans. May be derived from.
  • the cancer therapeutic and / or prophylactic agents described herein, or MAIT-like cells exert an anti-cancer effect without the intervention of CD8 cells.
  • the cancer therapeutic and / or prophylactic agents described herein, or MAIT-like cells exert an anti-cancer effect via NK cells.
  • the therapeutic and / or prophylactic agents for cancer of the present invention are known pharmaceutically acceptable carriers such as excipients, bulking agents, binders and lubricants, in addition to the MAI T-like cells described herein.
  • One or more components selected from known additives may be included.
  • the type of cancer is not limited, but for example, cancer in the skin, lung, stomach, pancreas, colonic rectum, liver, prostate, pancreas, esophagus, bladder, gallbladder / bile duct, breast, uterus, thyroid, and ovary. It may be a metastatic cancer.
  • the route of administration of the MAIT-like cells or cancer treatment and / or prophylactic agents described herein is not limited, but may be oral or parenteral, eg, intravenous, muscle, intraperitoneal. It can also be administered to a living body (target cells or organs) by intratumoral or subcutaneous injection; inhalation from the nasal cavity, oral cavity or lungs; or by a suppository, external preparation, or the like.
  • therapeutic and / or prophylactic agents for cancer described herein are not limited, and are tablets, capsules, powders, granules, pills, solutions, syrups, injections, topical agents, suppositories, eye drops. It may be an agent.
  • the dose of the therapeutic and / or prophylactic agent for cancer described in the present specification is appropriately selected depending on the type of active ingredient, the route of administration, the subject of administration, the age, weight, sex, symptoms and other conditions of the patient.
  • the number of administrations is not limited, and it can be administered once a day or in several divided doses.
  • the target of administration of the MAIT-like cells or cancer treatment and / or preventive agent described herein is, for example, mammals, for example, primates such as humans, experimental animals such as rats, mice, and rats, pigs, cows, and the like. Examples include domestic animals such as horses and sheep, and are humans, for example.
  • the monocytes present in the intermediate layer were collected and washed with PBS. After centrifugation, mononuclear cells were suspended in MACS buffer (PBS containing 0.5% BSA, 2 mM EDTA), biotin-labeled CD19 (clone 6D5), CD62L (clone MEL-14), Gr1 (clone RB6-8C5), CD11b. (Clone M1 / 70) and TCR ⁇ antibody (clone GL3) (both from Biolegend, 4 ⁇ g / ml each) were added and reacted at 4 ° C. for 15 minutes.
  • MACS buffer PBS containing 0.5% BSA, 2 mM EDTA
  • biotin-labeled CD19 clone 6D5
  • CD62L clone MEL-14
  • Gr1 clone RB6-8C5
  • CD11b CD11b.
  • TCR ⁇ antibody clone GL3
  • FITC-labeled CD44 antibody (clone IM7, Biolegend)
  • PE-labeled B220 antibody (clone RA3-6B2)
  • PE-labeled mouse F4 / 80 antibody (clone BM8)
  • PE-Cy7-labeled TCR ⁇ antibody (clone H57-597) (either Biolegend) was added to each 0.25 ⁇ g / 1x10 6 cells / 100 ⁇ L, and the mixture was left in a dark place at room temperature for another 15 minutes.
  • IPS cellization of mouse MAIT cells by Sendai virus vector Sendai virus (KOSM302L) carrying iPS-forming factor was donated by Dr. Masato Nakanishi, National Institute of Advanced Industrial Science and Technology.
  • the MAIT cells obtained as described above were infected with KOSM302L (WO2012 / 063817) to obtain iPS cells.
  • Ly5.2 MAIT cells 6,300 purified MAIT cells were centrifuged and anti-CD3 / CD28 antibody (15 ⁇ g / ml CD3 ⁇ (clone 145-2C11), 20 ⁇ g / ml CD28 (clone 37.51), both from Biolegend). Transfer to a coated 96-well plate and suspend in 20 ⁇ L KOSM302L (8,000 pfu / ⁇ L) and 80 ⁇ L cRPMI (RPMI culture medium containing 10% (v / v) FBS, 10 mM HEPES-NaOH pH 7.4). Inked at 37 ° C for 16 hours.
  • virus-infected MAIT cells were transferred to a 6-well plate in which mitomycin (MMC) -treated mouse fetal fibroblasts (MEF) were seeded as feeder cells, and 4 mL of mouse ES cell medium (StemSure DMEM) was transferred.
