JP6570109B2 - Chimeric antigen receptor gene expression system - Google Patents

Description

  The present invention relates to a system for expressing a chimeric antigen receptor gene and use thereof. Specifically, the present invention relates to a system capable of controlling the expression of a target antigen-specific chimeric antigen receptor gene and a treatment using the system.

  Chimeric Antigen Receptor (hereinafter also referred to as “CAR”) uses a single-chain variable region of an antibody as an extracellular domain, and connects the transmembrane region and the intracellular domain of a molecule that transmits a costimulatory signal with CD3ζ. Is. By binding to the antigen according to the specificity of the antibody, a signal is transmitted to the T cell, and target cell damage, cytokine release, and T cell division after stimulation occur. It is possible to give specificity to T cells by gene transfer, and cell preparation is generally easier than in the case of culturing and amplifying endogenous tumor-specific T cell receptor (TCR) positive T cells. In addition, the treatment method using CAR (CAR-T therapy) shows higher cytotoxic activity than antibody therapy, and multiple treatments are required to autonomously amplify by antigen stimulation in one infusion. There is no point. TCR gene transfer targets a complex of HLA and the peptide presented on it, so it can be treated only if the patient has a specific HLA (HLA-restricted), but CAR is HLA-restricted It is more advantageous in that there is no property. In other words, if the target antigen is positive for the tumor cells, all patients who are positive for the target antigen can be treated. On the other hand, since all cells are targeted if the antigen is positive, normal tissues are also targeted when the antigen is expressed in addition to the target cells (on-target side reaction). Such an excessively strong immune response that cannot normally occur shows high clinical efficacy. However, there are cases in which a strong immune response cannot be controlled and an unfavorable outcome occurs. So far, no tumor cell-specific target antigen has been obtained other than differentiation antigens such as CD19 / 20, and expression of the target antigen in some normal tissues is inevitably seen. The possibility that such a strong immune response may occur in normal tissues can be a problem when CAR-T therapy is more versatile in the future.

  By connecting intracellular domains such as CD28 and 4-1BB with CD3ζ, CAR can be activated as if it had been co-stimulated only by the signal from CAR, even if it was not transmitted from the target cell. . Normally, tumor cells do not express ligands such as CD80 / 86, so even if endogenous tumor-specific T cells are present, only incomplete stimuli can be transmitted, resulting in energy. CAR can provide costimulation with only one signal: binding to the target antigen. This is an advantage compared to TCR gene transfer therapy, and at the same time, it is the cause of the excessive immune reaction as described above.

  In CAR-T therapy, as mentioned above, if the target antigen is expressed as much as possible other than the tumor, an on-target side reaction occurs, resulting in significant side effects, and sometimes fatal. Therefore, as described above, selection of a target antigen is most important, and B-cell differentiation antigens such as CD20 / CD19 are the most advanced in the clinical trials of CAR-T therapy that have been conducted so far. A group at the University of Pennsylvania created a CAR by connecting a mouse-derived CD19 single-chain antibody and the intracellular domain of 4-1BB as described above, and introduced the gene into peripheral blood T cells of chemoresistant chronic lymphocytic leukemia. A surprising effect has been reported that when this is administered to a patient, remission is obtained in most cases (Non-patent Document 1). The effectiveness of CD19CAR-T therapy is also reported in Patent Document 1 and Non-Patent Document 2.

Japanese Patent No. 5312721

N Engl J Med. 2011; 365: 725-733. Cancer Res 2006; 66: 10995-11004. Hum Mol Genet 20 (R1): R93-99, 2011 N Engl J Med 2011; 365 (18): 1673-1683.

In CAR gene-transferred T cell therapy, it is advantageous that an antigen-specific and very strong reaction occurs with respect to a target antigen-positive cell as described above. On the other hand, there are problems that the antitumor reaction is too strong to control and CAR cannot be applied to target antigens expressed ectopically other than tumor cells. In order to deal with such problems, a technique that uses a suicide gene in addition to the introduction of a CAR gene has been adopted. Introducing a herpesvirus / thymidine kinase (HSV-TK) gene to induce ganciclovir-specific cell death HSV-TK gene transfer or iCaspase9 gene transfer therapy (mutant caspase 9 is introduced into target cells in advance, In addition, for example, dimerization is caused by administration of a drug called AP1903 and target cell death is induced by activating mutant caspase 9). HSV-TK has also been reported to have clinical usefulness (see, for example, Non-Patent Documents 3 and 4). However, in these suicide gene introductions, in order to show the effectiveness, CAR gene-introduced T cells are killed, and when there is a need for treatment again in the case of subsequent recurrence, the gene introduction T It may be necessary to infuse cells. In other words, such a method is effective for improving safety, but attenuates the therapeutic effect of CAR gene-transferred T cell therapy.
Therefore, an object of the present invention is to provide a new means capable of reducing side effects while maintaining the original therapeutic effect of CAR.

