Engineered human T cells comprising a switchable chimeric antigen cell surface receptor and methods for making them

Abstract

The present invention relates to engineered human T cells comprising a switchable chimeric antigen cell surface receptor, a pharmaceutical composition comprising the engineered human T cells, a kit comprising the engineered human T cells and a targeting module, and a method for producing the engineered human T cells.

Description

Technical Field

[0001] The present invention relates to engineered human T cells comprising a switchable chimeric antigen cell surface receptor, a pharmaceutical composition comprising the engineered human T cells, a kit comprising the engineered human T cells and a targeting module, and a method for producing the engineered human T cells.

Background Art

[0002] A chimeric antigen receptor (CAR) is an artificial receptor composed of a binding moiety that confers antigen specificity and one or several signaling chains derived from immunoreceptors (Cartellieri et al., 2010). Immunocytes genetically modified to express a CAR can be used to bind to cells or tissue structures expressing an appropriate target of the CAR binding moiety. By crosslinking, a signaling pathway via the CAR signaling chain is induced, which changes the biological properties of the CAR-grafted immunocytes. Conversely, activation of CAR in genetically modified regulatory T cells (Tregs) results in activation of Treg-specific immunomodulatory and immunosuppressive mechanisms such as secretion of interleukin (IL)-10 or tumor growth factor beta (TGF-β). Adoptive transfer of immunocytes engineered with a chimeric antigen receptor (CAR) is currently considered a very promising therapeutic option for treating malignant, infectious or autoimmune diseases that are not curable by other methods.

[0003] However, conventional CAR technology is associated with several important problems that need to be solved before this treatment method can be widely applied in clinical treatment. First, several safety issues need to be addressed. So far, the immune response of T cells engineered with conventional CARs has been difficult to control after injection into patients. The severe adverse event rate is high (Titov et al., 2018). In particular, unexpected target gene expression in normal tissues can trigger a rapid and severe immune reaction of engineered T cells against normal cells, which can result in severe side effects (Morgan et al., 2010). Furthermore, since CAR-T cells are a new class of self-propagating cell drugs, the injected T cells can proliferate actively in the presence of a large tumor burden, causing tumor lysis syndrome, cytokine release syndrome, and macrophage activation syndrome (Brudno and Kochenderfer, 2016). Another drawback of conventional CAR technology is the limited retargeting of engineered T cells to a single antigen. Such a monotherapy approach carries the risk of generating tumor escape variants that have lost their target antigen during treatment. The emergence of tumor escape variants several months after conventional CAR T cell therapy has already been observed in clinical trials (Sotillo et al., 2015). Collectively, these obstacles limit the application of CAR T cells to a very narrow indication. In fact, examples of clinical efficacy have, so far, been mostly seen in CD19-targeted CAR T cells and BCMA-targeted CAR T cells.

[0004] The modular, switchable "universal" chimeric antigen receptor T cell (UniCAR) approach can overcome these limitations by separating the antigen recognition and activation domains of the CAR into two distinct operating units. T cells are engineered to express a CAR with a universal binding domain that recognizes a tag (Cartellieri et al., 2016). Antigen specificity is provided by a soluble adapter molecule consisting of an antigen-binding domain fused to a tag recognized by the universal CAR. Cartellieri et al. have described the in vitro and in vivo treatment of CD33-positive and / or CD123-positive acute myeloid leukemia cells.

[0005] Subsequent to the UniCAR approach (EP2990416A1 (Patent Document 1)) for recognizing various antigens, the reversed universal CAR (RevCAR) approach is known, which promotes the binding of immune cells engineered to express a RevCAR containing a tag to target cells via an adapter molecule containing a tag-binding domain and a target cell-binding domain (EP3581200A1 (Patent Document 2) and WO2019 / 238722A1 (Patent Document 3)). WO2019 / 238722A1 (Patent Document 3) discloses an extracellular LA / SSB-derived tag and an adapter molecule containing a CD123 scFv and an scFv (5B9 or 7B6) that binds to the tag, which is added to crosslink the CAR and tumor cells to produce antigen-specific cytotoxicity.

[0006] Liu et al. have described a switchable and programmable universal CAR for CAR T therapy. D3 outlines different approaches for engineering a switchable CAR that includes an anti-5B9 UniCAR that targets the 5B9 tag on a bispecific switch molecule that targets cancer antigens such as CD123 or CD33 (Liu et al., 2019).

[0007] Furthermore, switchable CAR-T approaches such as UniCAR or RevCAR offer the possibility of pausing CAR T cells between activation and stimulation cycles by interrupting the administration of soluble adapter molecules. This is expected to prevent the exhaustion seen with continuous stimulation of conventional CAR T cells and thereby improve survival (Weber et al., 2021).

[0008] To avoid the problem of immune rejection, an autologous transplantation approach using the subject's own cells as a cell source for treatment is applied in the manipulation of immune cells with UniCAR or RevCAR. However, this approach is time-consuming and costly, and patients usually undergo several pretreatment lines, which can have an adverse effect on the functional characteristics of immune cells.

[0009] To solve this problem, the transfer of allogeneic cells to the subject can be utilized. However, the use of allogeneic cells is limited due to the problem of rejection by the recipient subject's immune cells, which recognize the transplanted cells as foreign and initiate an attack. Allogeneic cell immune rejection usually results from a mismatch of major histocompatibility complex (MHC) molecules between the donor and the recipient. MHC molecules exist within the human population in the form of many genetic variants of any given MHC gene, which encode different forms of MHC proteins. MHC class I molecules in humans include HLA-A molecules, HLA-B molecules, and HLA-C molecules, which are expressed on all nucleated cells and also on platelets, presenting epitopes and activating cytotoxic T cells. MHC class II molecules include HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules, which are expressed only on specialized antigen-presenting cells such as macrophages, B cells, and dendritic cells (DCs), presenting antigens and activating helper T cells, which then transmit signals to B cells to produce antibodies.

[0010] Minor differences in MHC alleles between individuals can activate recipient T cells upon transfer of donor T cells. During T cell development, an individual's T cell repertoire becomes tolerant to its own MHC molecules, but T cells that recognize MHC molecules of another individual can persist in the circulation and are called alloreactive T cells. Alloreactive T cells can be activated by the presence of cells of another individual expressing MHC molecules in the body, causing graft-versus-host disease and graft rejection.

[0011] Methods and compositions for reducing the susceptibility of allogeneic cells to rejection, including reducing the MHC protein expression of cells to avoid recipient T cell responses, are of interest. Indeed, genetically modifying allogeneic cells for transplantation into a subject is not possible because multiple gene edits are required to reduce the expression of all MHC proteins. At the same time, however, other harmful recipient immune responses are also avoided. One approach is to utilize extended immunosuppression to avoid immune rejection and increase survival (Neelapu et al., 2020). Although responses have been shown in the clinic, this approach increases the risk of infection, the persistence of adoptive T cells is uncertain, and it depends on ongoing immunosuppression. Other strategies include knocking out the β2M gene to remove all HLA class I molecules (HLA-A, HLA-B, and HLA-C) from the T cell surface and avoid recognition by host CD8+ T cells. However, the absence of HLA class I sends a "missing self" signal to host NK cells, and these cells immediately eliminate the β2M KO T cells. To address this, researchers have investigated the overexpression of the non-polymorphic HLA-E gene, which can provide a partial and not complete defense from NK cell-mediated lysis (Zhang, 2021). Ellis et al. have described the genetic manipulation of T cells for immunotherapy, particularly the genetic regulation of engineered T cells aimed at improving safety and efficacy, particularly the genetic deletion of the expression of both HLA class I and HLA class II by targeting B2M and CIITA respectively to extend the survival of chimeric antigen receptor (CAR) T cells in preclinical models (Ellis et al., 2021). However, allogeneic CART cells without HLA molecules may be killed by the recipient's natural killer cells, and therefore it has been disclosed that it may be necessary to overexpress HLA-E or other non-classical major histocompatibility complex (MHC) molecules as therapeutics.

[0012] Qasim et al. described T cells engineered to express a chimeric antigen receptor (CAR19) against the B cell antigen CD19 by lentiviral transduction of donor cells with matched non-human leukocyte antigens and simultaneous transcriptional activator-like effector nuclease (TALEN)-mediated gene editing of the T cell receptor α-chain and CD52 locus (Qasim et al., 2017).

Prior Art Documents

Patent Documents

[0013]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0014] Accordingly, an object of the present invention is to provide engineered allogeneic immune cells containing a switchable chimeric antigen cell surface receptor that preferably has better survival by reducing rejection by immune cells of a recipient subject and combining avoidance of rejection with avoidance of continuous stimulation that causes exhaustion.

[0015] According to the present invention, the problem is solved by the engineered human T cells, kits, and methods for producing engineered human T cells described in the independent claims.

[0016] Advantageous embodiments of the present invention are indicated in the dependent claims.

Means for Solving the Problems

[0017] A first aspect of the present invention is i. Reduced or eliminated surface expression of the endogenous T cell receptor alpha chain due to genetic modification in the T cell receptor alpha chain (TRAC) gene ii. Reduced or eliminated surface expression of HLA-A compared to unmodified T cells due to genetic modification in the HLA-A gene iii. Reduced or eliminated surface expression of HLA class II due to genetic modification in the CIITA gene, and iv. A switchable chimeric antigen cell surface receptor comprising: · A tag-binding domain or tag · An extracellular hinge and transmembrane domain, and · A signaling domain Provided are engineered human T cells comprising the above.

[0018] Advantageously, the engineered human T cells according to the invention, when used in combination with a targeting module, actively target antigens expressed by tumor cells and can induce a significant anti-tumor response. The anti-tumor response of the switchable chimeric antigen receptor is induced only in the presence of the targeting module. The effect can be reversibly interrupted by stopping the administration of the targeting module. More advantageously, the pharmacokinetic and pharmacodynamic half-lives of the targeting module are short, resulting in a rapid and reversible switch-off mechanism of the intervening immune response. More advantageously, the three knockouts (TRAC, HLA-A gene, CIITA) according to the invention reduce rejection by the recipient subject's immune cells and, in particular, reduce the chance of graft-versus-host disease (GvHD) of allogeneic T cells.

[0019] As used herein, the term "domain" refers to a portion of a protein sequence that can exist and function independently of the rest of the protein.

[0020] According to the present invention, the engineered human T cells have reduced or eliminated surface expression of endogenous TRAC due to genetic modification in the T cell receptor alpha chain (TRAC) gene. Preferably, the reduced or eliminated surface expression of endogenous TRAC prevents graft-versus-host disease.

[0021] In a plurality of embodiments, the genetic modification in the TRAC gene comprises at least one nucleotide among genomic coordinates chr14:22547524 to chr14:22547544. In a plurality of embodiments, the genetic modification in the TRAC gene comprises at least 10 or at least 15 consecutive nucleotides among the genomic coordinates.

[0022] In a plurality of embodiments, the genetic modification in the TRAC gene comprises at least one nucleotide of an exon of the TRAC gene.

[0023] In a plurality of embodiments, the genetic modification in the TRAC gene comprises at least one insertion, deletion, substitution, or deamination of at least one nucleotide among the genomic coordinates.

[0024] In a plurality of embodiments, the genetic modification in the TRAC gene comprises indels.

[0025] In a plurality of embodiments, TRAC gene expression is reduced or eliminated by a gene editing system that binds to a TRAC genomic target sequence comprising at least 5 consecutive nucleotides among the genomic coordinates, preferably among genomic coordinates chr14:22547524 to chr14:22547544.

[0026] According to the present invention, the engineered human T cells have reduced or eliminated surface expression of HLA-A compared to unmodified T cells due to genetic modification in the HLA-A gene.

[0027] In multiple embodiments, the genetic modification in the HLA-A gene comprises at least one nucleotide among genomic coordinates selected from chr6:29942854 to chr6:29942913 and chr6:29943518 to chr6:29943619.

[0028] In multiple embodiments, the T cells are homozygous for the HLA-B genotype and / or homozygous for the HLA-C genotype. Preferably, the T cells are homozygous for both the HLA-B genotype and the HLA-C genotype.

[0029] In multiple embodiments, the expression of at least one HLA-A allele selected from HLA-A1, HLA-A2, HLA-A3, HLA-A11, and HLA-A24 is reduced or eliminated in the T cells.

[0030] In multiple embodiments, the genetic modification in the HLA-A gene comprises at least one nucleotide among genomic coordinates chr6:29942864 to chr6:29942903, preferably chr6:29942876 to chr6:29942897.

[0031] In multiple embodiments, the genetic modification in the HLA-A gene comprises at least one nucleotide among genomic coordinates chr6:29943528 to chr6:29943609, preferably chr6:29943528 to chr6:29943550.

[0032] In multiple embodiments, the genetic modification in the HLA-A gene comprises at least 5, 6, 7, 8, 9, or 10 consecutive nucleotides among the genomic coordinates, preferably at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides among the genomic coordinates.

[0033] In multiple embodiments, the genetic modification in the HLA-A gene comprises at least one C to T substitution or at least one A to G substitution among the genomic coordinates.

[0034] In multiple embodiments, the genetic modification in the HLA-A gene comprises an indel.

