WO2021100585A1 - Method for producing chimeric antigen receptor gene-modified lymphocytes - Google Patents

Abstract

Provided is a novel production (culture) means that expands a CAR cell population. Specifically provided is a method for producing genetically modified lymphocytes that express a chimeric antigen receptor, by (1) a step for preparing gene-modified lymphocytes into which a gene of a target antigen-specific chimeric antigen receptor has been introduced; (2) a step for culturing the gene-modified lymphocytes prepared in step (1) in the presence of anti-idiotype antibody against the antigen recognition region of the chimeric antigen receptor expressed by the gene-modified lymphocytes, or in the presence of cells that express the target antigen for the chimeric antigen receptor expressed by the genetically modified lymphocytes; and (3) a step for recovering cultured gene-modified lymphocytes.

Description

Method for preparing chimeric antigen receptor genetically modified lymphocytes

The present invention relates to genetically modified lymphocytes expressing chimeric antigen receptors (CAR transgenic lymphocytes). Specifically, the present invention relates to a method for producing CAR gene-introduced lymphocytes, uses of the cells, and the like.

Genetically modified T cell therapy (CAR-T therapy) and genetically modified NK cell therapy (CAR-NK therapy) using a chimeric antigen receptor (hereinafter also referred to as "CAR") have been clinically applied. .. CAR typically comprises a structure in which the single-chain variable region of an antibody is the extracellular domain, which is linked to the transmembrane domain, the CD3ζ, and the intracellular domain of the molecule that transmits the co-stimulation signal. CAR transgenic lymphocytes are activated by binding to the antigen according to the specificity of the antibody, damaging target cells (such as cancer cells). CAR therapy has advantages such as relatively easy cell preparation, high cytotoxic activity, and long-lasting effect, and is particularly resistant to refractory and conventional treatments. It is expected as a new therapeutic means for cases. In fact, in patients with chemotherapy-resistant acute lymphoblastic leukemia, CAR for the CD19 antigen expressed on the cell surface is gene-introduced into peripheral blood T cells collected from the patients, cultured, and infused. The test has been conducted in Europe and the United States, and good results with a remission rate of 80 to 90% have been reported (Non-Patent Documents 1 to 3). CAR therapy is attracting attention as one of the most promising treatments for refractory cancer in the United States.

Conventionally, cells used for CAR therapy (typically CAR-T cells) have been prepared using a viral vector. However, commonly used retroviruses have a high frequency of insertion mutations into protooncogenes (leukemia occurs frequently in gene therapy using hematopoietic stem cells), and safety becomes a problem. In addition, since a dedicated cell culture facility is required when using a viral vector, there is also an economical problem that the treatment cost is very high (Non-Patent Document 4).

In order to solve the problems in conventional CAR therapy using viral vectors, the use of the transposon method, which is one of the gene modification techniques using non-viral vectors, is being studied. Although the transposon method enables permanent gene transfer like the viral vector method, the gene transfer efficiency is lower than that of the viral vector method, and it is suitable for gene transfer operations (electroporation and its improvement methods, etc.). As a result, the cells are damaged, and there is a problem that the cell viability and the cell proliferation rate decrease. As one of the solutions to this problem, the research group of the present inventors said, "Protecting lymphocytes after CAR gene transfer by co-culturing with activated T cells prepared separately, gene transfer efficiency and cells. Strategy to improve survival / cell proliferation rate ”and“ Virus-specific CAR by co-culturing lymphocytes after CAR gene transfer with activated T cells or mononuclear cells carrying viral peptides -A strategy for efficiently preparing T cells "was proposed (Patent Document 1).

International Publication No. 2017/061615 Pamphlet

Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, Teachey DT, Chew A, Hauck B, Wright JF, Milone MC, Levine BL, June CH. Chimeric antigen receptor-modified . N Engl J Med, 368 (16): 1509-18. 2013 Maude SL, Frey N, Shaw PA, Applenc R, Barrett DM, Bunin NJ, Chew A, Gonzalez VE, Zheng Z, Lacey SF, Mahnke YD, Melenhorst JJ, Rheingold SR, Shen A, T , Porter DL, Grupp SA. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med, 371 (16): 1507-17. 2014 Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Delbrook C, Feldman SA, Fry TJ, Orentas R, Sabatino M, Shah NN, Steinberg SM, Strong Zhang Rosenberg SA, Wayne AS, Mackall CL. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Morgan RA. Faster, cheaper, safer, T-cell engineering. J Immunother, 36 (1): 1-2. 2013

Although CAR therapy using the transposon method is promising, it is assumed that the number of cells required for treatment cannot be prepared due to problems of gene transfer efficiency and cell viability / cell proliferation rate. Even when CAR cells are prepared by the viral vector method, it is sufficient for some patients (donors) because there are few target cells (cells into which the CAR gene is introduced) in the blood and the cell activity / survival rate is low. The number of CAR cells may not be available. That is, the above problem is not limited to the transposon method. In addition, if the cell surface antigen of leukocytes such as CD19 is the target, it is expected that the survival rate / proliferation rate of CAR cells will be improved by stimulation from coexisting cells when culturing cells after CAR gene transfer. Cannot expect such an effect.
Therefore, the main object of the present invention is to provide a novel preparation (culture) means for increasing the number of CAR cells, and to contribute to the progress of clinical application of CAR therapy and the improvement of therapeutic results.

As a result of repeated studies to solve the above problems, the present inventors have two strategies, that is, a method of stimulating cells after CAR gene transfer (CAR cells) with an anti-iditope antibody (first strategy), and We have created a method (second strategy) for stimulating cells after CAR gene transfer (CAR cells) with cells expressing the target antigen. The first strategy is to directly stimulate CAR cells with anti-iditope antibodies, and CAR cell-specific proliferation can be expected. In fact, a significant increase in the number of CAR cells (improvement in gene transfer efficiency / cell proliferation rate) was observed as compared with the conventional method (stimulation with anti-CD3 antibody / anti-CD28 antibody) (see Examples described later). On the other hand, according to the second strategy, stimulation by the target antigen is added during the culture of CAR cells, and CAR cell-specific proliferation can be expected as in the first strategy. As described above, the two new strategies make it possible or facilitate to secure the number of cells required for treatment, and their clinical significance is extremely large. The inventions shown below are based on these two strategies. [1] A method for preparing a genetically modified lymphocyte expressing a chimeric antigen receptor, which comprises the following steps (1) to (3):
(1) A step of preparing a genetically modified lymphocyte into which a target antigen-specific chimeric antigen receptor gene has been introduced;
(2) The genetically modified lymphocyte prepared in step (1) is used in the presence of an anti-iditope antibody against the antigen recognition region of the chimeric antigen receptor on which it is expressed, or the target antigen of the chimeric antigen receptor on which it is expressed. The step of culturing in the presence of expressing cells; and (3) the step of collecting the genetically modified lymphocytes after culturing.
[2] The preparation method according to [1], wherein the transposon method is used for the introduction of the target antigen-specific chimeric antigen receptor gene in step (1).
[3] The preparation method according to [2], wherein the transposon method is the piggyBac transposon method.
[4] In any one of [1] to [3], step (2) is performed after 8 to 48 hours have passed from the operation of introducing the target antigen-specific chimeric antigen receptor gene in step (1). The preparation method described.
[5] The preparation method according to any one of [1] to [4], wherein the culture period of step (2) is 1 to 14 days.
[6] The preparation method according to any one of [1] to [5], wherein the culture in step (2) is not stimulated by the anti-CD3 antibody and the anti-CD28 antibody.
[7] The preparation method according to any one of [1] to [6], wherein the anti-iditope antibody is a solid phase antibody or a beaded antibody.
[8] Any of [1] to [7], wherein the genetically modified lymphocyte of step (1) can be obtained by introducing a target antigen-specific chimeric antigen receptor gene into a cell population containing T cells or progenitor cells thereof. The preparation method according to item 1.
[9] The preparation method according to [8], wherein the cell population is peripheral blood mononuclear cells (PBMCs).
[10] The cells expressing the target antigen in step (2) are prepared by introducing the gene encoding the target antigen into peripheral blood mononuclear cells (PBMCs), according to [1] to [9]. The preparation method according to any one item.
[10-1] The transposon method is used for the introduction of the target antigen-specific chimeric antigen receptor gene in step (1).
Any one of [1] to [10], wherein the cell expressing the target antigen in step (2) is prepared by introducing a gene encoding the target antigen into peripheral blood mononuclear cells (PBMCs). The preparation method described in the section.
[11] The preparation method according to any one of [1] to [10], wherein the cell expressing the target antigen and the genetically modified lymphocyte are derived from the same individual in step (2).
[12] The preparation method according to [11], wherein the individual is an individual different from the individual to be transplanted with the genetically modified lymphocyte recovered in step (3).
[13] The item according to any one of [1] to [12], wherein the step of culturing the cultured cells in the presence of T cell growth factor is performed between steps (2) and (3). Preparation method.
[14] The preparation method according to [13], wherein the T cell growth factor is IL-15.
[15] The preparation method according to [13], wherein IL-15 and IL-7 are used in combination as T cell growth factors.
[16] The preparation method according to any one of [1] to [15], wherein the following step (a) is performed between steps (1) and (2):
(A) Non-proliferative cells obtained by stimulating a cell population containing T cells with an anti-CD3 antibody and an anti-CD28 antibody and then performing a treatment for losing proliferative ability, and genetically modified lymphocytes prepared in step (1). A step of mixing lymphocytes and co-culturing while stimulating with anti-CD3 antibody and anti-CD28 antibody.
[17] The preparation method according to any one of [1] to [15], wherein the following step (b) is performed between steps (1) and (2):
(B) Non-proliferative PBMCs obtained by treating peripheral blood mononuclear cells (PBMCs) as they are or by stimulating them with anti-CD3 antibody and anti-CD28 antibody and then performing a treatment to lose proliferative ability, and step (1). The step of mixing and co-culturing the genetically modified lymphocytes prepared in.
[18] The preparation method according to any one of [1] to [15], wherein the following step (c) is performed between steps (1) and (2):
(C) A cell population containing T cells was stimulated with an anti-CD3 antibody and an anti-CD28 antibody, and then cultured in the presence of the viral peptide antigen and treated to lose the proliferative ability to retain the viral peptide antigen. A step of mixing non-proliferative cells with the transgenic lymphocytes prepared in step (1) and co-culturing them.
[19] The preparation method according to [16] or [18], wherein the cell population containing T cells is peripheral blood mononuclear cells (PBMCs).
[20] The preparation method according to any one of [16] to [19], wherein the treatment for losing the proliferative ability is irradiation.
[21] The preparation method according to any one of [16] to [20], wherein the non-proliferative cells and the genetically modified lymphocytes are derived from the same individual.
[22] A genetically modified lymphocyte expressing a chimeric antigen receptor obtained by the preparation method according to any one of [1] to [21].
[23] A cell preparation containing a therapeutically effective amount of the genetically modified lymphocyte according to [22].
[24] A method for treating cancer, which comprises a step of administering a therapeutically effective amount of the genetically modified lymphocyte according to [22] to a cancer patient.

