Cells expressing CARs and transcription factors, and their use

Abstract

To provide cells that express a CAR and a transcription factor, and uses thereof.SOLUTION: The inventors have found that, by co-expressing a chimeric antigen receptor (CAR) with a transcription factor in a cell, it is possible to prevent or reduce differentiation and / or exhaustion of the cell. This results in as the proportion of naive, central memory and stem-cell like memory cells in the cell composition for immunotherapy becomes greater, more effective the in vivo persistence and expansion of the cells becomes. The invention provides a first exogenous nucleic acid molecule encoding a CAR and a second exogenous nucleic acid molecule encoding a transcription factor.SELECTED DRAWING: None

Description

【Technical Field】 【0001】 Field of the Invention The present invention relates to cells that co-express a chimeric antigen receptor (CAR) and a transcription factor. Expression of the transcription factor can prevent or reduce cell differentiation and / or exhaustion in vitro and / or in vivo. 【Background Art】 【0002】 Background of the Invention A chimeric antigen receptor is a protein that transfers the specificity of a monoclonal antibody (mAb) to the effector function of T cells. Its normal form is a type I transmembrane domain protein having an amino-terminal antigen recognition region, a spacer, a transmembrane domain, and an intracellular T cell signaling domain. CAR technology enables the generation of a large number of T cells specific for any surface antigen. Cells are made, for example, by ex vivo viral vector transduction of a peripheral blood T cell population. The transduced cells express the CAR and can be used for adoptive immunotherapy for the treatment of diseases. 【0003】 To generate a clinically effective, therapeutic dose of T cells, activation of T cells is required prior to transduction and expansion. Existing T cell activation / expansion methods usually involve significant T cell differentiation and can result in transient effects including transient survival, lack of persistence, and lack of in vivo expansion of the transplanted T cells. To date, the clinical effectiveness of CAR-T cell immunotherapy has been limited by insufficient T cell expansion and insufficient persistence after injection into patients. Therefore, there is a need for improved therapeutic CAR-T cell compositions that survive, expand, and persist in vivo. 【Brief Description of the Drawings】 【0004】 [Figure 1]A schematic diagram illustrating a linear model of T cell differentiation, showing expression markers associated with each cell type. APC - antigen-presenting cell; TCM - central memory T cell; TEFF - effector T cell; TEM - effector memory T cell; TN - naive T cell; TSCM - T memory stem cell. [Figure 2] Graphs showing the proportions of effector cells, effector memory (EM) cells, central memory (CM) cells, and naive cells after transduction (day 0) and 6 days after 24 hours of co-culture with target cells expressing CD19 (day 7). T cells were either untransduced (NT), transduced using a vector expressing only CAR (HD37), or transduced using a vector expressing both CAR and the transcription factor FOXO1 (HD37-FOXO1) (wither). CD4+ and CD8+ subpopulations were analyzed separately. [Figure 3] Graphs showing the expression of CD27 and CD62L in CD4+ T cells and CD8+ T cells 6 days after 24 hours of co-culture with target cells expressing CD19. The T cells were either untransduced (NT), transduced using a vector expressing only CAR (HD37), transduced using a vector expressing CAR and the transcription factor EOMES (HD37-EOMES), or transduced using a vector expressing CAR and the transcription factor FOXO1 (HD37-FOXO1) (wither). [Figure 4] Graph showing CD62L expression in CD4+ T cells and CD8+ T cells 6 days after 24-hour co-culture with target cells expressing CD19. The T cells were either untransduced (NT), transduced using a vector expressing only CAR (HD37), or transduced using a vector expressing CAR, the transcription factor Runx3, and CBF beta (HD37-Runx3_CBF beta) (wither). [Figure 5]Graphs showing the proportions of effector cells, effector memory (EM) cells, central memory (CM) cells, and naive cells after transduction (day 0) and 6 days after 24 hours of co-culture with target cells expressing CD19 (day 7). T cells were either untransduced (NT), transduced using a vector expressing only CAR (HD37), transduced using a vector expressing CAR and transcription factor BACH2 (HD37-BACH2), or transduced using a vector expressing a mutant version of CAR and transcription factor BACH2 (HD37-BACH2_S520A) (wither). CD4+ and CD8+ subpopulations were analyzed separately. [Modes for carrying out the invention] 【0005】 Summary of aspects of the present invention For CAR-T cells to be effective, it is crucial that they persist and expand in vivo and resist excessively rapid differentiation and exhaustion. The persistence and engraftment of CAR T cells are related to the proportion of naive T cells, central memory T cells, and T stem cell-like memory T cells administered. 【0006】 The inventors have found that co-expression of transcription factors and CARs within cells can prevent or reduce cell differentiation and / or exhaustion. This results in increased proportions of naive cells, central memory cells, and stem cell-like memory cells in a cell composition for immunotherapy, leading to greater persistence and expansion of these cells in vivo. 【0007】 Therefore, in a first aspect, the present invention provides a cell comprising a first exogenous nucleic acid molecule encoding a chimeric antigen receptor (CAR) and a second exogenous nucleic acid molecule encoding a transcription factor. 【0008】 This transcription factor may prevent or reduce cell differentiation and / or exhaustion. 【0009】 The transcription factor in question may be an effector memory replacer such as BLIMP-1. 【0010】 Alternatively, the transcription factor could be a central memory repressor such as BCL6 or Bach2. 【0011】 The transcription factor may be Bach2, or a modified version of Bach2 in which the ability to be phosphorylated by ALK is reduced or removed, or may include these. For example, the modified Bach2 may contain mutations at one or more of the following positions with reference to the amino acid sequence shown in SEQ ID NO: Ser-535, Ser-509, Ser-520. 【0012】 The transcription factor in question may be FOXO1. 【0013】 The transcription factor in question may be EOMES. 【0014】 The transcription factor may include Runx3 and / or CBF beta. 【0015】 In a second aspect, the present invention provides a nucleic acid construct comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) and a second nucleic acid sequence encoding a transcription factor as defined in the first aspect of the present invention. 