[0035] In multiple embodiments, HLA-A expression is reduced or eliminated by a gene editing system that binds to an HLA-A genomic target sequence comprising at least 5 consecutive nucleotides among the genomic coordinates preferably selected from chr6:29942864~29942884, chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046.

[0036] In multiple embodiments, the HLA-B allele is selected from any one of the following HLA-B alleles: HLA-B*07:02; HLA-B*08:01; HLA-B*44:02; HLA-B*35:01; HLA-B*40:01; HLA-B*57:01; HLA-B*14:02; HLA-B*15:01; HLA-B*13:02; HLA-B*44:03; HLA-B*38:01; HLA-B*18:01; HLA-B*44:03; HLA-B*51:01; HLA-B*49:01; HLA-B*15:01; HLA-B*18:01; HLA-B*27:05; HLA-B*35:03; HLA-B*18:01; HLA-B*52:01; HLA-B*51:01; HLA-B*37:01; HLA-B*53:01; HLA-B*55:01; HLA-B*44:02; HLA-B*44:03; HLA-B*35:02; HLA-B*15:01; and HLA-B*40:02.

[0037] In multiple embodiments, the HLA-C allele is selected from any one of the following HLA-C alleles: HLA-C*07:02; HLA-C*07:01; HLA-C*05:01; HLA-C*04:01 HLA-C*03:04; HLA-C*06:02; HLA-C*08:02; HLA-C*03:03; HLA-C*06:02; HLA-C*16:01; HLA-C*12:03; HLA-C*07:01; HLA-C*04:01; HLA-C*15:02; HLA-C*07:01; HLA-C*03:04; HLA-C*12:03; HLA-C*02:02; HLA-C*04:01; HLA-C*05:01; HLA-C*12:02; HLA-C*14:02; HLA-C*06:02; HLA-C*04:01; HLA-C*03:03; HLA-C*07:04; HLA-C*07:01; HLA-C*04:01; HLA-C*04:01; and HLA-C*02:02.

[0038] In multiple embodiments, the HLA-B allele is selected from any one of the following HLA-B alleles: HLA-B*07:02; HLA-B*08:01; HLA-B*44:02; HLA-B*35:01; HLA-B*40:01; HLA-B*57:01; HLA-B*14:02; HLA-B*15:01; HLA-B*13:02; HLA-B*44:03; HLA-B*38:01; HLA-B*18:01; HLA-B*44:03; HLA-B*51:01; HLA-B*49:01; HLA-B*15:01; HLA-B*18:01; HLA-B*27:05; HLA-B*35:03; HLA-B*18:01; HLA-B*52:01; HLA-B*51:01; HLA-B*37:01; HLA-B*53:01; HLA-B*55:01; HLA-B*44:02; HLA-B*44:03; HLA-B*35:02; HLA-B*15:01; and HLA-B*40:02, and the HLA-C allele is selected from any one of the following HLA-C alleles: HLA-C*07:02; HLA-C*07:01; HLA-C*05:01; HLA-C*04:01 HLA-C*03:04; HLA-C*06:02; HLA-C*08:02; HLA-C*03:03; HLA-C*06:02; HLA-C*16:01; HLA-C*12:03; HLA-C*07:01; HLA-C*04:01; HLA-C*15:02; HLA-C*07:01; HLA-C*03:04; HLA-C*12:03; HLA-C*02:02; HLA-C*04:01; HLA-C*05:01; HLA-C*12:02; HLA-C*14:02; HLA-C*06:02; HLA-C*04:01; HLA-C*03:03; HLA-C*07:04; HLA-C*07:01; HLA-C*04:01; HLA-C*04:01; and HLA-C*02:02.

[0039] In a preferred embodiment, the HLA-B allele and the HLA-C allele are the following HLA-B allele and HLA-C allele: HLA-B * 07:02 and HLA-C *07:02, HLA-B*08:01 and HLA-C*07:01; HLA-B*44:02 and HLA-C*05:01; HLA-B*35:01 and HLA-C*04:01; HLA-B*40:01 and HLA-C*03:04; HLA-B*57:01 and HLA-C*06:02; HLA-B*14:02 and HLA-C*08:02; HLA-B*15:01 and HLA-C*03:03; HLA-B*13:02 and HLA-C*06:02; HLA-B*44:03 and HLA-C*16:01; HLA-B*38:01 and HLA-C*12:03; HLA-B*18:01 and HLA-C*07:01; HLA-B*44:03 and HLA-C*04:01; HLA-B*51:01 and HLA-C*15:02; HLA-B*49:01 and HLA-C*07:01; HLA-B*15:01 and HLA-C*03:04; HLA-B*18:01 and HLA-C*12:03; HLA-B*27:05 and HLA-C*02:02; HLA-B*35:03 and HLA-C*04:01; HLA-B*18:01 and HLA-C*05:01; HLA-B*52:01 and HLA-C*12:02; HLA-B*51:01 and HLA-C*14:02; HLA-B*37:01 and HLA-C*06:02; HLA-B*53:01 and HLA-C*04:01; HLA-B*55:01 and HLA-C*03:03; HLA-B*44:02 and HLA-C*07:04; HLA-B*44:03 and HLA-C*07:01; HLA-B*35:02 and HLA-C*04:01; HLA-B * 15:01 and HLA-C * 04:01, as well as HLA-B * 40:02 and HLA-C * is selected from any one of them.

[0040] In multiple embodiments, the T cells further have a gene modification in the HLA-B gene such that the surface expression of HLA-B is reduced or eliminated as compared to unmodified T cells, and / or have a gene modification in the HLA-C gene such that the surface expression of HLA-C is reduced or eliminated as compared to unmodified T cells.

[0041] In a further embodiment, the T cells further have a gene modification in the HLA-B gene such that the surface expression of HLA-B is reduced or eliminated as compared to unmodified T cells, and the T cells are homozygous for the HLA-C genotype.

[0042] In an alternative embodiment, the T cells further have a gene modification in the HLA-C gene such that the surface expression of HLA-C is reduced or eliminated as compared to unmodified T cells, and the T cells are homozygous for the HLA-B genotype.

[0043] In a further alternative embodiment, the T cells have a gene modification in the HLA-B gene such that the surface expression of HLA-B is reduced or eliminated as compared to unmodified T cells, and have a gene modification in the HLA-C gene such that the surface expression of HLA-C is reduced or eliminated as compared to unmodified T cells.

[0044] According to the present invention, the engineered human T cells have a gene modification in the CIITA gene such that the surface expression of HLA class II is reduced or eliminated.

[0045] In multiple embodiments, the gene modification in the CIITA gene comprises at least one nucleotide of a splice site within genomic coordinates chr16:10902171 to chr16:10923242.

[0046] In multiple embodiments, the genetic modification in the CIITA gene includes the modification of at least one nucleotide at the splice acceptor site, and preferably, one nucleotide is A or G or T.

[0047] In multiple embodiments, the genetic modification in the CIITA gene includes the modification of the splice site boundary nucleotides.

[0048] In multiple embodiments, the genetic modification in the CIITA gene includes at least 5, 6, 7, 8, 9, or 10 consecutive nucleotides within the genomic coordinates chr16:10902171~chr16:10923242.

[0049] In multiple embodiments, the genetic modification in the CIITA gene includes at least one C to T substitution or at least one A to G substitution within the genomic coordinates chr16:10902171~chr16:10923242.

[0050] In multiple embodiments, the gene modification in the CIITA gene is located at chr16:10895410-10895430, chr16:10898649-10898669, chr16:10898658-10898678, chr16:10902171-10902191, chr16:10902173-10902193, chr16:10902174-10902194, chr16:10902179-10902199, chr16:10902183-10902203, chr16:10902184-10902204, chr16:10902644-10902664, chr16:10902779-10902799, chr16:10902788-10902808, chr16:10902789-10902809, chr16:10902790-10902810, chr16:10902795-10902815, chr16:10902799-10902819, chr16:10903708-10903728, chr16:10903713-10903733, chr16:10903718-10903738, chr16:10903721-10903741, chr16:10903723-10903743, chr16:10903724-10903744, chr16:10903873-10903893, chr16:10903878-10903898, chr16:10903905-10903925, chr16:10903906-10903926, chr16:10904736-10904756, chr16:10904790-10904810, chr16:10904811-10904831, chr16:10906481-10906501, chr16:10906485-10906505, chr16:10906486-10906506, chr16:10906487-10906507, chr16:10906492-10906512, chr16:10908127-10908147, chr16:10908130-10908150, chr16:10908131-10908151, chr16:10908132-10908152, chr16:10908137-10908157, chr16:10908138-10908158,Genomic coordinates selected from chr16:10908139-10908159, chr16:10909006-10909026, chr16:10909007-10909027, chr16:10909018-10909038, chr16:10909021-10909041, chr16:10909022-10909042, chr16:10909172-10909192, chr16:10910165-10910185, chr16:10910176-10910196, chr16:10910186-10910206, chr16:10915547-10915567, chr16:10915551-10915571, chr16:10915552-10915572, chr16:10915567-10915587, chr16:10916348-10916368, chr16:10916359-10916379, chr16:10916362-10916382, chr16:10916449-10916469, chr16:10916450-10916470, chr16:10916455-10916475, chr16:10916456-10916476, chr16:10918423-10918443, chr16:10918504-10918524, chr16:10918511-10918531, chr16:10918512-10918532, chr16:10918539-10918559, chr16:10922153-10922173, chr16:10922478-10922498, chr16:10922487-10922507, chr16:10922499-10922519, chr16:10923205-10923225, chr16:10923214-10923234, chr16:10923218-10923238, chr16:10923219-10923239, chr16:10923220-10923240, chr16:10923221-10923241, and chr16:10923222-10923242 include indels, C to T substitutions, or A to G substitutions.,

[0051] In multiple embodiments, the genetic modification in the CIITA gene is located at chr16:10906485-10906505, chr16:10906486-10906506, chr16:10906487-10906507, chr16:10906492-10906512, chr16:10908127-10908147, chr16:10908130-10908150, chr16:1090813l-10908151, chr16:10908132-10908152, chr16:10908137-10908157, chr16:10908138-10908158, chr16:10908139-10908159, chr16:10909006-10909026, chr16:10909007-10909027, chr16:10909018-10909038, chr16:10909021-10909041, chr16:10909022-10909042, chr16:10909172-10909192, chr16:10910165-10910185, chr16:10910176-10910196, chr16:10910186-10910206, chr16:10915547-10915567, chr16:10915551-10915571, chr16:10915552-10915572, chr16:10915567-10915587, chr16:10916348-10916368, chr16:10916359-10916379, chr16:10916362-10916382, chr16:10916449-10916469, chr16:10916450-10916470, chr16:10916455-10916475, chr16:10916456-10916476, chr16:10918423-109l8443, chr16:10918504-10918524, chr16:10918511-10918531, chr16:10918512-10918532, chr16:10918539-10918559, chr16:10922153-10922173, chr16:10922478-10922498, chr16:10922487-10922507, chr16:10922499-10922519,Genomic coordinates selected from chr16:10923205-10923225, chr16:10923214-10923234, chr16:10923218-10923238, chr16:10923219-10923239, chr16:10923220-10923240, chr16:10923221-10923241, and chr16:10923222-10923242, preferably chr16:10908132-10908152, chr16:10908131-10908151, chr16:10916456-10916476, chr16:10918504-10918524, chr16:10909022-10909042, chr16:10918512-10918532, chr16:10918511-10918531, chr16:10895742-10895762, chr16:10916362-10916382, chr16:10916455-10916475, chr16:10909172-10909192, chr16:10906492-10906512, chr16:10909006-10909026, chr16:10922478-10922498, chr16:10895747-10895767, chr16:10916348-10916368, chr16:10910186-10910206, chr16:10906481-10906501, chr16:10909007-10909027, chr16:10895410-10895430, and chr16:10908130-10908150, and contains at least five consecutive nucleotides among the genomic coordinates.

[0052] In a plurality of embodiments, the genetic modification in the CIITA gene comprises at least 10 or at least 15 consecutive nucleotides among the genomic coordinates.

[0053] In a plurality of embodiments, the genetic modification in the CIITA gene comprises an indel.

[0054] In multiple embodiments, the genetic modification in the CIITA gene includes at least one nucleotide of a splice site within the genomic coordinates chr16:10902171 to chr16:10923242.

[0055] In multiple embodiments, the genetic modification in the CIITA gene includes the modification of at least one nucleotide of a splice acceptor site, preferably, one nucleotide is A or G or T.

[0056] In multiple embodiments, the genetic modification in the CIITA gene includes the modification of splice site boundary nucleotides.

[0057] In multiple embodiments, the genetic modification in the CIITA gene includes at least 5, 6, 7, 8, 9, or 10 consecutive nucleotides within the genomic coordinates chr16:10902171 to chr16:10923242.

[0058] In multiple embodiments, the genetic modification in the CIITA gene includes at least one C to T substitution or at least one A to G substitution within the genomic coordinates chr16:10902171 to chr16:10923242.

