WO2020004337A1 - Récepteur antigénique chimérique spécifique à cd37 - Google Patents

Récepteur antigénique chimérique spécifique à cd37 Download PDF

Info

Publication number
WO2020004337A1
WO2020004337A1 PCT/JP2019/024974 JP2019024974W WO2020004337A1 WO 2020004337 A1 WO2020004337 A1 WO 2020004337A1 JP 2019024974 W JP2019024974 W JP 2019024974W WO 2020004337 A1 WO2020004337 A1 WO 2020004337A1
Authority
WO
WIPO (PCT)
Prior art keywords
car
cells
chimeric antigen
antigen receptor
region
Prior art date
Application number
PCT/JP2019/024974
Other languages
English (en)
Japanese (ja)
Inventor
精太郎 寺倉
仁 清井
真吾 奥野
Original Assignee
国立大学法人名古屋大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人名古屋大学 filed Critical 国立大学法人名古屋大学
Priority to JP2020527516A priority Critical patent/JPWO2020004337A1/ja
Publication of WO2020004337A1 publication Critical patent/WO2020004337A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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 a chimeric antigen receptor. Specifically, the present invention relates to a CD37-specific chimeric antigen receptor and its application, and further to a means for designing a chimeric antigen receptor.
  • This application claims the priority based on Japanese Patent Application No. 2018-122519 filed on June 27, 2018, the entire content of which is incorporated by reference.
  • the chimeric antigen receptor (hereinafter referred to as “CAR”) has a single-chain variable region of an antibody as an extracellular domain, and a transmembrane region and an intracellular domain of a molecule that transmits a costimulatory signal with CD3 ⁇ . Things.
  • CAR chimeric antigen receptor
  • CAR-based therapy shows higher cytotoxic activity than antibody therapy and requires multiple treatments to autonomously amplify by antigen stimulation in one infusion Excellent in none. Since TCR gene transduction targets the complex of HLA and the peptide presented on it, it can be treated only if the patient has a specific HLA (HLA-restricted), but CAR has HLA-restricted It is more advantageous in that there is no property. That is, if the target antigen is positive for the tumor cells, treatment can be performed for all patients who are positive for the target antigen.
  • Chimeric antigen receptor gene-introduced T cell (CAR-T cell) therapy has recently been approved as a drug in the United States for CD19CAR-T, and its clinical effects are very powerful, and it has been used in various B cell hematologic malignancies. Clinical usefulness is expected (for example, refer to Patent Document 1, Non-Patent Documents 1 and 2).
  • CD19CAR-T targets CD19, it may become negative at certain frequencies and recur. This is a so-called escape phenomenon.
  • CAR-T cells against other antigens expressed on the surface of B cells at the same time as CD19 In fact, attempts have been made to develop CAR-T cells targeting B cell differentiation markers other than CD19, such as CD20 and CD22, but no satisfactory results have been obtained. Under such circumstances, an object of the present invention is to provide a new CAR suitable for clinical application.
  • Another object of the present invention is to provide a solution to this problem.
  • CD37 is strongly expressed on mature B-cell tumors, but also weakly expressed on granulocytes and T lymphocytes. Therefore, we focused on the CD37 expression level of target tumor cells and the CD37 expression level of normal cells, and examined the reactivity of CAR-T cells in detail.
  • the length of the extracellular domain spacer involved in antigen recognition is important for the reactivity of CAR-T cells, etc. It was revealed that CD37-specific CAR-T cells whose reactivity to normal cells was suppressed (the antigen recognition ability was moderately regulated) by shortening the spacer were reduced. CAR-T cells produced by introducing a CAR gene with a long extracellular domain spacer reacted with CD37-positive cells present in the culture solution after gene transfer, causing a mutual elimination phenomenon, but the spacer was shortened. In this case, it was possible to suppress the phenomenon.
  • CD37 is expressed on some of the normal cells, but CD37CAR-T cells that have been successfully produced have high specificity for tumor cells that highly express CD37 (in other words, normal (Low reactivity to cells), indicating a clinically applicable profile.
  • a genetically modified lymphocyte that expresses the CD37-specific chimeric antigen receptor according to any one of [1] to [7] on a cell surface.
  • a method for determining the structure of a chimeric antigen receptor whose target recognition ability is regulated comprising the following steps (1) to (3): (1) designing a plurality of chimeric antigen receptors having different distances between the antigen recognition region and the transmembrane region, and preparing a genetically modified lymphocyte expressing each of them; (2) evaluating the target recognition level of each genetically modified lymphocyte in vitro; (3) a step of selecting the structure of the chimeric antigen receptor expressed by the genetically modified lymphocyte showing the optimal recognition level.
  • [15] A chimeric antigen receptor whose target recognition ability is regulated, comprising a structure determined by the method according to [13] or [14].
  • [16] A gene encoding the chimeric antigen receptor according to [15].
  • CD37 expression in various tumor cell lines were stained with CD37-FITC antibody (black line) and FICT-isotype antibody (gray shaded) and analyzed by flow cytometry. CD37 expression in normal cells. Analyzed with leukocytes only. Summary of CD37 expression. Data was evaluated by the ratio of the mean fluorescence intensity (Mean fluorescence intensity) of the staining of the isotype antibody and the CD37 antibody.
  • AML acute myeloid leukemia: acute myeloid leukemia
  • B-ALL B-cell acute lymphoblastic leukemia: B-cell acute lymphoblastic leukemia
  • BCL B-cell lymphoma; B-cell lymphoma
  • Burkitt Burkitt's lymphoma
  • MM multiple myeloma: multiple myeloma
  • T-ALL T-cell acute lymphoblastic leukemia: T-cell acute lymphoblastic leukemia.
  • Time course analysis of CD37 expression after CD3 + T cell stimulation with CD3 / 28 beads (time course analysis). CD3-positive cells isolated from healthy donors were stimulated with CD3 / 28 beads, and the transition of CD37 expression was examined.
  • MFIR is a value obtained by dividing the MFI of the CD37 antibody by the MFI of the isotype antibody.