【0016】 The nucleic acid construct in question has the following structure: CAR-coexpr-TF; or TF-coexpr-CAR It may have, in the formula: CAR is the nucleic acid sequence that encodes the CAR; coexpr is a nucleic acid sequence that enables the co-expression of CARs and transcription factors; and TF is a nucleic acid construct, which is a nucleic acid sequence that encodes a transcription factor. 【0017】 The nucleic acid sequence "coexpr" may encode a sequence containing a self-cleaving peptide. 【0018】 In a third aspect, the present invention provides a kit of nucleic acid sequences comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) and a second nucleic acid sequence encoding a transcription factor as defined in the first aspect of the present invention. 【0019】 In a fourth aspect, the present invention provides a vector comprising the nucleic acid construct described in the second aspect of the present invention. 【0020】 In a fifth aspect, the present invention provides a kit of vectors comprising a first vector comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR); and a second vector comprising a second nucleic acid sequence encoding a transcription factor as defined in the first aspect of the present invention. 【0021】 In a sixth aspect, the present invention provides a method for creating the cells described in the first aspect of the present invention, the method comprising introducing into a cell the nucleic acid construct described in the second aspect of the present invention, the kit of nucleic acid sequences described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, or the kit of vectors described in the fifth aspect of the present invention. 【0022】 The cell may be derived from a sample isolated from a subject. 【0023】 In a seventh aspect, the present invention provides a pharmaceutical composition comprising a plurality of the cells described in the first aspect of the present invention. 【0024】 In an eighth aspect, the present invention provides a method for treating and / or preventing a disease, the method comprising administering to a subject the pharmaceutical composition described in the seventh aspect of the present invention. 【0025】 The method may comprise the following steps: (i) isolating a sample containing cells from a subject; (ii) Transducing or transfecting cells using a nucleic acid construct described in the second aspect of the present invention, a nucleic acid sequence kit described in the third aspect of the present invention, a vector described in the fourth aspect of the present invention, or a vector kit described in the fifth aspect of the present invention; (iii)(ii) Administer cells derived from (iii)(ii) to the subject. 【0026】 In its ninth aspect, the present invention provides a pharmaceutical composition according to its seventh aspect for use in the treatment and / or prevention of disease. 【0027】 In a tenth aspect, the use of cells according to the first aspect of the present invention is provided in the manufacture of a medicament for the treatment and / or prevention of disease. 【0028】 With respect to the eighth, ninth, and tenth aspects of the present invention, the disease may be cancer. 【0029】 Detailed description Chimeric antigen receptor (CAR) The cells of the present invention contain an exogenous nucleic acid molecule encoding a chimeric antigen receptor (CAR). 【0030】 Classical CARs are chimeric type I transmembrane proteins that link an extracellular antigen-recognition domain (binder) to an intracellular signaling domain (endodomain). The binder is typically a single-strand variable fragment (scFv) derived from a monoclonal antibody (mAb), but can also be based on other forms, including an antibody-like antigen-binding site. The spacer domain is usually necessary to isolate the binder from the cell membrane and to properly orient it. A commonly used spacer domain is the Fc of IgG1. Depending on the antigen, a more compact spacer (e.g., only the axis derived from CD8α and the hinge of IgG1) may be sufficient. The transmembrane domain anchors the protein within the cell membrane and connects the spacer to the endodomain. 【0031】 Early CAR designs had endodomains derived from the intracellular portion of either the γ chain of FcεR1 or CD3ζ. As a result, these first-generation receptors transmitted immune signals 1 sufficient to induce T cell killing of cognate target cells, but not enough to fully activate T cells for proliferation and survival. To overcome this limitation, hybrid endodomains were constructed: fusions of the intracellular portion of a T cell costimulatory molecule with the intracellular portion of CD3ζ resulted in second-generation receptors capable of simultaneously transmitting activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is that of CD28, which delivers the most potent costimulatory signal—the so-called immune signal 2 that induces T cell proliferation. Several receptors containing endodomains of the TNF receptor family (such as OX40 and 41BB, which transmit survival signals and are closely related) have also been described. Even more potent third-generation CARs with endodomains capable of transmitting activation, proliferation, and survival signals are now being described. 【0032】 Nucleic acids encoding CARs can be transported to T cells, for example, using retroviral vectors. Lentiviral vectors can also be used. In this method, a large number of cancer-specific T cells can be generated for adoptive cell transplantation. When a CAR binds to a target antigen, this results in the transmission of an activation signal to the T cells expressing the CAR. Thus, the CAR leads to the specificity and cytotoxicity of T cells against tumor cells expressing the target antigen. 【0033】 Therefore, a typical CAR includes (i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iii) a signaling domain, or an associated intracellular domain. 【0034】 antigen-binding domain The antigen-binding domain is the portion of the CAR that recognizes the antigen. Numerous antigen-binding domains (including those based on antigen-binding sites of antibodies, antibody mimetic compounds, and T cell receptors) are known in this field. Antigen-binding domains may include, for example, single-strand variable fragments (scFv) derived from monoclonal antibodies; natural ligands of the target antigen; peptides with sufficient affinity for the target; single-domain antibodies; artificial single binders such as Darpin (designed ankyrin repeat protein); or single strands derived from T cell receptors. 【0035】 The antigen-binding domain may include domains that are not based on the antigen-binding site of the antibody. For example, the antigen-binding domain may include a domain based on a protein / peptide (e.g., a soluble peptide such as a cytokine or chemokine) that is a soluble ligand for a surface receptor of tumor cells; or an extracellular domain of a membrane-anchored ligand or receptor, the extracellular domain to which its binding pair counterpart is expressed on tumor cells. 