[0059] In multiple embodiments, the gene modification in the CIITA gene is at chr16:10895410-10895430, chr16:10898649-10898669, chr16:10898658-10898678, chr16:10902171-10902191, chr16:10902173-10902193, chr16:10902174-10902194, chr16:10902179-10902199, chr16:10902183-10902203, chr16:10902184-10902204, chr16:10902644-10902664, chr16:10902779-10902799, chr16:10902788-10902808, chr16:10902789-10902809, chr16:10902790-10902810, chr16:10902795-10902815, chr16:10902799-10902819, chr16:10903708-10903728, chr16:10903713-10903733, chr16:10903718-10903738, chr16:10903721-10903741, chr16:10903723-10903743, chr16:10903724-10903744, chr16:10903873-10903893, chr16:10903878-10903898, chr16:10903905-10903925, chr16:10903906-10903926, chr16:10904736-10904756, chr16:10904790-10904810, chr16:10904811-10904831, chr16:10906481-10906501, chr16:10906485-10906505, chr16:10906486-10906506, chr16:10906487-10906507, chr16:10906492-10906512, chr16:10908127-10908147, chr16:10908130-10908150, chr16:10908131-10908151, chr16:10908132-10908152, chr16:10908137-10908157, chr16:10908138-10908158,Genomic coordinates selected from chr16:10908139-10908159, chr16:10909006-10909026, chr16:10909007-10909027, chr16:10909018-10909038, chr16:10909021-10909041, chr16:10909022-10909042, chr16:10909172-10909192, chr16:10910165-10910185, chr16:10910176-10910196, chr16:10910186-10910206, chr16:10915547-10915567, chr16:10915551-10915571, chr16:10915552-10915572, chr16:10915567-10915587, chr16:10916348-10916368, chr16:10916359-10916379, chr16:10916362-10916382, chr16:10916449-10916469, chr16:10916450-10916470, chr16:10916455-10916475, chr16:10916456-10916476, chr16:10918423-10918443, chr16:10918504-10918524, chr16:10918511-10918531, chr16:10918512-10918532, chr16:10918539-10918559, chr16:10922153-10922173, chr16:10922478-10922498, chr16:10922487-10922507, chr16:10922499-10922519, chr16:10923205-10923225, chr16:10923214-10923234, chr16:10923218-10923238, chr16:10923219-10923239, chr16:10923220-10923240, chr16:10923221-10923241, and chr16:10923222-10923242 include indels, C to T substitutions, or A to G substitutions.,

[0060] In multiple embodiments, the genetic modification in the CIITA gene is at chr16:10906485-10906505, chr16:10906486-10906506, chr16:10906487-10906507, chr16:10906492-10906512, chr16:10908127-10908147, chr16:10908130-10908150, chr16:1090813l-10908151, chr16:10908132-10908152, chr16:10908137-10908157, chr16:10908138-10908158, chr16:10908139-10908159, chr16:10909006-10909026, chr16:10909007-10909027, chr16:10909018-10909038, chr16:10909021-10909041, chr16:10909022-10909042, chr16:10909172-10909192, chr16:10910165-10910185, chr16:10910176-10910196, chr16:10910186-10910206, chr16:10915547-10915567, chr16:10915551-10915571, chr16:10915552-10915572, chr16:10915567-10915587, chr16:10916348-10916368, chr16:10916359-10916379, chr16:10916362-10916382, chr16:10916449-10916469, chr16:10916450-10916470, chr16:10916455-10916475, chr16:10916456-10916476, chr16:10918423-109l8443, chr16:10918504-10918524, chr16:10918511-10918531, chr16:10918512-10918532, chr16:10918539-10918559, chr16:10922153-10922173, chr16:10922478-10922498, chr16:10922487-10922507, chr16:10922499-10922519,Genomic coordinates selected from chr16:10923205-10923225, chr16:10923214-10923234, chr16:10923218-10923238, chr16:10923219-10923239, chr16:10923220-10923240, chr16:10923221-10923241, and chr16:10923222-10923242, preferably chr16:10908132-10908152, chr16:10908131-10908151, chr16:10916456-10916476, chr16:10918504-10918524, chr16:10909022-10909042, chr16:10918512-10918532, chr16:10918511-10918531, chr16:10895742-10895762, chr16:10916362-10916382, chr16:10916455-10916475, chr16:10909172-10909192, chr16:10906492-10906512, chr16:10909006-10909026, chr16:10922478-10922498, chr16:10895747-10895767, chr16:10916348-10916368, chr16:10910186-10910206, chr16:10906481-10906501, chr16:10909007-10909027, chr16:10895410-10895430, and chr16:10908130-10908150, and contain at least five consecutive nucleotides among the genomic coordinates.

[0061] In a plurality of embodiments, the genetic modification in the CIITA gene contains at least 10 or at least 15 consecutive nucleotides among the genomic coordinates.

[0062] According to the present invention, the engineered human T cells contain a switchable chimeric antigen cell surface receptor that includes a tag-binding domain or tag, an extracellular hinge and transmembrane domain, and a signaling domain.

[0063] As used herein, the term "switchable chimeric antigen receptor" refers to an artificial chimeric fusion protein, particularly a receptor comprising a tag-binding domain or tag, an extracellular hinge and transmembrane domain, and a signaling domain (Figure 1). The domains can be derived from a plurality of different sources, and thus the receptor is called chimeric. Advantageously, the receptor can bind to different targeting modules via a tag-binding domain or tag.

[0064] In a plurality of embodiments, the engineered human T cells comprise an exogenous nucleotide sequence encoding a switchable CAR expressed on the surface of the engineered T cells.

[0065] Advantageously, the engineered human T cells comprising a nucleotide sequence encoding a switchable CAR express the switchable CAR and have binding specificity for the tag-binding domain or tag of the targeting module, which in turn binds to an antigen on the target cell.

[0066] In a preferred embodiment, the switchable chimeric antigen cell surface receptor is a reversed universal chimeric antigen cell surface receptor comprising a tag, an extracellular hinge and transmembrane domain, and a signaling domain.

[0067] Advantageously, the engineered human T cells comprising the reversed universal chimeric antigen cell surface receptor are less exhausted and show less phenotypic differentiation after generation.

[0068] As used herein, the term "tag" refers to a marker, particularly a peptide sequence or organic molecule, that binds to a peptide or protein and enables them to bind to specific atoms, ions or molecules, particularly a tag-binding domain.

[0069] In multiple embodiments, the tag is selected from a fluorescent label, such as an organic molecule including FITC (fluorescein isothiocyanate), and biotin.

[0070] In multiple embodiments, the tag is a peptide epitope tag. In further embodiments, the tag comprises from 10 to 20 amino acids.

[0071] In multiple embodiments, the peptide epitope tag is a short linear peptide sequence preferably according to the myc tag, His tag, SEQ ID NO: 7, SEQ ID NO: 8 or mutants thereof, derived from the yeast transcription factor GCN4; a leucine zipper sequence, preferably SYNZIP 1 - SYNZIP48, BATF, FOS, ATF4, ATF3, BACH1, JUND, NFE2L3, HEPTAD (Reinke et al., 2010), SEQ ID NO: 9 or SEQ ID NO: 10 or mutants thereof; or a short linear peptide sequence preferably according to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or mutants thereof, derived from a human nuclear protein, more preferably derived from the human La protein.

[0072] As used herein, the term "nuclear protein" refers to a protein found in the cell nucleus. Advantageously, the tag is a peptide sequence derived from a nuclear antigen, but under the circumstances of the native protein in a physiological state, it cannot be accessed or bound by the corresponding tag-binding domain. More advantageously, the tag is non-immunogenic. This minimizes the risk of uncontrolled on-target off-site toxicity by immune cells expressing CAR, such as the release of toxic levels of cytokines variously called cytokine storm or cytokine release syndrome (CRS).

[0073] In multiple embodiments, the His tag is an amino acid sequence composed of histidine residues, preferably in the range of 6 to 14 histidine residues.

[0074] In a preferred embodiment, the peptide epitope tag is a short linear peptide sequence derived from the myc tag, His tag, or the yeast transcription factor GCN4 preferably according to SEQ ID NO: 7 or SEQ ID NO: 8; a leucine zipper sequence, preferably the SYNZIP1 - SYNZIP48, BATF, FOS, ATF4, ATF3, BACH1, JUND, NFE2L3, HEPTAD (Reinke et al., 2010), or a sequence according to SEQ ID NO: 9 or SEQ ID NO: 10; or a short linear peptide sequence derived from a human nuclear protein, preferably from the human La protein, more preferably according to SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

[0075] Preferably, the peptide epitope tag derived from the human La protein is the human La epitope E5B9 according to SEQ ID NO: 11 or E7B6 according to SEQ ID NO: 12 or SEQ ID NO: 13, most preferably the human La epitope E5B9 according to SEQ ID NO: 11 or E7B6 according to SEQ ID NO: 13.

[0076] As used herein, the term "mutant" refers to a peptide or protein having at least 90% sequence identity, preferably at least 95% sequence identity, to a specified peptide or protein. Advantageously, the mutant may have one or more activities of the specified peptide or protein, and in particular, binds to the same tag - binding domain as the peptide epitope tag.

[0077] In multiple embodiments, the mutant is a truncated peptide or protein. As used herein, the term "truncated versions" refers to a shortened peptide or protein having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 90% chain length and 100% sequence identity, most preferably at least 95% chain length and 100% sequence identity to the designated peptide or protein. Advantageously, the truncated version has at least 90%, preferably at least 95% of the activity of the designated peptide or protein.

[0078] In multiple embodiments, the tag-binding domain is an antibody, antibody fragment, protein or peptide.

[0079] As used herein, the term "antibody" refers to a protein that binds an antigen via an antigen-binding fragment variable region (Fab). This is composed of one constant domain and one variable domain each of the heavy chain (V H ) and the light chain (V L ). As used herein, the term "antibody fragment or antigen-binding fragment" refers to a protein that includes at least V L or V H of an antibody. In one embodiment, the antibody fragment is selected from single-chain variable fragments (scFv), single-chain antibodies, F(ab’)2 fragments, Fab fragments, and fragments generated by Fab expression libraries or single-domain antibodies (nanobodies).

[0080] As used herein, the term "single-chain variable fragment (scFv)" refers to an artificial antibody fragment that includes the variable domain of the light chain and the variable domain of the heavy chain of an antibody that are covalently bound. In multiple embodiments, V L and V H of the antibody are covalently bound by a short-chain peptide of 10-25 amino acids. In a further embodiment, the short-chain peptide is between the N-terminus of V H and V LConnect to the C-terminus of or vice versa.

[0081] Preferably, V H and V L are linked via a glycine-serine linker having the structure (G x S y ), where x and y are selected from 1 to 10, preferably 1 to 5. Most preferred are repeats from 1 to 10 of the sequence G4S1 (SEQ ID NO: 4). Additionally, a linker composed of a peptide sequence that can increase the protease resistance of the antibody derivative is preferred. As used herein, the term "derivative" refers to a molecule having a high degree of structural identity to that molecule, preferably having the same scaffold, in which at least one atom, group of atoms, functional group or substructure is replaced by another atom, group of atoms, functional group or substructure, such as a hydroxy group. Advantageously, the derivative may have one or more activities of the designated molecule.

[0082] In multiple embodiments, the linker is SEQ ID NO: 5 or SEQ ID NO: 6.

[0083] In multiple embodiments, the tag-binding domain is an antibody or antibody fragment, protein or peptide that binds to a myc tag, His tag, short linear peptide sequence derived from the yeast transcription factor GCN4, leucine zipper sequence, or short linear peptide sequence derived from a human nuclear protein, preferably a human La protein.

[0084] In a preferred embodiment, the tag-binding domain is an antibody or antibody fragment.

[0085] In multiple embodiments, the antibody is obtained from an animal species, preferably a mammal such as a human, monkey, mouse, rat, rabbit, guinea pig, horse, cow, sheep, goat, pig, dog, or cat. Preferably, the antibody or antibody fragment is a human antibody, humanized antibody, or deimmunized antibody. Humanized antibodies can be prepared in various ways, such as by resurfacing and CDR grafting. In the case of resurfacing, a combination of molecular modeling, statistical analysis, and mutagenesis is used to modify all non-CDR regions on the surface of the antibody to resemble the surface of the antibody of the target organism. In CDR grafting, CDR regions according to the present invention with sequences similar to the original CDR regions are introduced into known human framework regions. Deimmunized antibodies can be obtained by specifically mutating residues to generate immunogenic hotspots predicted based on in silico peptide-MHC affinity prediction.

[0086] As used herein, the term "CDR (complementary determining region)" refers to the portion of the variable chain of an antibody or antibody fragment that binds to its specific antigen. An antibody contains three CDRs (CDR1, CDR2, and CDR3) that are non-contiguously arranged in the amino acid sequence of each variable domain, and thus, two variable domains (V H and V L ) contain six CDRs, which can contact the antigen.

[0087] In multiple embodiments, the antibody or antibody fragment is a polyclonal antibody, monoclonal antibody, or chimeric antibody, and in a chimeric antibody, the antigen-binding region of a non-human antibody has been transferred into the framework of a human antibody by recombinant DNA technology including in silico design.

[0088] In multiple embodiments, antibodies against selected tags or antigens can be produced by immunizing various hosts including, but not limited to, goats, rabbits, rats, mice, and humans, via injection of cells expressing a specific protein, DNA or RNA encoding the protein, the protein itself, or any portion, fragment or oligopeptide that retains the immunogenic properties of the protein.