Preparation of CAR-T cells by the conventional method (culture method 1). Preparation of CAR-T cells by a novel method (culture method 2). Preparation of CAR-T cells by a novel method (culture method 3). Composition of pIRII-CAR.CD19.28z vector (SEQ ID NO: 1). The CD19CAR gene has a structure sandwiched between a 5'inverted repeat sequence (5'IR) and a 3'inverted repeat sequence (3'IR). CD19CAR has a leader sequence (SEQ ID NO: 2), light chain variable region (VL) (SEQ ID NO: 3), heavy chain variable region (VH) (SEQ ID NO: 4), Fc region (CH2, CH3) (SEQ ID NO: 5), Includes the transmembrane region of CD28 and the intracellular domain (SEQ ID NO: 6) and CD3ζ (SEQ ID NO: 7). Composition of pCMV-pigBac vector (SEQ ID NO: 8). The piggyBac transposase gene is located under the control of the CMV earliest promoter (CMV immediate ear1y promoter). Improvement of gene transfer efficiency by a new method (culture method 2). The cells on the 14th day were evaluated by flow cytometry, first cultured for 7 days by the conventional method (culture method 1), then cultured for 7 days by the new method (culture method 2) (upper right), and first 7 After culturing by the conventional method (culture method 1) for 1 day, the gene transfer efficiency was further compared with that cultivated by the conventional method (culture method 1) for 7 days (lower right). Improvement of gene transfer efficiency by a new method (culture method 2). First cultivated by the conventional method (culture method 1) for 7 days, then cultivated by the new method (culture method 2) for 7 days (left), and first cultivated by the conventional method (culture method 1) for 7 days, and then. Furthermore, the proliferation rates of CAR-T cells from the 7th day were compared with those cultured for 7 days by the conventional method (culture method 1) (right). Composition of pIRII-CAR.CD19_optimized vector (SEQ ID NO: 9), which is an optimized CAR gene transfer vector. Compared with the structure of the pIRII-CAR.CD19.28z vector, the Fc region (CH2, CH3) has been deleted. Improvement of gene transfer efficiency by a new method (culture method 2). The cells on day 7 were evaluated by flow cytometry, and the gene transfer efficiency was compared between the novel method (culture method 2) (upper right) and the conventional method (culture method 1) (lower right). Improvement of gene transfer efficiency by a new method (culture method 2). The number of CAR-T cells was compared between the new method (culture method 2) (left) and the conventional method (culture method 1) (right).

1. 1. Method for preparing genetically modified lymphocytes expressing chimeric antigen receptor 1-1. Specific Stimulation by Cells Expressing Anti-Idiotopal Antibodies / Target Antigens The present invention refers to genetically modified lymphocytes expressing chimeric antigen receptors (ie, CAR transgenic lymphocytes; hereinafter, referred to as "CAR cells" for convenience of explanation. There is) regarding the preparation method. CAR cells (typically CAR-T cells) obtained by the preparation method of the present invention can be used for CAR therapy. In the preparation method of the present invention, the following steps (1) to (3) are performed. Unless otherwise specified, various cells (for example, T cells) in the present specification are human cells.
(1) Step of preparing a genetically modified lymphocyte into which a target antigen-specific chimeric antigen receptor gene has been introduced (2) The gene-modified lymphocyte prepared in step (1) is expressed by the chimeric antigen receptor antigen. Steps of culturing in the presence of an anti-idiotype antibody against the recognition region or in the presence of cells expressing the target antigen of the chimeric antigen receptor on which it is expressed (3) Steps of collecting genetically modified lymphocytes after culturing

Step (1)
The step (1) is a step of preparing a cell genetically engineered to forcibly express the chimeric antigen receptor gene (CAR gene), and the target antigen-specific CAR gene is introduced into the target cell.

The CAR gene encodes a chimeric antigen receptor (CAR) that recognizes a specific target antigen. CAR is a structure that contains a target-specific extracellular domain, a transmembrane domain, and an intracellular signal domain for the effector function of immune cells. Hereinafter, each domain will be described.

(A) Extracellular domain The extracellular domain contains an antigen recognition region and exhibits target-specific binding. For example, the extracellular domain contains a scFv fragment of an antitargeted monoclonal antibody. As the monoclonal antibody here, for example, a rodent (mouse, rat, rabbit, etc.) antibody, a human antibody, a humanized antibody, or the like is used. A humanized monoclonal antibody is an antibody in which the structure of a monoclonal antibody of another animal species (for example, 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. Human-type CDR-grafted antibody (PT Johons et al., Nature 321,522 (1986) in which the chimeric antibody and the portion other than the CDR (complementarity determining region) existing in the constant region and the variable region are replaced with those of the human antibody. ))including. A method of selecting a human antibody framework (FR) that is highly homologous to a mouse antibody, a method of producing a highly homologous humanized antibody, and a mouse CDR to a human antibody in order to enhance the antigen-binding activity of the human CDR transplanted antibody. Further improvements have been made to the method of substituting amino acids in the FR region after transplantation (US Pat. No. 5585089, US Pat. No. 5697361, US Pat. No. 5693762, US Pat. No. 6180370, European Patent No. 451216 , European Patent No. 682040, Patent No. 2828340, etc.), which can also be used to produce humanized antibodies.

The scFv fragment is a structure in which the light chain variable region (VL) and heavy chain variable region (VH) of immunoglobulin 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 composed of peptides in which amino acids are linearly linked. A typical example of a peptide linker is a linker composed of glycine and serine (GGS linker or GS linker). The GGS linker and the amino acids that make up the GS linker, glycine and serine, are small in size and 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 number of amino acid residues constituting the linker is preferably 8 to 25, more preferably 15 to 20.

The target is typically an antigen whose expression is specific to tumor cells. The term "specific expression" as used herein means that significant or remarkable expression is observed as compared with cells other than tumor, and there is no intention of limiting the expression to those having no expression in cells other than tumor. Examples of target antigens are CD19 antigen, CD20 antigen, GD2 antigen, CD22 antigen, CD30 antigen, CD33 antigen, CD44variant7 / 8 antigen, CD123 antigen, CEA antigen, Her2 / neu antigen, MUC1 antigen, MUC4 antigen, MUC6 antigen, IL. -13 Receptor-alpha2, immunoglobulin light chain, PSMA antigen, VEGF receptor2, mesothelin antigen, EGFRvIII, EphA2 antigen, IGFR and the like can be mentioned.

It is also possible to target the GM-CSF (granulocyte-macricular colony stimulating factor) receptor expressed in leukemic stem cells / progenitor cells. In this case, GM-CSF, which is a ligand for the GM-CSF receptor, is used as the extracellular domain constituting CAR. Leukemia stem cells, leukemia precursor cells, leukemia cells, etc. of myelogenous tumors are targeted by CAR cells, and myeloproliferative tumors, myelodysplastic / myeloproliferative tumors (CMML, JMML, CML, MDS / MPN-UC) Cells applicable to the prevention and treatment of myelodysplastic syndrome, acute myelogenous leukemia, etc. are prepared.

(B) Transmembrane domain The transmembrane domain intervenes 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. A transmembrane domain consisting of an artificially constructed polypeptide may be used.

(C) Intracellular signal domain The intracellular signal domain transmits signals necessary for exerting the effector function of immune cells. That is, when the extracellular domain binds to the target antigen, an intracellular signal domain capable of transmitting a signal necessary for activation of immune cells is used. The intracellular signal domain includes a domain for transmitting a signal via the TCR complex (referred to as "first domain" for convenience) and a domain for transmitting a co-stimulation signal (for convenience, "second domain"). Called) is included. As the first domain, an intracellular domain such as FcεRIγ can be used in addition to CD3ζ. Preferably, CD3ζ is used. The intracellular domain of the co-stimulating molecule is used as the second domain. Examples of co-stimulatory molecules include CD28, 4-1BB (CD137), CD2, CD4, CD5, CD134, OX-40 or ICOS. Preferably, the intracellular domain of CD28 or 4-1BB is adopted.

The mode of connection between the first domain and the second domain is not particularly limited, but preferably, the transmembrane domain is preferably transmitted because it is known that the co-stimulation was strongly transmitted when the CD3ζ was connected distally in the past cases. Place the second domain on the side. A plurality of intracellular domains of the same or different species may be linked in a tandem manner to form a 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. As the linker, for example, a peptide linker can be used. A peptide linker is a linker composed of peptides in which amino acids are linearly linked. The structure, characteristics, etc. of the peptide linker are as described above. However, as the linker here, a linker composed of only glycine may be used. The length of the linker is not particularly limited. For example, a linker having 2 to 15 amino acid residues can be used.

(D) Other elements Leader sequences (signal peptides) are used to facilitate the transport of CAR onto the cell membrane. For example, the leader sequence of the GM-CSF receptor can be used. In addition, it is preferable to have a structure in which the extracellular domain and the transmembrane domain are linked via a spacer domain. That is, the CAR of the preferred embodiment comprises a spacer domain between the extracellular domain and the transmembrane domain. The spacer domain is used to promote the binding of CAR to the target antigen. For example, an Fc fragment of human IgG (eg, human IgG1, human IgG4) can be used as a spacer domain. In addition, a part of the extracellular domain of CD28, a part of the extracellular domain of CD8α, and the like can also be used as the spacer domain. A spacer domain can also be provided between the transmembrane domain and the intracellular signal domain.

There have been some reports on experiments and clinical studies using CAR (for example, Rossig C, et al. Mol Ther 10: 5-18, 2004; Dotti G, et al. Hum Gene Ther 20: 1229. -1239, 2009; Ngo MC, et al. Hum Mol Genet 20 (R1): R93-99, 2011; Ahmed N, et al. Mol Ther 17: 1779-1787, 2009; Pule MA, et al. Nat Med 14 : 1264-1270, 2008; Louis CU, et al. Blood 118: 6050-6056, 2011; Kochenderfer JN, et al. Blood 116: 4099-4102, 2010; Kochenderfer JN, et al. Blood 119: 2709-2720, 2012; Porter DL, et al. N Engl J Med 365: 725-733, 2011; Kalos M, et al. Sci Transl Med 3: 95ra73, 2011; Brentjens RJ, et al. Blood 118: 4817-4828, 2011; Brentjens RJ, et al. Sci Transl Med 5: 177 ra38, 2013), the CAR in the present invention can be constructed with reference to these reports.