【0036】 The antigen-binding domain may be based on the antigen's natural ligand. 【0037】 The antigen-binding domain may contain affinity peptides derived from a combinatorial library, or novel affinity proteins / peptides. 【0038】 Spacer Domain CARs include a spacer sequence that ligates the antigen-binding domain to the transmembrane domain and spatially separates the antigen-binding domain from the endodomain. The flexible spacer allows the antigen-binding domain to coordinate in different directions to facilitate binding. 【0039】 transmembrane domain The transmembrane domain is the sequence of CARs that spans the membrane. 【0040】 A transmembrane domain can be any protein structure that is thermodynamically stable within a membrane. Typically, this is an alpha-helix containing several hydrophobic residues. A transmembrane domain of any transmembrane protein can be used to supply the transmembrane portion of the present invention. The presence and width of a protein's transmembrane domain can be determined by those skilled in the art using the TMHMM algorithm (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ). Furthermore, if the protein's transmembrane domain is a relatively simple structure (i.e., a polypeptide sequence expected to form a hydrophobic alpha-helix of sufficient length to traverse the membrane), an artificially designed TM domain can also be used (U.S. Patent No. 7052906B1 describes a synthetic transmembrane component). 【0041】 The transmembrane domain may be derived from CD28, which provides excellent receptor stability. 【0042】 End domain The endodomain is the signaling portion of a CAR. It is either part of or related to the intracellular domain of the CAR. After antigen recognition, the receptors cluster, excluding native CD45 and CD148 from synapses, and then the signal is transmitted to the cell. The most commonly used endodomain component is that of the CD3 zeta, which contains three ITAMs. This transmits an activation signal to T cells after antigen binding. The CD3 zeta may not provide a fully competent activation signal and additional co-stimulatory signals may be required. For example, chimeric CD28 and OX40 may be used with the CD3 zeta to transmit proliferation / survival signals, or all three may be used together. 【0043】 The CAR or TanCAR end domain of the present invention may include the CD28 end domain as well as the OX40 and CD3 zeta end domains. 【0044】 The end domain in question is: (i) End domains containing ITAM such as CD3 zeta-derived end domains; and / or (ii) Co-stimulatory domains such as CD28-derived endodomains; and / or (iii) Survival signaling domains, such as the endodomain of the TNF receptor family (OX-40 or 4-1BB, etc.) It may include. 【0045】 Numerous systems have been described in which the antigen recognition portion is located on a separate molecule from the signal transduction portion (e.g., those described in International Publication No. 015 / 150771; International Publication No. 2016 / 124930 and International Publication No. 2016 / 030691). Therefore, a CAR expressed in the cells of the present invention may include an antigen-binding component comprising an antigen-binding domain and a transmembrane domain; this antigen-binding component may interact with a separate intracellular signaling component comprising a signaling domain. The cells of the present invention may include a CAR signaling system comprising an antigen-binding component and an intracellular signaling component, etc. 【0046】 Signal peptide The cells of the present invention may contain a signal peptide, and therefore, when CAR is expressed within the cell, the newly synthesized protein is guided to the endoplasmic reticulum and then to the cell surface, which is its expression site. 【0047】 Signal peptides can be located at the amino terminus of a molecule. 【0048】 The CAR of the present invention has the general formula: Signal peptide - antigen-binding domain - spacer domain - transmembrane domain - intracellular T cell signaling domain (endodomain) It may have. 【0049】 Transcription factor The cells of the present invention contain exogenous nucleic acid molecules encoding transcription factors. 【0050】 Transcription factors are proteins that bind to specific DNA sequences and regulate the rate of transcription of genetic information from DNA to messenger RNA by adjusting the expression of genes that contain or are adjacent to that sequence. 【0051】 Transcription factors work by either promoting (as activators) or inhibiting (as repressors) the recruitment of RNA polymerase. 【0052】 Transcription factors contain at least one DNA-binding domain (DBD) that attaches to either the enhancer or promoter region of DNA. Transcription factors can upregulate or downregulate the transcription of adjacent genes in a transcription factor-dependent manner. Transcription factors also contain a transactivation domain (TAD) that has a binding site for other proteins (such as transcription coregulators). 【0053】 Transcription factors use various means to regulate gene expression, including stabilizing or interfering with the binding of RNA polymerase to DNA, or catalyzing the acetylation or deacetylation of histone proteins. Transcription factors may have histone acetyltransferase (HAT) activity, weakening the binding of histones to DNA by acetylating histone proteins, thereby facilitating DNA transcription and thus upregulating transcription. Alternatively, transcription factors may have histone deacetylase (HDAC) activity, strengthening the binding of histones to DNA by deacetylating histone proteins, thereby facilitating DNA transcription and thus downregulating transcription. Another mechanism by which transcription factors can function is through the recruitment of coactivator or corepressor proteins to the transcription factor DNA complex. 【0054】 There are two classes of transcription factors: basic transcription factors and upstream transcription factors. 【0055】 Basic transcription factors are involved in the formation of the pre-transcriptional complex. The most common ones are abbreviated as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. These are ubiquitous and interact with the core promoter region surrounding the transcription start site(s) of all class II genes. 【0056】 Upstream transcription factors are proteins that bind upstream of the start site to stimulate or repress transcription. Because upstream transcription factors are considerably diverse, depending on the recognition sequences present near the gene, these are often synonymous with specific transcription factors. 【0057】 Some examples of specific transcription factors are shown in the table below: [Table 1] 【0058】 Transcription factors are often classified based on sequence similarity and the resulting tertiary structure of their DNA-binding domains. 