[0089] In multiple embodiments, the tag-binding domain binds to a tag derived from the human nuclear La protein, and preferably, the tag-binding domain has the following sequences: DIVMTQSPDSLAVSLGERATINCX 24 SSQSLLNSRTX 35 KNYLAWYQQKPGQPPKLLIYWASTRX 61 SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLX 101 TFGGGTKVElK (SEQ ID NO: 14) (X 24 , X 35 , X 61 and X 101 are independently selected from the alpha amino acid residues that make up the protein), or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, to one of the sequences of SEQ ID NO: 16 or SEQ ID NO: 18 and is an antibody or antigen-binding fragment comprising V L .

[0090] In multiple embodiments, the tag-binding domain comprises an anti-La epitope scFv.

[0091] In some embodiments, X 24 to X 101 are selected as follows: X 24is selected from polar and / or positively charged residues such as serine, threonine, asparagine, glutamine, histidine, lysine and arginine, preferably lysine or arginine, X 35 is preferably selected from lysine and proline, X 61 is selected from polar and charged residues such as asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine and arginine, preferably glutamic acid and lysine, X 101 is selected from hydrophobic residues such as isoleucine, leucine, valine, alanine, methionine, phenylalanine, proline and tryptophan, preferably leucine or proline.

[0092] In a further embodiment, the tag-binding domain is an antibody or antigen-binding fragment comprising a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, to one of the sequences of SEQ ID NO: 15 or SEQ ID NO: 17.

[0093] Preferably, the tag-binding domain comprises a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, to the sequence according to SEQ ID NO: 15 (V H ) and SEQ ID NO: 16 (V L ) or to the sequence according to SEQ ID NO: 17 (V H ) and SEQ ID NO: 18 (V L ).

[0094] Most preferably, the tag-binding domain comprises an anti-La 5B9 scFv according to SEQ ID NO: 15 (V H ) and SEQ ID NO: 16 (V L ) or an anti-La 7B6 scFv according to SEQ ID NO: 17 (V H ) and SEQ ID NO: 18 (V L ).

[0095] As used herein, the term "extracellular hinge and transmembrane domain" refers to a tag-binding domain or a flexible peptide sequence linked to a tag, which anchors the switchable CAR within the cell membrane of a cell and protrudes from the surface of the cell for optimal binding to its specific targeting module.

[0096] In multiple embodiments, the extracellular hinge and transmembrane domain is selected from the hinge and transmembrane domains of the human CD28 molecule, CD8a chain, NK cell receptor, preferably the natural killer group NKG2D; or a portion of the constant region of an antibody, and combinations thereof. As used herein, the term "combinations thereof" refers to combinations of different hinge and transmembrane domains.

[0097] Examples of combinations of extracellular hinge and transmembrane domains include, but are not limited to, the extracellular hinge and transmembrane domain of CD28, the extracellular hinge and transmembrane domain of CD8 alpha, the transmembrane domain of CD8 alpha, CD28 or CD137 combined with the constant region of IgG1 or IgG4.

[0098] In a preferred embodiment, the extracellular hinge and transmembrane domain includes the extracellular domain of CD28, more preferably the extracellular domain in which the B7-binding site is mutated. Advantageously, the mutation of the B7-binding site renders ligand binding impossible. In a more preferred embodiment, the extracellular hinge and transmembrane domain includes the CD28 transmembrane domain.

[0099] As used herein, the term "signaling domain" refers to a peptide sequence that transmits a signal to a cell by cross-linking a cell (effector cell) expressing a switchable CAR to a human cell surface protein or protein complex (target cell). The cross-linking between the effector and target cells is mediated by a targeting module according to the present invention.

[0100] In multiple embodiments, the signaling domain is selected from the cytoplasmic regions of CD28, CD137 (4-1BB), CD134 (OX40), CD278 (ICOS); DAP10 and CD27, programmed cell death-1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), the cytoplasmic region of the CD3 chain, DAP12, CD122 (interleukin-2 receptor β), CD132 (interleukin-2 receptor γ), CD127 (interleukin-7 receptor α), CD360 (interleukin-21 receptor), activating Fc receptors, mutants thereof, and combinations thereof.

[0101] As used herein, the term "mutant" refers to a protein having at least 90% sequence identity, preferably at least 95% sequence identity, to a signaling domain. Advantageously, the mutant transmits a signal to a cell by cross-linking a cell expressing a switchable CAR (effector cell) to a human cell surface protein or protein complex (target cell) in the same manner as the designated signaling domain.

[0102] In multiple embodiments, the mutant is a truncated form. As used herein, the term "truncated form" refers to a shortened protein having at least 90% sequence identity, preferably at least 95% sequence identity, to a signaling domain, more preferably at least 90% chain length and 100% sequence identity, most preferably at least 95% chain length and 100% sequence identity. Advantageously, the truncated form has at least 90%, more preferably at least 95% activity of the designated signaling domain.

[0103] Guedan et al. describe the use of a mutant of the cytoplasmic region of CD28 as a signaling domain (Guedan et al., 2020).

[0104] In a preferred embodiment, the signaling domain is selected from the cytoplasmic regions of CD28, CD137 (4-1BB), CD134 (OX40), CD278 (ICOS), DAP10 and CD27, programmed cell death-1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), the cytoplasmic region of the CD3 chain, DAP12, CD122 (interleukin-2 receptor β), CD132 (interleukin-2 receptor γ), CD127 (interleukin-7 receptor α) and CD360 (interleukin-21 receptor), and activating Fc receptors.

[0105] In the most preferred embodiment, the signaling domain comprises the CD28 intracellular domain, more preferably the CD28 intracellular domain in which the internalization motif is mutated and fused to the CD3ζ intracellular domain.

[0106] In a plurality of embodiments, the switchable chimeric antigen receptor comprises at least one signaling domain, preferably 2, 3, 4 or more signaling domains, particularly preferably the cytoplasmic regions of CD28, CD137, CD134, CD278; DAP10 and CD27, PD-1, CTLA-4, the cytoplasmic region of the CD3 chain, DAP12, CD122, CD132, CD127, CD360, activating Fc receptors, signaling domains selected from mutants thereof and combinations thereof.

[0107] In a preferred embodiment, the switchable chimeric antigen receptor comprises at least one signaling domain, preferably 2, 3, 4 or more signaling domains, particularly preferably the cytoplasmic regions of CD28, CD137, CD134, CD278; DAP10 and CD27, PD-1, CTLA-4, the cytoplasmic region of the CD3 chain, DAP12, CD122, CD132, CD127, CD360 and activating Fc receptors, signaling domains selected from these.

[0108] In a further embodiment, the switchable chimeric antigen receptor further comprises another domain, which is a short-chain peptide linker in the extracellular portion of the receptor that can serve to detect the chimeric antigen receptor on the cell surface or stimulate chimeric antigen receptor T cells.

[0109] In a preferred embodiment, the further domain forms a linear epitope for a monoclonal antibody (mab) that specifically binds to the further domain. In some embodiments, the further domain comprises at least one linear epitope, preferably E7B6 according to SEQ ID NO: 12 or SEQ ID NO: 13.

[0110] In some embodiments, the further domain is located between a tag-binding domain or tag and the extracellular hinge domain or an essential part of the extracellular hinge domain.

[0111] Advantageously, the switchable CAR-grafted cells having the further domain can be specifically stimulated to preferentially proliferate and survive longer than non-grafted cells, either in vitro or in vivo. Even more advantageously, the further domain can also be used to purify switchable CAR-grafted cells from a mixed cell population or, in vivo, to suppress the immune response mediated by switchable CAR-grafted cells and to eliminate switchable CAR-grafted cells.

[0112] In a further embodiment, the switchable CAR comprises a signal peptide. Advantageously, the signal peptide enables expression on the cell surface of effector cells. In a plurality of embodiments, the signal peptide is located at the N-terminus of the nucleotide sequence of the switchable CAR in front of the tag-binding domain or the tag. In some embodiments, the signal peptide targets the protein to the secretory pathway either co-translationally or post-translationally and is selected from leader peptides from proteins such as CD28, CD8 alpha, IL-2, lysozyme C or the heavy or light chain of an antibody of human origin to avoid an immunogenic response.

[0113] In a plurality of embodiments, the tag-binding domain or the tag is present at the amino terminus of the polypeptide comprising the switchable CAR. Advantageously, the location of the tag-binding domain or the tag at the amino terminus obviates the need to impede access of the tag-binding domain or the tag to the targeting module bound to the target cell.

[0114] In a plurality of embodiments, the switchable CAR comprises a sequence according to one of the sequences of SEQ ID NOs: 33 to 36. Preferably, the switchable CAR has a sequence according to one of the sequences of SEQ ID NOs: 33 to 36.

[0115] In a preferred embodiment, the switchable CAR comprises a sequence according to SEQ ID NO: 33. More preferably, the switchable CAR has a sequence according to SEQ ID NO: 33.

[0116] In a plurality of embodiments, the engineered human T cells are preferably used in the treatment of cancer, infectious diseases or autoimmune diseases in a method for stimulating a chimeric antigen receptor-mediated immune response in a mammal.

[0117] The term "autoimmune disorder" refers to an abnormal immune response of the body against substances and tissues that are normally present in the body (autoimmunity).

[0118] In multiple embodiments, the engineered human T cells are used in combination with a targeting module comprising a tag-binding domain or a tag and at least one target cell-binding domain, or a nucleic acid, vector or cell encoding the targeting module, wherein the tag-binding domain of the targeting module binds to the tag of the switchable chimeric antigen receptor, or the tag of the targeting module binds to the tag-binding domain of the switchable chimeric antigen receptor, and the engineered human T cells and the targeting module are administered in combination.

[0119] As used herein, the term "administered in combination" refers to a treatment in which the targeting module is administered before, simultaneously with and / or after the administration of a vector or cell comprising a nucleotide sequence encoding a switchable chimeric antigen receptor.

[0120] In multiple embodiments, the targeting module is administered 1 hour to 2 days before, preferably 4 to 24 hours before, the administration of the engineered human T cells. Advantageously, administering the targeting module before the administration of the engineered human T cells stimulates the switchable chimeric antigen receptor, increasing T cell proliferation and their accumulation at the target site.

[0121] In a further embodiment, the engineered human T cells are administered simultaneously with the targeting module.

[0122] In a further embodiment, the targeting module is administered up to after the administration of the engineered human T cells, preferably in the range of 3 days to 30 days later. Furthermore, such additional doses of the targeting module can be administered after a rest period to reactivate effector cells having a switchable CAR.

[0123] A further aspect of the invention provides a pharmaceutical composition comprising engineered human T cells according to the invention and a pharmaceutically acceptable diluent or carrier.

[0124] The pharmaceutical composition is preferably administered parenterally, particularly preferably intravenously. In a plurality of embodiments, the pharmaceutical composition is in a form suitable for intravenous administration. Preferably, the pharmaceutical composition is a solution, emulsion or suspension.

[0125] In a plurality of embodiments, the pharmaceutical composition is an injectable buffered solution containing engineered human T cells at a concentration in the range of 1×10 5 ~1×10 8 per mL.

[0126] The pharmaceutical composition contains a pharmaceutically acceptable thinner or dilution agent or carrier. In a plurality of embodiments, the carrier is selected from water, aqueous buffer solution, 0.9% saline, 5% glucose, 5% xylitol, 0.3% glycine solution, Ringer's solution or amino acid solution. In a further embodiment, the aqueous buffer solution is selected from aqueous buffer solutions of histidine, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate with a pH value in the range from pH 5.0 to pH 7.0. In a plurality of embodiments, the aqueous buffer solution has a buffer concentration in the range of 1 mmol / l (mM) to 500 mM, preferably in the range of 5 mM to 20 mM, particularly preferably in the range of 5 mM to 10 mM.

[0127] In a plurality of embodiments, the carrier preferably contains sodium chloride at a concentration in the range of 1 mM to 300 mM, particularly preferably 150 mM.

[0128] In a plurality of embodiments, the pharmaceutical composition further contains a stabilizer at a concentration preferably in the range of 1 mM to 900 mM, particularly preferably in the range of 50 mM to 600 mM. In a plurality of embodiments, the stabilizer is sucrose, trehalose or L-methionine.

[0129] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. The term "pharmaceutically acceptable excipient" refers to compounds that provide approximately physiological conditions and / or increase stability, such as agents and buffers for adjusting the pH value, agents for adjusting toxicity, and the like. In a plurality of embodiments, the pharmaceutically acceptable excipient is selected from sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and polysorbate-80 in the range of 0.0001% (w / v) to 1% (w / v), particularly preferably in the range of 0.001% (w / v) to 0.1% (w / v), and preferably is polysorbate-80.

[0130] In a further embodiment, the pharmaceutical composition is sterile. The pharmaceutical composition is sterilized by conventional well-known techniques including, but not limited to, sterile filtration.

[0131] In a plurality of embodiments, the pharmaceutical composition is used for administration to a subject.

[0132] In a plurality of embodiments, the pharmaceutical composition is lyophilized before storage or stored as a solution at or below ambient temperature, including, but not limited to, frozen storage.

[0133] In a plurality of embodiments, the pharmaceutical composition is reconstituted and / or diluted in an infusion and stabilization solution before administration to a subject. The solution used for reconstitution or infusion / stabilization may contain any of the components described for the pharmaceutical composition or similar components.