Various gene transfer methods can be used to introduce the CAR gene. The gene transfer method is roughly classified into a method using a viral vector and a method using a non-viral vector. The former skillfully utilizes the phenomenon that the virus infects cells, and high gene transfer efficiency can be obtained. As viral vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated virus vectors, herpesvirus vectors, Sendai viral vectors and the like have been developed. Among these, in the retroviral vector, lentiviral vector, and adeno-associated virus vector, the target gene incorporated into 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 previously reported methods or using commercially available dedicated kits. Examples of non-viral vectors include plasmid vectors, liposome vectors, and positively charged liposome vectors (Felgner, PL, Gadek, TR, Holm, M. et al., Proc. Natl. Acad. Sci., 84: 7413-7417. , 1987), YAC vector, BAC vector can be mentioned.

Preferably, gene transfer is performed by the transposon method. The transposon method is one of the non-viral gene transfer methods. Transposon is a general term for short gene sequences that cause gene translocations that have been conserved during evolution. A pair of gene enzyme (transposase) and its specific recognition sequence causes gene translocation. As the transposon method, for example, the piggyBac transposon method can be used. The PiggyBac transposon method utilizes transposons isolated from insects (Fraser MJ et al., Insect Mol Biol. 1996 May; 5 (2): 141-51 .; Wilson MH et al., Mol Ther. . 2007 Jan; 15 (1): 139-45.), Enables highly efficient integration into mammalian chromosomes. The PiggyBac transposon method is actually used for the introduction of CAR genes (for example, Nakazawa Y, et al., J Immunother 32: 826-836, 2009; Nakazawa Y et al., J Immunother 6: 3-10, 2013, etc. See). The transposon method applicable to the present invention is not limited to the one using piggyBac, for example, Sleeping Beauty (Ivics Z, Hackett PB, Plasterk RH, Izsvak Z (1997) Cell 91: 501-510.), Frog Prince (Miskey C, Izsvak Z, Plasterk RH, Ivics Z (2003) Nucleic Acids Res 31: 6873-6881.), Tol1 (Koga A, Inagaki H, Bessho Y, Hori H. 249 (4): 400-5 .; Koga A, Shimada A, Kuroki T, Hori H, Kusumi J, Kyono-Hamaguchi Y, Hamaguchi S. J Hum Genet. 2007; 52 (7): 628-35. Epub 2007 Jun 7.), Tol2 (Koga A, Hori H, Sakaizumi M (2002) Mar Biotechnol 4: 6-11 .; Johnson Hamlet MR, Yergeau DA, Kulyev E, Takeda M, Taira M, Kawakami K, Mead PE ) Genesis 44: 438-445 .; Choo BG, Kondrichin I, Parinov S, Emelyanov A, Go W, Toh WC, Korzh V (2006) BMC Dev Biol 6: 5.) You may decide.

The CAR gene transfer operation by the transposon method may be performed by a conventional method, and past literature (for example, for the piggyBac transposon method, Nakazawa Y, et al., J Immunother 32: 826-836, 2009, Nakazawa Y et al., J Immunother 6: 3-10, 2013, Saha S, Nakazawa Y, Huye LE, Doherty JE, Galvan DL, Rooney CM, Wilson MH. J Vis Exp. 2012 Nov 5; (69): e4235, Saito S, Nakazawa Y, Sueki A, et al. Anti-leukemic potential of piggyBac-mediated CD19-specific T cells against refractory Philadelphia chromasome-positive acute lymphoblastic leukemia. Cytotherapy. 2014; 16: 1257-69.) Is helpful.

In a preferred embodiment of the present invention, the piggyBac transposon method is adopted. Typically, in the piggyBac transposon method, a vector carrying a gene encoding the piggyBac transposase (transposase plasmid) and a desired nucleic acid construct (CAR expression cassette and / or siRNA expression cassette) are sandwiched between piggyBac reverse repeat sequences. Vectors having a structure (transposon plasmid) are prepared, and these vectors are introduced (transfected) into target cells. For transfection, various methods such as electroporation, nucleofection, lipofection, and calcium phosphate method can be used.

In the transposon plasmid, a poly A addition signal sequence is placed downstream of the CAR gene. Transcription is terminated by the use of the poly A addition signal sequence. As the poly A addition signal sequence, a poly A addition sequence of SV40, a poly A addition sequence of a bovine growth hormone gene, or the like can be used.

The transposon plasmid may include a detection gene (reporter gene, cell or tissue-specific gene, selectable marker gene, etc.), enhancer sequence, WRPE sequence, and the like. The detection gene is used for determining the success or failure and efficiency of the introduction of the expression cassette, detecting the expression of the CAR gene or determining the expression efficiency, selecting and sorting the cells expressing the CAR gene, and the like. On the other hand, the expression efficiency can be improved by using the enhancer sequence. The genes for detection include the neo gene that imparts resistance to neomycin, the npt gene that imparts resistance to kanamycin, etc. (Herrera Estrella, EMBO J. 2 (1983), 987-995) and the npt II gene (Messing & Vierra. Gene 1). 9: 259-268 (1982)), hph gene conferring resistance to neomycin (Blochinger & Digglmann, Mol Cell Bio 4: 2929-2931), dhfr gene conferring resistance to metatrexate (Bourouis et al. , EMBO J.2 (7)), etc. (marker gene), luciferase gene (Giacomin, P1. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), β -Glucronidase (GUS) gene, genes for fluorescent proteins such as GFP (Gerdes, FEBS Lett. 389 (1996), 44-47) and their variants (EGFP, d2EGFP, etc.), intracellular domains Genes such as the missing epithelial growth factor receptor (EGFR) gene can be used. The detection gene is linked to the CAR gene via, for example, a bicistronic control sequence (eg, ribosome internal recognition sequence (IRES)) or a sequence encoding a self-cleaving peptide. An example of a self-cleaving peptide is, but is not limited to, a 2A peptide (T2A) derived from Thosea signa virus. 2A peptide (F2A) derived from foot-and-mouth disease virus (FMDV), 2A peptide (E2A) derived from horse rhinitis A virus (ERAV), 2A peptide (P2A) derived from porcine teschovirus (PTV-1), etc. are known as self-cleaving peptides. Has been done.

Target cells (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, αβ-T cells, γδ-T cells, NK cells, and NKT cells. Can be mentioned. Various cell populations can be used as long as they contain lymphocytes or progenitor cells as described above. Peripheral blood mononuclear cells (PBMCs) collected from peripheral blood are one of the preferred target cells. That is, in a preferred embodiment, a gene transfer operation is performed on PBMCs, which are a cell population containing T cells or progenitor cells. PBMCs may be prepared by a conventional method. For the method of preparing PBMCs, refer to, for example, Saha S, Nakazawa Y, Huye LE, Doherty JE, Galvan DL, Rooney CM, Wilson MH. J Vis Exp. 2012 Nov 5; (69): e4235. it can.

Step (2)
The genetically modified lymphocytes (CAR cells) prepared in step (1) are subjected to culturing under specific conditions (step (2)). In one aspect (first aspect), the genetically modified lymphocyte prepared in step (1) is cultured in the presence of an anti-iditope antibody against the antigen recognition region of the chimeric antigen receptor on which it is expressed. In another aspect (second aspect), the genetically modified lymphocyte prepared in step (1) is cultured in the presence of cells expressing the target antigen of the chimeric antigen receptor on which it is expressed (target antigen expressing cells). In the present invention, the target CAR cells (that is, those into which the target antigen-specific chimeric antigen receptor gene has been introduced) are specifically stimulated by culturing in the presence of anti-iditope antibody or target antigen-expressing cells. , To improve the proliferation rate.

The anti-iditope antibody used in the first aspect may be prepared by using an immunological method, a phage display method, a ribosome display method, or the like. Specifically, it can be produced according to a known method such as the method described in PLoS One. 2013; 8 (3): e57838. Further, an anti-iditope antibody may be prepared by using a contract service (for example, GenScript, Creative Biolabs, Immuno-Biological Laboratories, Inc., GeneFrontier Corp.). The anti-iditope antibody may be either a polyclonal antibody or a monoclonal antibody. The anti-iditope antibody is an antibody that recognizes the antigen recognition region (variable domain) of an antibody molecule and binds to an epitope (idiotope) in a specific idiotype. Specifically, the anti-iditope antibody includes an anti-iditope antibody against CD19CAR, an anti-iditopes antibody against GD2CAR, an anti-iditopes antibody against CD22CAR, an anti-iditopes antibody against BCMACAR, and an anti-iditopes antibody against CD5CAR. Examples thereof include an anti-iditope antibody against CD123CAR and an anti-iditopes antibody against HER2CAR. Preferred examples thereof include an anti-iditope antibody against CD19CAR and an anti-iditopes antibody against GD2CAR.

The anti-iditope antibody may contain a constant region of immunoglobulin or may not contain a constant region of immunoglobulin. When the constant region is included, the constant region of the heavy chain (CH1, CH2, and CH3) and the constant region of the light chain (CL) may be all included, and any one or more of these may be included. May include a combination of. Specific examples of anti-iditope antibodies include immunoglobulin structure, Fab structure, F (ab') ₂ structure, minibody structure, scFv-Fc structure, Fv structure, scFv structure, and diabody structure. , Triabody structure, tetrabody structure and the like. In addition, anti-iditope antibodies also include low molecular weight antibodies such as Nanobodies and monobodies.

By culturing in the presence of anti-iditope antibody, CAR cells and anti-iditope antibody can be brought into contact with each other. For example, if an anti-iditopes antibody is immobilized on the culture surface of a culture vessel, the contact state can be formed during the culture. Examples of the substrate used for solid phase formation include a substrate containing plastic such as polystyrene, glass, nitrocellulose and the like as a main component. By "solid phase" is meant that the anti-iditopes antibody is immobilized by binding directly or indirectly to the substrate. Fixation of the anti-iditopes antibody to the substrate can be performed according to or according to a conventional method. Specific embodiments of fixation are not particularly limited, and examples thereof include fixation via a covalent bond, fixation via a bond between avidin or streptavidin and biotin, and fixation by physical adsorption. Further, by adding an anti-iditope antibody (beaded antibody) bound to beads (for example, magnetic beads) to the culture medium, it is possible to form a state in which CAR cells and the anti-iditope antibody can be contacted. The material of the beads is not particularly limited, and for example, metal particles such as gold, silver, copper, iron, aluminum, nickel, manganese, titanium, and oxides thereof; resin particles such as polystyrene and latex; silica particles and the like. Can be mentioned. The shape of the bead is not particularly limited, and examples thereof include a sphere, a rectangular parallelepiped, a cube, a triangular pyramid, and the like, or a shape close to these. The bead preferably has a substance on its surface to make the binding of the anti-iditope antibody easier and / or stronger. Examples of such substances include substances having a reactive group such as an epoxy group, an amino group, a carboxy group and an azide group; and substances having an affinity for other molecules such as avidin, protein A and protein B. Can be mentioned.