【0059】 Transcription factors possessing a basic domain include leucine zipper factors (e.g., bZIP, c-Fos / c-Jun, CREB, and plant G-box binding factors); helix-loop-helix factors (e.g., ubiquitous (class A) factors; myogenic transcription factors (MyoD); Achaete-Scute and Tal / Twist / Atonal / Hen); and helix-loop-helix / leucine zipper factors (e.g., bHLH-ZIP, c-Myc, NF-1(A,B,C,X), RF-X(1,2,3,4,5,ANK), and bHSH). 【0060】 Transcription factors containing a zinc-coordinated DNA-binding domain include nuclear receptor-type Cys4 zinc fingers (such as steroid hormone receptor and thyroid hormone receptor analogs); divergent Cys4 zinc fingers (such as GATA factor); Cys2His2 zinc finger domains (such as ubiquitous factors including TFIIIA and Sp1); developmental / cell cycle regulators including Kruppel; large factors with NF-6B-like binding properties; and Cys6 cysteine-zinc clusters and alternating zinc fingers. 【0061】 Transcription factors containing a helix-turn helix domain include those with a homeodomain; paired box; forkhead / winged helix; heat shock factors; tryptophan clusters; and TEA (transcriptional enhancer factor) domains such as TEAD1, TEAD2, TEAD3, and TEAD4. 【0062】 Finally, there are beta scaffold factors that involve contact with substructures, including the RHR (Rel homologous region) class; STAT; p53; MADS box; beta-barrel alpha-helix transcription factor; TATA-binding protein; HMG box; heteromeric CCAAT factor; Grainyhead; cold shock domain factor; and Runt. 【0063】 The transcription factor of the present invention may be constitutively activated or conditionally activated (i.e., requires activation). 【0064】 Transcription factors can be naturally occurring or artificially created. 【0065】 Suppression of T cell differentiation Following activation, T cells differentiate into various T cell subtypes as shown in Figure 1. We demonstrate that the persistence and engraftment of CAR T cells in the subject are related to the proportion of naive T cells, central memory T cells, and stem cell-like memory T cells administered to the subject. 【0066】 The cells of the present invention may include exogenous nucleic acid molecules encoding transcription factors that effectively increase the proportion of naive T cells, central memory T cells, and / or stem cell-like memory T cells in a composition for administration to a patient. 【0067】 The transcription factor in question may be, for example, a transcription factor that reppresses central memory (such as BCL6 or BACH2). Central memory repressors inhibit the differentiation of T cells into effector memory cells, so that T cells remain as less differentiated T cell subtypes (such as naive T cells and stem cell-like memory T cells). These repressors interfere with or reduce the differentiation rate of T cells throughout the various stages shown in Figure 1, biasing the T cell population toward a more naive phenotype. 【0068】 Alternatively, the transcription factor in question may be a transcription factor that reprises effector memory (such as BLIMP-1). 【0069】 BCL6 B cell lymphoma protein (BCL6) is an evolutionarily conserved zinc finger transcription factor containing a POZ / BTB domain at its N-terminus. BCL6 has been shown to act as a sequence-specific transcriptional repressor and to modulate the STAT-dependent interleukin-4 (IL-4) response in B cells. BCL6 interacts with several corepressor complexes to inhibit transcription. 【0070】 The amino acid sequence of BCL6 can be obtained from UniProt (accession number P41182) and is shown below as sequence number 1. Sequence ID 1-BCL6 [ka] [ka] 【0071】 BCL6 contains six zinc fingers at the following amino acid positions: 518-541, 546-568, 574-596, 602-624, 630-652, and 658-681. 【0072】 BACH2 The Broad complex and cap'n'collar homology (Bach)2 protein, also known as bric-a-brac and tramtrack, is a 92 kDa transcription factor. The Bach2 protein heterodimerizes with proteins of the musculoaponeurotic fibrosarcoma (Maf) family via its basic leucine zipper domain. The Bach2 locus is located within a superenhancer (SE) and regulates the expression of genes regulated by SEs. SEs are crucial for the expression of cell lineage genes. In T cells, the majority of SE-regulated genes are cytokine and cytokine receptor genes. Bach2 is a major SE-associated gene across all T cell lineages. 【0073】 The Bach2 protein consists of 72 phosphorylation sites. Of these sites, Ser-335 contains the Akt target consensus sequence (RXRXX(S / T)X). Eleven sites (Ser-260, Ser-314, Thr-318, Thr-321, Ser-336, Ser-408, Thr-442, Ser-509, Ser-535, Ser-547, and Ser-718) generate the mTOR target consensus sequence (proline at position +1). Substitution of Ser-535 and Ser-509 with Ala increases the nuclear localization of Bach2 and enhances the downregulation of its target genes. 【0074】 The Ser-520 site was identified as a substrate for Akt for phosphorylation. Substitution of Ser-520 with Ala also increases Bach2's repressor ability. Fusion of eGFP to WT or mutant Bach2 revealed enhanced nuclear localization of S520A Bach2. ​​Phosphorylation of Bach2 during T cell activation leads to the segregation of Bach2 into the cytoplasm. Mutations at the phosphorylation site confer resistance to such segregation to Bach2, and therefore increase its nuclear localization. 【0075】 The cells of the present invention may contain an exogenous nucleic acid molecule expressing a Bach2 variant with increased nuclear localization compared to the wild-type protein. This variant may have mutations at Ser-535, Ser-520, or Ser-509 with reference to the sequence shown in SEQ ID NO: 2. These mutations may be substitutions such as a substitution of Ser to Ala. 【0076】 Bach2 binds to the consensus motif (5'-TGA(C / G)TCAGC-3'), which is part of the motif (5'-TGA(C / G)TCA-3') recognized by the AP-1 family. The AP-1 family of transcription factors is involved in inducing the expression of genes downstream of TCR activation. Examples of AP-1 transcription factors include c-Jun, JunB, and c-Fos. AP-1 factors are phosphorylated upon TCR activation and subsequently regulate genes involved in effector differentiation. Bach2 competes with AP-1 for binding to the overlapping motif, thereby repressing the activation of these genes. 【0077】 Bach2 mRNA expression is high in naive CD8 T cells and gradually downregulated in central memory cells (CD62L+KLRG1-), effector cells (CD62L-KLRG1-), and terminally differentiated effector cells (CD62L-KLRG1+). Bach2 deficiency leads to terminally differentiated T cells and increases apoptosis. 【0078】 The amino acid sequence of Bac2 can be obtained from UniProt (accession number Q9BYV9) and is shown below as sequence number 2. Sequence ID 2-Bach-2 wild type [ka] 【0079】 The sequence of the mutant Bach2, which has an S-to-A substitution at position 520, is shown as sequence number 3. The S520A substitution is shown in bold and underlined. Sequence ID 3-S520A Bach2 mutant (insensitive to AKT) [ka] 【0080】 BLIMP-1 B-lymphocyte-induced maturation protein 1(BLIMP1) acts as a repressor for beta-interferon (β-IFN) gene expression. This protein specifically binds to the PRDI (positive regulatory domain I element) of the β-IFN gene promoter. 