In view of the above problems, the present inventors aimed to develop a system that controls the expression of CAR itself by drug administration. During the study, we focused on a system that controls gene expression by administering a tetracycline compound (eg, doxycycline) (tetracycline gene expression induction system), and examined its effectiveness. As a result of the study, it became clear that the tetracycline gene expression induction system functions effectively, and that CAR expression can be controlled with good responsiveness. It was also found that the same antitumor effect can be exhibited as compared with the case where CAR is constantly expressed by the conventional method. Furthermore, important and interesting knowledge was obtained for practical use.
The following invention is provided mainly based on the above results.
[1] a first expression cassette comprising a tet operator sequence, a promoter sequence under the control of the tet operator sequence, and a target antigen-specific chimeric antigen receptor gene arranged downstream of the promoter sequence;
A second expression cassette comprising a promoter sequence that functions in immune cells, and a sequence encoding a reverse tetracycline-regulated transactivator disposed downstream of the promoter sequence;
A target antigen-specific chimeric antigen receptor gene expression system comprising:
[2] The system according to [1], wherein the target antigen-specific chimeric antigen receptor gene is a CD19 chimeric antigen receptor gene.
[3] The CD19 chimeric antigen receptor comprises an extracellular domain containing an scFv fragment of an anti-CD19 monoclonal antibody, a transmembrane domain, and an intracellular signal domain for effector functions of immune cells. System.
[4] The system according to any one of [1] to [3], wherein the first expression cassette further includes a detection gene.
[5] The system according to [4], wherein the detection gene is linked to the target antigen-specific chimeric antigen receptor gene via a sequence encoding a self-cleaving peptide.
[6] The system according to [4] or [5], wherein the detection gene is an epidermal growth factor receptor (EGFR) gene lacking an intracellular domain.
[7] The system according to any one of [1] to [6], wherein the first expression cassette is held in a first vector and the second expression cassette is held in a second vector.
[8] The system according to any one of [1] to [6], wherein the first expression cassette and the second expression cassette are held in one vector.
[9] The system according to any one of [1] to [8], wherein the vector is a retroviral vector or a lentiviral vector.
[10] An immune cell into which the system according to any one of [1] to [9] is introduced.
[11] An immune cell into which the system according to any one of [1] to [6] is introduced using a transposon.
[12] The immune cell according to [10] or [11], wherein the immune cell is a T cell.
[13] A cell preparation comprising a therapeutically effective amount of the immune cells according to any one of [10] to [12].
[14] A first expression cassette comprising a tet operator sequence, a promoter sequence under the control of the tet operator sequence, and a target antigen-specific chimeric antigen receptor gene arranged downstream of the promoter sequence, and functions in immune cells And a second expression cassette comprising a sequence encoding a reverse tetracycline-regulated transactivator disposed downstream of the promoter sequence.
[15] The vector according to [14], which is a retrovirus vector or a lentivirus vector.
[16] A method for treating cancer, comprising administering a therapeutically effective amount of the immune cell according to any one of [10] to [12] to a cancer patient.

Design of CD19-CAR (Tet-CD19CAR) subject to Tet-on control. The structure of the vector used for gene transfer is shown. Results of Tet-CD19CAR gene transfer experiment. untdx represents no gene transfer. CD19CAR-OR represents a CD19CAR-OR produced using a system that constantly expresses CD19CAR. Examination of doxycycline (Dox) concentration necessary to induce CAR expression. EGFR represents the result of EGFR expression, and Fc represents the result of Fc expression. Examination of CAR expression dynamics after the start of Dox administration. The change of the expression of CAR after Dox administration start is shown. EGFR represents the result of EGFR expression, and Fc represents the result of Fc expression. Examination of CAR expression kinetics after Dox administration was discontinued. The change in the expression of CAR after Dox administration is stopped is shown. Scheme and results of gene transfer experiments for human peripheral blood-derived CD8-positive T cells. Purification using tEGFR after Tet-CD19CAR gene introduction. Scheme of experiments using Tet-CD19CAR T cells. Tet-CD19CAR T cell cytotoxic activity. untdx represents no gene transfer. Results of intracellular interferon gamma (IFN-γ) staining. untdx represents no gene transfer. Results of IL-2 ELISA. untdx represents no gene transfer. NS: No significant difference. ****: Significant difference (p <0.0001) Proliferation of Tet-CD19CAR T cells after CD19 stimulation. untdx represents no gene transfer.

1. Target antigen-specific chimeric antigen receptor gene expression system The first aspect of the present invention relates to a target antigen-specific chimeric antigen receptor gene expression system (hereinafter also referred to as “system of the present invention”). The present invention makes it possible to control the expression of a target antigen-specific chimeric antigen receptor, and utilizes a tetracycline gene expression induction system (hereinafter also referred to as “TC gene expression induction system”). The TC gene expression induction system is a system in which the expression state of a target gene can be controlled by tetracycline or an analog thereof (hereinafter collectively referred to as “TC compound”). By utilizing the TC gene expression induction system, the target antigen-specific chimeric antigen receptor gene (hereinafter also referred to as “CAR gene”), which is the target gene, is expressed in response to the presence of the TC compound. It is also possible to induce concentration-dependent expression of TC compounds. In the TC gene expression induction system used in the present invention, a reverse tetracycline-regulated transactivator (hereinafter referred to as “rtTA”) is used, and when a tetracycline compound is present, rtTA binds to the tet operator sequence, Induces CAR gene expression.

  Examples of the TC compound are tetracycline, doxycycline, deoxycycline, anhydrotetracycline, cyanotetracycline, and chlorotetracycline. For tetracycline compounds, see Hlavka and Boothe, "The Tetracyclines," in Handbook of Experimental Pharmacology 78, RK. Blackwood et al. (Eds.), SpringerVerlag, Berlin-New York, 1985 and LA Mitscher "The Chemistry of the Tetracycline Details on Antibiotics, Medicinal Research 9, Dekker, New York, 1978 etc.

  The system of the present invention includes two characteristic expression cassettes, namely a first expression cassette and a second expression cassette, in order to realize the expression control of the CAR gene by the TC compound. The first expression cassette includes a tet operator sequence, a promoter sequence under the control of the tet operator sequence, and a CAR gene arranged downstream of the promoter sequence.