The target antigen-expressing cell used in the second aspect is not particularly limited as long as it is a cell in which the antigen (particularly the CAR recognition portion) is exposed on the cell surface. Typically, it can be prepared by introducing a gene encoding a target antigen into peripheral blood mononuclear cells (PBMCs). By fractionating PBMCs, a specific cell population (for example, T cells, B cells, NK cells, dendritic cells, or a combination of two or more of these) is prepared and targeted to the cell population. A gene encoding an antigen may be introduced. Further, as a cell into which a gene encoding a target antigen is introduced, a cell such as K562 can also be used. The expression of the target antigen may be transient or constant. For gene transfer, for example, various vectors such as a plasmid may be used. More specifically, gene transfer can be carried out in the same manner as, for example, the above-mentioned CAR gene transfer.

Preferably, the origin of the genetically modified lymphocyte (CAR cell) prepared in step (1) and the target antigen-expressing cell are the same. In other words, CAR cells and target antigen-expressing cells are prepared using cells isolated from the same individual. In this way, it is possible to avoid the addition of unintended stimuli to CAR cells, the problem of immune rejection, infectious diseases, and the like. In this embodiment, the individual from which the CAR cells and the target antigen-expressing cells are derived is a patient (recipient) (autologous transplantation) or another person (allogeneic transplantation) who receives the CAR cells obtained by the preparation method of the present invention. is there. The latter (ie, in the case of allogeneic transplantation) is applied to patients who have difficulty in preparing the cells necessary for the preparation of CAR cells, for example, when there are few lymphocytes in the blood or the activity of lymphocytes is low. It can be said that it is suitable for preparing.

In order to promote recovery from damage / injury caused by the gene transfer operation when preparing CAR cells, 2 hours to 72 hours (preferably 8 hours to 8 hours) after the gene transfer operation, not immediately after the gene transfer operation in step (1). Step (2) may be performed after 48 hours, more preferably 16 to 24 hours) have elapsed.

In the present invention, in order to suppress non-specific cell proliferation and proliferate the target CAR cells specifically and efficiently, in principle, stimulation with anti-CD3 antibody and anti-CD28 antibody is performed during the culture in step (2). Do not add. On the other hand, in order to increase the cell viability / proliferation rate, it is advisable to use a culture medium to which a T cell growth factor has been added during the culture in step (2). Examples of T cell growth factors include IL-2, IL-7 and IL-15, of which IL-15 is particularly useful. Preferably, a culture solution to which IL-7 is added in addition to IL-15 is used. The amount of IL-15 added is, for example, 5 ng / ml to 10 ng / ml. Similarly, the amount of IL-7 added is, for example, 5 ng / ml to 10 ng / ml. T cell growth factors such as IL-15 and IL-7 can be prepared according to a conventional method. In addition, a commercially available product can also be used. Although the use of T cell growth factors in non-human animal species is not excluded, T cell growth factors are usually derived from humans (may be recombinants). Growth factors such as human IL-15 and human IL-7 can be easily obtained (for example, provided by Miltenyi Biotec, R & D Systems, etc.).

A medium supplemented with serum (human serum, fetal bovine serum, etc.) may be used, but by adopting a serum-free medium, it is highly safe for clinical application and the culture efficiency due to the difference between serum lots is high. It is possible to prepare cells that have the advantage of being less likely to make a difference. Specific examples of serum-free media for lymphocytes are TexMACS ^TM (Miltenyi Biotec) and AIM V® (Thermo Fisher Scientific). When the CAR cells obtained by the preparation method of the present invention are used for autotransplantation, a medium to which autologous serum (recipient's serum) is added may be used. A medium suitable for lymphocyte culture may be used as the basal medium, and specific examples thereof are TexMACS ^TM and AIM V (registered trademark) described above. Other culture conditions may be any one suitable for the survival and proliferation of lymphocytes, and general ones may be adopted. _{For example, the cells may be cultured in a CO 2} incubator (CO ₂ concentration 5%) set at 37 ° C.

The culture period of step (2) is, for example, 1 to 14 days, preferably 1 to 7 days, and more preferably 2 to 7 days. If the culturing period is too short, a sufficient effect cannot be expected, and if the culturing period is too long, the cell activity (life force) may decrease.

In a preferred embodiment, the transposon method is used to introduce the target antigen-specific chimeric antigen receptor gene in step (1), and the cells expressing the target antigen in step (2) encode the target antigen. It is prepared by introducing the gene into peripheral blood mononuclear cells (PBMCs). In a more preferred embodiment, the piggyBac transposon method is used to introduce the target antigen-specific chimeric antigen receptor gene in step (1), and the cells expressing the target antigen in step (2) use the target antigen. It is prepared by introducing the encoding gene into peripheral blood mononuclear cells (PBMCs).

Step (3)
In step (3) following step (2), the genetically modified lymphocytes (CAR cells) after culturing are collected. The collection operation may be performed by a conventional method. For example, it is collected by pipetting, centrifugation, or the like.

In a preferred embodiment, a step of culturing the cultured CAR cells in the presence of T cell growth factor is performed between steps (2) and (3). This step enables efficient expansion culture and also has the advantage of increasing cell viability.

IL-15, IL-7, etc. can be used as T cell growth factors. Preferably, the cells are cultured in a medium supplemented with IL-15 and IL-7 in the same manner as in step (2). The culture period is, for example, 1 to 21 days, preferably 5 to 18 days, and more preferably 10 to 14 days. If the culture period is too short, a sufficient increase in the number of cells cannot be expected, and if the culture period is too long, there is a risk of a decrease in cell activity (life force), cell exhaustion / fatigue, and the like. It may be subcultured in the middle of culturing. In addition, the medium is changed as necessary during the culture. For example, replace about 1/3 to 2/3 of the culture medium with a new medium once every 3 days.

1-2. Protection of cells after CAR gene transfer operation by co-culture with activated T cells CAR gene transfer operation (especially electroporation in the case of transposon method) can cause damage to target cells. In general, the cells into which the CAR gene has been introduced (that is, CAR cells) are in a damaged state after the gene transfer operation. Therefore, in one aspect of the present invention, in order to protect CAR cells damaged by the gene transfer operation in step (1) and improve gene transfer efficiency and / or survival rate or proliferation rate, step (1) and During step (2), the following step (a) is performed.
(A) Non-proliferative cells obtained by stimulating a cell population containing T cells with an anti-CD3 antibody and an anti-CD28 antibody and then performing a treatment for losing proliferative ability, and genetically modified lymphocytes prepared in step (1). Steps of mixing lymphocytes and co-culturing while stimulating with anti-CD3 antibody and anti-CD28 antibody

In step (a), non-proliferative cells used for protection of CAR cells (CAR cells used in step (2)) after the transfer operation of the target antigen-specific chimeric antigen receptor gene are prepared, and in step (1). It is used for co-culture with the prepared CAR cells.

To prepare non-proliferative cells, first stimulate the cell population containing T cells with anti-CD3 antibody and anti-CD28 antibody. This treatment gives activated T cells. As the "cell population containing T cells", peripheral blood mononuclear cells (PBMCs) collected from peripheral blood are preferably used. It is also possible to use PBMCs purified to increase the T cell content, mononuclear cells collected from peripheral blood by feresis, or the like as the "cell population containing T cells" here.

For example, by culturing in a culture vessel (for example, a culture dish) whose culture surface is coated with anti-CD3 antibody and anti-CD28 antibody for 3 hours to 3 days, preferably 6 hours to 2 days, and more preferably 12 hours to 1 day. , T cells in the cell population can be stimulated with anti-CD3 antibody and anti-CD28 antibody. Anti-CD3 antibody (for example, the trade name CD3pure antibody provided by Miltenyi Biotec) and anti-CD28 antibody (for example, the trade name CD28pure antibody provided by Miltenyi Biotec can be used) are also commercially available. It is easily available. It is also possible to perform the stimulation in step (a) using magnetic beads coated with anti-CD3 antibody and anti-CD28 antibody (for example, Dynabeads T-Activator CD3 / CD28 provided by VERITAS). It is preferable to use an "OKT3" clone as the anti-CD3 antibody.

Cells stimulated with anti-CD3 antibody and anti-CD28 antibody are subjected to a treatment to lose proliferative ability, but before that, it is advisable to culture in the presence of T cell growth factor. This culture enhances the activity of the cells after the stimulation treatment. The culture period here is, for example, 1 to 10 days, preferably 2 to 7 days, and more preferably 3 to 4 days. If the culture period is too short, sufficient activation cannot be expected, and if the culture period is too long, co-stimulatory molecules may be attenuated. The cultured cells may be cryopreserved once. In this case, the cells may be thawed at the time of use, stimulated again with anti-CD3 antibody and CD28 antibody (conditions conform to the above), and then subjected to "treatment to lose proliferative ability".

Activated T cells (non-proliferative cells) that have lost their proliferative ability can be obtained by undergoing the "treatment that causes them to lose their proliferative ability". The treatment that causes the loss of proliferative capacity is typically irradiation, but agents may also be used. An example of irradiation conditions is a treatment using gamma rays at an intensity of 25 Gy to 50 Gy for 15 to 30 minutes.

The non-proliferative cells prepared as described above and the CAR cells prepared in step (1) are mixed and co-cultured while stimulating with anti-CD3 antibody and anti-CD28 antibody. As a result, stimulation by co-stimulatory molecules by non-proliferative cells and stimulation by anti-CD3 antibody and anti-CD28 antibody are added, CAR cells are activated, and their survival and proliferation are promoted. This co-culture is preferably carried out immediately after the CAR gene transfer operation when preparing CAR cells. For example, co-culture is started immediately after the CAR gene transfer operation or within 1 day. The co-culture (that is, step (a)) and the above step (2) may be performed at the same time, in which case the start time of step (2) is the same as the start time of step (a) (for example). , Immediately after the CAR gene transfer operation or within 1 day).

The ratio of the number of non-proliferative cells to the number of genetically modified lymphocytes (CAR cells) used for co-culture (number of non-proliferative cells / number of genetically modified lymphocytes) is not particularly limited, but is, for example, 0.025 to 0.5. And.

