WO2020175366A1 - キメラ抗原受容体 - Google Patents

キメラ抗原受容体 Download PDF

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Publication number
WO2020175366A1
WO2020175366A1 PCT/JP2020/007038 JP2020007038W WO2020175366A1 WO 2020175366 A1 WO2020175366 A1 WO 2020175366A1 JP 2020007038 W JP2020007038 W JP 2020007038W WO 2020175366 A1 WO2020175366 A1 WO 2020175366A1
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cells
cell
car
antigen
vector
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PCT/JP2020/007038
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English (en)
French (fr)
Japanese (ja)
Inventor
紀彦 川又
慧 青山
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国立大学法人東京医科歯科大学
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Priority to JP2021502186A priority Critical patent/JP7684691B2/ja
Publication of WO2020175366A1 publication Critical patent/WO2020175366A1/ja

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4205—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
    • A61K2239/28—Expressing multiple CARs, TCRs or antigens

Definitions

  • the present invention relates to a chimeric antigen receptor, a nucleotide encoding the chimeric antigen receptor, a vector containing the nucleotide, a cell expressing the chimeric antigen receptor, and a pharmaceutical composition containing the cell.
  • C CAR chimeric antigen receptor
  • CAR used in clinical practice has a single-chain antigen receptor (single-chain antibody, single chain F vfragment: sc F v) that serves as an antigen-binding site in the extracellular region. It has a structure in which a costimulatory molecule for activating T cells (CD 28, 4-1 BB, etc.) and a molecule that constitutes the T cell receptor signal, CD3 zeta (CD3 H intracellular domain, are linked.
  • CD3 zeta CD3 H intracellular domain
  • CAR-T cells recognize only a single antigen, and thus the target diseases are limited from the viewpoint of specificity.
  • the single antigen is also expressed in normal cells, CAR-T cells cause cytotoxicity and cause side effects.
  • attack on normal tissues cannot be suppressed, which may cause serious organ damage.
  • Non-Patent Document 1 attempts have been made to develop CAR-T cells that recognize multiple antigens, such as a system in which the intracellular structure of CAR is divided and linked to multiple ScFVs. ..
  • CAR-T cell system has not yet been put to practical use.
  • Non-Patent Document 1 K l os s s CC. et al., B i o t e c h n o l ., Jan. 2013, Vol.
  • the present invention has been made in view of the above problems of the prior art, and expresses a chimeric antigen receptor (CAR) that recognizes a plurality of antigens and exerts cell damage and the like specifically in target cells.
  • the purpose is to provide cells. It is also an object to provide a CAR-expressing cell capable of controlling the activity such as cytotoxicity.
  • cleavage CAR a chimeric antigen receptor
  • activating CAR a chimeric antigen receptor
  • a system using a free signal transduction factor that functions when released from the cell membrane as the signal transduction molecule shown in Fig. 1 was envisioned.
  • the activated protease causes It is assumed that the activation activation is cleaved and the signal transduction factor functions by being released from the cell membrane. Then, it is considered that these 0-expressing cells are activated and it is possible to bring about cytotoxicity or the like to the target cells.
  • the cell expressing the cleavage 0 and the activation 0 is not activated, and therefore the cell is not activated. It is also assumed that the cell damage will not be caused.
  • a system using a membrane-localized signal transduction factor that functions when present inside the cell membrane as the signal transduction molecule was also envisioned.
  • the signal transduction factor is activated by the activated protease. It is assumed that the cells are inactivated by being released from the cell membrane by cleavage of the activating circle, and that normal cells will not be damaged.
  • the target cells when the antigen recognized by cleavage 0 is not expressed and the antigen recognized by activation 0 is expressed, the protease is not activated and the activity is the same as in conventional 0.
  • the signal transduction molecule functions inside the cell membrane, and cells expressing these O are activated, and it becomes possible to bring about cytotoxicity or the like in the target cell. ⁇ 02020/175366 4 (:171? 2020 /007038
  • the present inventors have proposed an antigen-binding region corresponding to activation [08] as an antigen-binding region.
  • ⁇ 1 — 001 9 — 01 also referred to as “3116” “SO8 [3 ⁇ 4”
  • 293 cells were co-expressed with cells expressing the target antigen, 001 9.
  • the localization of the so signal has changed. That is, as shown on the left side of FIG. 2, it was suggested that the target antigen, 019, was recognized, and that the 0 could be assembled on the cell membrane.
  • the present invention relates to a chimeric antigen receptor, a nucleotide encoding the chimeric antigen receptor, a vector containing the nucleotide, a cell expressing the chimeric antigen receptor, and a pharmaceutical composition containing the cell, More specifically, it is as follows. ⁇ 02020/175366 6 ⁇ (: 171-1? 2020 /007038
  • a chimeric antigen receptor comprising a region that binds to a first antigen, a transmembrane region, and a protease, and does not include a site that is cleaved by the protease.
  • a chimeric antigen receptor comprising a second antigen-binding region, a transmembrane region, a site cleaved by the protease according to ⁇ 1>, and a signal transduction factor.
  • ⁇ 3> The chimeric antigen receptor according to ⁇ 2>, which is a membrane-localized signal transduction factor, which functions when the signal transduction factor is present inside the cell membrane.
  • ⁇ 4> The chimeric antigen according to ⁇ 2>, wherein the signal transduction factor is a free signal transduction factor that functions when the site is cleaved by the protease described in ⁇ 1> and is released from the cell membrane. Receptor.
  • ⁇ 5> A nucleotide encoding the chimeric antigen receptor according to any one of ⁇ 1> to ⁇ 4>.
  • ⁇ 6> A vector containing the nucleotide according to ⁇ 5>.
  • ⁇ 7> A cell expressing the chimeric antigen receptor described in ⁇ 1> and the chimeric antigen receptor described in any one of ⁇ 2> to ⁇ 4>.
  • ⁇ 8> The cell according to ⁇ 7>, which is a Ding cell or a natural killer cell.
  • ⁇ 9> A pharmaceutical composition comprising the cell according to ⁇ 7> or ⁇ 8>.
  • two antigens expressed in target cells or normal cells are provided.
  • Fig. 1 is a schematic view showing an embodiment (positive system) of the chimeric antigen receptor (08 [3 ⁇ 4]) of the present invention.
  • a single-chain antibody (30) that recognizes antigen 8 is connected to a signal transduction factor (free signal transduction factor) that is active in the cytoplasm with a protease cleavage sequence sandwiched between them [08 (3 ⁇ 4 (3 ⁇ 4
  • the activation [08] and the expression [8] (cleavage 08 [3 ⁇ 4), which is obtained by connecting the protease to 30 V that recognizes the antigen, are expressed in Ding cells.
  • the protease When both the antigen and the antigen are expressed in the target cell (for example, tumor cell), the protease is activated and activated when each of the 30 V recognizes each of the antigens in the D cell.
  • the sequence in the chemical formula will be cleaved. Then, due to the cleavage, the T cell activation factor is released from the cell membrane and released into the cytoplasm, thereby activating the T cell.
  • FIG. 2 is a schematic diagram showing one embodiment (negative system) of the chimeric antigen receptor (08 [3 ⁇ 4]) of the present invention.
  • a signal transduction factor membrane-localized signal transduction factor
  • the 30 V that recognizes the target antigen 8 with a protease cleavage sequence sandwiched between them.
  • cleavage 08 [3 ⁇ 4] which is obtained by connecting the protease to 30 V that recognizes the target antigen, are expressed in Ding cells.
  • the D cell has a signal by recognizing the antigen by the 30 V of 0 for activation. The transfer factor is activated, and the Ding cell is activated.