【0081】 Increased expression of the Blimp-1 protein in B lymphocytes, T lymphocytes, NK cells, and other immune system cells triggers an immune response through the proliferation and differentiation of antibody-secreting plasma cells. Blimp-1 is also thought to be a "master regulator" of hematopoietic stem cells. 【0082】 BLIMP-1 is involved in regulating the terminal differentiation of antibody-secreting cells (ASCs) and plays an important role in maintaining homeostasis of effector T cells. 【0083】 The amino acid sequence of BLIMP-1 can be obtained from UniProt (accession number O75626) and is shown below as sequence number 4. Sequence ID 4-BLIMP-1 [ka] 【0084】 FOXO1 Forkhead box protein O1 (FOXO1), also known as forkhead in rhabdomyosarcoma, is a protein encoded by the FOXO1 gene in humans. FOXO1 is a transcription factor that plays a crucial role in regulating gluconeogenesis and glycogenolysis mediated by insulin signaling, and is also central to determining the involvement of preadipocytes in adipogenesis. FOXO1 is primarily regulated by phosphorylation at multiple residues; its transcriptional activity depends on its phosphorylation state. 【0085】 The amino acid sequence of FOXO1 can be obtained from UniProt (accession number Q12778) and is shown below as sequence number 5. Sequence ID 5-FOXO1 [ka] [ka] 【0086】 EOMES EOMES, also known as Eomesodermin and T-box brain protein 2 (Tbr2), is a protein encoded by the EOMES gene in humans. EOMES is a member of a conserved family of proteins that share a common DNA-binding domain (T-box). T-box genes encode transcription factors that regulate gene expression involved in the regulation of developmental processes. Eomes itself regulates radial glial cells and other related cells. Eomes has been found to be involved in immune responses, and there is some weak evidence of its involvement in other systems. 【0087】 The amino acid sequence of EOMES can be obtained from UniProt (accession number 095936) and is shown below as sequence number 6. Sequence ID 6-EOMES [ka] 【0088】 RUNX3 Runx3, or Runt-related transcription factor 3, is a member of the family of transcription factors containing the runt domain. Heterodimers of this protein and its beta subunit bind to a core DNA sequence (5'-YGYGGT-3') found in numerous enhancers and promoters, forming complexes that can activate or repress transcription. Runx3 also interacts with other transcription factors. Runx3 functions as a tumor suppressor, and this gene is frequently deleted or silenced in cancer. Multiple transcriptional variants encoding various isoforms have been found for this gene. 【0089】 The amino acid sequence of RUNX3 can be obtained from UniProt (accession number Q13761) and is shown below as sequence number 7. Sequence ID 7-RUNX3 [ka] 【0090】 CBF Beta Core-binding factor subunit beta (CBF beta) is the beta subunit of a heterodimeric core-binding transcription factor and belongs to the PEBP2 / CBF transcription factor family, which master-regulates numerous genes specific to hematopoiesis (e.g., RUNX1) and osteogenicity (e.g., RUNX2). This beta subunit is a DNA-non-binding regulatory subunit; when the complex binds to the core site of various enhancers and promoters (including murine leukemia virus, polyomavirus enhancers, T cell receptor enhancers, and GM-CSF promoters), this beta subunit allosterically enhances DNA binding by the alpha subunit. Alternative splicing generates two mRNA variants encoding different carboxyl ends. 【0091】 The amino acid sequence of CBF beta is available from (accession number Q13951) and is shown below as sequence number 8. Sequence ID 8 - CBF Beta [ka] 【0092】 exogenous nucleic acid molecule This invention provides cells containing an exogenous nucleic acid molecule encoding a transcription factor. The term "exogenous" means that the nucleic acid molecule is produced by a recombinant technique and introduced into the cell using a vector. The cells contain the nucleic acid molecule and are manipulated to express (or overexpress) the transcription factor. 【0093】 nucleic acid As used herein, the terms “polynucleotide,” “nucleotide,” and “nucleic acid” are intended to be synonymous with each other. 【0094】 Those skilled in the art will understand that numerous different polynucleotides and nucleic acids may encode the same polypeptide as a result of the degeneracy of the genetic code. Furthermore, those skilled in the art will understand that, using common techniques, it may be possible to create nucleotide substitutions that do not affect the sequence of the polypeptide encoded by the polynucleotides described herein in order to reflect the codon usage frequency of a particular host organism in which the polypeptide is expressed. 【0095】 The nucleic acid according to the present invention may include DNA or RNA. This nucleic acid may be single-stranded or double-stranded. This nucleic acid may be a polynucleotide containing synthesized or modified nucleotides. Numerous different types of modifications to oligonucleotides are known in the art. These modifications include the addition of methylphosphonate and phosphorothioate backbones, and acridine or polylysine chains at the 3' and / or 5' ends of the molecule. It is understood that for the purposes of use described herein, polynucleotides may be modified using any method available in the art. Such modifications may be made to enhance the in vivo activity or lifetime of the polynucleotide in question. 【0096】 The terms “variant,” “homogram,” or “derivative” include, in relation to a nucleotide sequence, any substitution, variation, modification, exchange, deletion, or addition of one (or more) nucleic acids to or from a sequence. 【0097】 Nucleic acid construct The present invention provides a nucleic acid construct comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) and a second nucleic acid sequence encoding a transcription factor. 【0098】 A nucleic acid construct may also include nucleic acid sequences capable of expressing two or more proteins. For example, a nucleic acid construct may include a sequence encoding a cleavage site between two nucleic acid sequences. This cleavage site may be self-cleaving, so that when a nascent polypeptide is produced, it is immediately cleaved into two proteins without requiring any external cleavage activity. 【0099】 Various autocleavage sites are known, including the autocleavage peptide of foot-and-mouth disease virus (FMDV) 2a with the following sequence: Sequence ID 9 RAEGRGSLLTCGDVEENPGP or Sequence ID 10 QCTNYALLKLAGDVESNPGP 【0100】 Alternatively, the co-expression sequence may be an internal ribosome entry sequence (IRES) or an internal promoter. 