  • CD37-CAR design In the upper (long spacer type) configuration, the scFv is linked to the CD28 transmembrane region via the Fc hinge, CH2 domain and CH3 domain. In the lower configuration (short spacer type), the scFv is connected to the CD28 transmembrane region via the Fc hinge.
  • CD37 VH Anti-CD37 antibody heavy chain variable region
  • CD37 VL Anti-CD37 antibody light chain variable region
  • Fc Fc hinge
  • CH2 CH2 domain
  • CH3 CH3 domain
  • CD28TM CD28 transmembrane region
  • CD28IC CD28 intracellular domain
  • TEGFR truncated EGFR (gene transfer marker).
  • Intracellular cytokine assay results The ability to produce the cytokine IFN- ⁇ was evaluated by flow cytometry (upper) and summarized in the graph (lower). None: No target cells, K562: Coculture with K562 (CD37 negative target), Ramos: Coculture with Ramos (CD37 positive target). Intracellular cytokine assay results. The production ability of cytokine IL-2 was evaluated by flow cytometry (upper), and the results were summarized in a graph (lower).
  • Effector cells Primary CD3 + T cells (Plain, 37S-HL, 37S-LH), Target cells (Target): Ramos (CD37 + , CFSE + ), E: T ratio: 1: 1, 1: 2 , 1: 4, 1: 8. Sorting of peripheral blood samples from healthy donors. Whole blood of a healthy donor was lysed and cells were sorted by FACS according to the level of CD37 expression. CD37 high-expressing cells and CD37 low-expressing cells were respectively reacted with CD37-CAR-T cells. Effect of CD37S-LH CAR-T cells on CD37-high normal cells and CD37-low normal cells (result of intracellular cytokine staining). CD37-CAR design.
  • CD37_00 no spacer
  • CD37_15 15 amino acid spacer
  • CD37_30 (30 amino acid spacer); CD37_60 (60 amino acid spacer); CD37_125 (125 amino acid spacer); CD37_232 (232 amino acid spacer).
  • VL Anti-CD37 antibody light chain variable region
  • VH Anti-CD37 antibody heavy chain variable region
  • TM CD28 transmembrane region
  • CD28 CD28 intracellular domain
  • T2A 2A peptide derived from Thosea asigna virus
  • tEGFR truncated EGFR Comparison of cell proliferation rate and gene transfer efficiency of six CD37-CARs.
  • the number of cells after CD37-CAR gene transfer (A), the gene transfer efficiency (B), and the number of gene transfer cells (C) are shown.
  • the middle row is a flow plot showing the ratio of the number of living cells.
  • the lower row shows the gene transfer efficiency.
  • Amplification efficiency when CD37CAR-T cells were stimulated with Ramos The time-dependent change in the number of cells after stimulation (upper row) and the number of cells on day 13 (lower row) are shown.
  • CD37-Specific Chimeric Antigen Receptor The first aspect of the present invention relates to a CD37-specific chimeric antigen receptor (conventionally, the chimeric antigen receptor is referred to as "CAR").
  • the CD37-specific chimeric antigen receptor is a structure containing an extracellular domain exhibiting CD37-specific binding ability (binding specificity), a transmembrane region, and an intracellular signal domain for effector functions of immune cells.
  • CD37-specific chimeric antigen receptor is a structure containing an extracellular domain exhibiting CD37-specific binding ability (binding specificity), a transmembrane region, and an intracellular signal domain for effector functions of immune cells.
  • each domain will be described.
  • Extracellular domain In the present invention, the extracellular domain shows CD37-specific binding.
  • the extracellular domain is roughly classified into an antigen recognition region responsible for antigen recognition and a spacer domain.
  • antigen recognition region responsible for antigen recognition
  • spacer domain the details of the antigen recognition region will be described.
  • the spacer domain will be described in the next section “(b) Transmembrane region”.
  • the antigen recognition region is not limited to this, but preferably has a structure in which the scFv of the anti-CD37 antibody, that is, the light chain variable region (VL) and the heavy chain variable region (VH) are linked by a linker.
  • a linker for example, a peptide linker can be used.
  • the peptide linker is a linker composed of a peptide in which amino acids are connected in a linear manner.
  • a typical example of a peptide linker is a linker composed of glycine and serine (GGS linker or GS linker).
  • GGS linker and glycine and serine which are amino acids constituting the GS linker, are small in their own size, and are unlikely to form a higher-order structure in the linker.
  • the length of the linker is not particularly limited. For example, a linker having 5 to 25 amino acid residues can be used.
  • Non-Patent Document 3 US Patent No. 8,333,966 (Non-Patent Document 3) can be referred to for the anti-CD37 antibody.
  • VL and VH constituting the scFv is not particularly limited. That is, an embodiment in which VL is arranged on the N-terminal side (in this case, the C-terminal of VL and the N-terminal of VH are linked by a linker), and an embodiment in which VH is arranged on the C-terminal side (in this case, (The C-terminus of VH and the N-terminus of VL are linked by a linker).
  • Examples of the amino acid sequences of VL and VH that constitute the scFv of the anti-CD37 antibody are shown in SEQ ID NO: 1 (VL) and SEQ ID NO: 2 (VH).
  • the scFv of an anti-CD37 humanized monoclonal antibody is used as the antigen recognition region.
  • the anti-CD37 humanized monoclonal antibody is a humanized monoclonal antibody using CD37 as an antigen.
  • a humanized monoclonal antibody is an antibody in which the structure of a monoclonal antibody of another animal species (eg, mouse or rat) is similar to the structure of a human antibody, and is a humanized antibody in which only the constant region of the antibody is replaced with that of a human antibody.
  • transmembrane domain The transmembrane domain is interposed between the extracellular domain and the intracellular signal domain.
  • a transmembrane region such as CD28, CD3 ⁇ , CD8 ⁇ , CD3, CD4 or 4-1BB can be used.
  • a transmembrane region composed of an artificially constructed polypeptide may be used. Incidentally, a specific example of the transmembrane region will be described in Examples described later. Those functionally equivalent to those shown in the embodiments can also be used.