  The tet operator sequence (tetO) is a sequence to which rtTA can bind. When rtTA binds, the promoter under the control of tetO is activated. The sequence of tetO is not particularly limited as long as it exhibits such characteristics. For example, the sequence of tetO shown in Gossen et al. (Gossen M. and Bujard M., (1992) Proc. Natl. Acad. Sci. USA, 89, 5547-5551) can be used. Any class (eg A, B, C, D, E) of tetO can be used. Preferably, a plurality of tetO is used. In that case, the plurality of tetOs are usually arranged consecutively. However, other sequences may be interposed between adjacent tetOs. The number of tetO (number of copies) is not particularly limited, but is 2 to 10, for example. A vector containing tetO is commercially available (for example, provided by Clontec), and a desired tetO can be prepared from the vector.

  A promoter sequence exists under the control of tetO. That is, the promoter sequence is operably linked to tetO. Usually, a promoter sequence is linked to the downstream side (3 ′ side) of tetO. The distance between tetO and the promoter sequence is not particularly limited as long as the promoter sequence is under the control of tetO. Typically, a promoter sequence is placed immediately after tetO (with no gaps). As the promoter sequence, a minimal promoter sequence is typically used. A “minimal promoter sequence” is a partial promoter sequence that defines a transcription initiation site but cannot initiate efficient transcription by itself. The activity of the minimal promoter sequence depends on the binding of rtTA to tetO controlled by tetracycline compounds. Specific examples of the minimal promoter sequence include a promoter derived from human cytomegalovirus (CMV).

  Transcription is initiated from the TATA box within the minimal promoter. In order to increase the stability of the transcript, an intron is usually present upstream of the gene of interest. Specific examples of intron sequences include rabbit-derived beta globin introns.

  A target antigen-specific chimeric antigen receptor gene (CAR gene) is present downstream of the intron sequence. That is, in the present invention, a gene encoding a target antigen-specific chimeric antigen receptor (CAR) is used as a gene to be expressed (target gene). CAR is a structure containing an extracellular domain derived from an antibody specific for a target antigen, a transmembrane domain, and an intracellular signal domain. The CAR of the present invention includes an extracellular domain containing an scFv fragment of an anti-target antigen monoclonal antibody, a transmembrane domain, and an intracellular signal domain for effector functions of immune cells. Hereinafter, each domain will be described.

(1) Extracellular domain The extracellular domain exhibits specific binding properties to the target antigen. The extracellular domain contains scFv fragments of anti-target antigen monoclonal antibodies. For example, anti-target antigen monoclonal antibodies such as rodent (mouse, rat, rabbit, etc.) antibodies, human antibodies, and humanized antibodies are used. 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 that of a human antibody, and only the constant region of the antibody is replaced with that of a human antibody. A chimeric antibody and a human-type CDR-grafted antibody (PTJohons et al., Nature 321,522 (1986) in which parts other than CDRs (complementarity determining regions) present in constant regions and variable regions are substituted with those of human antibodies. ))including. In order to increase the antigen-binding activity of human CDR-grafted antibodies, a method for selecting a human antibody framework (FR) highly homologous to a mouse antibody, a method for producing a humanized antibody having a high homology, a mouse CDR for a human antibody Techniques for further substitution of amino acids in the FR region after transplantation have already been developed (US Patent No. 5585089, US Patent No. 5693761, US Patent No. 5693762, US Patent No. 6180370, European Patent No. 451216) , European Patent No. 682040, Patent No. 2828340, etc.) and can also be used for the production of humanized antibodies.

  As the target antigen, an antigen that is specifically expressed in tumor cells is used. Here, “specific expression” means that significant or significant expression is observed as compared with cells other than tumor, and is not intended to be limited to those having no expression at all in cells other than tumor. Examples of target antigens include CD19 antigen, CD20 antigen, GD2 antigen, CD22 antigen, CD30 antigen, CD33 antigen, CD44 variant7 / 8 antigen, CEA antigen, Her2 / neu antigen, MUC1 antigen, MUC4 antigen, MUC6 antigen, IL-13 receptor -alpha2, immunoglobulin light chain, anti-PSMA, VEGF receptor2, etc.

  In the present invention, the scFv fragment of the anti-target antigen monoclonal antibody is used for the extracellular domain. An scFv fragment is a structure in which an immunoglobulin light chain variable region (VL) and heavy chain variable region (VH) are linked via a linker, and retains the ability to bind to an antigen. As the linker, for example, a peptide linker can be used. A peptide linker is a linker consisting of a peptide in which amino acids are linked in a straight chain. A typical example of a peptide linker is a linker composed of glycine and serine (GGS linker or GS linker). The GGS linker and glycine and serine, which are amino acids constituting the GS linker, have a small size per se and are difficult to form higher-order structures 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. The length of the linker is preferably 8-25, more preferably 15-20.

(2) Transmembrane domain The transmembrane domain is interposed between the extracellular domain and the intracellular signal domain. As the transmembrane domain, a transmembrane domain such as CD28, CD3ε, CD8α, CD3, CD4 or 4-1BB can be used. You may decide to use the transmembrane domain which consists of the polypeptide constructed artificially.