In order to increase the cell viability / proliferation rate, it is advisable to use a culture medium to which T cell growth factor has been added during co-culture. Examples of T cell growth factors include IL-2, IL-7 and IL-15, of which IL-15 is particularly useful. Preferably, a culture solution to which IL-7 is added in addition to IL-15 is used. The amount of IL-15 added is, for example, 5 ng / ml to 10 ng / ml. Similarly, the amount of IL-7 added is, for example, 5 ng / ml to 10 ng / ml.

A medium supplemented with serum (human serum, fetal bovine serum, etc.) may be used, but by adopting a serum-free medium, it is highly safe for clinical application and the culture efficiency due to the difference between serum lots is high. It is possible to prepare cells that have the advantage of being less likely to make a difference. Specific examples of serum-free media for lymphocytes are TexMACS ^TM (Miltenyi Biotec) and AIM V® (Thermo Fisher Scientific). When the CAR cells obtained by the preparation method of the present invention are used for autotransplantation, a medium to which autologous serum (recipient's serum) is added may be used. A medium suitable for lymphocyte culture may be used as the basal medium, and specific examples thereof are TexMACS ^TM and AIM V (registered trademark) described above. Other culture conditions may be any one suitable for the survival and proliferation of lymphocytes, and general ones may be adopted. _{For example, the cells may be cultured in a CO 2} incubator (CO ₂ concentration 5%) set at 37 ° C.

The co-culture period is, for example, 1 to 10 days, preferably 1 to 7 days, and more preferably 2 to 4 days. If the culturing period is too short, a sufficient effect cannot be expected, and if the culturing period is too long, the cell activity (life force) may decrease.

1-3. Protection of cells after CAR gene transfer operation by co-culture with non-proliferative PBMCs In another aspect of the present invention, the above 1-2. Alternatively, the following step (b) is performed between steps (1) and (2).
(B) Non-proliferative PBMCs obtained by stimulating PBMCs as they are or with anti-CD3 antibody and anti-CD28 antibody and then performing a treatment to lose proliferative ability, and genetically modified lymphocytes prepared in step (1). Steps to mix and co-culture

In step (b), non-proliferative PBMCs used for protection of CAR cells (CAR cells used in step (2)) after the transfer operation of the target antigen-specific chimeric antigen receptor gene are prepared, and in step (1). It is used for co-culture with the prepared CAR cells.

The method for stimulating PBMCs with anti-CD3 antibody and anti-CD28 antibody is as described in 1-2 above. Is similar to. The treatment for losing the proliferative ability, and prior to that, culturing in the presence of T cell growth factor and the method thereof are also described in 1-2. Is similar to. In addition, "as is PBMCs" means that PBMCs are not stimulated by anti-CD3 antibody and anti-CD28 antibody.

The non-proliferative PBMCs prepared as described above are mixed with the genetically modified lymphocytes (CAR cells) prepared in step (1) and co-cultured. Co-culture may be performed while stimulating with an anti-CD3 antibody and an anti-CD28 antibody. This co-culture is preferably carried out immediately after the CAR gene transfer operation when preparing CAR cells. For example, co-culture is started immediately after the CAR gene transfer operation or within 1 day. The co-culture (that is, step (b)) and the above step (2) may be performed at the same time, in which case the start time of step (2) is the same as the start time of step (b) (for example). , Immediately after the CAR gene transfer operation or within 1 day).

The ratio of the number of non-proliferative PBMCs used for co-culture to the number of genetically modified lymphocytes (CAR cells) (number of non-proliferative PBMCs / number of genetically modified lymphocytes) is not particularly limited, but is, for example, 0.025 to 0.5. , Preferably 0.05 to 1.0.

In order to increase the cell viability / proliferation rate, it is advisable to use a culture medium to which T cell growth factor has been added during co-culture. Examples of T cell growth factors include IL-2, IL-7 and IL-15, of which IL-15 is particularly useful. Preferably, a culture solution to which IL-7 is added in addition to IL-15 is used. The amount of IL-15 added is, for example, 5 ng / ml to 10 ng / ml. Similarly, the amount of IL-7 added is, for example, 5 ng / ml to 10 ng / ml. Conditions not mentioned (possibility of serum utilization, basal medium, culture temperature, etc.) are described in 1-2. Is the same as in the case of the above aspect.

Non-proliferative PBMCs may be added in the middle of co-culture (for example, 3 to 11 days after the start of co-culture, preferably 5 to 9 days, and more preferably 7 days). Alternatively, the cells after co-culture may be collected, mixed with other non-proliferative PBMCs, and then cultured again. These operations may be repeated twice or more. In this way, if stimulation or activation using non-proliferative PBMCs is performed a plurality of times, it is expected that the induction rate of CAR cells will be improved and the number of CAR cells will be increased. In addition, those prepared again, or those prepared by culturing a part of the cells prepared at the beginning or those stored in a cryopreservation can be used as the non-proliferative PBMCs here.

If non-proliferative PBMCs are prepared using a part of PBMCs isolated from peripheral blood obtained by one blood sampling and CAR cells are prepared from the other part, the present invention can be carried out. The number of blood samplings involved can be reduced, which is an extremely great advantage in clinical application. In particular, prepare additional non-proliferative PBMCs using the remaining PBMCs, or store some of the non-proliferative PBMCs prepared using some of the PBMCs (typically cryopreservation). , If it is used as additional non-proliferative PBMCs, then the three required cells, namely CAR cells, non-proliferative PBMCs used for co-culture with the cells, in the middle of co-culture Since non-proliferative PBMCs for addition can be prepared by a single blood collection, the burden on the patient in the treatment using the CAR cells obtained in the present invention is greatly reduced.

1-4. Preparation of virus-specific chimeric antigen receptor gene-modified lymphocytes by co-culture with T cells carrying a virus peptide In another aspect of the present invention, virus-specific chimeric antigen receptor gene-modified lymphocytes (virus-specific). CAR cells) are prepared. Virus-specific CAR cells can be expected to improve internal persistence by stimulation from viral T cell receptors when used for autologous transplantation, and further reduce allogeneic immune response (GVHD) when used for allogeneic transplantation. This has important advantages in clinical application, such as the possibility of producing CAR cells from a transplant donor and the possibility of formulating CAR cells from a third-party donor. In fact, virus-specific CAR cells have been reported to persist in the body for longer periods of time (Pule MA, et al. Nat Med. 2008 Nov; 14 (11): 1264-70.). In addition, according to a report of a third-party EBV-specific CTL clinical study (Annual Review Blood 2015, published in January 2015, Chugai Medical Co., Ltd.), the safety of virus-specific cytotoxic T cells (CTL) is high. It is backed up.

In this aspect, the following step (c) is performed to prepare virus-specific CAR cells. Matters not mentioned (for example, method for preparing a cell population containing T cells, basic operation for stimulation with anti-CD3 antibody and anti-CD28 antibody, method for processing to lose proliferative ability, basic operation for co-culture, etc. ) Is described in 1-2. Since it is the same as the aspect of the above, duplicate explanations are omitted and the corresponding explanations are used.
(C) A cell population containing T cells was stimulated with an anti-CD3 antibody and an anti-CD28 antibody, and then cultured in the presence of the viral peptide antigen and treated to lose the proliferative ability to retain the viral peptide antigen. The step of mixing non-proliferative cells with the genetically modified lymphocytes prepared in step (1) and co-culturing them.

In step (c), first, a cell population containing T cells is stimulated with an anti-CD3 antibody and an anti-CD28 antibody to obtain activated T cells. Then, the cells are cultured in the presence of the viral peptide antigen and treated to lose their proliferative ability. As a result, non-proliferative "activated T cells holding a viral peptide antigen on the cell surface" (hereinafter referred to as "viral peptide-retaining non-proliferative cells") are obtained. The order of culturing in the presence of the viral peptide antigen and the treatment for losing the proliferative ability is not particularly limited. Therefore, the growth ability may be lost after culturing in the presence of the virus peptide antigen, or the culture may be performed in the presence of the virus peptide antigen after the loss of the growth ability. Preferably, the former order is adopted because of the expectation that the uptake of the viral peptide antigen will be better before the loss of proliferative capacity. In order to culture in the presence of the viral peptide antigen, for example, a medium to which the viral peptide antigen has been added may be used. Alternatively, the viral peptide antigen may be added to the medium during culturing. The concentration of the viral peptide antigen added is, for example, 0.5 μg / ml to 1 μg / ml. The culture period is, for example, 10 minutes to 5 hours, preferably 20 minutes to 3 hours. As used herein, the term "viral peptide antigen" refers to an epitope peptide or a long peptide containing an epitope capable of inducing cytotoxic T cells (CTL) specific to a specific virus. The viral peptide antigen is not limited to these, but is, for example, an adenovirus (AdV) antigen peptide (see, for example, WO2007015540 A1) and a cytomegalovirus (CMV) antigen peptide (for example, JP-A-2002). -255997, 2004-242599, 2012-87126), Epstein-Barr virus (EBV) antigen peptide (for example, WO 2007049737 A1, Japanese Patent Application No. 2011-177487, Japanese Patent Application Laid-Open No. (See 2006-188513), etc. can be used. The viral peptide antigen can be prepared by a conventional method (for example, liquid phase synthesis method, solid phase synthesis method) based on the sequence information. In addition, some viral peptide antigens are commercially available (for example, provided by Medical & Biological Laboratories, Ltd., Takara Bio, Miltenyi Biotec, etc.).

Although it is possible to use one type of antigen peptide, usually two or more types of antigen peptides (antigen peptide mixture) are used. For example, AdV antigen peptide mixture, CMV antigen peptide mixture or EBV antigen peptide mixture, or a combination of two or more of these antigen peptide mixture (for example, AdV antigen peptide mixture, CMV antigen peptide mixture and EBV antigen peptide mixture). Use a mixture). By using two or more kinds of antigen peptides in combination, a plurality of activated T cells having different targets (antigen peptides) can be obtained, and the virus-specific CAR cells obtained by the preparation method of the present invention are effective therapeutic targets ( We can expect an increase in the number of patients (improvement of coverage). In deciding which virus-derived antigen peptide to use, the use of the virus-specific CAR cells obtained by the preparation method of the present invention, specifically, the disease to be treated and the pathological condition of the patient should be considered. Good. For example, when the purpose is to treat recurrent leukemia after hematopoietic stem cell transplantation, the antigen-peptide mixture of EBV virus may be used alone or in combination with an antigen-peptide mixture of other viruses. AdV antigen-peptide mixture, CMV antigen-peptide mixture, and EBV antigen-peptide mixture are also commercially available (for example, PepTivator (registered trademark) AdV5 Hexon, PepTivator (registered trademark) CMV pp65, PepTivator (for example, provided by Milteny Biotech). Registered trademarks) EBV EBNA-1, PepTivator (registered trademark) EBV BZLF1, PepMix ^TM Collection HCMV ^{provided by JPT Peptide Technologies, PepMix TM} EBV (EBNA1), etc.) can be easily obtained.