  • the transmembrane region is linked via a spacer domain to the antigen recognition region of the extracellular domain. That is, the spacer provides a distance (space) between the antigen recognition region and the transmembrane region.
  • the antigen recognition region and the transmembrane region may be directly linked. In this case, the distance between the antigen recognition region and the transmembrane region becomes zero (0), and when the CAR having the structure is expressed in a cell, a state where the antigen recognition region is directly bound to the cell membrane is usually formed.
  • the “spacer” is a member (linker) that connects the transmembrane region to the antigen recognition region, and is used to distinguish it from the linker that constitutes the antigen recognition region in the case of scFv, and to separate the antigen recognition region and the transmembrane region.
  • linker a member that connects the transmembrane region to the antigen recognition region, and is used to distinguish it from the linker that constitutes the antigen recognition region in the case of scFv, and to separate the antigen recognition region and the transmembrane region.
  • spacer is used.
  • the spacer is composed of 1 to 30 amino acids and is linear (if the number of constituent amino acids is 2 or more, it is a linear peptide).
  • the sequence of the spacer is not particularly limited.
  • a molecule to be used for a hinge portion of IgG preferably human IgG (for example, subtype IgG1 or IgG4) or a part thereof, a hinge portion and a portion of CH2, or a transmembrane region (for example, A sequence such as a part of CD28) is used as a spacer.
  • a highly flexible spacer can be expected to be formed by using the hinge portion or a part thereof.
  • a general CAR has a structure in which a transmembrane region is linked to an antigen recognition region via a heavy chain Fc region (hinge, CH2, CH3) of an antibody.
  • the heavy chain Fc region is composed of about 220 amino acid residues. Therefore, in the case of a general CAR, a transmembrane region is separated from an antigen recognition region by a spacer consisting of about 220 amino acid residues.
  • the number of amino acid residues constituting the spacer is 1 to 30, and the distance between the antigen recognition region and the transmembrane region is overwhelmingly shorter than that of a general CAR.
  • the antigen-recognition ability (target recognition level) is appropriately regulated, and the effect of reducing the reactivity to normal cells is exhibited, which is unique to the present invention.
  • Reducing the length of the spacer can advantageously act on the effects of the present invention. Therefore, the number of amino acid residues constituting the spacer is preferably 1 to 25, and more preferably 1 to 20.
  • the structure other than the spacer particularly, the antigen recognition region
  • the cell into which the CAR gene is introduced referred to as “CAR-expressing cell” for convenience of explanation
  • the length of the spacer at which the optimum target recognition level can be obtained may be determined by verification or confirmation by experiment.
  • the intracellular signal domain transmits a signal necessary for exerting effector functions of immune cells. That is, when the extracellular domain binds to CD37, an intracellular signal domain capable of transmitting a signal necessary for activating immune cells is used.
  • the intracellular signal domain includes a domain for transmitting a signal through the TCR complex (referred to as “first domain” for convenience) and a domain for transmitting a costimulatory signal (for convenience, “second domain”). ) are included.
  • an intracellular domain such as Fc ⁇ RI ⁇ can be used as the first domain.
  • CD3 ⁇ is used.
  • an intracellular domain of a costimulatory molecule is used as the second domain. Examples of costimulatory molecules include CD28, 4-1BB (CD137), CD2, CD4, CD5, CD134, OX-40 and ICOS.
  • the intracellular domain of CD28 or 4-1BB is employed.
  • the mode of connection between the first domain and the second domain is not particularly limited, but is preferably a transmembrane region since it has been known in the past that co-stimulation was strongly transmitted when CD3 ⁇ was distantly connected.
  • the second domain on the side. A plurality of the same or different intracellular domains may be linked in tandem to form the first domain. The same applies to the second domain.
  • the first domain and the second domain may be directly linked to each other, or a linker may be interposed between them.
  • a linker for example, a peptide linker can be used.
  • the peptide linker is a linker composed of a peptide in which amino acids are connected in a linear manner. The structure, characteristics, and the like of the peptide linker are as described above. However, a linker composed only of glycine may be used here.
  • the length of the linker is not particularly limited. For example, a linker having 2 to 15 amino acid residues can be used.
  • specific examples of the intracellular signal domain will be described in Examples below. Those functionally equivalent to those shown in the embodiments can also be used.
  • a leader sequence (signal peptide) is used to promote the transport of CAR onto the cell membrane.
  • a leader sequence of a GM-CSF receptor can be used.
  • a spacer domain may be provided between the transmembrane region and the intracellular signal domain. In other words, instead of directly connecting the intracellular signal domain to the transmembrane region, the intracellular signal domain may be connected via a linker (usually a peptide linker).
  • a second aspect of the present invention relates to a gene encoding CAR (hereinafter sometimes referred to as “CAR gene”) and its use (expression cassette, vector, A method of preparing a genetically modified lymphocyte that expresses CAR, a genetically modified lymphocyte that expresses CAR, and uses thereof.
  • the CAR gene of the present invention encodes a CAR having the above structure. Therefore, a gene-modified lymphocyte (CAR gene-introduced lymphocyte) that expresses the CAR of the present invention on the cell surface can be obtained by introducing it into an appropriate cell (a cell for CAR expression) and expressing it.
  • CAR-transfected lymphocytes can be used for CAR therapy.
  • a specific example of the CAR gene sequence is shown in SEQ ID NO: 3.
  • the CAR gene comprises a GM-CSF receptor leader sequence (SEQ ID NO: 4), a region encoding VL (SEQ ID NO: 5), a linker sequence (SEQ ID NO: 6), from the 5 'end to the 3' end.