[0134] In a plurality of embodiments, the pharmaceutical composition is preferably used in the treatment of cancer, infectious diseases, or autoimmune diseases in a method for stimulating a chimeric antigen receptor-mediated immune response in mammals.

[0135] In multiple embodiments, the pharmaceutical composition is used in the treatment of cancer, infectious diseases or autoimmune diseases, in which case the engineered human T cells have HLA-B alleles and HLA-C alleles that are compatible with the patient.

[0136] A further aspect of the invention is a population of cells comprising engineered human T cells according to the invention.

[0137] In multiple embodiments, when measured by flow cytometry, at least 65%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or most preferably at least 99% of the cell population is negative for TRAC, HLA-A and / or TCR protein.

[0138] Another aspect of the invention is a) Engineered human T cells comprising: i. Reduced or eliminated surface expression of the endogenous T cell receptor alpha chain by genetic modification in the gene of the T cell receptor alpha chain, ii. Reduced or eliminated surface expression of HLA-A compared to unmodified T cells by genetic modification in the HLA-A gene, iii. Reduced or eliminated surface expression of HLA class II by genetic modification in the CIITA gene, and iv. A switchable chimeric antigen cell surface receptor comprising: · A tag-binding domain or tag, · An extracellular hinge and transmembrane domain, and · A signaling domain, and b) A targeting module comprising a tag-binding domain or tag and at least one target cell-binding domain, or a nucleic acid, vector or cell encoding said targeting module comprising a kit, A kit in which the tag-binding domain of a targeting module binds to the tag of a switchable chimeric antigen receptor, or the tag of the targeting module binds to the tag-binding domain of a switchable chimeric antigen receptor.

[0139] As used herein, the term "targeting module" refers to a polypeptide or protein having at least two different domains, each domain being specific for a target or a homogeneous group of targets, at least one domain being specific for a target cell, and one domain being specific for a switchable chimeric antigen receptor, particularly a tag-binding domain or a tag. In a plurality of embodiments, the targeting module is isolated. As used herein, the term "isolated" means being changed or removed from its natural state.

[0140] Preferably, the targeting module is expressed as a recombinant protein. In a further embodiment, the targeting module is chemically synthesized.

[0141] As used herein, the term "specific" refers to the ability of an antibody or antibody fragment, or a protein, peptide or low molecular weight organic ligand, to recognize and bind to a binding partner (e.g., tumor antigen) protein present in a sample, but not substantially recognize or bind to other molecules in the sample.

[0142] As used herein, the term "bind" or "binding" refers to non-covalent bonds, particularly ionic bonds, hydrogen bonds, van der Waals forces and / or hydrophobic interactions.

[0143] In a plurality of embodiments, the targeting module is in monomeric, dimeric or polymeric form, preferably monomeric form.

[0144] In a further embodiment, the targeting module is monovalent, divalent or multivalent.

[0145] As used herein, the term "target cell binding domain" refers to a peptide, protein, or low molecular weight organic ligand that specifically binds to a protein or protein complex (antigen) on the surface of a target cell, preferably a cancer cell, T cell, infected cell, pathogen, or parasite.

[0146] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can be included in the sequence of the protein or peptide.

[0147] As used herein, the term "low molecular weight organic ligand" refers to an organic molecule having a molecular weight of up to 10 kilodaltons, preferably up to 3 kilodaltons, that specifically binds to a protein or protein complex (antigen) on the surface of a target cell, preferably a cancer cell, T cell, infected cell, or pathogen or parasite.

[0148] In several embodiments, at least one target cell binding domain is CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6 CD52, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD90, CD99, CD123, CD133, CD135, CD150 CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366 and CD371, cytokine receptors, preferably interleukin receptors, particularly preferably IL-8Rα, IL-8Rβ, IL-11Rα, IL-11Rβ, IL13Rα1; CXCR4, c-Met, mesothelin, members of the epidermal growth factor receptor family and mutants thereof, particularly preferably ErbB1, ErbB2, ErbB3, ErbB4 or mutants thereof; members of the tumor necrosis factor receptor superfamily, claudin, ephrin, ephrin receptor, particularly preferably EphA1-10, EphA5 or EphB1-6; fucosyltransferase, prostate specific antigen, preferably PSCA and PSMA; fetal antigens, preferably CEA and fetal acetylcholine receptor;An antibody, antibody fragment, protein, peptide or low molecular weight organic ligand that binds to a surface antigen selected from the group consisting of members of the vascular endothelial growth factor family, EpCAM, AFP, members of the intercellular adhesion molecule family, C-type lectins, integrins, members of the mucin protein family, FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, ligands of the NKG2D receptor, members of the epithelial glycoprotein family, diasialogangliosides, glypicans, G-protein coupled receptors, human papillomavirus proteins, cancer testicular antigens, fibroblast activation proteins, members of the carbonic anhydrase family, members of the carbohydrate antigen family, Notch ligands, MCSP, glycoprotein A33, guanylate cyclase 2C and tumor-specific glycans, including mutants and analogs of the specified antibody, antibody fragment, protein, peptide or low molecular weight organic ligand.;

[0149] As used herein, the term "mutant" refers to a peptide or protein having at least 90% sequence identity, preferably at least 95% sequence identity, to the specified antibody, antibody fragment, protein or peptide. Advantageously, the mutant binds the same antigen as the specified antibody, antibody fragment, protein or peptide.

[0150] As used herein, the term "analog" refers to a molecule having a high degree of structural identity to the specified antibody, antibody fragment, protein, peptide or low molecular weight organic ligand, preferably with at least one atom, group of atoms, functional group or substructure replaced by another group of atoms, such as a hydroxy group. In a plurality of embodiments, an analog of somatostatin (SRIF14) is octreotide or pasireotide. Advantageously, the analog binds the same antigen as the specified antibody, antibody fragment, protein, peptide or low molecular weight organic ligand.

[0151] In several embodiments, analogs of a specified antibody, antibody fragment, protein or peptide include modifications selected from the group consisting of D-amino acids, peptidomimetic bonds, amino alcohols, amino acids not constituting proteins, unnatural amino acids, amino acids with modified side chains and / or cyclic proteins. Advantageously, these analogs exhibit increased stability.

[0152] The term "target cell binding domain" also includes soluble T cell receptors composed of the alpha and beta chains or gamma and delta chains of a T cell receptor (TCR), fragments or mutants thereof. Binding moieties derived from such TCRs recognize and bind peptides presented by human leukocyte antigen class (HLA) I and II protein complexes. Examples include, but are not limited to, TCRs specific for peptides derived from proteins such as the EGFR family, survivin, sry-like high mobility group box (SOX) protein family, melanoma-associated antigens (e.g., the autoimmunogenic cancer / testis antigen NY-ESO-1, members of the melanoma antigen family A MAGEA, antigens preferentially expressed in melanoma PRAME), and leukemia-associated antigens (e.g., the Wilms tumor gene 1 WT1).

[0153] In a further embodiment, the target cell binding domain is a soluble T cell receptor consisting of the alpha and beta chains or gamma and delta chains of a T cell receptor (TCR).

[0154] In multiple embodiments, at least one target cell binding domain binds to a surface antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6 CD52, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD90, CD99, CD123, CD133, CD135, CD150 CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366 and CD371, cytokine receptors, CXCR4, c-Met, mesothelin, members of the epidermal growth factor receptor family and mutants thereof, members of the tumor necrosis factor receptor superfamily, claudin, ephrin, ephrin receptor, fucosyltransferase, prostate specific antigen, fetal antigen, members of the vascular endothelial growth factor family, EpCAM, AFP, members of the intercellular adhesion molecule family, C-type lectin, integrin, members of the mucin protein family, FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, ligands of the NKG2D receptor, members of the epithelial glycoprotein family, disialoganglioside, glypican, G protein-coupled receptor, human papillomavirus protein, cancer testis antigen, fibroblast activation protein, members of the carbonic anhydrase family, members of the carbohydrate antigen family, Notch ligand, MCSP, glycoprotein A33, guanylate cyclase 2C and tumor-specific glycans, and is an antibody, antibody fragment, protein, peptide or low molecular weight organic ligand.

[0155] In a preferred embodiment, at least one target cell binding domain is an antibody, antibody fragment, protein, peptide or low molecular weight organic ligand that binds to CD123.

[0156] In multiple embodiments, the variable region of at least one target cell binding domain, preferably the CD123 binding domain, contains a humanized amino acid sequence.

[0157] In a further preferred embodiment, at least one target cell binding domain is an antibody fragment that binds to CD123.

[0158] In some embodiments, the targeting module according to the invention is bivalent or multivalent and comprises at least one CD123 binding domain.

[0159] In multiple embodiments, the different domains of the targeting module are linked to each other by a linker. The linker preferably comprises a short chain sequence of 20 to 30 amino acid residues. In multiple embodiments, the targeting module comprises a flexible peptide sequence selected such that the domains have a three-dimensional folding that can exhibit specificity for effector cells and target cell binding.

[0160] A preferred linker is a glycine-serine linker having a structure (G x S y ) in which x and y are selected from 1 to 10, preferably 1 to 5. Most preferred are 2 to 5 repeats of the sequence G4S1 (SEQ ID NO: 4). Furthermore, a linker composed of a peptide sequence that can increase the protease resistance of the antibody derivative is preferred.

[0161] In multiple embodiments, the linker of the tag binding domain comprises 20 to 30 amino acids, preferably 25 amino acids.

[0162] In multiple embodiments, the linker has a sequence according to SEQ ID NO: 5 or SEQ ID NO: 6.

[0163] In multiple embodiments, the targeting module comprises one of the sequences according to SEQ ID NO: 19 to SEQ ID NO: 32.

[0164] In a preferred embodiment, the targeting module comprises one of the sequences according to SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32. More preferably, the targeting module comprises one of the sequences according to SEQ ID NO: 24 or SEQ ID NO: 31. Most preferably, the targeting module has one of the sequences according to SEQ ID NO: 24 or SEQ ID NO: 31.

[0165] In a plurality of embodiments, the targeting module comprises yet another domain selected from the group consisting of a co-stimulatory ligand, a radionuclide, a cell death-inducing chemical compound and a half-life increasing domain, preferably IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, HSA, an FcRn-binding peptide or a mutant thereof. As used herein, the term "mutant" refers to a protein having at least 90% sequence identity, preferably at least 95% sequence identity, to the half-life increasing domain. Advantageously, the mutant may have one or more activities of the designated peptide or protein, and in particular, the mutant increases the half-life, such as the half-life increasing domain.

[0166] In a preferred embodiment, the targeting module comprises yet another domain selected from the group consisting of a co-stimulatory ligand, a radionuclide, a cell death-inducing chemical compound and a half-life increasing domain, preferably IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, HSA or an FcRn-binding peptide.

[0167] In a plurality of embodiments, the length of the targeting module ranges from 20 to 1600 amino acids, preferably from 200 to 800 amino acids.

[0168] In a plurality of embodiments, the kit further comprises at least one yet another targeting module, or at least one yet another nucleic acid, vector or cell encoding the yet another targeting module, At least one further targeting module comprises at least one target cell binding domain and a tag binding domain or tag, The at least one target cell binding domain binds to a surface antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6 CD52, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD90, CD99, CD133, CD135, CD150 CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366 and CD371, cytokine receptors, preferably interleukin receptors, particularly preferably IL-8Rα, IL-8Rβ, IL-11Rα, IL-11Rβ, IL13Rα1; CXCR4, c-Met, mesothelin, members of the epidermal growth factor receptor family and mutants thereof, particularly preferably ErbB1, ErbB2, ErbB3, ErbB4 or mutants thereof; members of the tumor necrosis factor receptor superfamily, claudin, ephrin, ephrin receptor, particularly preferably EphA1-10, EphA5 or EphB1-6; fucosyl transferase, prostate specific antigen, preferably PSCA and PSMA; fetal antigens, preferably CEA and fetal acetylcholine receptor; members of the vascular endothelial growth factor family, EpCAM, AFP, members of the intercellular adhesion molecule family, C-type lectin, integrin, members of the mucin protein family, FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, ligands of the NKG2D receptor, members of the epithelial glycoprotein family, disialoganglioside, glypican, G protein-coupled receptor, human papillomavirus protein, cancer testis antigen, fibroblast activation protein, members of the carbonic anhydrase family, members of the carbohydrate antigen family, Notch ligand, MCSP, glycoprotein A33, guanylate cyclase 2C and tumor-specific glycans, and is an antibody, antibody fragment, protein, peptide or low molecular weight organic ligand, including mutants and analogs of the specified antibody, antibody fragment, protein, peptide or low molecular weight organic ligand, The targeting module and at least one further distinct targeting module comprise different target cell binding domains, and an identical tag binding domain or tag.

[0169] In a further embodiment, at least one further distinct targeting module comprises two target cell binding domains that bind to surface antigens selected from PSCA and PSMA, ErbB-1 and ErbB-2, PSCA and ErbB-2, PSMA and CEA, IL13Rα2 and ErbB-2, CD38 and CD269, CD19 and CD20, mesothelin and mucin 16, PD-L1 and ErbB-2.

[0170] In a plurality of embodiments, the kit comprises from one to three targeting modules that bind to CD123, preferably one targeting module, and one or two further distinct targeting modules.