  • MMC mitomycin
  • MEF mouse fetal fibroblasts
  • Fujifilm Wako Pure Chemical Industries, Ltd. 15% (v / v) FBS (BioSera), x1 Non-essential amino acids (NEAA) (Fujifilm Wako Pure Chemical Industries, Ltd.), 2 mM glutamate (Fujifilm Wako Pure Chemical Industries, Ltd.), 100 U / mL penicillin / IPS colonies were formed by culturing in 100 ⁇ g / mL streptomycin (Lonza), 1,000 U / mL mouse LIF (Fujifilm Wako Pure Chemical Industries, Ltd.), 0.1 mM 2-mercaptoethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) for 4 weeks. ..
  • the medium was changed every 2 days, and CHIR99021 (Fujifilm Wako Pure Chemical Industries, Ltd., final concentration 3 ⁇ M) and PD0325901 (Fujifilm Wako Pure Chemical Industries, Ltd., final concentration 1 ⁇ M) were added from the 3rd day of culture for the purpose of naive iPS cells. Finally, 46 iPS colonies were acquired.
  • Ly5.1 MAIT cells Centrifuge 31,390 purified MAIT cells, suspend in 20 ⁇ L KOSM302L (8,000 pfu / ⁇ L) and 240 ⁇ L cRPMI, and shake slowly at 37 ° C for 2 hours and 45 minutes. did. After centrifugation, iPS colonies were formed in the same manner as in (1), and finally 36 iPS colonies were obtained.
  • iPS colonies were physically peeled from the MEF using a 27-gauge needle, aspirated with a P20 pipette (Nichiryo), and dispensed into a 96-well plate at 0.25% ( w / v) Trypsin-1 mM EDTA (TE) (Fujifilm Wako Pure Chemical Industries, Ltd.) Transferred to 80 ⁇ L. Incubate at 37 ° C. for 30 minutes, add 120 ⁇ L of cRPMI, suspend well and centrifuge. After centrifugation, iPS cells were transferred to a 12-well plate seeded with MMC-treated MEF, proliferated and cryopreserved.
  • TE Trypsin-1 mM EDTA
  • MAIT cell-derived iPS cells have a genetically rearranged MAIT cell-specific TCR locus genome arrangement (Fig. 7).
  • the TCR of MAIT cells is composed of ⁇ and ⁇ chains, and the ⁇ chain is limited to V ⁇ 19-J ⁇ 33.
  • the ⁇ chain has limited repatova such as V ⁇ 8 and V ⁇ 6, but is not uniquely determined. Therefore, by using the following primer set 1, V ⁇ 19-J ⁇ 33 for which gene rearrangement has been completed is detected by PCR.
  • primer set 2 P1 and P2 sequence
  • P1 5'-TCAACTGCACATACAGCACCTC-3' (SEQ ID NO: 1)
  • P2 5'-AGCTGCAGAGGTTAGCACAG-3' (SEQ ID NO: 2)
  • m-reMAIT cells differentiation of mouse MAIT-like cells was induced according to the method for inducing T cell differentiation from pluripotent stem cells (hereinafter referred to as m-reMAIT cells).
  • MAIT-iPS cells derived from Ly5.2 or Ly5.1
  • TE pluripotent stem cells
  • 1.2 x 10 5 cells were seeded on two confluent OP9 / dlk-1 (10 cm culture dishes).
  • 10% (v / v) FBS was cultured in ⁇ MEM (Fujifilm Wako Pure Chemical Industries, Ltd.) for 5 days to induce mesoderm.
  • mesoderm containing OP9 / dlk-1 was treated with 2 ml TE for 10 minutes to make it unicellular. 8 ml of the same medium was added, suspended well, and allowed to stand at 37 ° C. for 45 minutes. The mesoderm contained in the supernatant was collected by centrifugation, human FLT3 ligand (Biolegend) was added to a concentration of 5 ng / m, and two OP9 / dlk-1 (10 cm culture dishes) became confluent. It was sown on top and cultured.
  • lymphocyte immature cells were collected by pipetting and re-sown on confluent OP9 / dlk-1 cells (6-well plate), 20% (v / v) FBS (BioSerum), human FLT3 ligand (5 ng). / mL, Biolegend), cultivated in ⁇ MEM (Fujifilm Wako Pure Chemical Industries, Ltd.) containing mouse IL-7 (1 ng / mL, Biolegend). Then, when the lymphocyte immature cells proliferated, these cells were transferred onto the confluent OP9 / dlk-1 in a 10 cm culture dish, further proliferated, and flow cytometric analysis was performed.