The viral peptide-retaining non-proliferative cells prepared as described above and the genetically modified lymphocytes (CAR cells) prepared in step (1) are mixed and co-cultured. As a result, stimulation via a co-stimulating molecule and a virus antigen peptide by a virus peptide-carrying non-proliferative cell is applied, and virus antigen-specific genetically modified lymphocytes are activated and their survival and proliferation are promoted. In order to protect CAR cells damaged by the gene transfer operation in step (1) and improve gene transfer efficiency and / or survival rate or proliferation rate, this co-culture is used for CAR genes in preparing CAR cells. It is preferable to carry out immediately after the introduction operation. For example, co-culture is started immediately after the CAR gene transfer operation or within 1 day. The co-culture (that is, step (c)) and the above step (2) may be performed at the same time, in which case the start time of step (2) is the same as the start time of step (c) (for example). , Immediately after the CAR gene transfer operation or within 1 day).

The ratio of the number of viral peptide-carrying non-proliferative cells used for co-culture to the number of genetically modified lymphocytes (CAR cells) (number of viral peptide-carrying nonproliferative cells / number of genetically modified lymphocytes) is not particularly limited. For example, 0.025 to 0.5, preferably 0.05 to 1.0.

In principle, this step is to stimulate with anti-CD3 antibody and anti-CD28 antibody for reasons such as selectively proliferating virus-specific CAR cells, avoiding strong stimulation and preventing T cell exhaustion / fatigue. Do not add. On the other hand, in order to increase the cell viability / proliferation rate, it is advisable to use a culture medium to which a T cell growth factor has been added during co-culture. Examples of T cell growth factors include IL-2, IL-7 and IL-15, of which IL-15 is particularly useful. Preferably, a culture solution to which IL-7 is added in addition to IL-15 is used. The amount of IL-15 added is, for example, 5 ng / ml to 10 ng / ml. Similarly, the amount of IL-7 added is, for example, 5 ng / ml to 10 ng / ml. Conditions not mentioned (possibility of serum utilization, basal medium, culture temperature, etc.) are described in 1-2. Is the same as in the case of the above aspect.

Viral peptide-carrying non-proliferative cells may be added during co-culture. Alternatively, the cells after co-culture may be collected, mixed with another virus peptide-carrying non-proliferative cell, and then co-cultured again. These operations may be repeated twice or more. As described above, if the activation without stimulation using the virus peptide-retaining non-proliferative cells is performed a plurality of times, the induction rate of the virus-specific CAR cells can be improved and the number of virus-specific CAR cells can be expected to increase. In addition, a cell prepared again or a cell in which a part of the cell prepared at the beginning is preserved can be used as "another viral peptide-carrying non-proliferative cell" here.

The co-culture period is, for example, 1 to 21 days, preferably 5 to 18 days, and more preferably 10 to 14 days. If the culturing period is too short, a sufficient effect cannot be expected, and if the culturing period is too long, there is a risk of a decrease in cell activity (life force), cell exhaustion / fatigue, and the like.

Prior to co-culturing with viral peptide-carrying non-proliferative cells, the genetically modified lymphocytes (CAR cells) prepared in step (1) are co-cultured with viral peptide-carrying non-proliferative peripheral blood mononuclear cells (PBMCs). It may be. The period of co-culture here is, for example, 1 to 21 days, preferably 5 to 18 days, and more preferably 10 to 14 days. In the case of this embodiment, the cells obtained by co-culturing the genetically modified lymphocytes (CAR cells) prepared in step (1) and the viral peptide-carrying non-proliferative PBMCs (co-culture in the first step) are obtained by the above method. It will be mixed with the prepared viral peptide-carrying non-proliferative cells and co-cultured in the second stage. The viral peptide-retaining non-proliferative PBMCs here can be prepared by subjecting the PBMCs to a culture in the presence of a viral peptide antigen and a treatment for losing the ability to proliferate. Specifically, for example, PBMCs isolated from peripheral blood are radiation-treated and then cultured in the presence of a viral peptide antigen to obtain viral peptide-retaining non-proliferative PBMCs. In addition, if it is decided to prepare viral peptide-retaining non-proliferative PBMCs using a part of PBMCs isolated from peripheral blood obtained by one blood sampling and to prepare CAR cells from the other part, the present invention. It is possible to reduce the number of blood samplings associated with the implementation of the above, which is an extremely great advantage in clinical application. In particular, the remaining PBMCs are used to prepare viral peptide-retaining non-proliferative cells (cells used for the second stage co-culture), or the viral peptide-retaining non-proliferative PBMCs prepared using a part of PBMCs. If a part is preserved (typically cryopreserved) and used as a viral peptide-carrying non-proliferative cell (cell used for the second stage co-culture), the necessary three types Cells, that is, CAR cells, viral peptide-retaining non-proliferative PBMCs used for co-culture with the cells (first-stage co-culture), and viral peptide-retaining non-proliferative cells used for second-stage co-culture. Since it can be prepared by collecting blood once, the burden on the patient in the treatment using the CAR cells obtained in the present invention is greatly reduced.

2. Gene-modified lymphocytes expressing chimeric antigen receptor and their uses Further aspects of the present invention are the genetically modified lymphocytes expressing chimeric antigen receptor obtained by the preparation method of the present invention (hereinafter, "the present invention". "CAR cells") and their uses. The CAR cells of the present invention can be used for the treatment, prevention or amelioration of various diseases (hereinafter referred to as "target diseases") for which CAR therapy is considered to be effective. The representative of the target disease is cancer, but it is not limited to this. Examples of target diseases include various B-cell lymphomas (follicle malignant lymphoma, diffuse malignant lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive hodgkin lymphoma, etc.), myeloproliferative neoplasm, bone marrow. Hypoplastic / myeloproliferative neoplasms (CMML, JMML, CML, MDS / MPN-UC), myelodysplastic syndrome, acute myeloproliferative leukemia, neuroblastoma, brain tumor, Ewing sarcoma, osteosarcoma, retinoblastoma, small lung Celloma, melanoma, ovarian cancer, horizontal print myeloma, kidney cancer, pancreatic cancer, malignant mesoderma, prostate cancer, etc. "Treatment" includes alleviating (mitigating) the symptoms characteristic of the target disease or associated symptoms, preventing or delaying the exacerbation of the symptoms, and the like. "Prevention" means preventing or delaying the onset / delay of a disease (disorder) or its symptoms, or reducing the risk of onset / onset. On the other hand, "improvement" means that the disease (disorder) or its symptoms are alleviated (mild), improved, ameliorated, or cured (including partial cure).

The CAR cells 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 cells of the present invention. For example, it ^{contains 10 4} to 10 ¹⁰ cells for a single dose. Various components (vitamins) such as dimethylsulfoxide (DMSO) and serum albumin for the purpose of protecting cells, antibiotics for the purpose of preventing bacterial contamination, and various components (vitamins) for the purpose of activating, proliferating or inducing differentiation of cells. , Cytokines, growth factors, steroids, etc.) may be contained in the cell preparation.

The administration route of the CAR cell or cell preparation of the present invention is not particularly limited. For example, it is administered by intravenous injection, intraarterial injection, intraportal injection, intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. Topical administration may be used instead of systemic administration. As local administration, direct injection into a target tissue / organ / organ can be exemplified. The administration schedule may be prepared in consideration of the gender, age, body weight, pathological condition, etc. of the subject (patient). In addition to a single dose, multiple doses may be administered continuously or periodically.

<New CAR-T preparation method that improves gene transfer efficiency / cell proliferation rate>
The following new preparation methods (culture methods 2 and 3) were created with the aim of improving the gene transfer efficiency / cell proliferation rate when preparing CAR-T cells. For comparison, the conventional preparation method (culture method 1) is also described.

1. 1. Material (1) Antibody Anti-CD3 antibody (Miltenyi Biotec)
Anti-CD28 antibody (Miltenyi Biotec)
Anti-iditope antibody (anti-iditope antibody against CD19CAR derived from L cells prepared according to a known method (for example, the method described in PLoS One. 2013; 8 (3): e57838.))
(2) Medium TexMACS ^TM (Miltenyi Biotec)
(3) Cytokine recombinant human IL-7 (Miltenyi Biotec)
Recombinant Human IL-15 (Miltenyi Biotec)
(4) Viral Peptide Mix PepTivator® CMV pp65-premium grade, Human (Miltenyi Biotec) PepTivator® AdV5 Hexon-premium grade, Human (Miltenyi Biotec) PepTivator® EBV EBNA -1-premium grade, human (Miltenyi Biotec) PepTivator® EBV BZLF1-premium grade, human (Miltenyi Biotec)
(5) plasmid pIRII-CD19CAR vector (expressing CAR)
pCMV-piggyBac vector (expressing piggyBac transposase)
(6) Cell culture container 24-well uncoated tissue culture plate (Falcon)
24-well tissue culture plate (Falcon)
G-Rex10 (Wilson Wolf)

2. Method (1) Preparation of anti-CD3 antibody / anti-CD28 antibody coated (sensitized) plate (used in the conventional culture method 1) Anti-CD3 antibody and anti-CD28 antibody were diluted with PBS to 1 mg / ml, and 24 Add 0.5 ml / well to the uncoated plate of the well. The plate is allowed to stand in an incubator at 37 ° C for 2-4 hours. Aspirate antibody-diluted PBS and wash once with 1 ml PBS per well.

(2) Preparation of anti-ideotype antibody-coated (sensitized) plate (used in culture method 2)
The anti-iditope antibody is diluted with PBS to 0.5 mg / ml and added to a 24-well uncoated plate to 0.5 ml / well. The plate is allowed to stand in an incubator at 37 ° C for 2-4 hours. Aspirate antibody-diluted PBS and wash once with 1 ml PBS per well.

(3) Preparation of target antigen gene-introduced PBMCs (used in culture method 3)
Day 0: Isolate mononuclear cells (PBMCs) from peripheral blood. For PBMCs, 5 μg of a plasmid vector expressing the CAR target antigen is introduced by electroporation (nucleofection), and culture is started in an incubator.
Day 1: After irradiation, it is used as a feeder cell.