(3) Intracellular signal domain The intracellular signal domain transmits a signal necessary for exerting an effector function of an immune cell. That is, an intracellular signal domain that can transmit a signal necessary for activation of immune cells when an extracellular domain binds to a target antigen is used. The intracellular signal domain includes a domain for transmitting a signal via the TCR complex (for convenience, referred to as “first domain”) and a domain for transmitting a costimulatory signal (for convenience, “second domain”). Called). In addition to CD3ζ, an intracellular domain such as FcεRIγ can be used as the first domain. CD3ζ is preferably used. As the second domain, an intracellular domain of a costimulatory molecule is used. Examples of costimulatory molecules include CD28, 4-1BB (CD137), CD2, CD4, CD5, CD134, OX-40, or ICOS. Preferably, the intracellular domain of CD28 or 4-1BB is employed.

  The connection mode of the first domain and the second domain is not particularly limited, but preferably, it is known that costimulation was strongly transmitted when CD3ζ was connected distally in the past case. The second domain is arranged on the side. A plurality of identical 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 linked directly or a linker may be interposed between them. As the linker, for example, a peptide linker can be used. A peptide linker is a linker consisting of a peptide in which amino acids are linked in a straight chain. The structure, characteristics, etc. of the peptide linker are as described above. However, as a linker here, you may use what consists only of glycine. The length of the linker is not particularly limited. For example, a linker having 2 to 15 amino acid residues can be used.

(4) Other factors
A leader sequence (signal peptide) is used to promote the secretion of CAR. For example, the leader sequence of GM-CSF receptor can be used. In addition, a structure in which an extracellular domain and a transmembrane domain are linked via a spacer domain is preferable. That is, the CAR of a preferred embodiment includes a spacer domain between the extracellular domain and the transmembrane domain. The spacer domain is used to promote binding between CAR and the target antigen. For example, an Fc fragment of human IgG (eg, human IgG1, human IgG4) can be used as the spacer domain. In addition, a part of the extracellular domain of CD28, a part of the extracellular domain of CD8α, and the like can be used as the spacer domain. A spacer domain can also be provided between the transmembrane domain and the intracellular signal domain.

  In addition, there are some reports such as experiments and clinical studies using CAR (Non-Patent Documents 1 to 4, Patent No. 5312721, JP 2010-189402, etc.), and these reports The CAR of the present invention can be constructed with reference to FIG.

  A poly A addition signal sequence is arranged downstream of the CAR gene. Transcription is terminated by the use of a poly A addition signal sequence. As the poly A addition signal sequence, the poly A addition sequence of the target gene itself, the poly A addition sequence of SV40, the poly A addition sequence of the bovine-derived growth hormone gene, and the like can be used.

  A detection gene (reporter gene, cell or tissue specific gene, selection marker gene, etc.), enhancer sequence, WRPE sequence, etc. may be included in the expression cassette. The detection gene is used for determining the success or failure of the introduction of the expression cassette and determining the efficiency, detecting the CAR gene expression or determining the expression efficiency, selecting and sorting the cells in which the CAR gene is expressed, and the like. On the other hand, the use of an enhancer sequence can improve the expression efficiency. The detection gene includes a neo gene conferring resistance to neomycin, an npt gene conferring resistance to kanamycin and the like (Herrera Estrella, EMBO J. 2 (1983), 987-995) and nptII gene (Messing & Vierra. Gene 1 9: 259-268 (1982)), hph gene conferring resistance to hygromycin (Blochinger & Diggl mann, Mol Cell Bio 4: 2929-2931), dhfr gene conferring resistance to metatrexate (Bourouis et al. , EMBO J.2 (7)), etc. (above, marker gene), luciferase gene (Giacomin, P1. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), β -Glucuronidase (GUS) gene, GFP (Gerdes, FEBS Lett. 389 (1996), 44-47) and its variants (EGFP, d2EGFP, etc.) and other fluorescent protein genes (reporter genes), intracellular domains Using genes such as the epidermal growth factor receptor (EGFR) gene You can. The detection gene is linked to the CAR gene via, for example, a bicistronic regulatory sequence (for example, a ribosome internal recognition sequence (IRES)) or a sequence encoding a self-cleaving peptide. An example of a self-cleaving peptide is 2A peptide (T2A) derived from Thosea asigna virus, but is not limited thereto. Known as self-cleaving peptides are 2A peptide (F2A) from horseshoe disease virus (FMDV), 2A peptide (E2A) from equine rhinitis virus A (ERAV), 2A peptide (P2A) from porcine teschovirus (PTV-1) It has been.

  Specific examples of the first expression cassette in the present invention include, in order from the upstream side (5 ′ side), tetO (7 repeats), CMV minimal promoter, intron, CAR gene, T2A, tEGFR (truncated EGFR) gene, poly Structure in which an A-added signal sequence is arranged, or the reverse order (ie, tetO (7 repeats), CMV minimal promoter, intron, CAR gene, T2A, tEGFR (truncated EGFR) gene in reverse order from the 3 'side) , Poly A addition signal sequence).