  • Ly5.2m-reMAIT cells 1.0 x10 8 or more can be acquired (Fig. 1). Ly5.1m-reMAIT cells also showed a similar differentiation pattern.
  • mice 4. Anti-cancer effect using m-reMAIT cells
  • Ly5.2 m-reMAIT cells (1.0 x 10 6 ) obtained above were adopted into the abdominal cavity of wild-type mice (C57BL / 6), and 5 days later, mouse melanoma (B16F10) was transplanted via the tail vein. Eighteen days after B16F10 transplantation, mice were euthanized and the number of cancer nodules metastasized to the lungs was measured. As a result, cancer metastasis was significantly suppressed compared to the control group transplanted with B16F10 alone (Fig. 2A-). 2B).
  • mice transferred with Ly5.2m-reMAIT cells showed a significant prolongation of survival compared with non-transferred mice, which was also observed with Ly5.1m-reMAIT cell transfer. It was observed (Fig. 3).
  • prolongation of survival by Ly5.2 m-reMAIT cell transfer was also observed in mouse lung cancer Lewis lung carcinoma (LLC) (Fig. 4).
  • a new model mouse from MAIT cell-derived iPS cells (MAIT-iPS cells) via a chimeric mouse MAIT-iPS cells were used to prepare chimeric mice as follows. Three clones were selected from the obtained 46 (clones) Ly5.2 MAIT-iPS cells and injected into 8-cell embryos prepared from ICR. This injection was performed by the Nonprofit Organization Developmental Engineering Study Group (Osaka University Microbial Research Institute). Each clone was injected into 10-20 embryos and returned to 3 fostered female mice, resulting in a total of 11 male chimeric mice.
  • iPS cells are germline chimeras that contribute to the differentiation into primordial germ cells (sperm) in one mouse with a chimera rate of 60-90%, and this was confirmed as an 8-week-old female C57BL / 6 (Japan). It was mated with Claire) and black offspring were selected.
  • the black offspring may have inherited the reconstituted TCR locus, which is a gene derived from C57BL / 6 MAIT cells, and this was confirmed by PCR.
  • mice carrying V ⁇ 19-J ⁇ 33 or V ⁇ 8.2-D1-J1.2 produced wild-type and mice with these gene arrangements as offspring in a 1: 1 ratio according to Mendel's laws when mated with C57BL / 6. ..
  • mice having both V ⁇ 19-J ⁇ 33 and V ⁇ 8.2-D1-J1.2 were produced.
  • Mice carrying the V ⁇ 8.2-D1-J1.2 locus can be detected by PCR using the following primer set 3 (primers P4 and P6).
  • Primer set 3 Primer 4: 5'-GTACTGGTATCGGCAGGAC-3' (SEQ ID NO: 4)
  • Primer 6 5'-GAGCCGAAGGTGTAGTCGG-3' (SEQ ID NO: 6)
  • the wild type (not completed gene rearrangement) TCR ⁇ locus can be detected by PCR using the following primer set 4 (P4 and P5) (Fig. 7).
  • Primer set 4 Primer 4: 5'-GTACTGGTATCGGCAGGAC-3' (SEQ ID NO: 4)
  • 50 ⁇ L of blood was drawn from the buccal vein of V ⁇ 19, V ⁇ 8, and wild-type C57BL / 6 mice and transferred to a 1.5 ml tube containing 5 ⁇ L EDTA (100 mM). After stirring, 20 ⁇ L was placed in another 1.5 ml tube, PE-labeled anti-B220 antibody (0.05 ⁇ g), PE-labeled anti-Gr1 antibody (0.05 ⁇ g), APC-labeled mMR1tet (0.012 ⁇ g), PE-Cy7-labeled mouse TCR ⁇ antibody (0.05 ⁇ g). It was mixed with ⁇ g) and inked for 45 minutes in a dark place at room temperature.
  • mice had tens to hundreds of times more MAIT cells than C57BL / 6 (Fig. 5).
  • MAIT cells have an anticancer effect (not specific to cancer antigens)
  • m-reMAIT cells can be used as a cell therapy method for cancer
  • a mouse POC Proof of Concept
  • 1.0 x10 6 L7-1 was administered 5 days before LLC administration, and 200 ⁇ g of anti-CD8 antibody (clone 2.43: Bio X cell) or the same amount of rat IgG2b isotype control (clone LTF2:) was administered the day before LLC administration. Bio X cell) was administered intraperitoneally. The same amount of anti-CD8 antibody or control IgG was administered 7 days and 14 days after the administration of LLC, and the survival time was measured.