  • the non-target cell normal cell
  • the 30 V possessed by cleavage 08 recognizes the antigen and activates the protease and activates it. The sequence inside will be truncated. Then, due to the cleavage, the signal transduction factor is released from the cell membrane, thereby suppressing the activation of Ding cells relative to non-target cells.
  • Fig. 3 is a schematic view showing the outline of 0 for verification of the present invention.
  • the upper part shows the structure of a typical publicly known second generation ⁇
  • the lower part shows the structure for demonstrating the present invention ⁇ [3 ⁇ 4 (an I ⁇ - ⁇ 0 1 9- ⁇ 0 2 8- ⁇ 3- ⁇ 1 ⁇ 6 1 ⁇ "Shows the structure of So'08. ⁇ 02020/175366 8 ⁇ (: 171? 2020 /007038
  • the ⁇ [3 ⁇ 4 has a structure of the second generation 0 based, sandwich ⁇ 028 downstream anchor sequence comprising the transmembrane region (Ding IV!) (9 4 1), in place of Rei_03 ⁇ 6 3, 1 to 1 V protease cleavage site consisting of 8 amino acids (amino acid sequence set forth in SEQ ID NO: 9) and fluorescent protein ( ⁇
  • 2 is a fluorescence micrograph showing the results of observing 293 cells.
  • the vector encoding the gene was introduced into the gene and observed 24 hours later. “The results of the detection of Seo alone are shown, and the overlay image with nuclear staining is shown on the right. The observation was performed with an all-in-one fluorescence microscope 67-81 00 (60 times), and the sensitivity of 30 was set to 200. 01 (3116 ““The exposure time of the camera was taken at 3.5 seconds (observation and shooting conditions are the same in FIGS. 5 and 6).”
  • FIG. 8 is a fluorescence micrograph showing the results of observing the co-culture of 293 cells expressing E.coli and ⁇ 562 cells or [3 ⁇ 4 3 ]cells. After transfecting the vector encoding the XVIII gene into 293 cells, 24 hours later, 562 cells or 3 ” ⁇ cells are added,
  • the black arrowhead is the total length of 31 ⁇ 1: * Indicates the band derived from Eighteen (about 701 ⁇ 03).
  • the white arrowhead is a band (1) that was cut and released by 1 to 1 IV (3 6 "" a band derived from simple substance (about 301 ⁇ 0 ⁇ ):.
  • FIG. 9 is a schematic view showing an outline of a 08[3 ⁇ 4 (cutting 08[3 ⁇ 4]) of the present invention. On the top ⁇ 02020/175366 10 ⁇ (: 171? 2020 /007038
  • the white line (scale bar) at the bottom right represents 20.
  • the vertical axis represents the intensity of the signal
  • the horizontal axis represents the position on the line from the left to the right in the red line (under the color display) of the photograph.
  • FIG. 14 3 1-001 9- ⁇ 1 ⁇ 6 ““Shows the results of observing 293 cells that co-expressed So 008 and ⁇ 028 ⁇ I ⁇ 0 _1 ⁇ 1 ⁇ y P RA4 808. It is a fluorescence micrograph. 293 cells are treated with ant ⁇ 1 010 9- ⁇ 11 & rr ⁇ ⁇ 8 and ⁇ 028 ⁇ I ⁇ 0-1 to 1 IV [3 ⁇ 4 8 4 0 8 [3 ⁇ 4, respectively. — Transfecting vector was introduced at a weight ratio of 10: 1 (molar ratio of 9.34: 1), and observed 24 hours later. 20 times) and the sensitivity was set to 200. The exposure time was 1.1 seconds.
  • So cells and Established cells. 1 to 1 on the cell surface The expression levels of 2 and 00109 were analyzed by a flow cytometer.
  • FIG. 3 is a micrograph showing the result of co-culture of 293 cells that overexpressed both [3 ⁇ 4] and 3 ⁇ __ [3 ⁇ 4_3 cells by fluorescent immunostaining.
  • 293 cells 0 0 8 3-3 ⁇ 1 -1 to 1 [[2 (405-3)- ⁇ 028 — ⁇ I 63 V 6 3 II ⁇ 1 01 ⁇ 116 ""So ⁇ 8 [ 3 ⁇ 4 vector and ⁇ I -001 9-0028 ( ⁇ ! ⁇ 0) -1 ⁇ 1 1?
  • [3 (8) vector was introduced into the vector at a weight ratio of 5:1, and coculture was started 24 hours later.
  • the co-culture was performed on 100 dishes in 2 XI 0 5 cells, 293 cells and 3 ⁇ _, respectively. _ 3 cells. After 4 days of co-culture The cells were observed. These observations were made with BZ-81 00 at a magnification of 60, and the IS sensitivity was set to 200. The exposure time of mC herry was 3.5 seconds.
  • Fig. 18 is a schematic diagram showing the outline of the coding region in the vector expressing annt i -CD 19 E f f e c t o r CAR-T 2A-Y F P.
  • the intracellular domain of CD3-zeta was connected to the H VPR recognition sequence downstream of CD28.
  • the Y F P gene was connected via the T 2 A sequence.
  • FIG. 19 Expression of CD 69 and YFP in J urkat cells co-cultured with K 562 cells or Raji cells and transfected with anti-CD 19 E ffector CAR-T 2A-YFP expression vector was analyzed by flow cytometry. It is a dot plot diagram showing the results of analysis in a tree.
  • pc DNA3— anti-CD 19 Effector CAR_T 2 A— YFP was introduced into J urkat cells and coculture was started 24 hours later. With 96 we I ⁇ bottom plates were seeded by 1 X 1 ⁇ 5 cells per well. After 24 hours of co-culture, cells were collected and analyzed using FACS ari all.
  • CD69 is a CD19-positive Raji cell-specific T cell activation marker.
  • YFP reflects the expression of anti-CD 19 Effector CAR, which is expressed cistronicly via the T 2 A sequence.
  • FIG. 20 IL-2 mRNA expression level in J urkat cells co-cultured with K562 cells or Raji cells and introduced with anti-CD 19 Effector CAR-T 2A-YFP expression vector. It is a graph. In addition, the gate shown in Fig. 19 was installed, and Positive .069_?mi-positive cells were isolated and evaluated for L-2 gene expression by qRT-PCR. Analysis was performed by the AACt method using GAPDH as the internal control. Taking the K562 co-culture group as 1, the relative L-2 mRN A expression level was shown.
  • FIG. 4 is a dot plot diagram showing the results of flow cytometric analysis of the expression of CD69 and YFP in r CAR/J urkat).
  • Jurkat cells transduced with pc DNA 3-anti -CD 19 E ffector CAR-T2A-YFP were cultured in the presence of 1 000 ⁇ g/mL G418, and anti -CD 19 E ffector CAR/J urkat cells were established.
  • K562 cells Raji cells, SK-BR-3/empty cells or SK_BR-3/CD19 cells at a cell number of 1:1 for 24 hours, the cells are harvested, It was analyzed using FACS Ca libur.
  • FIG. 22 anti — CD 19 E ffector CAR/J urkat cells and anti -HER 2 (4 D 5-3) -S cissor s-CAR-T2 A — mC herry expression vector or anti — HE R2 ( 4 D5-8) — S cissors- CAR- T2A- mC herry expression vector
  • the expression of YFP and m C herry in the Jurkat cells introduced with the vector is shown by the results of the site-cytometry analysis. It is a dot plot diagram.
  • FIG. 23 anti-CD 19 E ffector CAR + empty/J urkat cells or anti — CD 19 E ffector CAR + anti — co-cultured with Raji cells or SK_BR ⁇ 3/CD 19 cells It is a histogram which shows the result of having analyzed CD69 expression in HER2(4D5-3)Scissors/Jurkat cell.