(4) Preparation and culturing of CAR-T by the conventional method (see culturing method 1 and FIG. 1)
Day 0: Mononuclear cells are separated from peripheral blood and counted. To ^{1x10 7} monocytes, add 5 ng each of pIRII-CAR.CD19.28z vector (Fig. 4) and pCMV-pigBac vector (Fig. 5), and electroporate using 4D nucleofector (Lonza). Gene transfer by ration (nucleofection). ^{Then, the cells are floated on TexMACS TM} supplemented with IL-7 10 ng / ml and IL15 5 ng / ml, and culture is started in a 37 ° C. incubator on a 24-well plate.
Day 1: Stimulate with anti-CD3 antibody / anti-CD28 antibody coated plate.
Day 4: Transfer cells to G-Rex 10 and incubate with ^{TexMACS TM} supplemented with IL-7 10 ng / ml and IL 15 5 ng / ml.
Day 7: Replace half of the culture with ^{TexMACS TM} with IL-7 10 ng / ml and IL 15 5 ng / ml.
Day 10: Replace half of the culture with IL-7 10 ng / ml and IL 15 5 ng / ml TexMACS ^TM.
Day 14: The culture is finished.

(5) Preparation and culture of CAR-T using anti-iditope antibody (see culture method 2 and FIG. 2) Day 0: Mononuclear cells were isolated from peripheral blood, and 1x10 ⁷ mononuclear cells were treated. , PIRII-CAR.CD19.28z vector (Fig. 4) and pCMV-pigBac vector (Fig. 5) are added 5 ng each and gene is introduced by electroporation (nucleofection). ^{Then, the cells are floated on TexMACS TM} supplemented with IL-7 10 ng / ml and IL15 5 ng / ml, and culture is started in a 37 ° C. incubator on a 24-well plate.
Day 1: Stimulate with an anti-iditope antibody coated plate.
Day 4: Transfer cells to G-Rex 10 and incubate with ^{TexMACS TM} supplemented with IL-7 10 ng / ml and IL 15 5 ng / ml.
Day 7: Replace half of the culture with ^{TexMACS TM} with IL-7 10 ng / ml and IL 15 5 ng / ml.
Day 10: Replace half of the culture with IL-7 10 ng / ml and IL 15 5 ng / ml TexMACS ^TM.
Day 14: The culture is finished.

(6) Preparation and culture of CAR-T using target antigen-expressing cells (target antigen gene-introduced PBMCs) (see culture method 3 and FIG. 3).
Day 0: Mononuclear cells were isolated from peripheral blood, and 5 ng each of pIRII-CAR.CD19.28z vector (Fig. 4) and pCMV-pigBac vector (Fig. 5) were added to 1x10 ^{7 mononuclear cells.} Then, the gene is introduced by electroporation (nucleofection). ^{Then, the cells are floated on TexMACS TM} supplemented with IL-7 10 ng / ml and IL15 5 ng / ml, and culture is started in a 37 ° C. incubator on a 24-well plate.
Day 1: Stimulate with target antigen gene-introduced PBMCs (feeder cells) (eg, culture in a culture dish inoculated with feeder cells).
Day 4: Transfer cells to G-Rex 10 and incubate with ^{TexMACS TM} supplemented with IL-7 10 ng / ml and IL 15 5 ng / ml.
Day 7: Replace half of the culture with ^{TexMACS TM} with IL-7 10 ng / ml and IL 15 5 ng / ml.
Day 10: Replace half of the culture with IL-7 10 ng / ml and IL 15 5 ng / ml TexMACS ^TM.
Day 14: The culture is finished.

3. 3. Results Preparation and culture of CAR-T using conventional methods or anti-iditope antibodies (A)
2. above. (1) An anti-CD3 antibody / anti-CD28 antibody-coated (sensitized) plate was prepared, and (2) an anti-iditopes antibody-coated (sensitized) plate was prepared by the same method as described in the method.
First, the cells were cultured by the conventional method (culture method 1) for 7 days, then further cultured by the conventional method (culture method 1) for 7 days (A-1), and first cultured by the conventional method (culture method 1) for 7 days. After that, the gene transfer efficiency was compared with that of the one (A-2) cultured for 7 days by the new method (culture method 2).

After the completion of culturing (14th day), for culture method 1 (conventional method) (A-1) and culture method 2 (A-2), gene transfer efficiency (CAR-T cells / whole viable cells), CAR-T cells The numbers were calculated and compared. The gene transfer efficiency was calculated from the results of flow cytometry analysis, and the number of CAR-T cells was evaluated by the proliferation rate from the 7th day.

The gene transfer efficiency was 8% for culture method 1 (A-1) and 18.1% for culture method 2 (A-2) (Fig. 6A), and culture method 2 was significantly higher than that of conventional culture method 1. Improvement of gene transfer efficiency was observed. That is, it was demonstrated that the novel culture method is effective in improving the efficiency of gene transfer. The proliferation rate of CAR-T cells from day 7 was 20-fold with anti-iditopes antibody stimulation (A-2) and 13-fold with conventional OKT3 / anti-CD28 antibody stimulation (A-1). (Fig. 6B). The new culture method overcomes the weaknesses of the transposon method (gene transfer efficiency and cell viability / cell proliferation rate are lower than those of the viral vector method), and will expand the clinical application of CAR therapy using the transposon method. I can say. In addition, the novel culture method is highly versatile and can be applied to the preparation of CAR cells by the viral vector method or the like, and its utility value is great.

2. (1) An anti-CD3 antibody / anti-CD28 antibody-coated (sensitized) plate and (2) an anti-ideotype antibody-coated (sensitized) plate were prepared by the same method as described in the method.

Preparation and culture of CAR-T using conventional methods or anti-iditope antibodies (B)
Day 0: Mononuclear cells were isolated from peripheral blood, and 5 ng each of pIRII-CAR.CD19.28z vector (Fig. 4) and pCMV-pigBac vector (Fig. 5) were added to 1x10 ^{7 mononuclear cells.} Then, the gene is introduced by electroporation (nucleofection). ^{Then, the cells are floated on TexMACS TM} supplemented with IL-7 10 ng / ml and IL15 5 ng / ml, and culture is started in a 37 ° C. incubator on a 24-well plate.
Day 1: Stimulate with anti-iditope antibody coated plate (B-2, culture method 2) or OKT3 / anti-CD28 antibody coated plate (B-1, culture method 1 (conventional method)).
^{Day 4: Transfer cells to a 24-well plate and incubate with TexMACS TM} supplemented with IL-7 10 ng / ml and IL 15 5 ng / ml.
Day 7: Culture finished

Results After the completion of culturing (7th day), the gene transfer efficiency (CAR-T cells / whole viable cells) and the number of CAR-T cells were determined and compared. The gene transfer efficiency was calculated from the results of flow cytometry analysis, and the number of CAR-T cells was evaluated by the proliferation rate on the 7th day.

The gene transfer efficiency was 20.5% for anti-iditope antibody stimulation (B-2, culture method 2) and 3.58% for OKT3 / anti-CD28 antibody stimulation (B-1, culture method 1 (conventional method)) (Fig. 8A). ), Compared with the conventional method of OKT3 / anti-CD28 antibody stimulation, anti-iditopes antibody stimulation showed a significant improvement in gene transfer efficiency. The number of CAR-T cells was 550,000 for anti-iditopes antibody stimulation (B-2) and 180,000 for OKT3 / anti-CD28 antibody stimulation (B-1) (Fig. 8B). / Compared with anti-CD28 antibody stimulation, anti-iditope antibody stimulation showed a significant improvement in CAR-T cell number.

<Protection of CAR-T cells after gene transfer and preparation of virus-specific CAR-T cells>
As applications of the above culture methods 2 and 3, a method combining stimulation with activated T cells (culture method 4) and a method combining stimulation with virus peptide-added activated T cells (culture method 5) are shown below.

1. 1. Material (1) Antibody Anti-CD3 antibody (Miltenyi Biotec)
Anti-CD28 antibody (Miltenyi Biotec)
Anti-iditope antibody (anti-iditope antibody against CD19CAR derived from L cells prepared according to a known method (for example, the method described in PLoS One. 2013; 8 (3): e57838.))
(2) Medium TexMACS (Miltenyi Biotec)
(3) Cytokine recombinant human IL-7 (Miltenyi Biotec)
Recombinant Human IL-15 (Miltenyi Biotec)
(4) Viral Peptide Mix PepTivator® CMV pp65-premium grade, Human (Miltenyi Biotec) PepTivator® AdV5 Hexon-premium grade, Human (Miltenyi Biotec) PepTivator® EBV EBNA -1-premium grade, human (Miltenyi Biotec) PepTivator® EBV BZLF1-premium grade, human (Miltenyi Biotec)
(5) plasmid pIRII-CD19CAR vector (expressing CAR)
pCMV-piggyBac vector (expressing piggyBac transposase)
(6) Cell culture container 24-well uncoated tissue culture plate (Falcon)
24-well tissue culture plate (Falcon)
G-Rex10 (Wilson Wolf)

2. Method (1) Preparation of activated T cells (1-1) Preparation of anti-CD3 antibody / anti-CD28 antibody coated (sensitized) plate Anti-CD3 antibody and anti-CD28 antibody were diluted with PBS to 1 mg / ml and diluted with PBS. Add to a 24-well uncoated plate to 0.5 ml / well. The plate is allowed to stand in an incubator at 37 ° C for 2-4 hours. Aspirate antibody-diluted PBS and wash once with 1 ml PBS per well.

(1-2) Culture on anti-CD3 antibody / anti-CD28 antibody coated plate Day 0: PBMC isolated from peripheral blood was added to IL-15 5 ng / ml to 5 ^{x 10 5} / ml with ^{TexMACS TM.} And dispense 2 ml per well into anti-CD3 antibody / anti-CD28 antibody coated plates.
Day 1: Transfer cells to a 24-well tissue culture plate. Replace half of the culture solution and add to IL-15 5 ng / ml.
Day 4: Add IL-15 to 5 ng / ml.
Day 7: Cells are collected, dispensed and cryopreserved.

(1-3) Restimulation of activated T cells Day 0: Thaw the cryopreserved cells, wash twice, and dilute with IL15 5 ng / ml-added TexMACS ^{TM to 1x10 6 cells / ml.} , Dispense 1 well 2 ml each into anti-CD3 antibody / anti-CD28 antibody coated plates.
Day 3: Collect cells and use for CAR-T culture.

(2) Preparation of anti-iditope antibody-coated (sensitized) plate Dilute the anti-iditope antibody with PBS to 0.5 mg / ml, and add to a 24-well uncoated plate to 0.5 ml / well. The plate is allowed to stand in an incubator at 37 ° C for 2-4 hours. Aspirate antibody-diluted PBS and wash once with 1 ml PBS per well.

(3) Preparation of target antigen gene-introduced PBMCs Day 0: Mononuclear cells (PBMCs) are isolated from peripheral blood. For PBMCs, 5 μg of a plasmid vector expressing the CAR target antigen is introduced by electroporation (nucleofection), and culture is started in an incubator.
Day 1: After irradiation, it is used as a feeder cell.