  • VH-encoding region (SEQ ID NO: 7), anti-CD37 antibody-derived sequence (gatcag), hinge-encoding region (spacer sequence) (SEQ ID NO: 8), CD28 transmembrane region (SEQ ID NO: 9) , A region encoding a CD28 intracellular domain (SEQ ID NO: 10), a linker sequence (GGCGGAGGG), and a region encoding a CD3 ⁇ intracellular domain (SEQ ID NO: 11).
  • an expression cassette for CAR expression (hereinafter, also referred to as “CAR expression cassette”) can be constructed.
  • the CAR expression cassette includes a promoter and a CAR gene under the control of the promoter.
  • the CAR gene is placed downstream of the promoter so as to be under the control of the promoter.
  • promoters include CMV-IE (a promoter derived from the cytomegalovirus early gene), SV40ori, retrovirus LTP, SR ⁇ , EF1 ⁇ , and ⁇ -actin promoter.
  • the promoter is operably linked to the CAR gene.
  • the promoter is operably linked to the CAR gene
  • the CAR gene is placed under the control of the promoter” and is usually directly attached to the 3 'end of the promoter.
  • the CAR genes will be linked via another sequence.
  • a polyA addition signal sequence is arranged downstream of the CAR gene. Transcription is terminated by use of a polyA addition signal sequence.
  • the poly-A addition signal sequence an SV40 poly-A addition sequence, a bovine-derived growth hormone gene poly-A addition sequence and the like can be used.
  • a gene for detection reporter gene, cell or tissue-specific gene, selection marker gene, etc.
  • an enhancer sequence a WRPE sequence, etc.
  • the detection gene is used for determining the success or failure and efficiency of introduction of the expression cassette, detecting the expression of the CAR gene or determining the expression efficiency, selecting and sorting cells in which the CAR gene has been expressed, and the like.
  • expression efficiency is improved by using an enhancer sequence.
  • Genes for detection include the neo gene which confers resistance to neomycin, the npt gene (Herrera ⁇ Estrella, EMBO J.H2 (1983), 987-995) which confers resistance to kanamycin and the like, and the nptII gene (Messing & Vierra.Gene 1). 9: 259-268 (1982)), the hph gene (Blochinger ⁇ ⁇ ⁇ ⁇ & igDiggl mann, Mol Cell Bio 4: 2929-2931) that confers resistance to hygromycin, and the dhfr gene (Bourouis et al. EMBO J.2 (7)), etc.
  • luciferase gene (Giacomin, P1. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), ⁇ -Glucuronidase (GUS) gene, GFP (Gerdes, FEBS Lett. 389 (1996), 44-47) and its variants (EGFP, d2EGFP, etc.) and other fluorescent protein genes (reporter genes) and intracellular domains Lacking epidermal growth factor receptor (EGFR) Genes such gene can be used.
  • the detection gene is linked to the CAR gene via, for example, a bicistronic control sequence (eg, internal ribosome recognition sequence (IRES)) or a sequence encoding a self-cleaving peptide.
  • a self-cleaving peptide is, but is not limited to, the 2A peptide (T2A) from Thosea asigna virus.
  • Known self-cleaving peptides include 2A peptide (F2A) derived from hoof disease virus (FMDV), 2A peptide (E2A) derived from equine rhinitis A virus (ERAV), and 2A peptide (P2A) derived from porcine teschovirus (PTV-1). Have been.
  • the CAR expression cassette is mounted on a vector for introduction into CAR expression cells.
  • the term "vector” refers to a nucleic acid molecule capable of transporting a nucleic acid molecule inserted therein into a target (target cell), and its form, origin, and the like are not particularly limited.
  • a suitable vector is a viral vector, but non-viral vectors can also be used. The method using a viral vector skillfully utilizes the phenomenon that a virus infects a cell, and can achieve high gene transfer efficiency.
  • Retrovirus vectors lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, Sendai virus vectors, and the like have been developed as virus vectors.
  • retrovirus vectors lentivirus vectors, and adeno-associated virus vectors
  • the target gene incorporated in the vector is integrated into the host chromosome, and stable and long-term expression can be expected.
  • Each viral vector can be prepared according to a previously reported method or using a commercially available dedicated kit.
  • non-viral vectors include plasmid vectors, liposome vectors, positively charged liposome vectors (Felgner, PL, Gadek, TR, Holm, M. et al., Proc. Natl. Acad. Sci., 84: 7413-7417 , 1987), a YAC vector and a BAC vector.
  • the recombination operation (construction of a CAR expression cassette, mounting of the CAR expression cassette on a vector, etc.) includes standard recombinant DNA techniques such as a method using restriction enzymes and DNA ligase (for example, Molecular Cloning, Third Edition, 1.84, see Cold Spring Harbor Laboratory Press, New York).
  • Cells into which the CAR gene is introduced include CD4-positive CD8-negative T cells, CD4-negative CD8-positive T cells, T cells prepared from iPS cells, ⁇ -T cells, ⁇ -T cells, and NK cells , NKT cells and the like.
  • Various cell populations can be used as long as they contain lymphocytes or progenitor cells as described above.
  • PBMC peripheral blood mononuclear cells collected from peripheral blood is one of the preferred target cells. That is, in a preferred embodiment, a gene transfer operation is performed on PBMC.
  • PBMC may be prepared by a conventional method.
  • the cells are stimulated with an anti-CD3 antibody and an anti-CD28 antibody to activate cells for CAR expression.
  • stimulation with an anti-CD3 antibody and an anti-CD28 antibody can be performed by culturing in a culture vessel (eg, a culture dish) whose culture surface is coated with an anti-CD3 antibody and an anti-CD28 antibody.
  • An anti-CD3 antibody for example, a trade name CD3pure antibody provided by Miltenyi Biotech
  • an anti-CD28 antibody for example, a trade name CD28pure antibody provided by Miltenyi Biotech can be used
  • the stimulation can be performed using magnetic beads coated with an anti-CD3 antibody and an anti-CD28 antibody (for example, Dynabeads T-Activator CD3 / CD28 provided by VERITAS). Note that it is preferable to use the “OKT3” clone as the anti-CD3 antibody.
  • T cell growth factor IL-2, IL-15, IL-7 and the like can be used.