  • the gray histogram shows the analysis results of anti — CD 19 E ffector CAR + empty/J urkat cells
  • the histogram represented by the solid line shows anti — CD 19 E ffector CAR + anti -HER 2 (4 D 5 -3) Shows the analysis results of S cissors/J urkat cells.
  • the cells were collected, and the expression of CD69 in YFP-positive cells was analyzed by using FACS Caliburr.
  • FIG. 24 anti-CD 19 Effector CAR + empty/Jurkat cells, anti-CD co-cultured with Raji cells, SK-BR-3 cells or SKB R-3/CD 19 cells 1 9 E ffector CAR + anti — HER 2 (4 D 5 — 3) S cissors/J urkat cells, or anti — CD 1 9 E ffector CAR + anti — HER 2 (4 D 5 — 8) S cissors/J It is a histogram showing the results of analysis of CD69 expression in urkat cells.
  • the gray histogram shows the result of co-culturing with SK_BR ⁇ 3 cells (below the color display), the histogram represented by the solid red line (second from the left). Histogram) shows the result of co-culture with SK_BR — 3/CD 19 cells, and the black solid histogram (third histogram from the left) is R a
  • the results of co-culturing with ji cells are shown. The cells were collected after co-cultivation at a cell number of 1:1 for 24 hours, and the expression of CD69 in YFP positive cells was analyzed using FACS Calilibur.
  • T cells expressing the following two types of chimeric antigen receptors are recognized by recognizing the two types of antigens through the respective chimeric antigen receptors. It is possible to cause cell damage and the like to target cells such as cancer cells without causing cell damage and the like to normal cells.
  • a chimeric antigen receptor (hereinafter also referred to as "cleavage CAR") containing a region that binds to a first antigen, a transmembrane region, and a protease, and does not include a site that is cleaved by the protease.
  • a chimeric antigen receptor (hereinafter, also referred to as "activating CAR") containing a region that binds to a second antigen, a transmembrane region, a site that is cleaved by the protease, and a signal transduction factor.
  • the present invention provides these chimeric antigen receptors, and combinations thereof.
  • the “chimeric antigen receptor (Chimeric Antigen Receptor: CAR)” is an extracellular region that binds to an antigen, a transmembrane region, and an intracellular region in that order. Means a chimeric protein that is arranged from the N-terminal side and is directly or indirectly linked.
  • a region that binds to an antigen means a region that specifically binds to an epitope of an antigen, and an antibody or a functional fragment thereof can be used.
  • the “functional fragment” of an antibody may be a part of an antibody as long as it can specifically bind to an antigen, for example, a single chain antibody (s c F v)
  • Single domain antibody (sd Ab, VH H, VNAR), sc (F v) 2, F ab, F ab', F (ab') 2, variable region fragment (F v), disulfide-bonded F v, Diabodies and multispecific antibodies are usually used for CAR.
  • rs c F v is a structure in which a light chain variable region (VL) and a heavy chain variable region (VH) of a monoclonal antibody (immunoglobulin) are linked via a linker, and binds to an antigen. Holds Noh.
  • a linker for example, a peptide linker can be used.
  • the length of the linker is not particularly limited.
  • a linker having 5 to 25 amino acids can be used.
  • the length of the linker is preferably 8 to 25 amino acids, more preferably 15 to 20 amino acids.
  • Preferable examples of peptide linkers include linkers composed of glycine and serine (GGS linker, GS linker, etc.).
  • the amino acids that compose the GGS linker and GS linker, glycine and serine have the advantage that their size is small and that higher-order structures are less likely to be formed in the linker.
  • the monoclonal antibody serving as the basis of scFV is not particularly limited, and examples thereof include antibodies derived from non-human animals (rodents such as mouse, rat, and rabbit).
  • rodents such as mouse, rat, and rabbit.
  • a method for producing such a monoclonal antibody a method using a hybridoma, a method in which a host is transformed with an expression vector containing an antibody gene by a genetic engineering technique, or a transgenic animal is desired. The method of producing by immunizing with the antigen of.
  • the monoclonal antibody according to the present invention may be a human antibody, a humanized antibody or the like.
  • a humanized antibody is an antibody in which the structure of a monoclonal antibody of another animal species (for example, mouse or rat) is similar to the structure of human antibody, and only the constant region of the antibody is replaced with that of human antibody.
  • the "antigen", the first antigen and the second antigen are not particularly limited and, for example, are specifically expressed in cancer cells or their precursor cells (cancer stem cells etc.).
  • Biomolecules tumor-specific antigens, Tumor-specificantigens: TSA
  • biomolecules whose expression is increased in cancer cells compared to normal cells tumor-associated antigens, Tumor-associatedantigens: TAA
  • Cancer antigens such as
  • cancer antigens include CD 19 and H ER 2 (ER B B 2, N EU)
  • the antigen according to the present invention also includes an antigen specific to an infectious disease.
  • an antigen may be any substance that is specifically expressed in viruses or bacteria that cause infectious diseases, for example, 1 to 1 V V-specific antigen (1 to 1 V V 9 1 20 etc.), 1 to 1 specific antigen, Minami V specific antigen, 0 IV! V specific antigen, 1 to 1 specific antigen, Lassa virus specific antigen, influenza virus specific antigen, fungal specific antigen To be ⁇ 02020/175366 20 ⁇ (: 171? 2020 /007038
  • the antigen according to the present invention also includes an antigen specific to an inflammatory disease.
  • antigens include, for example, 803 (8? 1 1 ), 808 1 ⁇ /1-30 01, ⁇ 1_ 1 1, 001 25, 001 47 % 001 54 ( ⁇ 040 !_),
  • those skilled in the art can express not only in myeloma but also in normal cells suitable for each disease, based on publicly known information on the difference in expression between target cells such as cancer cells and normal cells.
  • the antigen to be selected can be selected.
  • the first antigen and the second antigen are usually different antigens.
  • the "transmembrane region” is not particularly limited as long as it is a polypeptide that has a function of penetrating the cell membrane and can retain 0.8% in the cell membrane.
  • proteins that are the origin of the polypeptide that carries the transmembrane region include, for example, 0028, 0 03 ⁇ , 003 £, 0045, 004, 005, 008/3, 00
  • the ⁇ chain and / 3 chain of the receptor are included.
  • the "intracellular region” has different modes of 0 for cleavage and 0 for activation.
  • the "intracellular region" in Cleavage 08 [3 ⁇ 4 has at least a protease.
  • protease As a “protease”, when cleavage 08 and activation 08 bind to their corresponding antigens and assemble at the cell membrane, they are activated in close proximity to the transmembrane domain and signal.
  • protease that can be cleaved with a transfer factor
  • examples thereof include aspartate protease (1 to 1 V V protease (1 to 1 [3 ⁇ 4), secretase, etc.), cysteine protease (Caspase, Tobacco Etch Virus (Chomi) Protease, etc.), Serine Protease (Factor X3, Fuulin, etc.), and Metalloprotease (8881 ⁇ /1 protein, Matrix metalloprotease, etc.).
  • protease according to the present invention from the viewpoint that cleavage for activation [8] easily occurs depending on the antigen binding of cleavage for activation, activation occurs when the antigen-binding region binds to an antigen.
  • Protease is preferred. Examples of such a protease include a protease that exhibits protease activity by multimerization, and more specifically, 1 to 1 V protease and caspase 9 are mentioned.
  • the protease according to the present invention is derived from an organism (including a virus) different from the origin of the cell in which the present invention 88 is expressed. Proteases are preferred. When the cell is derived from human, a non-human-derived protease is preferable, and more specifically, Examples include V-protease and Domi-V protease.