  The second expression cassette includes a promoter sequence that functions in immune cells and a sequence that encodes a reverse tetracycline-regulated transactivator (rtTA) arranged downstream of the promoter sequence. As the promoter, a promoter that functions in immune cells is used. A constitutive promoter, T cell specific promoter, minimal promoter, etc. can be used. Specific examples of promoters include phosphoglycerate kinase (PGK) promoter, CMV promoter, CAG promoter, SV40ori, retrovirus LTP, SRα, EF1α, β actin promoter, nuclear factor of activated T cells (NFAT) -regulated promoter Etc.

  rtTA does not bind to tetO in the absence of a TC compound, but binds to tetO in the presence of a TC compound. rtTA is a fusion protein containing an rtet repressor (rTetR) and a transcriptional activation domain. rTetR was found as a variant of TetR (Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H. Transcriptional activation by tetracyclines in mammalian cells. Science 1995; 268: 1766-1769.). As the transcription activation domain, an acidic activation domain, a proline-rich transcription activation domain, a serine / threonine-rich transcription activation domain, a glutamine-rich activation domain, or the like is used. Preferably, the herpes simplex virus virion protein 16 (VP16) which is an acidic activation domain (Labow et al., (1990) Mol. Cell Biol. 10, 3343-3356; Baim et al., (1991) Proc. Natl Acad. Sci. USA 8, 5072-5076 etc.) is adopted as a transcription activation domain.

  rtTA is not particularly limited. Several rtTAs have been reported (Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89 (12): 5547-5551; Gossen, M. et al., ( 1995) Science 268 (5218): 1766-1769 .; Urlinger et al. (2000) Proc. Natl. Acad. Sci. USA 97 (14): 7963-7968.), These previously reported ones can be used. Preferably, a modified rtTA (Zhou, X. et al. (2006) Gene Ther. 13 (19): 1382-1390.) With enhanced reactivity to doxycycline is used.

  The sequence encoding rtTA is placed under the control of a promoter. That is, in the second expression cassette, the promoter sequence and the sequence encoding rtTA are operably linked.

  As in the case of the first expression cassette, a detection gene (reporter gene, cell or tissue-specific gene, selection marker gene, etc.), enhancer sequence, WRPE sequence, etc. may be included in the second expression cassette. Good.

  A specific example of the second expression cassette in the present invention has a structure in which a constitutive promoter (for example, PGK promoter), a sequence encoding rtTA, and a poly A addition signal sequence are arranged in this order from the upstream side (5 ′ side). Things can be mentioned.

  The first expression cassette and the second expression cassette are mounted on a vector for introduction into a target cell. As used herein, the term “vector” refers to a nucleic acid molecule that can transport a nucleic acid molecule inserted therein into a target (ie, an immune cell or a progenitor cell thereof), and its form, origin, etc. are particularly limited. It is not a thing. Various types of vectors are available. An example of a suitable vector is a viral vector, but non-viral vectors can also be used. The gene transfer method using a virus vector skillfully utilizes the phenomenon that a virus infects cells, and high gene transfer efficiency can be obtained. 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. Among these, retrovirus vectors, lentivirus vectors, and adeno-associated virus vectors, the target gene incorporated into the vector is incorporated 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. Examples of 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), YAC vectors and BAC vectors. The system of the present invention may be constructed using a transposon. Actually, a method using a transposon as a method for introducing a CAR gene (see, for example, Nakazawa Y, et al., J Immunother 32: 826-836, 2009, Nakazawa Y et al., J Immunother 6: 3-10, 2013) Has been developed and is useful for realizing the system of the present invention.

  In one embodiment of the system of the present invention, the first expression cassette and the second expression cassette are mounted on separate vectors. That is, a vector for a first expression cassette (referred to as “first expression vector”) and a vector for a second expression cassette (referred to as “second expression vector”) are prepared. In this embodiment, the first expression vector and the second expression vector are introduced into the target cell. The order of introduction is not particularly limited, but preferably the introduction of the second expression vector is preceded. In the case of adopting this order, the target cell into which the second expression vector is appropriately introduced is selected or concentrated or purified, and then the first expression vector is introduced to improve the introduction rate of the system of the present invention.

In another embodiment of the system of the present invention, the first expression cassette and the second expression cassette are mounted on one vector. In other words, a vector holding both the first expression cassette and the second expression cassette is prepared. In this embodiment, if one vector is introduced into the target cell, the target cell into which the system of the present invention has been introduced can be obtained. The fact that the system of the present invention can be introduced by an operation of introducing one vector (typically a single introduction operation) is a great merit for the clinical application of the present invention. In the vector of this aspect, a tet operator sequence, a promoter sequence under the control of the tet operator sequence, a first expression cassette containing a CAR gene arranged downstream of the promoter sequence, a promoter sequence that functions in T cells, A second expression cassette containing rtTA arranged downstream of the promoter sequence will be retained. Currently, a vector equipped with the necessary elements for the TC gene expression induction system (rtTA is expressed in the forward direction and the target gene is expressed in the reverse direction) is commercially available (Tet-One ^TM System: Clontech). Using this vector, the system of this embodiment in which the first expression cassette and the second expression cassette are mounted on one vector can be constructed.

2. Cells Introduced with Target Antigen-Specific Chimeric Antigen Receptor Gene Expression System The second aspect of the present invention provides immune cells into which the system of the present invention has been introduced (hereinafter referred to as “cells of the present invention”). When the system of the present invention functions, CAR is expressed on the surface of the cell of the present invention. The cell of the present invention is activated by transmitting a signal into the cell when the CAR present on the cell surface recognizes and binds to the target antigen. The cell of the present invention is produced by introducing the system of the present invention into a target cell. Usually, a recombinant expression vector comprising the system of the present invention (a combination of a first expression vector carrying a first expression cassette and a second expression vector carrying a second expression cassette, or a first expression cassette and a second expression cassette) A target cell). T cells, NK cells, or progenitor cells thereof (hematopoietic stem cells, lymphocyte progenitor cells, etc.) can be used as target cells. As T cells here, CD4 positive CD8 negative T cells, CD4 negative CD8 positive T cells, CD4 negative CD8 negative T cells, CD4 positive CD8 positive T cells, T cells prepared from iPS cells, αβ-T cells, γδ -T cells can be mentioned. Various cell populations can be used as long as they contain T cells or progenitor cells as described above. PBMC (peripheral blood mononuclear cells) collected from peripheral blood is one of the preferred target cells. A cell population obtained by passage and / or purification of a cell population collected from a living body can also be used as a target cell.