  • T cell growth factors such as IL-2, IL-15 and IL-7 can be prepared according to a conventional method. Also, commercially available products can be used. Although it does not preclude the use of T cell growth factors from animal species other than humans, usually, T cell growth factors are of human origin (or may be recombinant). T cell growth factors such as human IL-2, IL-15, and human IL-7 can be easily obtained (for example, provided by Miltenyi Biotech, R & D Systems, etc.).
  • CAR transfected lymphocytes typically, cells after CAR transfection (CAR transfected lymphocytes) are expanded for their application (administration to patients).
  • CAR gene-introduced lymphocytes are cultured using a culture solution to which T cell growth factor has been added (subculture is performed if necessary).
  • the same treatment (reactivation) as in the case of activating the CAR-expressing cells may be performed.
  • a medium supplemented with serum human serum, fetal bovine serum, etc.
  • serum-free medium human serum, fetal bovine serum, etc.
  • serum-free media for lymphocytes are TexMACS TM (Miltenyi Biotech), AIM V® (Thermo Fisher Scientific).
  • autologous serum that is, serum collected from a patient who receives CAR gene-transduced lymphocytes.
  • a medium suitable for culturing lymphocytes may be used. Specific examples include TexMACS TM and AIMV (registered trademark) described above.
  • Other culture conditions may be any conditions suitable for survival and proliferation of lymphocytes, and general conditions may be employed. For example, culture may be performed in a CO 2 incubator (CO 2 concentration: 5%) set at 37 ° C.
  • a further aspect of the present invention is a genetically modified lymphocyte expressing a chimeric antigen receptor obtained by the preparation method of the present invention (hereinafter referred to as “the CAR gene-transduced lymphocyte of the present invention”). And its use.
  • the CAR transfected lymphocytes of the present invention can be typically used for the treatment, prevention or amelioration of tumors / cancers that overexpress CD37 (hereinafter referred to as “target diseases”).
  • target diseases include various B-cell malignant lymphomas (B-cell acute lymphocytic leukemia, follicular lymphoma, diffuse lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin lymphoma, etc.) ), Myeloproliferative disorders, myelodysplastic / myeloproliferative tumors (CMML, JMML, CML, MDS / MPN-UC), myelodysplastic syndrome, acute myeloid leukemia, multiple myeloma, and the like.
  • B-cell malignant lymphomas B-cell acute lymphocytic leukemia, follicular lymphoma, diffuse lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin lymphoma, etc.
  • Myeloproliferative disorders myelodysplastic
  • Treatment includes alleviation (mildness) of symptoms or accompanying symptoms characteristic of the target disease, prevention or delay of worsening of symptoms, and the like. “Prevention” refers to preventing or delaying the onset / expression of a disease (disorder) or its symptoms, or reducing the risk of onset / expression. On the other hand, “improvement” means that a disease (disorder) or a symptom thereof is alleviated (mild), improved, ameliorated, or cured (including partial cure).
  • the CAR transfected lymphocytes of the present invention can also be provided in the form of a cell preparation.
  • the cell preparation of the present invention contains a therapeutically effective amount of the CAR transgenic lymphocyte of the present invention.
  • a therapeutically effective amount of the CAR transgenic lymphocyte of the present invention For example, for a single administration, 1 ⁇ 10 4 to 1 ⁇ 10 10 cells are contained.
  • Various components such as dimethyl sulfoxide (DMSO) and serum albumin to protect cells, antibiotics to prevent bacterial contamination, and various components (vitamins, etc.) to activate, proliferate or differentiate cells , Cytokines, growth factors, steroids, etc.) may be included in the cell preparation.
  • administration route of the CAR gene-introduced lymphocyte or cell preparation of the present invention is not particularly limited.
  • administration may be by intravenous, intraarterial, intraportal, intradermal, subcutaneous, intramuscular, or intraperitoneal injection.
  • local administration may be performed. Examples of the local administration include direct injection into a target tissue / organ / organ.
  • the administration schedule may be created in consideration of the sex, age, weight, disease state, and the like of the subject (patient). In addition to single administration, continuous or periodic administration may be performed multiple times.
  • a therapeutically effective amount of the CAR transgenic lymphocytes is administered to the patient.
  • the CAR gene-introduced lymphocytes of the present invention exhibit a characteristic of exhibiting an antitumor effect on a tumor expressing CD37 on the cell surface due to its characteristic structure. Therefore, application to the treatment of B-cell malignant lymphoma, part of acute myeloid leukemia, multiple myeloma, and the like is expected.
  • CAR structure determination method a method for determining the structure of a CAR whose target recognition ability is regulated.
  • the CAR structure determination method of the present invention enables highly safe preparation of CAR with reduced reactivity to normal cells.
  • the following steps (1) to (3) are performed.
  • Target of each genetically modified lymphocyte Step of evaluating recognition level in vitro
  • step (1) first, a plurality of CARs having different distances between the antigen recognition region and the transmembrane region constituting the extracellular domain are designed.
  • a plurality of CARs with different lengths of the spacer connecting the antigen recognition region and the transmembrane region are designed.
  • the structure / configuration other than the spacer is the same.
  • a difference may be provided in the structure / configuration other than the spacer except for the exception.
  • scFv is used as an antigen recognition region, but is not limited thereto as long as it has a structure that does not hinder the function as a CAR and can obtain the required specificity and reactivity.
  • the target molecule is a receptor
  • its ligand for example, a natural ligand
  • a part thereof, or a variant thereof which exhibits binding ability to the receptor
  • the method of the present invention is excellent in versatility, and the target molecule (antigen) recognized by the antigen recognition region is not particularly limited.