  • protease inhibition Agent-controllable proteases are preferred.
  • protease include saquinavir, ⁇ 0 2020/175 366 22 ⁇ (: 171? 2020 /007038
  • protease corresponding to protease inhibitors such as nelfinavir, atazanavir, fosamprenavir, lopinavir, darunavir, ritonavir, tipranavir, amprenavir and indinavir.
  • a secretase corresponding to protease inhibitors such as
  • 1 to 1 V V protease is particularly preferable as the protease according to the present invention, and further, as shown in Examples described later, multimerization (dimerization Necessary for "1 to 1 V V protease in which the sequence is excluded is more preferable.
  • the "intracellular region" in Activator 08 [3 ⁇ 4 has at least a site cleaved by a protease and a signal transduction factor.
  • the "site that is cleaved by a protease” is located between the above-mentioned transmembrane region and the signal transduction factor in the activation gene.
  • Those skilled in the art can appropriately understand the sequences recognized and cleaved by the above-mentioned protease based on known information, and can arrange the sequences between the region factors.
  • the ⁇ 0 2 8 intracellular domain used in conventional ⁇ eight cleavage sites of aspartic proteases, such as 1 ⁇ 1 IV protease is present. In such a case, it can be suitably used as the activation reagent of the present invention without newly inserting the protease cleavage site.
  • Ordinary 0 [3 ⁇ 4 means existing 0, and generally, as an intracellular region, the activation signal transduction of the Ding cell receptor (Ding [[]] and 0 0 3 complex is performed. Having only domain (1st generation 08 [3 ⁇ 4), in addition to activation signaling domain, costimulatory signaling domain derived from costimulatory molecule (eg, 0 0 2 8 or 4 _ _ _ _ _ _ _ _ _ intracellular Domain (2nd generation ⁇ [3 ⁇ 4), and multiple costimulatory signaling domains in addition to the activation signaling domain (for example, the intracellular domains of ⁇ 0 28 and 4 _ ⁇ ) Things (3rd generation There are eight [3 ⁇ 4).
  • the "signal transduction factor” means a factor capable of transducing a signal necessary for exerting an effector function in a cell when the above-mentioned antigen-binding region binds to an antigen.
  • the number of signal transduction factors contained in the activation CAR is not limited to one, and may include multiple signal transduction factors (for example, 1 to 5,
  • the plurality of signal transducing factors contained in the activating C AR may be the same or different.
  • the "signal transduction factor” includes a membrane-localized signal transduction factor and a free signal transduction factor.
  • Membrane-localized signal transduction factor means a membrane-localized signal transduction factor that functions when it is present inside the cell membrane, and is a primary signal used in normal CAR.
  • the activation signaling domain of the protein involved can be used. It is known that the immunoreceptor-activating tyrosine morphology (ITAM) is involved in primary signal transduction.
  • the proteins with ⁇ T AM for example, CD3 F c R r, F c R / S, CD 3 T s CD35, CD3 s, CD 5, CD 22, CD 79 a, CD 79 b, CD 66 d, Examples include DA P 10 and DAP 12.
  • Free signal transduction factor means a signal transduction factor that functions when it is released from the cell membrane, and includes, for example, mutant RHOA (p. Gly 17 V al variant etc.) and mutant VAV. (1 -67 amino acid deletion type, p. Asp 797 A sn mutant, etc.), mutant ZA P-70, RAS, NI CD (Noteh intracellular domain), NF AT (contributes to TCR signaling Transcription factor).
  • mutant RH0A S akata— Y anagi mo to M. et al., N at Ge net. 201 February 2014, Volume 46, No. 2, pp. 171-175, 9 S Y. et al. , Blood, 201 Aug.
  • the CAR of the present invention can contain other domains in addition to the above-mentioned antigen-binding region, transmembrane region, and protease or protease cleavage site and signal transduction factor.
  • other domains include, but are not limited to, costimulatory transduction domains, spacer sequences, signal peptides and the like.
  • the costimulatory molecule expressed on T cells transmits a costimulatory signal intracellularly by binding with a ligand specific to each costimulatory molecule expressed on antigen-presenting cells,
  • the “costimulatory signal transduction domain” means an intracellular domain involved in costimulatory signal transduction of the costimulatory molecule as described above.
  • costimulatory molecules include CD 28, 4-1 BB (CD 1 37), 0X040 (CD 1 34), I COS, CD 2, CD4, CD 5, CD8a, CD8/S, CD 1 54, etc.
  • the CAR of the present invention can include costimulatory signal transduction domains of these costimulatory molecules.
  • the number of costimulatory signaling domains that the CAR of the present invention may include is not limited to one and may be multiple.
  • the CAR of the present invention can contain, for example, 1 to 5, 1 to 3, or 1 or 2 costimulatory signaling domains.
  • the domains may be the same or different.
  • the "spacer sequence” means a sequence that connects regions or the like.
  • the number of amino acids in the spacer sequence is not particularly limited, but it is usually 2 to 300 amino acids, preferably 5 to 200 amino acids, and more preferably 7 to 100 amino acids. Connects the antigen binding region and transmembrane region
  • the sequence to be used is not particularly limited as long as it does not inhibit the antigen-binding ability of the antigen-binding region. For example, 9 amino acids derived from human rhodopsin (C 9 sequence),
  • Extracellular hinge domains such as IgG, IgA, IgD, CD8a, CD8/S, CD28, and CD4, and a linker composed of the above-mentioned glycine and serine are listed.
  • the sequence connecting the transmembrane region or costimulatory signal transduction domain and the protease or the site cleaved by the proteinase does not inhibit the activity of protease, signal transduction factor or costimulatory signal transduction domain.
  • an anchor sequence of HIV gp 41, and a linker composed of the above-mentioned glycine and serine though not particularly limited as long as it does not inhibit the berthing by the protease.
  • the "signal peptide” means a peptide for promoting the secretion of CAR or directing the localization to the cell membrane, and is also called a leader sequence.
  • the signal peptide will usually be attached directly or indirectly to the N-terminus of the antigen binding region.
  • the signal peptide may be cleaved from the antigen binding region during cell processing and localization of CAR to the cell membrane. Examples of such signal peptides include a chain and /S chain of T cell receptor, CD8a, CD8/S, CD3f, CD28, CD3s, CD45, CD4, CD5, CD9, CD1. 6.
  • Signal peptides such as CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR, and human GM-CSF receptor a can be mentioned.
  • the CAR of the present invention may have other functional proteins in addition to the above-mentioned costimulatory transduction domain, spacer sequence and signal peptide.
  • the other functional protein is not particularly limited and may be appropriately selected depending on the function to be imparted to C A R of the present invention.
  • functional proteins used for the purpose of facilitating the purification and detection of CAR include My c tag, His tag, HA tag, FLAG tag (registered trademark, Sigma—Aldrich), and fluorescent protein. Quality tags á G FP, etc.) and the like.
  • the "cleaving CAR" of the present invention is, as long as it comprises, in order from the N-terminal side, at least an antigen-binding region, a transmembrane region and a protease, which are directly or indirectly linked.
  • the signal peptide, the antigen binding region, the spacer sequence, the transmembrane region, the spacer sequence and the protease are linked in this order from the N-terminal side.
  • the "cleavage CAR" of the present invention Does not include a site cleaved by the protease. If the CAR does not include the relevant part, those skilled in the art can search for peptide databases (ME RO PS, https: //ww w. ebi .ac .uk/me rops / etc.), for example. By doing so, we can confirm.