  Transformation of the target cell may be performed by a conventional method. For example, infection (when using viral vectors), electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. USA 81, 7161-7165 (1984)), lipofection (Felgner, PL et al , Proc. Natl. Acad. Sci. USA 84, 7413-7417 (1987)) and the like. As is clear from the operation, the cells of the present invention are prepared ex vivo.

  The cells of the present invention can be used for the treatment, prevention, or improvement of various diseases for which CAR-T therapy is considered effective (hereinafter referred to as “target diseases”). A representative target disease is cancer, but is not limited thereto. Examples of target diseases include various B-cell lymphomas (follicular malignant lymphoma, diffuse malignant lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20 positive Hodgkin lymphoma, etc.), treatment-resistant chronic lymphoma Leukemia, treatment-resistant idiopathic thrombocytopenic purpura, treatment-resistant systemic lupus erythematosus, hemophilia with coagulation inhibitors, neuroblastoma, brain tumor, Ewing sarcoma, retinoblastoma, small cell lung tumor, melanoma, ovary These include cancer, rhabdomyosarcoma, kidney cancer, pancreatic cancer, malignant mesothelioma, prostate cancer, other adenocarcinoma, and aplastic anemia whose blood transfusion effect is no longer achieved by anti-HLA antibodies. “Treatment” includes alleviation of symptoms or concomitant symptoms characteristic of the target disease (mildness), prevention or delay of deterioration of symptoms, and the like. “Prevention” refers to preventing or delaying the onset / onset of a disease (disorder) or its symptoms, or reducing the risk of onset / onset. On the other hand, “improvement” means that a disease (disorder) or a symptom thereof is alleviated (lightened), improved, ameliorated, or cured (including partial healing).

The cells of the present invention can also be provided in the form of cell preparations. The cell preparation of the present invention contains a therapeutically effective amount of the cells of the present invention. For example, for single administration, 10 ⁴ to 10 ¹⁰ cells are contained. Various components (vitamins) for the purpose of cell activation, proliferation or differentiation induction, such as dimethyl sulfoxide (DMSO) and serum albumin for the purpose of cell protection, such as antibiotics for the purpose of blocking bacterial contamination , Cytokines, growth factors, steroids, etc.) may be included in the cell preparation.

  The administration route of the cell or cell preparation of the present invention is not particularly limited. For example, administration is by intravenous injection, intraarterial injection, intraportal injection, intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. Local administration may be used instead of systemic administration. Examples of the local administration include direct injection into a target tissue / organ / organ. The administration schedule may be prepared in consideration of the subject's (patient) sex, age, weight, disease state, and the like. In addition to single administration, multiple administration may be performed continuously or periodically.

  After administration of the cell or cell preparation, a tetracycline compound (TC compound) is administered to make the system of the present invention function. The dose of the TC compound is not particularly limited as long as the system of the present invention functions. It is known that when 200 mg of doxycycline tablets is administered to healthy adults, the maximum blood concentration is obtained after 2 to 4 hours and is about 3 μg / ml (from the Vibramycin tablet interview form). In the study by the present inventors, the CAR expression reaches a peak at a concentration of 100 ng / ml or more (Examples described later). For example, by administering one tablet of 200 mg of doxycycline tablet, the concentration at which the system of the present invention is functioned is adjusted. Can be easily achieved. Administration continuously or intermittently allows the system of the present invention to function continuously. When it is necessary to stop the system of the present invention, such as when signs of side effects are observed, administration of the TC compound is discontinued. It is advisable to make a dosing plan so that side effects do not occur, taking into account the case and patient condition. For example, a dosing method in which administration is started at a low dose and the dose is increased while observing the progress, an administration method in which the next administration is performed if necessary while observing the progress after the initial administration, or the first time A dosing method or the like in which the next administration is performed with a sufficient interval after administration can be employed.

Until now, Tet-On system / Tet-Off system has been known as a tetracycline gene expression induction system. In this system, a gene for expressing tetracycline expression regulatory protein (tet-on 3G protein) and target gene ( It was necessary to separately introduce a promoter region (located downstream of P _{TRE 3G} ) that binds tet-on 3G protein and causes transcription in a tetracycline-dependent manner. In recent years, it has become possible to introduce these two genes using a single viral vector (Tet-One ^TM System: Clontech), and the efficiency of floating cells such as T cells is dramatically improved. It was expected. Therefore, it was examined whether CAR expression can be controlled by drug administration by using the system. CD19-CAR was used as a model for this purpose.