  • target molecules include CD19, CD20, GD2, CD22, CD30, CD33, CD37, CD44variant7 / 8, CEA (carcinoembryonic antigen), Her2 / neu, MUC1 (Mucin 1), MUC4 (Mucin 4), and MUC6 (Mucin 6). ), IL-13 receptor-alpha2, immunoglobulin light chain, anti-PSMA, VEGF receptor2.
  • CAR transfected lymphocyte a genetically modified lymphocyte that expresses CAR.
  • Preparation and the like of the CAR gene may be performed by a conventional method (see the description of the above aspect).
  • step (2) the target recognition level of each genetically modified lymphocyte prepared in step (1) is evaluated in vitro.
  • the original target cell a tumor cell that expresses an antigen recognized by the antigen recognition region of the CAR on the cell surface and is targeted for CAR attack; tumors derived from cell lines or patients
  • the level of target recognition is assessed by comparing the reactivity to cells that can be used to challenge with other cells that should not be challenged (typically normal cells).
  • the evaluation here includes, for example, an intracellular cytokine production assay (eg, a method using flow cytometry), an extracellular cytokine production assay (eg, an ELISA (Enzyme-Linked ImmunoSorbent Assay) method, an ELISPOT (Enzyme-Linked ImmunoSpot) method) , A tumor cytotoxicity test (eg, 51 Cr release test) and the like.
  • an intracellular cytokine production assay eg, a method using flow cytometry
  • an extracellular cytokine production assay eg, an ELISA (Enzyme-Linked ImmunoSorbent Assay) method, an ELISPOT (Enzyme-Linked ImmunoSpot) method
  • a tumor cytotoxicity test eg, 51 Cr release test
  • the efficiency of introduction of CAR genes, the proliferation rate of genetically modified lymphocytes, the cell division response after stimulation, etc. should be compared and evaluated. Further, the effects on target cells may be evaluated in vivo using an animal model.
  • step (3) the structure of the CAR expressed by the genetically modified lymphocyte showing the optimal recognition level is selected. While maintaining reactivity with the target tumor cell (however, the higher the reactivity with the target tumor cell is, the better), the indicator or criteria that the reactivity is low or substantially non-reactive with normal cells Then, the level of the recognition level is evaluated, the genetically modified lymphocyte showing the optimum recognition level is determined, and the CAR structure expressed by the gene is selected as the optimum one.
  • the target-recognition level is regulated and the safety of the genetically modified lymphocytes (CAR gene-transferred lymphocytes) is improved. can get.
  • the genetically modified lymphocytes will be more suitable for clinical use.
  • the distance between the antigen recognition region and the transmembrane region may be optimized based on the selected structure (step (4)). In the optimization, a plurality of CARs having different distances (however, the degree of the difference in distance is smaller than in the case of step (1)) are designed, and evaluation and selection are performed according to steps (1) to (3). Will be.
  • the determined structure may be further modified with respect to points other than the distance between the antigen recognition region and the transmembrane region.
  • modification of the amino acid sequence constituting the antigen recognition region eg, deletion, substitution, addition, etc. of the amino acid residue constituting the amino acid sequence.
  • the number of amino acids to be modified is, for example, 50 or less, preferably 25 or less. , More preferably 15 or less, even more preferably 10 or less, and most preferably 5 or less.
  • Similar modification of the transmembrane region or replacement of another molecule with the transmembrane region, intracellular signal domain It is possible to add the same modification as described above, or modification such as replacement of another molecule with an intracellular signal domain, addition of an intracellular signal domain, and the like.
  • CD37 is a four-transmembrane protein mainly expressed in mature B cells, and is involved in both survival and apoptosis signals in the interaction with interleukin 6 receptor and integrin ⁇ 4 ⁇ 1.
  • CD37 is highly expressed on chronic lymphocytic leukemia and non-Hodgkin's lymphoma in hematological malignancies. In order to develop a new CAR molecule targeting CD37, the following studies were conducted.
  • CD37 expression CD37 expression in various tumor cell lines and normal cells was examined. CD37 has been reported to be highly expressed mainly in differentiated B lymphocytes. Expression of CD37 in various tumor cell lines including the B-cell malignant lymphoma cell line was confirmed by flow cytometry. FIG. 1 shows the results of flow cytometry. The B cell malignant lymphoma lines Raji, OCI-Ly1, SU-DHL10, Ramos, Daudi and KARPAS1106P are strongly positive. Weak expression was seen in the multiple myeloma cell lines RPMI8226, KMS12BM and MM1S (U266H was a multiple myeloma cell line but was not expressed).
  • K562 a cell line derived from chronic myeloid leukemia, and SUP-T1 and Jurkat, which are T cell leukemia cell lines (weak expression was observed in Molt-4).
  • B-ALL1 a B-cell acute lymphocytic leukemia cell line.
  • Weak expression was seen in acute myeloid leukemia cell lines HL-60, Kasumi-1, MOLM-13, MV4-11, NOMO-1 and OCI-AML3.
  • No expression was found in the Philadelphia chromosome-positive acute lymphocytic leukemia cell line NPhA1.
  • B-cell malignant lymphoma and B-cell acute lymphocytic leukemia are targeted diseases of CD37-specific CAR (CD37CAR).
  • CD37CAR CD37-specific CAR
  • part of acute myeloid leukemia and multiple myeloma can be candidates for the target disease.
  • Gate-C / D was considered to be granulocytes, and almost no expression was observed.
  • Gate-A is a small lymphocyte, which also showed only weak expression.
  • Gate-B is a fraction containing lymphocytes, and some B-cell-expressed cells were highly expressed.
  • CDCD37 expression of the above tumor cell line was quantified by the amount of CD37 antibody bound (QIFIKIT).
  • K562 / CD37 is K562 transfected with CD37, and Low, Int and High are cells fractionated into low, medium and high expression, respectively.
  • FIG. 5 Tumor cell lines showed more than 100,000 binding of CD37 antibody per cell.
  • FIG. 6 Peripheral blood cells of healthy donors were quantified by the gate shown in FIG. 2 (FIG. 6).