  • the "activating CAR" of the present invention comprises, in order from the N-terminal side, at least an antigen-binding region, a transmembrane region, a site cleaved by a protease and a signal transduction factor, which are directly or indirectly linked. Anything can be used, but from the N-terminal side, the signal peptide, the antigen-binding region, the spacer sequence, the transmembrane region, the costimulatory transduction domain, the spacer sequence, and the part cleaved by the protease. ⁇ 0 2020/175 366 27 ⁇ (: 171? 2020 /007038
  • the position and the signal transduction factor are directly or indirectly linked.
  • the present invention provides a polynucleotide encoding the XVIII of the present invention.
  • a monoclonal antibody that recognizes a target antigen is prepared, and the amino acid sequence of such a monoclonal antibody is determined by a known method such as Edman method. It can also be obtained based on the amino acid sequence determined by the method. It can also be obtained by analyzing the nucleotide sequence of a hybridoma producing the monoclonal antibody.
  • nucleotide sequences encoding each region and the like of the present invention including the antigen-binding region can be known to those skilled in the art by publicly known literature or 1 ⁇ 1 ⁇ (
  • nucleotide sequence encoding the above regions and the like is not limited to known ones, and any nucleotide sequence that encodes the above regions or the like can be used. Due to the degeneracy of the genetic code, there are multiple codons for an amino acid. Therefore, there are many nucleotide sequences that encode the same amino acid sequence.
  • the nucleotide sequence of the polynucleotide of the present invention may be any of a plurality of nucleotide sequences generated by the degeneracy of the genetic code, as long as it encodes the eighteenth of the present invention.
  • the selected codon for encoding the amino acid may be modified. Good.
  • the polynucleotide encoding XVIII of the present invention can be obtained.
  • the ligation of polynucleotides encoding each region can be carried out, for example, as described in the Examples below, by means of the burlap extension ⁇ [3 ⁇ 4 method ( ⁇ 0 ”1 3 6 father 6 ⁇ 3 I ⁇ n ⁇ [3 ⁇ 4). It can be performed using a known method such as.
  • the present invention provides a vector containing the above-mentioned polynucleotide.
  • the vector of the present invention may be linear or circular, and examples thereof include viral vectors, plasmid vectors, episomal vectors, artificial chromosome vectors, and transposon vectors.
  • viral vectors examples include retrovirus vectors such as lentivirus, Sendai virus vector, adenovirus vector, adeno-associated virus vector, herpes virus vector, vaccinia virus vector, box virus vector, polio. Viral vectors, Sylvis virus vectors, rhabdovirus vectors, paramyxovirus vectors, and rhusomyxovirus vectors.
  • the plasmid vector for example, 1, eight 1 _ 1 1
  • Episomal vector is a vector that is capable of autonomous replication outside the chromosome.
  • the specific means of using an episomal vector is known (see ⁇ Ri et al., 30 I 6 ⁇ 6, 2009, 324, 797 -801, page).
  • Examples of the episomal vector include a vector containing, as a vector element, a sequence required for autonomous replication, which is derived from £6V, 340 and the like.
  • the vector elements required for autonomous replication are, specifically, a replication origin and a gene encoding a protein that regulates replication by binding to the replication origin. In the case of /, the origin of replication is ⁇ “ ⁇ ?
  • Examples of artificial chromosome vectors include YAC (Ye astartificia I chr omo s ome) vector, BAC (B acteria I artificial chr omo s ome) vector, and PAC (P 1 — derivedartificial chr omo s ome) vector. Is mentioned.
  • a retrovirus vector is preferable, and a lentivirus is more preferable because it is easy to introduce a gene with high efficiency even in cells with slow growth and a stable expression strain can be easily established. preferable.
  • the vector of the present invention binds to an expression control sequence such as a promoter, an enhancer, a poly A addition signal, and an expression terminator, an origin of replication or an origin of replication. It may contain a nucleotide sequence encoding a protein that regulates replication, a nucleotide encoding another protein, and the like.
  • a polynucleotide encoding the above-mentioned CAR and a nucleotide encoding another protein described below can be efficiently transcribed by arranging the polynucleotide operably downstream of the promoter. ..
  • promoters include CMV (cytomegalovirus) promoter _, S Ra promoter, SV40 early promoter, retrovirus LTR, RSV (Rous sarcoma virus) promoter, HSV-TK (simple helix). Pesvirus thymidine kinase) promoter, EF 1 a promoter, metallothionein promoter, heat shock promoter, etc.
  • nucleotides encoding other proteins include, for example, fluorescent protein genes, reporter genes, marker genes such as drug resistance genes, and genes encoding molecules involved in T cell activation such as cytokines. Etc. can be mentioned.
  • 2 A peptide sequence for example, T hoseaasigna-derived 2A peptide (T 2A)
  • T 2A T hoseaasigna-derived 2A peptide
  • the suicide gene is defined as "nucleotides encoding other proteins".
  • the vector of the present invention may include: Examples of the suicide gene include thymidine kinase (H S V-T K) of herpes simplex virus and inducible caspase 9 (in d u c i b l e c a s p a s e 9 :i C a s p 9).
  • AP1 903 which is a ganciclovir for HSV-TK, and a dimer induction compound (CID) for iCasp9, etc.
  • CID dimer induction compound
  • the polynucleotide encoding the cleavage CAR and the polynucleotide encoding the activation CAR may be incorporated into a single vector or may be incorporated into separate vectors. However, it is preferable to incorporate them in separate vectors from the viewpoint of facilitating suppression of the antigen recognition-independent activation of T cells transformed with such a vector and expressing a cleavage CAR and an activation CAR. desirable.
  • IR ES, DN A encoding the 2 A peptide sequence, etc. are inserted into the vector to allow polycistronic cleavage CAR and activation. It becomes possible to express CAR. ⁇ 0 2020/175 366 31 ⁇ (: 171? 2020 /007038
  • the present invention provides a cell expressing O of the present invention.
  • the cell of the present invention can be obtained by introducing the polynucleotide or vector encoding the above-mentioned XVIII of the present invention into the cell.
  • the "cell” into which the polynucleotide or the vector of the present invention is introduced is preferably a cell derived from a mammal, for example, a cell derived from human, or a rodent (mouse, rat, etc.). Cells derived from non-human mammals such as cow, sheep, horse, dog, pig and monkey can be used.
  • the type of cells is not particularly limited, and it is possible to use body fluids such as blood and bone marrow fluid, tissues such as spleen, thymus, lymph nodes, cells collected from tumors and cancerous ascites, and the like.
  • Preferred examples include immune cells, and peripheral blood mononuclear cells isolated from peripheral blood can be preferably used.
  • Ding cells are Ding cells, Cells, and their progenitor cells.
  • the type of Ding cells is not particularly limited, and is ⁇ /3 Ding cells, 5 d cells, 08 positive D cells, 04 positive D cells, cytotoxic D cells ( ⁇ 3 1_), tumor invasion. It may be any type of Ding cell such as Ding cell, F-Ector memory Ding cell (Ding 1 ⁇ /1), Central memory Ding cell (Ding, Natural killer Ding cell, etc.
  • the cell according to the present invention may be a cell in which the function of a protein (such as an immune checkpoint substance) that suppresses an excessive immune reaction is suppressed. Such an evening
  • the cells according to the present invention contain endogenous cells to facilitate polymorphic transplantation.
  • [3 ⁇ 4,! ! !_ It may be a cell in which the functions of proteins such as Yachi are suppressed. ⁇ 0 2020/175 366 32 ⁇ (: 171? 2020 /007038
  • a knockout method targeting a gene encoding the protein, 3 complementary to the transcription product of the gene double-stranded [3 ⁇ 4, for example, 3 ⁇ [3 ⁇ 4]
  • Method of using, method of using antisense 8 complementary to the transcript, having ribozyme activity of specifically cleaving the transcript The method using eight is mentioned.