【0101】 vector The present invention also provides a vector or a vector kit comprising one or more nucleic acid sequences or constructs according to the present invention. Such a vector can be used to introduce nucleic acid sequences or constructs into a host cell, thereby causing the host cell to express a protein encoded by the nucleic acid sequence or construct. 【0102】 The vector may be, for example, a plasmid, a viral vector (such as a retroviral vector or lentiviral vector), a transposon-based vector, or synthetic mRNA. 【0103】 The vector can be transfected or transduced into T cells. 【0104】 cell This invention provides cells that co-express CARs and transcription factors. 【0105】 These cells may be cytolytic immune cells. 【0106】 Cytolytic immune cells can be T cells or T lymphocytes, a type of lymphocyte that plays a central role in cellular immunity. Cytolytic immune cells can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of T cell receptors (TCRs) on their cell surface. There are various types of T cells, as summarized below. 【0107】 Helper T cells (TH cells) assist other leukocytes in immune processes such as the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells become activated when presented with peptide antigens by MHC class II molecules on the surface of antigen-presenting cells (APCs). These TH cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or TFH (these secrete different cytokines to promote different types of immune responses). 【0108】 Cytolytic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells, and are also involved in transplant rejection. CTLs express CD8 on their surface. These cells recognize targets by binding to antigens associated with MHC class I (present on the surface of all nucleated cells). CD8+ cells can be inactivated into an anerious state by IL-10, adenosine, and other molecules secreted by regulatory T cells, preventing autoimmune diseases such as experimental autoimmune encephalomyelitis. 【0109】 Memory T cells are a subset of antigen-specific T cells that persist long after recovery from infection. Upon re-exposure to their allogeneic antigen, memory T cells immediately expand into a large number of effector T cells, thus providing the immune system with a "memory" of past infection. Memory T cells include three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Typically, memory T cells express the cell surface protein CD45RO. 【0110】 Regulatory T cells (Treg cells), formerly known as suppressor T cells, are crucial for maintaining immune tolerance. Their primary roles are to shut down T-cell immunity towards the end of the immune response and to suppress autoreactive T cells that have escaped the negative selection process in the thymus. 【0111】 Two major classes of CD4+ Treg cells have been described: endogenous Treg cells and adaptive Treg cells. 【0112】 Endogenous Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) originate in the thymus and are associated with interactions between developing T cells and both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells activated by TSLP. Endogenous Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations in the FOXP3 gene can prevent the development of regulatory T cells and lead to IPEX, a fatal immune disorder. 【0113】 Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can arise during a normal immune response. 【0114】 Natural killer cells (or NK cells) are a type of cytolytic cell that forms part of the innate immune system. NK cells provide a rapid response to innate signals from virus-infected cells in an MHC-independent manner. 【0115】 NK cells (belonging to the group of innate lymphocytes) are defined as large granular lymphocytes (LGLs) and constitute a third type of cell that differentiates from common lymphoid progenitor cells that produce B lymphocytes and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, and then enter the circulation from these locations. 【0116】 The cells of the present invention may be any of the above cell types. 【0117】 If a transcription factor reduces or inhibits T cell differentiation or exhaustion, the cell may preferentially be one of the following T cell subtypes: naive T cells; stem cell-like memory T cells; and central memory T cells. 【0118】 The cells of the present invention can be produced ex vivo from the patient's own peripheral blood (first party), in the context of hematopoietic stem cell transplantation from donor peripheral blood (second party), or from peripheral blood from an unrelated donor (third party). 【0119】 Alternatively, the cells may be derived from induced progenitor cells or embryonic progenitor cells through ex vivo differentiation, for example, into T cells. Or, immortalized cell lines that maintain lysis function and can act as therapeutic cells may be used. 【0120】 In all these embodiments, cells may be generated by the introduction of DNA or RNA encoding CARs and transcription factors, which may be one of a variety of methods including transduction using a viral vector or transfection using DNA or RNA. 【0121】 The cells of the present invention may be ex vivo cells derived from a subject. These cells may be derived from a peripheral blood mononuclear cell (PBMC) sample. The cells may be activated and / or expanded by treatment, for example, with an anti-CD3 monoclonal antibody, before being transduced with the nucleic acid sequence or construct of the present invention. 【0122】 The cells of this invention are: (i) Isolating a sample containing cells from the subject or any other source as described above; (ii) Transducing or transfecting cells using nucleic acid sequences or constructs according to the present invention It can be created by. 【0123】 composition The present invention also relates to a pharmaceutical composition comprising multiple types of cells of the present invention. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and / or pharmaceutically active compounds. Such formulations may be in a form suitable for, for example, intravenous infusion. 【0124】 Treatment method The cells of this invention may have the ability to kill target cells such as cancer cells. 【0125】 The cells of the present invention can be used for treating infections such as viral infections. 【0126】 The cells of the present invention can also be used to control pathogenic immune responses (e.g., autoimmune diseases, allergies, and graft-versus-host rejection). 【0127】 The cells of the present invention can also be used to treat cancerous diseases such as bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cells), leukemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, and thyroid cancer. 