  • the fraction containing B lymphocytes showed binding of more than 100,000 CD37 antibodies, but the other fractions had an expression level of about 3000-5000 in general.
  • the data of FIG. 6 is shown in a graph (FIG. 7).
  • CD CD37 expression on peripheral blood CD3-positive T cells from healthy donors was examined over time. Specifically, peripheral blood CD3-positive T cells were stimulated with CD3 / 28 beads, and the subsequent CD37 expression was observed over time. The expression was transiently increased the day after the stimulation, but thereafter CD37 expression was observed at an almost constant expression level (FIG. 8).
  • CD37-CAR A chimeric antigen receptor (CAR) for CD37, which is mainly expressed in normal B cells and B cell tumors, was designed and created.
  • CD37 chimeric antigen receptor
  • binding to CD37 according to the specificity of the anti-CD37 antibody causes a signal to be transmitted to T cells, and T cell division after target cell injury, cytokine release, and stimulation occurs. I expected.
  • CAR-T cell therapy is easy to prepare cells, exhibits higher cytotoxic activity than antibody therapy, and can be expected to have a sufficient effect even with a single treatment. Since there is no restriction, if the target antigen is positive for tumor cells, there is an advantage that treatment can be performed for all patients who are positive for the target antigen.
  • a plurality of sequences were prepared in which the downstream length of the single-chain antibody (scFv) was regulated (FIGS. 9 and 10). This was expected to control the degree of binding to the target antigen.
  • the anti-CD37 antibody scFv (using the sequence linked in the order of heavy chain variable region-light chain variable region and the sequence linked in the order of light chain variable region-heavy chain variable region) was used in the CD28 transmembrane region (CD28TM) And a CD28 intracellular domain (CD28IC) and a CD3 ⁇ intracellular domain (CD3z).
  • the details of the designed structure of the CD37-CAR will be described using 37S-LH (a structure at the right end in FIG. 10), which is one of the short spacer types, as an example.
  • the CAR is a light chain of an anti-CD37 humanized monoclonal antibody.
  • a hinge region (SEQ ID NO: 13) derived from human IgG4 to a scFv having a structure in which a heavy chain variable region (VH) (SEQ ID NO: 2) is linked to a variable region (VL) (SEQ ID NO: 1) by a linker (SEQ ID NO: 12); It has a structure in which a transmembrane region (SEQ ID NO: 14), a CD28 intracellular domain (SEQ ID NO: 15), a glycine linker (GGG), and CD3 ⁇ (SEQ ID NO: 16) are linked.
  • SEQ ID NO: 17 shows the sequence of the CAR expression construct.
  • the CAR gene (SEQ ID NO: 3) (the sequence gatcag (6 bp) interposed between the VH sequence and the hinge sequence is a sequence derived from an anti-CD37 antibody), the T2A sequence (SEQ ID NO: 18), a sequence encoding tEGF (truncated ⁇ EGF) (SEQ ID NO: 19), a stop codon, and a NotI site are arranged in this order.
  • the cytokine producing ability of the prepared CD37-CAR-T cells was evaluated by intracellular IFN- ⁇ staining (FIG. 13). Co-cultured with K562 (CD37-negative target) and Ramos (CD37-positive target) (control has no target cells), and the subsequent cytokine production was evaluated. In CD37L-HL and CD37L-LH, cytokine production was observed even in the case of control (no target cells) and K562, and significant cytokine production was observed in the case of CD37-positive target (Ramos).
  • CD37-CAR-T cells were stimulated with K562-CD37, and the subsequent cell proliferation was evaluated (FIG. 15).
  • the long spacer type (37L-LH, 37L-HL) showed almost no growth under the condition without IL-2 or under the condition with IL-2, but the short spacer type (37S-LH, 37S-HL) All showed good growth after stimulation.
  • the long spacer type CD37L-CAR-T cells it was thought that cytotoxicity occurred by targeting CD37 which is very slightly expressed on the T cells, and prevented the proliferation of CAR-T cells.
  • CD37 expressed on short spacer type CD37S-CAR-T cells was quantified using QIFIKIT (FIG. 16).
  • QIFIKIT QIFIKIT
  • the binding of about 1000-2000 CD37 antibody was observed, but the binding of CD37S-CAR-T cells was reduced to about 200 CD37 antibody. This suggests that CD37S-CAR-T cells may recognize cells with higher expression.
  • CD37L-CAR-T cells were observed to have a depressing phenomenon, suggesting that the target antigen recognition threshold would be lower.
  • CD37S-CAR-T cells were prepared, and their ability to produce cytokines against various CD37-positive targets was evaluated. Regarding the tumor cell line, even if the expression of CD37 was low (approximately 10,000 in CD37 antibody binding ability), favorable cytokine production ability was exhibited (FIG. 17).
  • CD37S-CAR-T cells did not produce cytokines when co-cultured with the CD37 low-expressing cell fraction, but showed significant cytokine production when co-cultured with the CD37-positive fraction.
  • the CD37 low-expressing fraction of the normal donor has a CD37 antibody binding site of about 3000 to 5000, so that the CD37 antibody-binding site on the CD37S-CAR-T cell shown in FIG. Many. It was suggested that recognition might or might not be determined solely by the number of CD37 antibody binding sites.
  • each CDR37-CAR was created using overlapping PCR and inserted into the pLZRS-BMN-Z vector.
  • a retrovirus for gene transfer was prepared using this vector, and the gene was transferred to CD3 purified lymphocytes derived from a healthy donor.
  • the sequence of each CAR37-CAR expression construct (the HindIII site and the Kozak sequence, followed by the CAR gene, the T2A sequence (SEQ ID NO: 18), the sequence encoding tEGF (truncated EGF) (SEQ ID NO: 19), and the stop codon , And NotI sites are arranged in order).
  • CD37_00 SEQ ID NO: 24 CD37_15: SEQ ID NO: 17 CD37_30: SEQ ID NO: 25 CD37_60: SEQ ID NO: 26 CD37_125: SEQ ID NO: 27 CD37_232: SEQ ID NO: 28
  • the present invention can be a solution to the on-target side reaction that is a problem in CAR therapy, and contributes to the establishment of CAR therapy with improved safety. Therefore, its clinical significance is great.
  • the CAR structure determination method of the present invention is excellent in versatility, can be applied to the design of various CARs having different target molecules, and its utility value is extremely large.