  • site-specific nucleases eg, zinc finger nuclease (1 ⁇ 1), transcription activation-like effector nuclease (Chohachi 1_Mi 1 ⁇ 1),
  • a genome editing method which is a method of modifying a target gene using a double-strand cleaving enzyme), is also suitably used for suppressing the function of the substance.
  • Examples include the 0-Hachimi-dextran method, the electroboration method, and the particle gun method.
  • the vector of the present invention is a retroviral vector
  • an appropriate packaging cell is selected based on the 1_c sequence and the packaging signal sequence of the vector, and this is used to select a retrovirus. Particles may be prepared.
  • packaging cells for example, ⁇ 13 ,8 3 17 , ⁇ + Michi 8 6 , ⁇ ?+ 6 880 1-1 2, 9 3 I
  • 293 cells and 293 cells with high transfection efficiency can also be used as packaging cells.
  • the ratio of vectors to be introduced is not limited to those skilled in the art. If so, it can be appropriately adjusted in consideration of the type of vector and the strength of the promoter used for expression of each 0, the type of protease, and the like. For example, from the viewpoint that the antigen recognition-independent activation of Ding cells transformed with these vectors and expressing Cleavage 08 [3 ⁇ 4 and Activation O is more easily suppressed, they are introduced into the cells.
  • Weight ratio or molar ratio of these vectors (weight or molar amount of vector coding for cleavage 0: activation
  • the weight or molar amount of the vector coding the CAR for use in the present invention is preferably 5:1 to 100:1, and more preferably 10:1 to 50:1.
  • the expression of CAR in the cells can be measured by flow cytometry, RT-PCR, Northern blotting, Western blotting, ELISA, fluorescent immunostaining, etc. It can be confirmed by a known method.
  • T cells that express the cleavage and activation CARs recognize normal cells by recognizing two types of antigens through each CAR. It is possible to cause cell damage or the like to the target cell without causing damage or the like.
  • the protease When the antigen recognized by the activation antigen is expressed, the protease is not activated, and the activation antigen is the target antigen of the activation ⁇ [8]
  • the signal transduction molecule functions inside the cell membrane, and the cells expressing O are activated, resulting in cytotoxicity or the like in the target cells.
  • the present invention provides a pharmaceutical composition comprising the cell of the present invention for treating or preventing a disease involving a target cell.
  • cancer
  • ⁇ infectious diseases'' include infectious diseases such as 1 to 1 V, 1 to 1, Minami, ⁇ 1 ⁇ /1, 1 to 1?, Lassa virus, influenza virus, and fungi.
  • infectious diseases include systemic lupus erythematosus (3!_M), diabetes, rheumatoid arthritis ([3 ⁇ 48), reactive arthritis, multiple sclerosis). , Pemphigus vulgaris, celiac disease, Crohn's disease, inflammatory bowel disease, ulcerative colitis, autoimmune thyroid disease, X-linked agammaglobulinemia.
  • the pharmaceutical composition of the present invention may contain other components such as a pharmaceutically acceptable carrier in addition to the cells of the present invention.
  • a pharmaceutically acceptable carrier include cell culture medium, physiological saline, phosphate buffer and citrate buffer. Also ⁇ 0 2020/175 366 35 ⁇ (: 171? 2020 /007038
  • Ding cell activating factor such as cytokine, immunostimulant, immune checkpoint inhibitor, other cells expressing O, anticancer agent, anti-inflammatory agent, etc., It is not limited to these.
  • the use of the cells of the present invention makes it possible to treat or prevent a disease involving target cells. Therefore, the present invention may also provide the following treatment or prevention method.
  • a disease involving a target cell that expresses at least the second antigen which comprises the step of administering to the subject a cell that expresses 0 for cleavage and 0 for activation, or a pharmaceutical composition containing the cell.
  • the administration of the cell or the pharmaceutical composition of the present invention to a subject is not particularly limited, and can be performed, for example, by injection (intravenous administration etc.) or infusion. Alternatively, it may be administered by direct injection or the like into the site where the target cells are present (for example, in a tumor).
  • the dose of the cell or the pharmaceutical composition of the present invention may be any amount that is therapeutically effective, and those skilled in the art will know the age, sex, weight, etc. of the administration subject, the type of disease, the degree of progression, and
  • the dose and administration interval can be appropriately adjusted depending on the symptoms and the administration method.
  • the dose is 1 ⁇ 10 3 to 1 ⁇ 10 10 cells/9 (body weight), preferably 1 ⁇ 10 4 to 1 ⁇ 10 1 as the number of cells to be administered per administration. 9/9 (weight), more preferably, to 1 X 1 ⁇ 5 ⁇ 1 X 1 ⁇ 8 (body weight).
  • the administration interval can be, for example, weekly, every 10 to 30 days, every month, every 3 to 6 months, every year, etc.
  • the cells of the present invention can autonomously proliferate in the body of the subject to be administered, and thus can be administered only once.
  • the number of cells of the present invention in the body may be monitored after administration, and the administration time may be determined according to the result.
  • the cells of the present invention that express a cleavage reagent having a protease that can be controlled by a protease inhibitor are used, excessive activation of the cells may cause adverse effects such as hypercytokineemia.
  • the subject's activity is inhibited by administering an inhibitor of protease to the subject. It can be suppressed.
  • 293 T cells and SKB R-3 cells were Dulbecco's modified Eagles supplemented with 10% fetal bovine serum (FBS; Sigma Aldrich, St Louis, M ⁇ , I ot # 14 M 0 67). It culture
  • K562 cells, Raji cells and Jurkat cells were cultured in RPMI-1640 (FUJIFI LM Wako Pure Chemical, Osaka, Japan) supplemented with 10% FBS. did.
  • HIV Protease (H ⁇ VPR) Inhibitor Saquinavir and Nelfinavir (NIHAIDSR esearchand R eterence Reagent P rogr am) were donated by the Department of Virus Control, Tokyo Medical and Dental University It was
  • the anti-RFP rabbit polyclonal antibody (PM005, ⁇ ⁇ t # 045) was used as an anti-My c T ag rabbit polyclonal antibody (06-549, lot) from Medical and Biological Laboratories (N agoya, Japan). #24 1 65) is from U pstate B iotechnology (Lake Placid, NY), and anti-Syntaxin 4 mouse monoclonal antibody (QQ- 1, 7, lot # F 2 1 1 8) is S anta C ruz B iotechnology.
  • PE-labeled anti-human CD 69 antibody (3 1 09 05, lot # B 258745) and Per CP/C y 5.5 labeled anti-human HER 2 antibody (3244 1 5, lot # B 236448) were labeled with B iolegend (S purchased from an Diego, CA).
  • APC mouse anti-human CD 19 antibody (5554 1 5, lot # 7 1 086 1 2) was purchased from BD P harmingen (S an Diego, CA).
  • p HRPGK anti CD 1 9 1 syn N otch 1 T et RV P 64 was prepared to construct a plasmid vector encoding each chimeric antigen receptor (CAR) shown in FIGS. 3, 9 and 18.
  • CAR chimeric antigen receptor
  • Fluorescent protein mC herry -C DNA plasmid was distributed by N athan CS haner (N athan CS haner et al., N at Biotechno 1., Jan. 2004, Vol. 22, No. 12, 1 567-1 572). See page).