【0128】 The cells of the present invention may be used in the following treatments: oral and pharyngeal cancers, including cancers of the tongue, mouth, and pharynx; gastrointestinal cancers, including cancers of the esophagus, stomach, and colorectal colon; and liver and bile duct cancers, including hepatocellular carcinoma and bile duct cancer. Cancers of the tree; cancers of the respiratory system, including cancers of the bronchial tubes and larynx; cancers of the bones and joints, including osteosarcoma; cancers of the skin, including melanoma; breast cancer; cancers of the reproductive system, including cancers of the uterus, ovaries and cervix in women, and cancers of the prostate and testes in men; cancers of the kidneys and urinary tract, including renal cell carcinoma and transitional cell carcinoma of the uterus (utterer) or bladder; cancers of the brain, including glioma, glioblastoma multiforme, and medulloblastoma; cancers of the endocrine system, including thyroid cancer, adrenal cancer, and cancers associated with multiple endocrine neoplasia syndrome; lymphomas, including Hodgkin lymphoma and non-Hodgkin lymphoma; multiple myeloma and plasmacytoma; both acute and chronic leukemia (myeloid or lymphoid); and other and site-unspecified cancers (including neuroblastoma). 【0129】 The present invention is now further described by examples. The examples are intended to assist those skilled in the art in carrying out the present invention and are not intended to limit the scope of the invention in any way. [Examples] 【0130】 Examples Example 1 - Co-expression of chimeric antigen receptor (CAR) and transcription factor (TF) In BW5T cells, a bicistronic construct was expressed as a single transcript. This transcript autocleaved at the 2A site to produce a chimeric antigen receptor (CAR) and a transcription factor (TF). Control constructs lacking either the 2A site and the transcription factor ("CAR only") or lacking both the CAR and the 2A site ("TF only") were also generated. 【0131】 The CAR in question was an anti-CD19 CAR containing an endodomain derived from CD3 zeta and the costimulatory receptor 41BB. 【0132】 The following constructs containing the transcription factors were tested: [Table 2] 【0133】 Example 2 - Phenotype Assay The expression of various CARs on the surface of T cells may influence the memory state of T cells in the absence of CAR antigens. Furthermore, when a CAR binds to its alloantigen, the T cell is activated, leading to further differentiation from a more naive central memory phenotype to a more differentiated effector memory phenotype / effector phenotype. It is expected that the expression of appropriate transcription factors / repressors will inhibit this CAR-mediated differentiation to varying degrees. 【0134】 T cells expressing various combinations of CAR-TFs, as well as controls expressing only the relevant CARs and only the TFs, were co-cultured with CD19-positive SKOV3 target cells for 24 hours. The T cells were then harvested and cultured until day 7. To determine whether cells expressing factors that bias cells towards central memory are more naive after transduction and remain more naive when stimulated with antigen-containing target cells, the following memory markers were analyzed by flow cytometry on day 0 and day 7 of co-culture. Memory markers - CCR7, CD45RA, CD62L, CD27 【0135】 Data for FOXO1 are shown in Figure 2. Co-expression of FOXO1 and CAR(HD37) increased the proportion of naive cells and central memory cells (CMs) in both the CD4+ and CD8+ subpopulations at both day 0 and day 7. This indicates that FOXO1 biases cells towards the naive / central memory phenotype both after transduction and after co-culture with target cells. 【0136】 Figure 3 shows data on CD27 and CD62L expression 6 days after 24 hours of co-culture with target cells. The transcription factor EOMES caused significant upregulation of CD27 in both CD4+ and CD8+ T cell subpopulations. FOXO1 caused upregulation of CD27, particularly in CD8+ cells. The transcription factor FOXO1 caused significant upregulation of CD62L in both CD4+ and CD8+ subpopulations. CD62L is a marker for naive cells / central memory cells, and phenotypic determination of memory correlates with CD62L levels for FOXO1: more naive and memory cells. Since CD27 is a marker for all cells except fully differentiated effector cells, cells expressing EOMES may primarily be a less differentiated effector memory subtype that does not show significant upregulation of CD62L. 【0137】 As shown in Figure 4, the presence of both Runx3 and CBF beta caused upregulation of CD62L both after transduction (day 0) and 6 days after 24 hours of co-culture. 【0138】 Data for BACH2 and the BACH2 mutant S520A are shown in Figure 5. Both BACH2 and BACH2S520A increase the proportion of naive cells and central memory cells (CMs) on day 0 and day 7. 【0139】 In separate assays, T cells expressing various combinations of CAR-TFs, as well as T cells possessing only the relevant CARs, were co-cultured with CD19-positive SupT1 target cells. To examine whether the cells expressed "exhaustion" markers to a weaker degree upon stimulation, the expression of the following exhaustion markers was analyzed by flow cytometry on days 0, 2, 4, and 7 of co-culture. Fatigue markers - PD1, Tim3, Lag3 【0140】 Cells were gated with various T cell and T cell subset markers (CD3 and CD8), depending on CAR expression (via the RQR8 transduction marker) and the target subpopulation. 【0141】 Example 3 - Functional Assay As cells differentiate, they acquire a stronger ability to lyse target cells in vitro and secrete more IFN-γ, but lose their proliferative capacity. This study investigates whether phenotypic bias alters these abilities. 【0142】 SupT1 cells (CD19-negative) are manipulated to become CD19-positive to create target CD19-negative and CD19-positive cell lineages. Transduced and untransduced T cells, as well as transduced T cells of the control construct, are exposed in a 1:1 ratio to either SupT1 cells or SupT1.CD19 cells. Target cell toxicity is analyzed by FACS at 24 and 72 hours. Toxin toxicity is also monitored by the Incucyte assay, which involves co-culturing transduced T cells on a monolayer of fluorescently labeled adherent cells expressing their respective CAR antigens. CAR-mediated cytotoxicity results in a decrease in the number of fluorescent target cells (which can be continuously monitored over time to allow for measurement of the kinetics of CAR-mediated cytotoxicity). 【0143】 To monitor cytokine release, supernatant samples were collected 48 hours after exposure and assayed for the following cytokines using a cytokine bead array: IL2, IL4, IL6, IL10, TNF-α, IFN-γ, and GzmB. 【0144】 To measure proliferation, T cells are labeled with the fluorescent dye Cell Trace Violet for 20 minutes. After labeling, a co-culture is set up in a 1:1 ratio (target cells:transduced T cells). Because the dye is distributed during the successive generation of daughter cells, CAR-mediated T cell proliferation results in a decrease in Cell Trace Violet fluorescence. The degree of this dilution, and therefore the degree of amplification, can be measured by flow cytometry on days 4 and 7 of the co-culture. 