(1) Design of CD19-CAR (Tet-CD19CAR) subject to Tet-on control (Figure 1)
Tet-on 3G protein is transcribed by the human PGK promoter. The transcribed tet-on 3G protein binds to the 3G tet response element promoter (PTRE 3G) by binding to doxycycline (Dox), and turns on transcription of the downstream gene. In order to reduce background transcription when Dox is not administered, the target gene (CD19-CAR-T2A-tEGFR gene) is incorporated into the vector in the opposite direction to normal. The thus prepared tet-on-CD19CAR plasmid vector was introduced into Phoenix-Ampho cells, which are cells for retroviral packaging, and then the culture supernatant was collected to obtain a retroviral vector. CD19-CAR-T2A-tEGFR comprises human GM-CSF receptor leader sequence (SEQ ID NO: 1), light chain variable region (VL) (SEQ ID NO: 2) of mouse CD19 antibody (FMC63), linker (SEQ ID NO: 3), Mouse CD19 antibody (FMC63) heavy chain variable region (VH) (SEQ ID NO: 4), human IgG4-derived Fc (hinge, CH2, CH3) (SEQ ID NO: 5), CD28 transmembrane region (SEQ ID NO: 6), CD28 cells It has a structure in which an inner domain (SEQ ID NO: 7), glycine linker (GGG), CD3ζ (SEQ ID NO: 8), T2A sequence (SEQ ID NO: 9), and tEGFR (truncated EGFR) (SEQ ID NO: 10) are linked. The nucleotide sequence (CD19-CAR-T2A-tEGFR gene) encoding CD19-CAR-T2A-tEGFR is shown in SEQ ID NO: 11.

(2) Tet-CD19CAR gene transfer In order to investigate whether gene transfer is possible using the created retroviral vector or whether expression can be induced by administration of Dox, we have introduced a human T-cell leukemia tumor line, SUPT1. The Tet-CD19CAR gene was introduced. The result of confirming gene expression after gene introduction and fractionating positive fractions is shown (FIG. 2). Tet-CD19CAR Dox (-) is the result of examining the CAR expression without Dox administration by separating the portion expressing CAR after Dox administration. Similarly, Tet-CD19CAR Dox (+) examined CAR expression after Dox administration. There is a difference in the expression level between Dox (+) and Dox (-).

(3) Examination of Dox concentration necessary to induce CAR expression
Using SUPT1-tet-CD19CAR cells, the Dox concentration required to induce CAR expression was examined. In both staining with EGFR antibody and staining with Fc, expression equal to or higher than that was observed by administration of Dox 100 ng / ml or more (FIG. 3).

(4) Examination of CAR expression kinetics after starting and ending Dox administration
Using SUPT1-tet-CD19CAR cells, the dynamics of CAR expression after Dox administration was started and after administration was discontinued. Both EGFR antibody staining and Fc antibody staining were examined, and CAR expression peaked 24 hours after the start of Dox administration (FIG. 4). In addition, CAR expression reached a background level 48 hours after discontinuation of Dox administration (FIG. 5).

(5) Gene transfer experiment for human peripheral blood-derived CD8-positive T cells The experimental procedure was as follows. CD8 positive cells were isolated from peripheral blood of healthy donors, and gene transfer was performed using Tet-CD19CAR retrovirus. The CAR expression was examined on the 8th day from the start of the culture. CAR expression was not observed in Tet-CD19CAR in the Dox non-administered group, but expression was observed in the Dox-administered group (FIG. 6). Even in the Dox (−) group, 6.9% of fractions exhibit low expression. In the Dox (+) group, there were 3.3% fractions showing weak expression and 5.8% fractions showing high expression.

(6) Purification using tEGFR after Tet-CD19CAR gene introduction
CD19CAR-OR and Tet-CD19CAR Dox (+) cells were purified using tEGFR. The gene transfer efficiency of Tet-CD19CAR was about 5 to 10%, which was poor compared to CD19CAR-OR cells, but showed a purity of about 80% after tEGFR purification (FIG. 7).

(7) Experiment schedule in the latter half of the culture (Figure 8)
On the 8th day from the start of the culture, purification was performed using tEGFR, and then stimulation was performed using CD3 / 28 beads. After 11 days from the start of the culture, the culture was divided into a Dox (+) group and a Dox (−) group, and the culture was continued with Dox (+) or Dox (−), respectively. Thereafter, the cell function was measured around the 15th day from the start of the culture.

(8) Cytotoxic activity of Tet-CD19CAR T cells K562 (K562-) into which CD19 was gene-transferred using the Tet-CD19CAR T cells purified after (6) after gene transfer as in (5). Standard chromium release tests were performed targeting CD19) and non-transfected K562 cells. CD19CAR (CD19CAR-OR) and Tet-CD19CAR Dox (+) that are constitutively expressed showed equivalent cytotoxic activity against K562-CD19 (FIG. 9). Cytotoxic activity was significantly attenuated against K562 (FIG. 9). On the other hand, Tet-CD19CAR Dox (−) did not injure K562 / K562-CD19 (FIG. 9). This shows that the weak CAR expression observed by flow cytometry in Tet-CD19CAR Dox (−) is weaker than the range in which cytotoxic activity is exerted.

(9) Intracellular interferon gamma (IFN-γ) staining Using the Tet-CD19CAR T cells after purification of (6), co-culture with K562 / K562-CD19 for 4 hours to obtain intracellular IFN-γ staining. went. No reaction was observed in the non-gene-introduced cells, and the conventional reaction was observed in the CD19CAR-OR gene-introduced cells (FIG. 10). Tet-CD19CAR Dox (−) cells had a very low IFN-γ positive cell ratio, but Tet-CD19CAR Dox (+) cells showed a reaction equivalent to CD19CAR-OR (FIG. 10).

(10) IL-2 ELISA
The Tet-CD19CAR T cells after purification in (6) were co-cultured with K562 / K562-CD19, and IL-2 was quantified by ELISA in the culture supernatant after 16 hours. No reaction was observed in the non-gene-introduced cells, and the conventional good response was observed in the CD19CAR-OR gene-introduced cells (FIG. 11). Only very low IL-2 release was seen in Tet-CD19CAR Dox (−) cells, whereas IL-2 production equivalent to CD19CAR-OR was seen in Tet-CD19CAR Dox (+) cells (FIG. 11). ).