  • p HRPGK anti CD 19 syn-Notch T et RVP 64 PCR is performed by using the plasmid as a hook and anti-CD 19 outer F primer and anti-CD 19-CD 28 inner R primer. It was In addition, pc DNA 3.1-PS 11-sc F v F c-CD 28- gp 4 1 (706- 7 1 3) plasmid was made into a claw shape, and anti-CD 1 9 — CD 28 inner F primer and PCR was performed using the CD 28- outer R primer.
  • PCR was carried out using the cDNA as a claw type, an anti-CD 19 outer F primer, and a CD 28-CV 1 inner R primer into which an H H VPR recognition sequence was incorporated. Further, PCR was carried out by using pCry2PHR-mCh-RhoA plasmid as a claw type and using CV1-mC heeryinner F primer and mC herryouter R primer. Then, the two types of amplification products obtained by these PC Rs were made into a claw type, and PCR using the anti-CD 19 outer F primer and the mC herryouter R primer was performed to detect the H ⁇ VPR recognition sequence.
  • the mC herry gene was ligated to the 3'end of the cDNA encoding the anti-CD19 scFv and CD28 intracellular domains with the encoding cDNA interposed.
  • overlap extension PCR using a primer containing a 15 mer overlapping sequence was used.
  • the 5'end of the cDNA encoding the obtained CD19 sc Fv, CD28 intracellular domain, HIVPR recognition sequence and mC herry was Eco RI, and the 3'end was X ho. Carry out restriction enzyme treatment on pc DNA 3.
  • the vector is referred to as “pc DNA3— anti — CD 19 — E ffector — CAR — T 2A — YFP vector” or “anti -CD 19 E ffector CAR R-T 2 A- YFP expression vector”. Also called.
  • the activation CAR coded by the vector is also referred to as rantti-CD19_Effector-CAR".
  • HIVPR To further delete the C-terminus of the intracellular domain of CD28, HIVPR In order to delete the N-terminal of the vector, the target sequence was removed by PCR based on pc DNA3-a nti -CD 19-CD 28 -HIVPR vector, and anti -CD 19-CD 28-HIVPRA 4 CAR and Vectors (pc DNA3-a nti -CD 1 9-CD 28-HIVPR (A4) vector and pc DNA3 that code anti -CD 19 -CD 28 AI CD-H IVPRA 4 CAR (see Figure 9 for both). -a nti -CD 19_CD 28 ( ⁇ I CD) -HIVPR (A4) vector) was prepared.
  • pc DNA3-a nti -CD 19-CD 28-HIVPR vector pc DNA3-a nti -CD 19-CD 28-HIVPR (A 4) vector
  • pc DNA3-a nti -CD 19-CD 28-HIVPR (A 4) vector pc DNA3-a nti -CD 19-.
  • A4 vector a vector was also prepared in which the YFP gene was inserted so as to connect to the 3'end of each cleavage CAR gene.
  • pc DNA3-anti-HER 2 (4 D 5-3) -CD 28-CS- mC herry vector and pc DNA3-anti-CD 19-CD 28 (AICD) -HIVPR (A4) vector.
  • pcDNA3— anti — HER 2 (4 D 5 — 3) — S cissors — CAR — T 2A — mC herry vector — was created using overlap extension PCR.
  • anti-HER 2 (4 D 5 -3) -S cissors -CAR-T 2A-mC herry gene fragment was prepared by restriction enzyme treatment with N he and X h ⁇ ⁇ , and pc DN A3 (-) -H ygr ⁇ By inserting into vector (T her mo Fisher Scientific), ⁇ 02020/175366 41 ⁇ (: 171? 2020 /007038
  • the gene was cloned and the 1 to 2 expression vector was prepared by inserting into the 088 (3)- 11 So 9" vector.
  • each CAR The prepared DNA sequences encoding each CAR are shown in SEQ ID NOs: 1, 3, 5, 7, 45, 47, 49 and 51.
  • the amino acid sequences of the respective C ARs are shown in SEQ ID NOs: 2.4.6, 8, 46, 48, 50 and 52.
  • the sequence consisting of amino acids 1 to 21 indicates the leader sequence, and the sequence consisting of amino acids 22 to 31 indicates IV!
  • the sequence consisting of amino acids from position 273 to 3 shows I 1 -001 9 _ 3 0, the sequence consisting of amino acids from 274 to 282 shows 9 sequence, and the sequence consisting of amino acids from 283 to 348 represents 0028. It shows the transmembrane region, and the sequence consisting of amino acids 349 to 36 1 represents the 028 intracellular domain, the sequence consisting of amino acids 362 to 369 represents the 9 41 sequence, and the sequence consisting of 3 0 7 to 377.
  • the sequence consisting of amino acids indicates the 1 to 1 V cleavage site, and the sequence consisting of amino acids at positions 378 to 61 3 is
  • amino acid sequence of "3 ⁇ 1 -001 9- ⁇ 028-1 to 1 ⁇ ? [3 ⁇ 4 ⁇ 8 [3 ⁇ 4]" shown in SEQ ID NO: 4 the amino acid sequence up to the 369th position is SEQ ID NO: 2 Same as those in.
  • amino acid sequence of SEQ ID NO: 4 the sequence consisting of amino acids 370 to 468 shows 1 to 1 V.
  • amino acid sequence of "3 ⁇ 1 -001 9-0028 ⁇ ⁇ 0-1 to 1 IV 8 4 808 [3 ⁇ 4] shown in SEQ ID NO: 8 the amino acid sequence up to the 348th position is SEQ ID NO: Same as those in 2.
  • sequence consisting of amino acids 349 to 356 shows 941 sequence, and the sequence consisting of amino acids 357 to 451 lacks 1 amino acid at the 1 ⁇ ! end. Have lost Indicates.
  • SEQ ID NO: 46 “3 ⁇ 1 ⁇ 1 ⁇ [3 ⁇ 4 2 (405-3) -0028- In the amino acid sequence of ⁇ 8[3 ⁇ 4], the sequence consisting of amino acids at positions 1 to 21 indicates the leader sequence, and the sequence consisting of amino acids at positions 22 to 31 indicates IV! Sequence consisting of amino acid at position 277 is 3 n ti-HER 2 (4 D 5-3) sc F v, the sequence consisting of amino acids 278 to 286 represents the C 9 sequence, and the sequence consisting of amino acids 287 to 352 represents the CD 28 transmembrane region.
  • sequence consisting of amino acids 353 to 365 represents the CD28 intracellular domain
  • sequence consisting of amino acids 366 to 373 represents the gP41 sequence
  • sequence consisting of amino acids 374 to 381 represents The sequence showing the HIVPR cleavage site and consisting of amino acids 382 to 617 is mC herry: ⁇
  • amino acid sequence of "ranti-CD 1 9-CD 28-CS-CD3 zeta (I CD) -T 2A-YFP" shown in SEQ ID NO: 48 the amino acid sequence up to position 377 is SEQ ID NO: 2 Same as those in.
  • amino acid sequence of SEQ ID NO: 48 the sequence consisting of amino acids 378 to 489 represents CD 3 zeta (I CD), and the sequence consisting of amino acids 490 to 5 10 represents T 2 A sequence.
  • the sequence consisting of amino acids 511 to 747 is YFP.
  • the sequence consisting of amino acids 1 to 21 is the leader sequence, and the sequence consisting of amino acids 22 to 31 is M It shows the yc tag, the sequence consisting of amino acids at positions 32 to 277 shows anti-HER 2 (4 D 5 -3) sc F v, and the sequence consisting of amino acids from 278 to 286 shows C 9 sequence,
  • the sequence consisting of amino acids 287 to 352 represents the CD28 transmembrane region, the sequence consisting of amino acids 353 to 360 represents the gP41 sequence, and the sequence consisting of amino acids 361 to 455 represents the N-terminal region. It shows the H H VPR cleavage site in which 4 amino acids are deleted, the sequence consisting of amino acids 456 to 476 represents the T 2 A sequence, and the sequence consisting of amino acids 477 to 71 12 represents m C herry. Show.