  • Fig. 8 The results are shown in Fig. 8.
  • the vertical axis represents the survival rate of mice, and the horizontal axis represents the number of days of survival.
  • Statistical calculations between each group were performed using the Log Rank test, and multiple comparisons were corrected by the Bonferroni method, and P ⁇ 0.05 was determined to be statistically significant (*). Similar to FIG. 4, the group in which reMAIT cells were adopted had a longer survival time than the group in which IgG was injected but did not transfer reMAIT cells (comparison between L7-1 + IgG and IgG).
  • Ly5.2m-reMAIT cells (1.0 x 10 6 ) were adopted into the abdominal cavity of wild-type mice (C57BL / 6), and the Ly5.2m-reMAIT cell transfer group (also referred to as "L7-1") was transferred.
  • Ly5.2m-reMAIT cell transfer group also referred to as "L7-1”
  • Inject a rabbit polyclonal antibody (rabbit anti-AsialoGM1: described as Fujifilm anti-AGM1) that removes natural killer (NK) cells from individuals into the (described) and non-implanted groups, respectively, and then add LLC (3.0 x 10 5 ). Rabbits were transplanted via a vein and the survival time was measured.
  • 1.0 x10 6 L7-1 was intraperitoneally administered 5 days before LLC administration, and 50 ⁇ l of anti-AGM1 (rabbit anti-asialoGM1: Fujifilm) was intraperitoneally administered the day before LLC administration. Fourteen days after the administration of LLC, the same amount of antibody was administered and the survival time was measured.
  • anti-AGM1 rabbit anti-asialoGM1: Fujifilm
  • FIG. 9 The results are shown in Fig. 9.
  • the vertical axis represents the survival rate of mice, and the horizontal axis represents the number of days of survival.
  • Statistical calculations between each group were performed using the Log Rank test, and multiple comparisons were corrected by the Bonferroni method, and P ⁇ 0.05 was determined to be statistically significant (*).
  • Figure 4 shows that the mouse survival extension observed by reMAIT cell adoption (comparison of none / none L7-1 / none) was canceled by injection of anti-AGM1 (L7-1 / none and L7-1). / anti-Comparison of AGM1) Therefore, it was shown that the anticancer effect (extension of survival time) by reMAIT cells is mediated by NK cells.

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Abstract

L'invention concerne une souris enrichie en cellules du type MAIT.　L'invention concerne également : une souris enrichie en cellules MAIT comportant une cellule du type MAIT obtenue par induction de la différenciation d'une cellule souche pluripotente artificielle dérivée par la reprogrammation d'une cellule MAIT dérivée de souris ; et une souris enrichie en cellules MAIT étant produite par l'intermédiaire d'une souris chimérique à l'aide d'une cellule souche pluripotente artificielle dérivée de cellules MAIT, et comprotant, dans un allèle, un gène de chaîne alpha de TCR ou un gène de chaîne bêta de TCR pour lequel un réarrangement de gène a été réalisé d'une manière spécifique de cellule MAIT.
PCT/JP2020/040361 2019-10-29 2020-10-28 Cellules du type mait chez les souris et souris riche en cellules mait WO2021085450A1 (fr)

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Title
CHIBA ASAKO, CHIBA ASAKO, TAJIMA RYOHSUKE, TOMI CHIHARU, MIYAZAKI YUSEI, YAMAMURA TAKASHI, MIYAKE SACHIKO: "Mucosal-associated invariant T cells promote inflammation and exacerbate disease in murine models of arthritis", ARTHRITIS & RHEUMATISM, WILEY INTERSCIENCE, US, vol. 64, no. 1, 1 January 2012 (2012-01-01), US , pages 153 - 161, XP055932063, ISSN: 0004-3591, DOI: 10.1002/art.33314 *
NAKAO, H.: "Reprogramming of MAIT Cells to Pluripotency and Redifferentiation.", METHODS IN MOLECULAR BIOLOGY, vol. 2098, 3 December 2019 (2019-12-03), pages 237 - 257, XP055932061, DOI: 10.1007/978-1-0716-0207-2_16 *
WAKAO, H. ET AL.: "Expansion of Functional Human Mucosal-Associated Invariant T Cells via Reprogramming to Pluripotency and Redifferentiation.", CELL STEM CELL, vol. 12, no. 5, pages 546 - 558, XP055243716, DOI: 10.1016/j.stem. 2013.03.00 1 *
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