Abstract

Le problème à résoudre par la présente invention concerne la fourniture : d'un récepteur antigénique chimérique qui a une sécurité améliorée et qui est approprié pour des utilisations cliniques ; ainsi que l'utilisation du récepteur antigénique chimérique. L'invention concerne un récepteur antigénique chimérique spécifique à CD37 ayant un niveau de reconnaissance d'antigène qui est régulé par l'ajustement d'un domaine espaceur de celui-ci.
PCT/JP2019/024974 2018-06-27 2019-06-24 Récepteur antigénique chimérique spécifique à cd37 WO2020004337A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2020527516A JPWO2020004337A1 (ja) 2018-06-27 2019-06-24 Ｃｄ３７特異的キメラ抗原レセプター

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2018-122519 2018-06-27
JP2018122519 2018-06-27

Publications (1)

Publication Number Publication Date
WO2020004337A1 true WO2020004337A1 (fr) 2020-01-02

Family

ID=68984992

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2019/024974 WO2020004337A1 (fr) 2018-06-27 2019-06-24 Récepteur antigénique chimérique spécifique à cd37

Country Status (2)

Country Link
JP (1) JPWO2020004337A1 (fr)
WO (1) WO2020004337A1 (fr)

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009502171A (ja) * 2005-07-25 2009-01-29 トゥルビオン・ファーマシューティカルズ・インコーポレーテッド Ｃｄ３７特異的及びｃｄ２０特異的結合分子を用いたｂ細胞の減少
JP2010535483A (ja) * 2007-08-09 2010-11-25 ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング 抗ｃｄ３７抗体
US20110158995A1 (en) * 2008-07-28 2011-06-30 Renault S.A.S Multi-Specific Binding Proteins Targeting B Cell Disorders
WO2016172703A2 (fr) * 2015-04-23 2016-10-27 Haemalogix Pty. Ltd. Récepteurs d'antigènes chimériques à antigène du myélome kappa et utilisations associées
WO2016176651A2 (fr) * 2015-04-29 2016-11-03 Fred Hutchinson Cancer Research Center Cellules souches/progénitrices hématopoïétiques et effectrices non t modifiées, et leurs utilisations
WO2017008019A1 (fr) * 2015-07-08 2017-01-12 The Johns Hopkins University Lymphocytes infiltrant la moelle (mil) en tant que source de lymphocytes t pour une thérapie par des récepteurs chimériques d'un antigène (car)
WO2017062628A1 (fr) * 2015-10-06 2017-04-13 City Of Hope Récepteurs d'antigènes chimériques ciblant les gènes psca
WO2017149515A1 (fr) * 2016-03-04 2017-09-08 Novartis Ag Cellules exprimant de multiples molécules de récepteur d'antigène chimère (car) et leurs utilisations
WO2017180993A1 (fr) * 2016-04-14 2017-10-19 Bluebird Bio, Inc. Systèmes de récepteur d'antigène chimère de récupération
WO2018132506A1 (fr) * 2017-01-10 2018-07-19 The General Hospital Corporation Récepteurs antigéniques chimériques basés sur des domaines de signal 1 alternatifs

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB201600328D0 (en) * 2016-01-08 2016-02-24 Univ Oslo Hf Anti-CD37 chimeric antigen receptors and immune cells expressing them

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009502171A (ja) * 2005-07-25 2009-01-29 トゥルビオン・ファーマシューティカルズ・インコーポレーテッド Ｃｄ３７特異的及びｃｄ２０特異的結合分子を用いたｂ細胞の減少
JP2010535483A (ja) * 2007-08-09 2010-11-25 ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング 抗ｃｄ３７抗体
US20110158995A1 (en) * 2008-07-28 2011-06-30 Renault S.A.S Multi-Specific Binding Proteins Targeting B Cell Disorders
WO2016172703A2 (fr) * 2015-04-23 2016-10-27 Haemalogix Pty. Ltd. Récepteurs d'antigènes chimériques à antigène du myélome kappa et utilisations associées
WO2016176651A2 (fr) * 2015-04-29 2016-11-03 Fred Hutchinson Cancer Research Center Cellules souches/progénitrices hématopoïétiques et effectrices non t modifiées, et leurs utilisations
WO2017008019A1 (fr) * 2015-07-08 2017-01-12 The Johns Hopkins University Lymphocytes infiltrant la moelle (mil) en tant que source de lymphocytes t pour une thérapie par des récepteurs chimériques d'un antigène (car)
WO2017062628A1 (fr) * 2015-10-06 2017-04-13 City Of Hope Récepteurs d'antigènes chimériques ciblant les gènes psca
WO2017149515A1 (fr) * 2016-03-04 2017-09-08 Novartis Ag Cellules exprimant de multiples molécules de récepteur d'antigène chimère (car) et leurs utilisations
WO2017180993A1 (fr) * 2016-04-14 2017-10-19 Bluebird Bio, Inc. Systèmes de récepteur d'antigène chimère de récupération
WO2018132506A1 (fr) * 2017-01-10 2018-07-19 The General Hospital Corporation Récepteurs antigéniques chimériques basés sur des domaines de signal 1 alternatifs

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HICKS, S. W. ET AL.: "The antitumor activity of IMGN529, a CD 37-Targeting antibody-drug conjugate, is potentiated by rituximab in non-hodgkin lymphoma models", NEOPLASIA, vol. 19, no. 9, September 2017 (2017-09-01), pages 661 - 671, XP055611917 *
TERAKURA, SEITARO: "CAR-T cell therapy in combination with allogeneic stem cell transplantation", JOURNAL OF HEMATOPOIETIC CELL TRANSPLANTATION, vol. 6, no. 1, 2017, pages 1 - 7, XP055668167 *

Also Published As

Publication number Publication date
JPWO2020004337A1 (ja) 2021-08-02

Similar Documents

Publication Publication Date Title
JP7379561B2 (ja) キメラ抗原及びｔ細胞受容体、並びに使用方法
KR102618231B1 (ko) 변형된 만능성 줄기 세포, 및 제조 및 사용 방법
CN107406517B (zh) 包含cd19结合域的嵌合抗原受体(car)
JP7303749B2 (ja) Ｔｉｍ－１を標的とするキメラ抗原受容体
AU2021201679A1 (en) Transgene genetic tags and methods of use
US20210038659A1 (en) Combination therapy using a chimeric antigen receptor
TW201930342A (zh) 包含nkg2d結構域的多特異性嵌合受體及其使用方法
CA3173394A1 (fr) Methodes de production de cellules exprimant un recepteur antigenique chimerique
US20230398216A1 (en) Chimeric activation receptors
JP2021531813A (ja) キメラ抗原受容体療法のｔ細胞の増殖動態及びその使用
CN115485369A (zh) γδT细胞及其用途
US20240009310A1 (en) A CHIMERIC ANTIGEN RECEPTOR CONSTRUCT ENCODING A CHECKPOINT INHIBITORY MOLECULE AND AN IMMUNE STIMULATORY CYTOKINE AND CAR-EXPRESSING CELLS RECOGNIZING CD44v6
US20230321242A1 (en) Chimeric antigen receptor (car)-expressing cells recognizing cea
WO2020004337A1 (fr) Récepteur antigénique chimérique spécifique à cd37
EP4242309A1 (fr) Récepteur antigénique chimérique
WO2021100585A1 (fr) Procédé de production de lymphocytes modifiés par un gène récepteur d'antigène chimère
JP2015092865A (ja) ヒト化抗ｃｄ２０キメラ抗原レセプター
CN115960257B (zh) 靶向IL13Rα2的经优化的嵌合抗原受体及其用途
US20240109978A1 (en) Chimeric antigen receptor (car) spacer modifications enhance car t cell functionality
IL308148A (en) Chimeric receptors and methods of their use
CN115704039A (zh) 包含编码抗原结合分子的多核苷酸和靶向ecm试剂的多核苷酸的多核苷酸及修饰细胞
WO2022165419A9 (fr) Méthodes pour augmenter la fonction des lymphocytes t

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19824986

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2020527516

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19824986

Country of ref document: EP

Kind code of ref document: A1