(11) Cell proliferation effect after stimulation with CD19 Using the Tet-CD19 CAR T cells after purification of (6), each cell was stimulated 1: 1 with K562-CD19, and cell proliferation after stimulation was examined. Non-transgenic cells and Tet-CD19CAR Dox (−) cells did not show a proliferative response after K562-CD19 stimulation (FIG. 12). On the other hand, CD19CAR-OR gene-introduced cells and Tet-CD19CAR Dox (+) cells showed an equivalent cell proliferation reaction (FIG. 12).

(12) Summary • A retroviral vector encoding Tet-CD19CAR was prepared, and Tet-CD19CAR T cells could be generated using this.
-The doxycycline required for expressing CAR in Tet-CD19CAR T cells was 100 ng / ml. That is, CAR expression was possible with a low concentration of drug. This point is important for clinical application.
-CAR expression after administration of doxycycline in Tet-CD19CAR T cells reached a peak at 24 hours and decreased to a background level 48 hours after doxycycline was discontinued. That is, good responsiveness was recognized. In particular, the responsiveness at the time of expression decline exceeds expectations and supports the effectiveness of this strategy.
-Dox (+) Tet-CD19CAR T cells showed CD19-specific cytotoxic activity, IL-2 production ability and proliferation ability after stimulation equivalent to CD19CAR-OR gene-transferred T cells upon CD19 stimulation. This fact supports the suitability of this strategy for clinical applications.
Dox (−) Tet-CD19CAR T cells showed cytotoxic activity that was significantly attenuated by CD19 stimulation, and showed little cytokine production and cell proliferation after stimulation.

(13) Advantages of target antigen-specific CAR expression (Tet-on CAR) using tetracycline gene expression induction system As described above, it is shown that the tetracycline gene expression induction system functions effectively for target antigen-specific CAR expression. It was done. This strategy has the following advantages: That is, CAR expression can be controlled without killing the CAR gene-transferred T cells. By stopping drug administration when an adverse event occurs, the immune response can be converged. Since Tet-CD19CAR T cells remain in the body, they can be reactivated by re-administration of the drug and treatment can be resumed.

  This strategy is not limited to CD19CAR but can be applied to other target antigens. No truly cancer-specific antigen has been obtained so far, and there is little prospect of obtaining it. In other words, it is predicted that some target antigens for CAR-T cell therapy should be expected to be expressed outside the target cells. In such a situation, the newly developed Tet-on CAR system can be used to ensure safety when developing a new CAR. The combined use of the Tet-on CAR system and the new CAR will enable safer clinical application.

  In the present invention, CAR is expressed using a tetracycline gene expression induction system system. According to the present invention, an excessive immune reaction can be avoided by expressing CAR when necessary and turning off the expression when an excessively strong reaction appears. The present invention is also effective when using CAR against a target antigen expressed in a small amount in normal tissues other than tumor cells, and can obtain a therapeutic effect while reducing side effects. Thus, the present invention dramatically increases the usefulness of CAR. By applying the present invention, it is possible to envisage a treatment that obtains an antitumor effect little by little only when a drug that induces expression is administered.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (15)

a first expression cassette comprising a tet operator sequence, a promoter sequence under the control of the tet operator sequence, and a target antigen-specific chimeric antigen receptor gene located downstream of the promoter sequence;
A second expression cassette comprising a promoter sequence that functions in immune cells, and a sequence encoding a reverse tetracycline-regulated transactivator disposed downstream of the promoter sequence;
A target antigen-specific chimeric antigen receptor gene expression system comprising:   The system according to claim 1, wherein the target antigen-specific chimeric antigen receptor gene is a CD19 chimeric antigen receptor gene.   The system of claim 2, wherein the CD19 chimeric antigen receptor comprises an extracellular domain comprising an scFv fragment of an anti-CD19 monoclonal antibody, a transmembrane domain, and an intracellular signal domain for effector function of immune cells.   The system according to any one of claims 1 to 3, wherein the first expression cassette further comprises a detection gene.   The system according to claim 4, wherein the detection gene is linked to the target antigen-specific chimeric antigen receptor gene via a sequence encoding a self-cleaving peptide.   The system according to claim 4 or 5, wherein the detection gene is an epidermal growth factor receptor (EGFR) gene lacking an intracellular domain.   The system according to any one of claims 1 to 6, wherein the first expression cassette is held in a first vector and the second expression cassette is held in a second vector.   The system according to any one of claims 1 to 6, wherein the first expression cassette and the second expression cassette are held in one vector.   The system according to any one of claims 1 to 8, wherein the vector is a retroviral vector or a lentiviral vector.   An immune cell into which the system according to any one of claims 1 to 9 has been introduced.   An immune cell into which the system according to any one of claims 1 to 6 has been introduced using a transposon.   The immune cell according to claim 10 or 11, wherein the immune cell is a T cell.   A cell preparation comprising a therapeutically effective amount of the immune cells according to any one of claims 10-12.   a first expression cassette comprising a tet operator sequence, a promoter sequence under the control of the tet operator sequence, and a target antigen-specific chimeric antigen receptor gene arranged downstream of the promoter sequence, and a promoter sequence that functions in immune cells And a second expression cassette comprising a sequence encoding a reverse tetracycline-regulated transactivator disposed downstream of the promoter sequence.   The vector according to claim 14, which is a retroviral vector or a lentiviral vector.

Images