[0171] In an alternative embodiment, the kit comprises a nucleic acid, vector or cell encoding the targeting module. The nucleic acid, vector and / or cell are isolated.

[0172] In a plurality of embodiments, the nucleic acid is cDNA. As used herein, the term "cDNA" (complementary DNA) refers to double-stranded DNA synthesized from single-stranded RNA, such as mRNA, in a reaction catalyzed by the enzyme reverse transcriptase. In a plurality of embodiments, the cDNA is synthetically derived. In a further embodiment, the cDNA is derived from mRNA and thus contains only exons and no introns, in contrast to genomic DNA.

[0173] The vector is preferably a plasmid, artificial chromosome, linear DNA or RNA, viral particle, or another vector containing an expression cassette that is stably integrated into the genome of a host cell or host organism.

[0174] In multiple embodiments, the cells are selected from immune cells preferably having cytolytic activity, phagocytic activity or immunosuppressive activity, such as T cells, natural killer (NK) cells and macrophages. In preferred embodiments, the cells are selected from T cells, including alpha / beta and gamma / delta T cells, or subpopulations of T cells such as stem cell memory T cells or central memory T cells, cytotoxic T cells or NK cells.

[0175] In multiple embodiments, the engineered human T cells and / or targeting modules are in the form of a pharmaceutical composition.

[0176] In multiple embodiments, the kit is preferably used in the treatment of cancer, infectious diseases or autoimmune diseases in a method for stimulating a chimeric antigen receptor-mediated immune response in a mammal.

[0177] In multiple embodiments, the engineered human T cells and / or targeting modules are used to manufacture a medicament for therapeutic and / or diagnostic use in the case of cancer, infection or autoimmune diseases.

[0178] In multiple embodiments, the engineered human T cells and / or targeting modules are used to treat tumors expressing CD123, preferably acute myeloid leukemia.

[0179] A further aspect of the present invention is a) A CRISPR system comprising: · A nucleic acid targeting a region of the endogenous T cell receptor alpha chain gene, · A nucleic acid targeting a region of the endogenous HLA-A gene, · A nucleic acid targeting a region of the endogenous CIITA gene, and b) A nucleic acid encoding a switchable chimeric antigen cell surface receptor or a vector containing the nucleic acid, comprising: - A tag-binding domain or tag, - An extracellular hinge and transmembrane domain, and - A signaling domain, and c) A targeting module comprising a tag-binding domain or a tag and at least one target cell-binding domain, or a nucleic acid, vector or cell encoding said targeting module A kit comprising wherein the tag-binding domain of the targeting module binds to the tag of the switchable chimeric antigen receptor, or the tag of the targeting module binds to the tag-binding domain of the switchable chimeric antigen receptor.

[0180] As used herein, the term "CRISPR" (clustered regularly interspaced short palindromic repeats) refers to a system that evolved in bacteria as an adaptive immune system to defend against viral attack. When exposed to a virus, short segments of viral DNA are integrated into the CRISPR locus of the bacterial genome. RNA is transcribed from a portion of the CRISPR locus that contains the viral sequence. This RNA has a sequence complementary to the viral genome and mediates the targeting of the Cas9 protein to sequences within the viral genome. The Cas9 protein cleaves the viral target, thereby silencing the viral target. The CRISPR / Cas system has been applied to genome editing in eukaryotic cells. Modification of target sequences is possible by introducing site-specific single-strand breaks (SSB) or double-strand breaks (DSB), for example via non-homologous end joining (NHEJ) or homologous recombination repair (HDR).

[0181] In a plurality of embodiments, the CRISPR system comprises mRNA of CRISPR-associated protein 9 and a target site-specific single-guide RNA targeting TRAC, HLA-A or CIITA, packaged in lipid nanoparticles.

[0182] In a plurality of embodiments, the target site-specific single-guide RNA targeting a region of the endogenous T cell receptor alpha chain gene comprises a nucleotide sequence according to SEQ ID NO: 1.

[0183] In multiple embodiments, a target site-specific single guide RNA targeting a region of HLA-A comprises a nucleotide sequence according to SEQ ID NO: 2.

[0184] In multiple embodiments, a target site-specific single guide RNA that causes downregulation of CIITA gene expression comprises a nucleotide sequence according to SEQ ID NO: 3.

[0185] [Table 1]

[0186] Another aspect of the present invention is a method for generating engineered human T cells that express a switchable chimeric antigen receptor, comprising: (1) providing human T cells; (2) introducing by transfection into the human T cells a CRISPR system comprising a nucleic acid that targets a region of the endogenous T cell receptor alpha chain within the T cells; (3) introducing by transfection into the human T cells a CRISPR system comprising a nucleic acid that targets a region of HLA-A within the T cells; (4) introducing by transfection into the human T cells a CRISPR system comprising a nucleic acid that targets a region of CIITA within the T cells to generate CRISPR-modified T cells, and (5) introducing by transduction into the human T cells a nucleic acid encoding a switchable chimeric antigen cell surface receptor or a vector comprising the nucleic acid, the switchable chimeric antigen cell surface receptor comprising: · a tag-binding domain or tag, · an extracellular hinge and transmembrane domain, and · a signaling domain This is the method.

[0187] In a preferred embodiment, the switchable chimeric antigen cell surface receptor is an inverted universal chimeric antigen cell surface receptor comprising a tag, an extracellular hinge and transmembrane domain, and a signaling domain.

[0188] In a plurality of embodiments, the CRISPR system comprises a lipid nucleic acid assembly composition comprising an endonuclease or mRNA encoding an endonuclease and a target site-specific single guide RNA.

[0189] In a plurality of embodiments, the lipid nucleic acid assembly composition comprises lipid nanoparticles. In a preferred embodiment, the lipid nucleic acid assembly composition is a lipid nanoparticle (LNP).

[0190] In a plurality of embodiments, the lipid nanoparticles are a mixture of cationic and / or ionizable lipids and helper lipids.

[0191] In a plurality of embodiments, the lipid nanoparticles are a mixture of ionizable lipids, helper lipids and PEG-DMG.

[0192] In a plurality of embodiments, the endonuclease is CRISPR-associated enzyme 9.

[0193] In a plurality of embodiments, the endonuclease is Streptococcus pyogenes (S. pyogenes) Cas9, Neisseria meningitidis (N. meningitidis) Cas9, Streptococcus thermophilus (S. thermophilus) Cas9 or Staphylococcus aureus (S. aureus) Cas9.

[0194] According to the present invention, the method comprises introduction by transfection of a CRISPR system comprising a nucleic acid that causes downregulation or removal of gene expression of TRAC in T cells.

[0195] In multiple embodiments, the genetic modification in the TRAC gene comprises at least one nucleotide within genomic coordinates chr14:22547524 to chr14:22547544. In multiple embodiments, the genetic modification in the TRAC gene comprises at least 10 or at least 15 consecutive nucleotides within the genomic coordinates.

[0196] In multiple embodiments, the genetic modification in the TRAC gene comprises at least one nucleotide of an exon of the TRAC gene.

[0197] In multiple embodiments, the genetic modification in the TRAC gene comprises at least one insertion, deletion, substitution, or deamination of at least one nucleotide within the genomic coordinates.

[0198] In multiple embodiments, the genetic modification in the TRAC gene comprises an indel.

[0199] In multiple embodiments, the expression of the TRAC gene is reduced or eliminated by a gene editing system that binds to a TRAC genomic target sequence comprising at least 5 consecutive nucleotides within the genomic coordinates, preferably within genomic coordinates chr14:22547524 to chr14:22547544.

[0200] In multiple embodiments, the target site-specific single-guide RNA in step (2) comprises a nucleotide sequence according to SEQ ID NO: 1, or a sequence that is at least 90%, preferably 95%, more preferably 99% identical to the sequence according to SEQ ID NO: 1.

[0201] According to the present invention, the method comprises introduction of a CRISPR system comprising a nucleic acid that targets a region of HLA-A in T cells by transfection, with downregulation of gene expression.

[0202] In multiple embodiments, the genetic modification in the HLA-A gene comprises at least one nucleotide among the genomic coordinates selected from chr6:29942854 to chr6:29942913 and chr6:29943518 to chr6:29943619.

[0203] In multiple embodiments, the T cells are homozygous for the HLA-B genotype and / or homozygous for the HLA-C genotype. Preferably, the T cells are homozygous for the HLA-B genotype and the HLA-C genotype.

[0204] In multiple embodiments, the expression of at least one HLA-A allele selected from HLA-A1, HLA-A2, HLA-A3, HLA-A11, and HLA-A24 is reduced or eliminated in the T cells.

[0205] In multiple embodiments, the genetic modification in the HLA-A gene comprises at least one nucleotide among the genomic coordinates chr6:29942864 to chr6:29942903, preferably chr6:29942876 to chr6:29942897.

[0206] In multiple embodiments, the genetic modification in the HLA-A gene comprises at least one nucleotide among the genomic coordinates chr6:29943528 to chr6:29943609, preferably chr6:29943528 to chr6:29943550.

[0207] In multiple embodiments, the genetic modification in the HLA-A gene comprises at least 5, 6, 7, 8, 9, or 10 consecutive nucleotides among the genomic coordinates, preferably at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides among the genomic coordinates.

[0208] In multiple embodiments, the genetic modification in the HLA-A gene comprises at least one C to T substitution or at least one A to G substitution among the genomic coordinates.

[0209] In multiple embodiments, the genetic modification in the HLA-A gene comprises an indel.

[0210] In multiple embodiments, the expression of HLA-A is reduced or eliminated by a gene editing system that binds to an HLA-A genomic target sequence comprising at least 5 consecutive nucleotides among the genomic coordinates preferably selected from chr6:29942864-29942884; chr6:29942868-29942888; chr6:29942876-29942896; chr6:29942877-29942897; chr6:29942883-29942903; chr6:29943126-29943146; chr6:29943528-29943548; chr6:29943529-29943549; chr6:29943530-29943550; chr6:29943537-29943557; chr6:29943549-29943569; chr6:29943589-29943609; and chr6:29944026-29944046.

[0211] In multiple embodiments, the HLA-B allele is selected from any one of the following HLA-B alleles: HLA-B*07:02; HLA-B*08:01; HLA-B*44:02; HLA-B*35:01; HLA-B*40:01; HLA-B*57:01; HLA-B*14:02; HLA-B*15:01; HLA-B*13:02; HLA-B*44:03; HLA-B*38:01; HLA-B*18:01; HLA-B*44:03; HLA-B*51:01; HLA-B*49:01; HLA-B*15:01; HLA-B*18:01; HLA-B*27:05; HLA-B*35:03; HLA-B*18:01; HLA-B*52:01; HLA-B*51:01; HLA-B*37:01; HLA-B*53:01; HLA-B*55:01; HLA-B*44:02; HLA-B*44:03; HLA-B*35:02; HLA-B*15:01; and HLA-B*40:02.

[0212] In multiple embodiments, the HLA-C allele is selected from any one of the following HLA-C alleles: HLA-C*07:02; HLA-C*07:01; HLA-C*05:01; HLA-C*04:01 HLA-C*03:04; HLA-C*06:02; HLA-C*08:02; HLA-C*03:03; HLA-C*06:02; HLA-C*16:01; HLA-C*12:03; HLA-C*07:01; HLA-C*04:01; HLA-C*15:02; HLA-C*07:01; HLA-C*03:04; HLA-C*12:03; HLA-C*02:02; HLA-C*04:01; HLA-C*05:01; HLA-C*12:02; HLA-C*14:02; HLA-C*06:02; HLA-C*04:01; HLA-C*03:03; HLA-C*07:04; HLA-C*07:01; HLA-C*04:01; HLA-C*04:01; and HLA-C*02:02.

[0213] In multiple embodiments, the HLA-B allele is selected from any one of the following HLA-B alleles: HLA-B*07:02; HLA-B*08:01; HLA-B*44:02; HLA-B*35:01; HLA-B*40:01; HLA-B*57:01; HLA-B*14:02; HLA-B*15:01; HLA-B*13:02; HLA-B*44:03; HLA-B*38:01; HLA-B*18:01; HLA-B*44:03; HLA-B*51:01; HLA-B*49:01; HLA-B*15:01; HLA-B*18:01; HLA-B*27:05; HLA-B*35:03; HLA-B*18:01; HLA-B*52:01; HLA-B*51:01; HLA-B*37:01; HLA-B*53:01; HLA-B*55:01; HLA-B*44:02; HLA-B*44:03; HLA-B*35:02; HLA-B*15:01; and HLA-B*40:02, and the HLA-C allele is selected from any one of the following HLA-C alleles: HLA-C*07:02; HLA-C*07:01; HLA-C*05:01; HLA-C*04:01 HLA-C*03:04; HLA-C*06:02; HLA-C*08:02; HLA-C*03:03; HLA-C*06:02; HLA-C*16:01; HLA-C*12:03; HLA-C*07:01; HLA-C*04:01; HLA-C*15:02; HLA-C*07:01; HLA-C*03:04; HLA-C*12:03; HLA-C*02:02; HLA-C*04:01; HLA-C*05:01; HLA-C*12:02; HLA-C*14:02; HLA-C*06:02; HLA-C*04:01; HLA-C*03:03; HLA-C*07:04; HLA-C*07:01; HLA-C*04:01; HLA-C*04:01; and HLA-C*02:02.