(4) Method combining stimulation with activated T cells (culture method 4)
Day 0: Mononuclear cells were isolated from peripheral blood, and 5 ng each of pIRII-CAR.CD19.28z vector (Fig. 4) and pCMV-pigBac vector (Fig. 5) were added to 1x10 ^{7 mononuclear cells.} Then, the gene is introduced by electroporation (nucleofection). Transgenic cells and irradiated with 5x10 ⁵ cells of activated T cells were mixed, cultured in an incubator of IL-7 10ng / ml and 37 ° C. at floated 24-well plates in IL15 5 ng / ml added TeXmacs ^TM Start.
Day 1: Stimulate with anti-iditope antibody coated plates or target antigen gene-introduced PBMCs (feeder cells).
Day 4: Transfer cells to G-Rex 10 and incubate with ^{TexMACS TM} supplemented with IL-7 10 ng / ml and IL 15 5 ng / ml.
Day 7: Replace half of the culture with ^{TexMACS TM} with IL-7 10 ng / ml and IL 15 5 ng / ml.
Day 10: Replace half of the culture with IL-7 10 ng / ml and IL 15 5 ng / ml TexMACS ^TM.
Day 14: The culture is finished.

In the above culture method 4, the number of CAR-T cells is increased by cell stimulation (protective action of transgenic cells damaged during electroporation) by expression of co-stimulating molecules, cytokine stimulation by culture microenvironment, and the like. It can be expected that the increase and the efficiency of gene transfer will increase further.

(5) Method combining stimulation with activated T cells added with viral peptide (culture method 5)
Day 0: Irradiated activated T cells 5x10 ⁵ cells to viral peptide (PepTivator CMV pp65, PepTivator AdV5 Hexon , the PepTivator EBV EBNA-1 and PepTivator EBV BZLFl each 50 ng) is added and incubated for 30 minutes at 37 ° C. .. To ^{1x10 7} peripheral blood mononuclear cells, add 5 ng each of pIRII-CAR.CD19.28z vector (Fig. 4) and pCMV-pigBac vector (Fig. 5) and electroporate (nucleofection). Introduce the gene. ^{The transgenic cells and irradiated viral peptide-added activated T cells are mixed, floated on TexMACS TM} supplemented with IL-7 10 ng / ml and IL15 5 ng / ml, and cultured in a 24-well plate in a 37 ° C incubator. To start.
Day 1: Stimulate with anti-iditope antibody coated plates or target antigen gene-introduced PBMCs (feeder cells).
Day 2-5: If necessary, replace half of the culture with IL-7 10 ng / ml and IL 15 5 ng / ml TexMACS ^TM.
Day 7: Cells are collected and counted. ⁶ virus peptide-added activated T cells prepared in the same manner as above and the recovered cells were suspended in 30 ml of IL-15 (5 ng / ml) and IL-7 (10 ng / ml) -added culture medium. Start culturing with G-Rex10.
Day 10: Replace half of the culture with IL-7 10 ng / ml and IL 15 5 ng / ml TexMACS ^TM.
Day 14: The culture is finished.

In the above culture method 5, the number of CAR-T cells is further increased by cell stimulation by the expression of co-stimulating molecules, cytokine stimulation by the culture microenvironment, and relatively gentle cell stimulation from virus-specific T cell receptors. And further increase in gene transfer efficiency can be expected. In addition, a decrease in allogeneic reactivity (effect that can be expected for virus-specific CTL), the possibility of using CAR-T cells derived from a third party due to this, and the stimulation of virus antigen receptors in the body by a virus in the body. Persistency may increase, and further improvement in safety and increase in therapeutic effect are expected.

<Culture method 6 (improvement of culture method 5)>
1. 1. Method Day 0: Isolate mononuclear cells (PBMCs) from peripheral blood. After irradiating a part ( ⁶ PBMC 1x10), add viral peptides (PepTivator CMV pp65, PepTivator AdV5 Hexon, PepTivator EBV EBNA-1 and PepTivator EBV BZLF1 50 ng each) and incubate at 37 ° C for 30 minutes. On the other hand, with respect to 1x10 ⁷ cells of PBMC, gene transfer by pIRII-CAR.CD19_optimized vector (Fig. 7) and pCMV-pigBac vector electroporation by addition of (Fig. 5) by 5 ng (nucleocapsid transfection). ^{Mix the transgenic cells with irradiated viral peptide-added PBMC, float on TexMACS TM} supplemented with IL-7 10 ng / ml and IL15 5 ng / ml, and start culturing in a 37 ° C incubator on a 24-well plate. .. In addition, the above 2. Activated T cells are prepared from the remaining PBMCs according to the method (1).
Day 1: Stimulate with anti-iditope antibody coated plates or target antigen gene-introduced PBMCs (feeder cells).
Day 2-5: If necessary, replace half of the culture with IL-7 10 ng / ml and IL 15 5 ng / ml TexMACS ^TM.
Day 7: Cells are collected and counted. ^{On the other hand, after irradiating 2x10 6} activated T cells prepared by the above method with virus peptides (PepTivator CMV pp65, PepTivator AdV5 Hexon, PepTivator EBV EBNA-1 and PepTivator EBV BZLF1 50 ng each), 37 Incubate at ° C for 30 minutes. The virus peptide-added activated T cells 2x10 ⁶ obtained in this manner and the recovered cells were suspended in 30 ml of IL-15 (5 ng / ml) and IL-7 (10 ng / ml) -added culture medium, and G. -Start culturing with Rex10.
Day 10: Replace half of the culture with IL-7 10 ng / ml and IL 15 5 ng / ml TexMACS ^TM.
Day 14: The culture is finished.

The above culture method 6 makes it possible to obtain CAT-T cells by collecting blood once, and has an advantage that the burden on the patient is reduced. Further, the culture method 6 can be expected to further improve the gene transfer efficiency.

According to the present invention, the gene transfer efficiency or cell proliferation rate when preparing CAR cells is improved. That is, the present invention makes it possible to efficiently prepare a larger number of CAR cells, and can contribute to improving the therapeutic results of CAR therapy, expanding the scope of application of CAR therapy, and the like.

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications are also 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 the papers, published patent gazettes, patent gazettes, etc. specified in this specification shall be cited by reference in their entirety.

Claims (24)

Method for preparing genetically modified lymphocytes expressing chimeric antigen receptor, including the following steps (1) to (3):
(1) A step of preparing a genetically modified lymphocyte into which a target antigen-specific chimeric antigen receptor gene has been introduced;
(2) The genetically modified lymphocyte prepared in step (1) is used in the presence of an anti-iditope antibody against the antigen recognition region of the chimeric antigen receptor on which it is expressed, or the target antigen of the chimeric antigen receptor on which it is expressed. The step of culturing in the presence of expressing cells; and (3) the step of collecting the genetically modified lymphocytes after culturing. The preparation method according to claim 1, wherein the transposon method is used for the introduction of the target antigen-specific chimeric antigen receptor gene in step (1). The preparation method according to claim 2, wherein the transposon method is the piggyBac transposon method. The preparation method according to any one of claims 1 to 3, wherein step (2) is performed after 8 to 48 hours have elapsed from the operation of introducing the target antigen-specific chimeric antigen receptor gene in step (1). The preparation method according to any one of claims 1 to 4, wherein the culture period of step (2) is 1 to 14 days. The preparation method according to any one of claims 1 to 5, wherein stimulation by an anti-CD3 antibody and an anti-CD28 antibody is not applied during culturing in step (2). The preparation method according to any one of claims 1 to 6, wherein the anti-iditope antibody is a solid phase antibody or a beaded antibody. The invention according to any one of claims 1 to 7, wherein the genetically modified lymphocyte of step (1) is obtained by introducing a target antigen-specific chimeric antigen receptor gene into a cell population containing T cells or progenitor cells thereof. Preparation method. The preparation method according to claim 8, wherein the cell population is peripheral blood mononuclear cells (PBMCs). The cell according to any one of claims 1 to 9, wherein the cell expressing the target antigen in step (2) is prepared by introducing a gene encoding the target antigen into peripheral blood mononuclear cells (PBMCs). The preparation method described. The preparation method according to any one of claims 1 to 10, wherein in step (2), the cell expressing the target antigen and the genetically modified lymphocyte are derived from the same individual. The preparation method according to claim 11, wherein the individual is an individual different from the individual to be transplanted with the genetically modified lymphocyte collected in step (3). The preparation method according to any one of claims 1 to 12, wherein a step of culturing the cultured cells in the presence of a T cell growth factor is performed between steps (2) and (3). The preparation method according to claim 13, wherein the T cell growth factor is IL-15. The preparation method according to claim 13, wherein IL-15 and IL-7 are used in combination as T cell growth factors. The preparation method according to any one of claims 1 to 15, wherein the following step (a) is performed between steps (1) and (2):
(A) Non-proliferative cells obtained by stimulating a cell population containing T cells with an anti-CD3 antibody and an anti-CD28 antibody and then performing a treatment for losing proliferative ability, and genetically modified lymphocytes prepared in step (1). A step of mixing lymphocytes and co-culturing while stimulating with anti-CD3 antibody and anti-CD28 antibody. The preparation method according to any one of claims 1 to 15, wherein the following step (b) is performed between steps (1) and (2):
(B) Non-proliferative PBMCs obtained by treating peripheral blood mononuclear cells (PBMCs) as they are or by stimulating them with anti-CD3 antibody and anti-CD28 antibody and then performing a treatment to lose proliferative ability, and step (1). The step of mixing and co-culturing the genetically modified lymphocytes prepared in. The preparation method according to any one of claims 1 to 15, wherein the following step (c) is performed between steps (1) and (2):
(C) A cell population containing T cells was stimulated with an anti-CD3 antibody and an anti-CD28 antibody, and then cultured in the presence of the viral peptide antigen and treated to lose the proliferative ability to retain the viral peptide antigen. A step of mixing non-proliferative cells with the genetically modified lymphocytes prepared in step (1) and co-culturing them. The preparation method according to claim 16 or 18, wherein the cell population containing T cells is peripheral blood mononuclear cells (PBMCs). The preparation method according to any one of claims 16 to 19, wherein the treatment for losing the proliferative ability is irradiation. The preparation method according to any one of claims 16 to 20, wherein the non-proliferative cells and the genetically modified lymphocytes are derived from the same individual. A genetically modified lymphocyte expressing a chimeric antigen receptor obtained by the preparation method according to any one of claims 1 to 21. A cell preparation containing a therapeutically effective amount of the genetically modified lymphocyte according to claim 22. A method for treating cancer, which comprises a step of administering the genetically modified lymphocyte according to claim 22 to a cancer patient in a therapeutically effective amount.

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