  • amino acid sequence of “ ⁇ CD-H IVPRA 4 T 2 A-m C herry” the sequence consisting of amino acids 32 to 277 is the anti-HER 2 (4 D 5 -8) sc F v, and other sequences are the same as those in SEQ ID NO: 50.
  • N u c I e o f e c t o r k i t V (L o n z a, B a se l, Sw i t z e r l a n d) was used, and each was carried out according to the attached protocol.
  • pc DNA3(-)-anti-HER2(4D5-8)-Scissors-CAR-T2A-mCherry-Hygro vector was introduced into the gene. Twenty-four hours after gene transfer, YFP and mCherry-positive cells were selected using FACS aria II and the final concentration of 1 mg/mL G4 18 and 200 g/mL Hygr om ycin were added to the medium.
  • a protein lysate of whole cells, cell membrane or cytoplasm was prepared.
  • the whole cell lysate was added to the collected cells by lysis buffer _ (1% T r i t o n -X, 50 mM T r i s -H C I ,0.5 M N aC I ,.
  • Each of the protein lysates was mixed with an equal amount of 2XL ae mm li sample buffer and reacted at 100 ° C for 5 minutes to prepare a sample for Western blot.
  • As the gel for electrophoresis 12.5% PAGE L (ATTO, Tokyo, Japan) was used. Blocking after blotting was performed using 5% skim milk (Meiji, Tokyo, Japan).
  • Western lightning plus ECL Perkin E ⁇ 02020/175 366 48 ⁇ (: 17 2020 /007038
  • the fluorescence microscope observation was performed using an all-in-one fluorescence microscope BZ-8100 or BZ-X800 (both are KEYE NCE, Osak a, Jap a n). Nuclear staining was performed using Ho e c h st 33258 (D o j i n d o, K u ma mo t o, J apa n).
  • TRI zolreagent (T her mo Fisher Scientific) was used for mRNA extraction from cells, and Super S cript IIR everse T ranscriptase (T her mo Fisher Scientific) was used for c DN A synthesis. I did it.
  • the reverse transcription reaction was performed using 250 ng of t o t a l RNA.
  • this vector was introduced into 293 cells, and a n t i _CD 19_C
  • saquinavir or nelfinavir which are 1 to 1 V inhibitors, are 1 to 1 V inhibitors.
  • Target domain here 01 (3116 ““so”) ⁇ 02020/175366 51 ⁇ (: 171? 2020 /007038
  • 1 ⁇ 1 V is a dimeric protease, It has been reported that the four amino acids at its 1 ⁇ 1 end, called the sequence (" ⁇ - ⁇ 1 ⁇ 1 16 6-chome" contributes to the self-dimerization. " ..
  • each of the vectors thus prepared was introduced into 293 cells, and ⁇ 028-1 ⁇ 1 ⁇ ? [3 ⁇ 4 0 8 1 0 028-1 ⁇ 1 ⁇ ? [3 ⁇ 44808 [3 ⁇ 4 and 02828 88 ⁇ ⁇ 0 to 1 to 1 ⁇ y P RA4 08] were each overexpressed and analyzed by a western blot.
  • the full-length band was detected only in the sample overexpressing 028888 I 0-1 to 1 ⁇ ZP RA4 08.
  • each cleavage 0 was expressed in 2 93 3 cells in a mode in which a cage was fused at the 0 end. Observed and analyzed with a fluorescence microscope. As a result, as shown in Figure 11, No localization to the cell membrane was observed in cells that expressed the fusion protein of Eight and eight. On the other hand, as shown in Figure 12, In the cells expressing the fusion protein of Eight and eight, although a signal was partially observed in the cytoplasm, it tended to be localized in the cell membrane. Furthermore, as shown in FIG. 13, no fluorescence signal derived from the cytoplasm was detected in cells expressing the fusion protein of ⁇ 0 2 8 8 ⁇ ⁇ 0-1 to 10 8 [3 ⁇ 4 and ⁇ , Localization to the complete cell membrane was observed.
  • the gnoll was detected.
  • the activated protease is cleaved to activate 0, so that the signal transduction factor (corresponding to 01 ⁇ 6““;/ above) may be released into the cytoplasm. It was revealed.
  • Example 3 in the 1-antigen recognition system (single arm system), which uses only 001 9 as the target antigen, the cleavage of the activation 08 by the cleavage 08 causes the antigen to be recognized in the target cells. Recognized to occur
  • Routing cells were prepared.
  • the activation O according to the present invention can induce the activation of Ding cells specific to a target cell.
  • a recognition sequence which is a costimulatory molecule, was interposed as an activation expression vector.
  • I -0 We created an expression vector (see Figure 18).
  • it is also used in actual clinical trials such as Chisagen Lecle (Kimria (registered trademark), manufactured by Novartis) 003 6
  • Chisagen Lecle Karlia (registered trademark), manufactured by Novartis) 003 6
  • the T_A sequence was mediated downstream of anti_CD19Effect ⁇ r CAR and YFP was inserted. The gene is integrated.
  • the P-expressing vector was gene-transfected into Jurkat cells and cocultured with K562 cells (CD19 negative cells) or Raji cells (CD19 positive cells).
  • K562 cells CD19 negative cells
  • Raji cells CD19 positive cells
  • inducing expression of CD69 which is a T cell activation marker specifically in CD19-positive Raji cells
  • FIG. 20 when CAR positive J u r k at cells were isolated and gene expression analysis was performed, induction of L _ 2 m R N A expression was confirmed by co-culture with R a j i.
  • anti-CD 19 Effector/Jurkat cells anti-CD 19 Jurkat cells stably expressing Effector CAR -T 2A-YFP (hereinafter also referred to as "anti-CD 19 Effector/Jurkat cells") were established. Then, also in this cell, as shown in Fig. 21, by co-culture with Raji cells or the modified SK_BR_3 cells into which the CD19 gene had been introduced, the cells of CD69 expression, It was confirmed that activation was induced
  • target cell-specific T cell activation can also be induced by activation C A R mediated by the H V VP recognition sequence.
  • Example 5 An anti-HER2(4D5-3)-Scissors-CAR-T2A-mCherry expression vector was introduced into r/J urkat cells.
  • anti -HER 2 (4 D 5 -3) -S cissors -CAR-T 2A-mC herry expression vector
  • anti -HER 2 (4 D 5-8) — S cissors— CAR— T 2A — MC herry expression vector was introduced into anti — CD 19 E ffector/J urkat cells
  • anti — CD 19 E ffector CAR and anti — HER 2 (4 D 5-8) -S cissors -Established J urkat cells that stably express both CAR (hereinafter also referred to as ranti — CD 19 Effector CAR + anti — HER 2 (4 D 5-8 )S cissors/J urkat cells) (See Figure 22).
  • the cells were co-cultured with R_3 cells or the modified SK-BR__3 cells, and T cell activation was analyzed using the expression level of CD69, which is a marker for early activation, as an index.
  • cleaving 08 [3 ⁇ 4 can regulate the activation of Ding cells by the activation 08 depending on the expression pattern of the surface antigen of the target cell. It was also demonstrated that cleavage 0, which has a high affinity for surface antigens, can more efficiently attenuate the activation 08 activation.
  • the present invention [08] treats diseases such as cancer while controlling side effects and controlling adverse events (such as hypercytokineemia) caused by excessively activated Ding cells. Or, it can be prevented, and is useful in cancer immunotherapy and the like.

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