[0214] In a preferred embodiment, the HLA-B allele and the HLA-C allele are the following HLA-B allele and HLA-C allele: HLA-B * 07:02 and HLA-C *07:02, HLA-B*08:01 and HLA-C*07:01; HLA-B*44:02 and HLA-C*05:01; HLA-B*35:01 and HLA-C*04:01; HLA-B*40:01 and HLA-C*03:04; HLA-B*57:01 and HLA-C*06:02; HLA-B*14:02 and HLA-C*08:02; HLA-B*15:01 and HLA-C*03:03; HLA-B*13:02 and HLA-C*06:02; HLA-B*44:03 and HLA-C*16:01; HLA-B*38:01 and HLA-C*12:03; HLA-B*18:01 and HLA-C*07:01; HLA-B*44:03 and HLA-C*04:01; HLA-B*51:01 and HLA-C*15:02; HLA-B*49:01 and HLA-C*07:01; HLA-B*15:01 and HLA-C*03:04; HLA-B*18:01 and HLA-C*12:03; HLA-B*27:05 and HLA-C*02:02; HLA-B*35:03 and HLA-C*04:01; HLA-B*18:01 and HLA-C*05:01; HLA-B*52:01 and HLA-C*12:02; HLA-B*51:01 and HLA-C*14:02; HLA-B*37:01 and HLA-C*06:02; HLA-B*53:01 and HLA-C*04:01; HLA-B*55:01 and HLA-C*03:03; HLA-B*44:02 and HLA-C*07:04; HLA-B*44:03 and HLA-C*07:01; HLA-B*35:02 and HLA-C*04:01; HLA-B * 15:01 and HLA-C * 04:01, as well as HLA-B * 40:02 and HLA-C * is selected from any one of 02:02.

[0215] In multiple embodiments, the T cells have reduced or eliminated surface expression of HLA-B, compared to unmodified T cells, by genetic modification in the HLA-B gene, and / or have reduced or eliminated surface expression of HLA-C, compared to unmodified T cells, by genetic modification in the HLA-C gene.

[0216] In a further embodiment, the T cells further have reduced or eliminated surface expression of HLA-B, compared to unmodified T cells, by genetic modification in the HLA-B gene, and the T cells are homozygous for the HLA-C genotype.

[0217] In an alternative embodiment, the T cells further have reduced or eliminated surface expression of HLA-C, compared to unmodified T cells, by genetic modification in the HLA-C gene, and the T cells are homozygous for the HLA-B genotype.

[0218] In a further alternative embodiment, the T cells have reduced or eliminated surface expression of HLA-B, compared to unmodified T cells, by genetic modification in the HLA-B gene, and have reduced or eliminated surface expression of HLA-C, compared to unmodified T cells, by genetic modification in the HLA-C gene.

[0219] In multiple embodiments, the target site-specific single guide RNA in step (3) comprises a nucleotide sequence according to SEQ ID NO: 2, or a sequence that is at least 90%, preferably 95%, more preferably 99% identical to the sequence according to SEQ ID NO: 2.

[0220] In multiple embodiments, the guide RNA for HLA-A comprises at least one modification preferably selected from the group consisting of 2'-O-methyl (2'-O-Me)-modified nucleotides, phosphorothioate (PS) bonds between nucleotides, 2'-fluoro (2'-F)-modified nucleotides, modification at one or more of the first 5 nucleotides at the 5' end of the guide RNA, modification at one or more of the last 5 nucleotides at the 3' end of the guide RNA, PS bonds between the first 4 nucleotides of the guide RNA, PS bonds between the last 4 nucleotides of the guide RNA, 2'-O-Me-modified nucleotides at the first 3 nucleotides at the 5' end of the guide RNA, 2'-O-Me-modified nucleotides at the last 3 nucleotides at the 3' end of the guide RNA, or combinations thereof.

[0221] In multiple embodiments, the method further comprises the step of matching the engineered human T cells to the patient for HLA-B alleles and HLA-C alleles.

[0222] In an alternative embodiment, the method further comprises the step of introducing into the human T cells a CRISPR system comprising a nucleic acid targeting a region of HLA-B in the T cells by downregulation of gene expression and / or introducing into the human T cells a CRISPR system comprising a nucleic acid targeting a region of HLA-C in the T cells by transfection.

[0223] According to the present invention, the method comprises introduction by transfection of a CRISPR system comprising a nucleic acid targeting a region of CIITA in the T cells.

[0224] In multiple embodiments, the genetic modification in the CIITA gene comprises at least one nucleotide of a splice site within genomic coordinates chr16:10902171 to chr16:10923242.

[0225] In multiple embodiments, the genetic modification in the CIITA gene comprises modification of at least one nucleotide at a splice acceptor site, preferably, one nucleotide is A or G or T.

[0226] In multiple embodiments, the genetic modification in the CIITA gene comprises modification of splice site boundary nucleotides.

[0227] In multiple embodiments, the genetic modification in the CIITA gene comprises at least 5, 6, 7, 8, 9, or 10 consecutive nucleotides within genomic coordinates chr16:10902171 to chr16:10923242.

[0228] In multiple embodiments, the genetic modification in the CIITA gene comprises at least one C to T substitution or at least one A to G substitution within genomic coordinates chr16:10902171 to chr16:10923242.

[0229] In multiple embodiments, the genetic modification in the CIITA gene is at chr16:10895410-10895430, chr16:10898649-10898669, chr16:10898658-10898678, chr16:10902171-10902191, chr16:10902173-10902193, chr16:10902174-10902194, chr16:10902179-10902199, chr16:10902183-10902203, chr16:10902184-10902204, chr16:10902644-10902664, chr16:10902779-10902799, chr16:10902788-10902808, chr16:10902789-10902809, chr16:10902790-10902810, chr16:10902795-10902815, chr16:10902799-10902819, chr16:10903708-10903728, chr16:10903713-10903733, chr16:10903718-10903738, chr16:10903721-10903741, chr16:10903723-10903743, chr16:10903724-10903744, chr16:10903873-10903893, chr16:10903878-10903898, chr16:10903905-10903925, chr16:10903906-10903926, chr16:10904736-10904756, chr16:10904790-10904810, chr16:10904811-10904831, chr16:10906481-10906501, chr16:10906485-10906505, chr16:10906486-10906506, chr16:10906487-10906507, chr16:10906492-10906512, chr16:10908127-10908147, chr16:10908130-10908150, chr16:10908131-10908151, chr16:10908132-10908152, chr16:10908137-10908157, chr16:10908138-10908158,Genomic coordinates selected from chr16:10908139-10908159, chr16:10909006-10909026, chr16:10909007-10909027, chr16:10909018-10909038, chr16:10909021-10909041, chr16:10909022-10909042, chr16:10909172-10909192, chr16:10910165-10910185, chr16:10910176-10910196, chr16:10910186-10910206, chr16:10915547-10915567, chr16:10915551-10915571, chr16:10915552-10915572, chr16:10915567-10915587, chr16:10916348-10916368, chr16:10916359-10916379, chr16:10916362-10916382, chr16:10916449-10916469, chr16:10916450-10916470, chr16:10916455-10916475, chr16:10916456-10916476, chr16:10918423-10918443, chr16:10918504-10918524, chr16:10918511-10918531, chr16:10918512-10918532, chr16:10918539-10918559, chr16:10922153-10922173, chr16:10922478-10922498, chr16:10922487-10922507, chr16:10922499-10922519, chr16:10923205-10923225, chr16:10923214-10923234, chr16:10923218-10923238, chr16:10923219-10923239, chr16:10923220-10923240, chr16:10923221-10923241, and chr16:10923222-10923242 include indels, C to T substitutions, or A to G substitutions.,

[0230] In multiple embodiments, the genetic modification in the CIITA gene is at chr16:10906485-10906505, chr16:10906486-10906506, chr16:10906487-10906507, chr16:10906492-10906512, chr16:10908127-10908147, chr16:10908130-10908150, chr16:1090813l-10908151, chr16:10908132-10908152, chr16:10908137-10908157, chr16:10908138-10908158, chr16:10908139-10908159, chr16:10909006-10909026, chr16:10909007-10909027, chr16:10909018-10909038, chr16:10909021-10909041, chr16:10909022-10909042, chr16:10909172-10909192, chr16:10910165-10910185, chr16:10910176-10910196, chr16:10910186-10910206, chr16:10915547-10915567, chr16:10915551-10915571, chr16:10915552-10915572, chr16:10915567-10915587, chr16:10916348-10916368, chr16:10916359-10916379, chr16:10916362-10916382, chr16:10916449-10916469, chr16:10916450-10916470, chr16:10916455-10916475, chr16:10916456-10916476, chr16:10918423-109l8443, chr16:10918504-10918524, chr16:10918511-10918531, chr16:10918512-10918532, chr16:10918539-10918559, chr16:10922153-10922173, chr16:10922478-10922498, chr16:10922487-10922507, chr16:10922499-10922519,Genomic coordinates selected from chr16:10923205-10923225, chr16:10923214-10923234, chr16:10923218-10923238, chr16:10923219-10923239, chr16:10923220-10923240, chr16:10923221-10923241, and chr16:10923222-10923242, preferably chr16:10908132-10908152, chr16:10908131-10908151, chr16:10916456-10916476, chr16:10918504-10918524, chr16:10909022-10909042, chr16:10918512-10918532, chr16:10918511-10918531, chr16:10895742-10895762, chr16:10916362-10916382, chr16:10916455-10916475, chr16:10909172-10909192, chr16:10906492-10906512, chr16:10909006-10909026, chr16:10922478-10922498, chr16:10895747-10895767, chr16:10916348-10916368, chr16:10910186-10910206, chr16:10906481-10906501, chr16:10909007-10909027, chr16:10895410-10895430, and chr16:10908130-10908150, and contain at least 5 consecutive nucleotides among the genomic coordinates selected.

[0231] In multiple embodiments, the genetic modification in the CIITA gene contains at least 10 or at least 15 consecutive nucleotides among the genomic coordinates.

[0232] In multiple embodiments, the target site-specific single-guide RNA in step (4) is a CIITA genomic target sequence that contains at least one nucleotide of a splice acceptor site or a splice donor site.

[0233] In a preferred embodiment, one nucleotide is a splice site boundary nucleotide of a splice acceptor site or a splice site boundary nucleotide of a splice donor site.

[0234] In multiple embodiments, the target site-specific single guide RNA in step (4) comprises a guide sequence that causes cleavage to be performed at a CIITA genomic target sequence that is 4 nucleotides or less, 3 nucleotides or less, 2 nucleotides or less, or 1 nucleotide from a splice site boundary nucleotide by an RNA-guided DNA binding agent.

[0235] In multiple embodiments, the target site-specific single guide RNA in step (4) comprises a nucleotide sequence according to SEQ ID NO: 3 or a sequence that is at least 90%, preferably 95%, more preferably 99% identical to the sequence according to SEQ ID NO: 3.

[0236] In multiple embodiments, the CIITA guide RNA preferably comprises at least one modification selected from the group consisting of 2'-O-methyl (2'-O-Me) modified nucleotides, phosphorothioate (PS) linkages between nucleotides, 2'-fluoro (2'-F) modified nucleotides, modification at one or more of the first 5 nucleotides at the 5' end of the guide RNA, modification at one or more of the last 5 nucleotides at the 3' end of the guide RNA, PS linkages between the first 4 nucleotides of the guide RNA, PS linkages between the last 4 nucleotides of the guide RNA, 2'-O-Me modified nucleotides at the first 3 nucleotides at the 5' end of the guide RNA, 2'-O-Me modified nucleotides at the last 3 nucleotides at the 3' end of the guide RNA, or combinations thereof.

[0237] In multiple embodiments, the genetic modification in the CIITA gene comprises an indel.

[0238] In multiple embodiments, the introduction of a nucleic acid encoding a switchable chimeric antigen cell surface receptor or a vector containing the nucleic acid according to step (5) is performed by adding a non-viral vector or a viral vector including, but not limited to, a lentiviral vector or an adenoviral vector. The introduction of a nucleic acid encoding a switchable chimeric antigen cell surface receptor or a vector containing the nucleic acid according to step (5) can be performed at random sites or in a site-specific manner including, but not limited to, homologous recombination repair (HDR) of a CRISPR-edited locus.

[0239] In a preferred embodiment, the introduction of a nucleic acid encoding a switchable chimeric antigen cell surface receptor or a vector containing the nucleic acid according to step (5) is performed by adding self-inactivating retroviral vector particles.

[0240] A further aspect of the invention is a method for stimulating a chimeric antigen receptor-mediated immune response in a mammal, preferably a human, having cancer, an infectious disease or an autoimmune disease, by administering an engineered human T cell and a targeting module according to the invention, a pharmaceutical composition or a kit according to the invention, preferably to a subject in need thereof.

[0241] A further aspect of the invention relates to the use of an engineered human T cell and a targeting module according to the invention, a pharmaceutical composition or a kit according to the invention for stimulating a chimeric antigen receptor-mediated immune response in a mammal, preferably a human, having cancer, an infectious disease or an autoimmune disease.