【0145】 All publications cited in the above specification are incorporated herein by reference. Various modifications and variations of the methods and systems described in the present invention will be obvious to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in relation to certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the described form that will be obvious to those skilled in the art in molecular biology or related fields for carrying out the present invention are intended to be within the scope of the following claims. The present invention provides, for example, the following items: (Item 1) A cell containing a first exogenous nucleic acid molecule encoding a chimeric antigen receptor (CAR) and a second exogenous nucleic acid molecule encoding a transcription factor. (Item 2) The cell according to item 1, wherein the transcription factor prevents or reduces the differentiation and / or exhaustion of the cell. (Item 3) The cell according to item 1 or 2, wherein the transcription factor is an effector memory repressor. (Item 4) The cells described in item 3, wherein the transcription factor is BLIMP-1. (Item 5) The cell according to item 1 or 2, wherein the transcription factor is a central memory repressor. (Item 6) The cells described in item 5, wherein the transcription factor is BCL6 or Bach2. (Item 7) The cells described in item 6, wherein the transcription factor is Bach2 or contains Bach2. ​​(Item 8) The cells described in item 5, comprising a modified version of Bach2 in which the transcription factor has reduced or eliminated ability to be phosphorylated by ALK. (Item 9) The cells described in item 8, which include a modified version of Bach2 having a mutation at one or more of the following positions with reference to the amino acid sequence shown in Sequence ID No. 2: Ser-535, Ser-509, Ser-520. (Item 10) A nucleic acid construct comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) and a second nucleic acid sequence encoding a transcription factor as defined in any of the above items. (Item 11) A nucleic acid construct as described in item 10, having the following structure: CAR-coexpr-TF; or TF-coexpr-CAR It has, in the formula: CAR is the nucleic acid sequence that encodes the CAR; coexpr is a nucleic acid sequence that enables the co-expression of the CAR and the transcription factor; and A nucleic acid construct in which TF is the nucleic acid sequence encoding the transcription factor. (Item 12) A nucleic acid construct as described in item 11, wherein coexpr encodes a sequence containing a self-cleaving peptide. (Item 13) A nucleic acid sequence kit comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) and a second nucleic acid sequence encoding a transcription factor as defined in any of items 1 through 9. (Item 14) A vector containing a nucleic acid construct as described in any of items 10 to 12. (Item 15) A vector kit comprising: a first vector containing a first nucleic acid sequence encoding a chimeric antigen receptor (CAR); and a second vector containing a second nucleic acid sequence encoding a transcription factor as defined in any of items 1 through 9. (Item 16) A method for producing cells as described in any of items 1 to 9, comprising the step of introducing into cells a nucleic acid construct as described in any of items 10 to 12, a nucleic acid sequence kit as described in item 13, a vector as described in item 14, or a vector kit as described in item 15. (Item 17) The method according to item 16, wherein the cells are derived from a sample isolated from the subject. (Item 18) A pharmaceutical composition comprising multiple types of cells as described in any of items 1 to 9. (Item 19) A method for treating and / or preventing a disease, comprising the step of administering a pharmaceutical composition described in item 18 to a subject. (Item 20) The following steps: (i) to isolate a sample containing cells from the subject; (ii) Transducing or transfecting the cells using a nucleic acid construct described in any of items 10 to 12, a nucleic acid sequence kit described in item 13, a vector described in item 14, or a vector kit described in item 15; (iii) Administering the cells derived from (ii) to the subject. The method described in item 19, including the method described in item 19. (Item 21) The method according to item 19 or 20, wherein the disease is cancer. (Item 22) A pharmaceutical composition as described in item 18 for use in the treatment and / or prevention of disease. (Item 23) Use of cells described in any of items 1 to 9 in the manufacture of a medicine for the treatment and / or prevention of disease.

Claims

[Claim 1] A cell containing a first exogenous nucleic acid molecule encoding a chimeric antigen receptor (CAR) and a second exogenous nucleic acid molecule encoding FOXO1. [Claim 2] A nucleic acid construct comprising a first nucleic acid encoding a chimeric antigen receptor (CAR) and a second nucleic acid encoding FOXO1. [Claim 3] The following structure: CAR-coexpr-TF, or TF-coexpr-CAR It has, in the formula: CAR is a nucleic acid that codes for the aforementioned CAR, coexpr is a nucleic acid that enables the co-expression of CAR and FOXO1. The nucleic acid construct according to claim 2, wherein TF is a nucleic acid encoding FOXO1. [Claim 4] The nucleic acid construct according to claim 3, wherein coexpr encodes an amino acid containing a self-cleaving peptide. [Claim 5] A nucleic acid kit containing a first nucleic acid encoding a chimeric antigen receptor (CAR) and a second nucleic acid encoding FOXO1. [Claim 6] A vector comprising the nucleic acid construct according to any one of claims 2 to 4. [Claim 7] A vector kit comprising a first vector containing a first nucleic acid encoding a chimeric antigen receptor (CAR), and a second vector containing a second nucleic acid encoding FOXO1. [Claim 8] An ex vivo method for producing the cells described in claim 1, comprising the step of introducing into cells a nucleic acid construct described in any of claims 2 to 4, a nucleic acid kit described in claim 5, a vector described in claim 6, or a vector kit described in claim 7. [Claim 9] The method according to claim 8, wherein the cells are derived from a sample isolated from a subject. [Claim 10] A pharmaceutical composition comprising multiple types of cells as described in claim 1. [Claim 11] The pharmaceutical composition according to claim 10 for use in the treatment and / or prevention of disease. [Claim 12] Use of the cells according to claim 1 in the manufacture of a pharmaceutical product for the treatment and / or prevention of disease. [Claim 13] The pharmaceutical composition according to claim 11, wherein the disease is cancer. [Claim 14] The use according to claim 12, wherein the disease is cancer.

Images