[0242] For therapeutic use, a sterile pharmaceutical composition according to the invention or a sterile kit according to the invention comprising a pharmacologically effective amount of an engineered human T cell and a targeting module according to the invention is administered to a subject for treating the aforementioned diseases.

[0243] In some embodiments, a method for stimulating a chimeric antigen receptor-mediated immune response in a mammal, preferably for the treatment of cancer, an infectious disease or an autoimmune disease, comprises the following steps: a) administering to the mammal an effective amount of a targeting module, and b) administering to the mammal an effective amount of engineered human T cells according to the invention, wherein the tag-binding domain of the targeting module binds to the tag of a switchable chimeric antigen receptor, or the tag of the targeting module binds to the tag-binding domain of a switchable chimeric antigen receptor, comprising, The targeting module is administered to the mammal before, simultaneously with or after the administration of the vector or cell.

[0244] In a plurality of embodiments, a method for stimulating a chimeric antigen receptor-mediated immune response in a mammal further comprises determining the HLA-B and HLA-C alleles of the recipient subject (patient), and matching the engineered human T cells to the patient for the HLA-B and HLA-C alleles (Furst et al., 2019).

[0245] The step of matching the engineered human T cells to the patient for the HLA-B and HLA-C alleles is an important step because (HLA) mismatch is a major risk factor for antibody-mediated and immune cell-mediated rejection and transplant failure. As used herein, a matched allele refers to the same allele as understood in the relevant art.

[0246] In a plurality of embodiments, the patient comprises the HLA-B and HLA-C alleles of the engineered human T cells.

[0247] In an alternative embodiment, the patient comprises one or more HLA-B and HLA-C alleles of the engineered human T cells.

[0248] In a further embodiment, a method for stimulating a chimeric antigen receptor-mediated immune response in a mammal, preferably for the treatment of cancer, an infectious disease or an autoimmune disease, comprises the following steps: a) administering to the mammal an effective amount of a targeting module comprising at least one target cell-binding domain and a tag-binding domain or tag, At least one target cell binding domain binds to a surface antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6, CD52, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD90, CD99, CD123, CD133, CD135, CD150, CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366 and CD371, cytokine receptors, preferably interleukin receptors, particularly preferably IL-8Rα, IL-8Rβ, IL-11Rα, IL-11Rβ, IL13Rα1; CXCR4, c-Met, mesothelin, members of the epidermal growth factor receptor family and mutants thereof, particularly preferably ErbB1, ErbB2, ErbB3, ErbB4 or mutants thereof; members of the tumor necrosis factor receptor superfamily, claudin, ephrin, ephrin receptor, particularly preferably EphA1-10, EphA5 or EphB1-6; fucosyl transferase, prostate specific antigen, preferably PSCA and PSMA; fetal antigens, preferably CEA and fetal acetylcholine receptor; members of the vascular endothelial growth factor family, EpCAM, AFP, members of the intercellular adhesion molecule family, C-type lectin, integrin, members of the mucin protein family, FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, ligands of the NKG2D receptor, members of the epithelial glycoprotein family, disialoganglioside, glypican, G protein-coupled receptor, human papillomavirus protein, cancer testis antigen, fibroblast activation protein, members of the carbonic anhydrase family, members of the carbohydrate antigen family, Notch ligand, MCSP, glycoprotein A33, guanylate cyclase 2C and tumor-specific glycans, an antibody, antibody fragment, protein, peptide or low molecular weight organic ligand, and mutants and analogs of the specified antibody, antibody fragment, protein, peptide or low molecular weight organic ligand, steps, and b) administering to a mammal an effective amount of engineered human T cells, and c) administering to a mammal an effective amount of at least one further targeting module, comprising the targeting module and the at least one further targeting module comprising different target cell binding domains and the same tag binding domain or tag, and the method being carried out in the order of steps a), b) and c).

[0249] In a preferred embodiment, the targeting module is administered alone, preferably 1 hour to 2 days, more preferably 4 to 24 hours, before administration of the engineered human T cells, and the targeting module is administered until after administration of the engineered human T cells, preferably in the range of 3 days to 30 days. Further, such additional doses of the targeting module can be administered after a rest period to reactivate effector cells having a switchable chimeric antigen receptor.

[0250] The present invention is not limited to the embodiments shown and described, but also includes all embodiments having the same effect within the scope of the gist of the present invention. Further, the present invention is not limited to the specifically described combinations of features, but can also be defined as a whole by any other combination of the specific features of all the individual features disclosed, provided that the individual features are not mutually exclusive or the specific combination of the individual features is not explicitly excluded.

[0251] Hereinafter, the present invention will be described in more detail using examples of embodiments. The examples of embodiments describe engineered human T cells and methods for producing such T cells and are for explaining the present invention without limiting the present invention.

[0252] The implementation of the present invention is described only by way of example with reference to the following attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0253]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Mode for Carrying Out the Invention

[0254] Gene Editing Gene editing is performed by transfecting cells to be edited with an sgRNA specific to the target gene together with Cas9 mRNA or protein. Any transfection method can be applied, including electroporation and lipofection or lipid-mediated delivery.

[0255] Guide Selection The CRISPR / Cas9 sgRNA target sequence is defined as the 5 prime (5’) 20 nucleotides of the NGG protospacer adjacent motif (PAM). sgRNAs were selected for genomic positions targeting cleavage sites within the coding sequences of TRAC (ENSG00000277734), CIITA (ENSG00000179583), and HLA-A (ENSG00000277734). This excluded sgRNAs that cleave within introns or within the 5’ or 3’ untranslated regions (UTRs). sgRNAs that were determined to overlap single nucleotide polymorphisms (SNPs) with high minor allele frequencies (MAF) as seen in GenomAD were excluded (Lek et al., 2016). Top in silico predicted guides for TRAC, CIITA, and HLA-A knockout were screened in primary T cells using target next-generation sequencing (NGS) of polymerase chain reaction (PCR) amplicons derived from the target sites, described herein as amplicon-seq, to quantify on-target insertions / deletions (indels). The editing percentage (e.g., “editing efficiency” or “percent editing” or “indel frequency”) is defined as the total number of sequencing reads with insertions / deletions (“indels”) or substitutions relative to the total number of sequencing reads, including wild type. Nucleotides within 10 base pairs of the intended cleavage site were evaluated for insertions, deletions, and mismatches relative to the reference sequence. Qualification of amplicon-seq for indel quantification using a control guide showed a low limit of detection of 0.5%. LNPs containing the most efficient guide for each target were tested in primary T cells, and knockout efficiency was quantified by NGS and flow cytometry to confirm loss of protein expression (Figure 3).

[0256] Switchable CAR For genetic manipulation to express a switchable CAR, a polynucleotide vector encoding the switchable CAR and all necessary elements to ensure vector expression in the genetically manipulated immune cells are introduced into the immune cells. In particular, the switchable CAR includes IL-2LP (modified human IL-2 leader peptide), RCE (RevCAR epitope, also a tag), G4S1 (glycine-serine linker), ECD (extracellular domain), TMD (transmembrane domain), and ICD (intracellular domain).

[0257] Vector introduction can be performed by nucleic acid electroporation or transfection, or by using viral vector systems such as gene transfer of adenovirus, adeno-associated virus, retrovirus, foamy virus, or lentivirus.

[0258] Lentiviral gene transfer is applied to the stable expression of a switchable CAR in immune cells by first constructing a lentiviral vector encoding the selected switchable CAR. The lentiviral vector is pLVX-EF1 alpha UniCAR 28 / ζ (Clontech, Takara Bio Group), where the lentiviral part of the vector is derived from human immunodeficiency virus (HIV) and the MSC / IRES / ZxGreenI part is replaced by the switchable CAR construct.

[0259] Lentiviral particles are produced by transient transfection of human embryonic kidney (HEK) 293T (ACC 635) cells with a switchable CAR encoding a lentiviral vector plasmid, and co-transfection with a plasmid (psPAX2) encoding group-specific antigen (gag) and polymerase (pol), and a plasmid (pMD2.G) encoding an envelope. After transfection, the packaging plasmid expresses the Gag and Pol proteins of HIV-1. Plasmid MD2.G encodes the glycoprotein of vesicular stomatitis virus (VSV-G). The VSV-G protein is used in lentiviral vectors to introduce a wide range of mammalian cells. A variety of envelopes derived from different viral species can be utilized for this purpose. Lentiviral vectors can be successfully pseudotyped with the envelope glycoprotein (Env) of bidirectional murine leukemia virus (MLV) or the G protein of vesicular stomatitis virus (VSV-G), a modified envelope of prototype foamy virus (PFV), or a chimeric envelope glycoprotein variant derived from gibbon ape leukemia virus (GaLV) and MLV.

[0260] The supernatant of transfected HEK293T cells is collected 24 to 96 hours after transfection, and virus particles are concentrated from the supernatant by ultracentrifugation or other methods. For lentiviral transduction of immune cells, peripheral blood mononuclear cells (PBMCs) or isolated T cells are activated with mabs specific for the CD3 complex, such as clone OKT3 or UCHT1, that are in solution or coated on plastic cell culture dishes or magnetic beads or biodegradable polymer matrices. Activation of PBMCs or isolated T cells is further enhanced by stimulating the co-stimulatory pathway with mabs or ligands specific for CD27, CD28, CD134 or CD137, alone or in combination, that are coated on plastic cell culture dishes or magnetic beads or biodegradable polymer matrices, and by adding exogenous recombinant cytokines such as interleukin (IL)-2, IL-7, IL-12, IL-15 and IL-21. Concentrated or non-concentrated virus particles are added to the culture of PBMCs or T cells 24 to 96 hours after first administration as a single dose or multiple doses of an activating CD3-specific antibody and / or an antibody specific for the co-stimulatory receptors CD27, CD28, CD134 or CD137 and / or a recombinant cytokine. Electroporation, transduction and proliferation of T cells can be performed manually in an open cell culture system or in a closed, partially or fully automated system.

[0261] Stable transduction of T cells can be determined by flow cytometry after staining with a tag-containing molecule for surface expression of a switchable CAR or a mab against the fourth domain of a switchable CAR, 3 days or more after the final administration of the virus supernatant. T cells transduced with a switchable CAR can be expanded in vitro by culturing the T cells in the presence of recombinant cytokines and activating anti-CD3 mab.

[0262] In cases where the switchable CAR has an optional fourth domain that is a peptide sequence forming a linear epitope for the mab, genetically modified immune cells expressing the switchable CAR are specifically grown in vitro by coating the surface of the culture dish, or any type of beads or biodegradable polymer matrix added to the cell culture at a predetermined ratio, with the mab or its antibody fragment that binds to the fourth switchable CAR domain. When the mab coated on the surface binds to the switchable CAR peptide domain, crosslinking of the switchable CAR expressed on the cell surface and formation of an immune synapse are induced, which results in activation of the signaling pathway specifically triggered by the signal domain of the switchable CAR. Depending on the induced signaling pathway, this can lead to enhanced proliferation of immune cells with the switchable CAR and persistence of resistance to activation-induced cell death, and ultimately enrichment of immune cells genetically modified with the switchable CAR in the mixed population.

[0263] The optional fourth domain, which is a peptide sequence forming a linear epitope for the mab, can be further utilized to enrich and purify immune cells expressing the switchable CAR from a mixed population. Enrichment and purification are performed by using the mab or its antibody fragment that binds to the fourth switchable CAR domain to mark the cells expressing the switchable CAR for cell sorting, or to temporarily link the immune cells expressing the switchable CAR to small particles that can be used for cell isolation. In one aspect, the switchable CAR-grafted immune cells are incubated with the mab that recognizes the fourth domain. Next, magnetic beads are added, which conjugate with the antibody or its fragment against the species-specific and isotype-specific heavy and light chains of the mab that binds to the optional fourth domain. In this way, the immune cells expressing the switchable CAR and the magnetic beads are linked, captured by a magnetic field, and separated from other immune cells.

[0264] Generation of engineered human T cells T cells are isolated from fresh leukapheresis products of healthy human donors of HLA - B / C type. The selected T cells are characterized with respect to purity (frequency of CD3+) and content (viability, number of viable cells). Knockout of the T cell receptor alpha chain (TRAC), HLA - A, and class II major histocompatibility complex transactivator (CIITA) is performed via lipid nanoparticles (LNPs) containing Cas9 mRNA and single - guide RNA (sgRNA) for HLA - A, CIITA, or TRAC. These LNPs are composed of a lipid mixture (ionizable lipid, helper lipid, cholesterol, and pegylated lipid) and a cargo of mRNA encoding Cas9 and single - guide RNA targeting the gene of interest. The LNPs are added continuously to the culture, which greatly reduces the risk of translocation. The T cell culture is genetically modified to express RevCAR by adding self - inactivating retroviral vector particles. After the genetic manipulation process, the culture is grown for an additional 5 - 6 days and then harvested. TCRα / β+ cells may be depleted.

[0265] Characterization of engineered human T cells according to the present invention The editing efficiency and the transduction efficiency of RevCAR or UniCAR were evaluated by flow cytometry (Figure 3). The functionality of the engineered human T cells described in the present invention was evaluated with respect to protein expression and phenotype of proliferation markers, exhaustion markers, and additional phenotypic markers after the generation of human primary T cells.

[0266] Figure 4 shows a comparison of protein expression of the proliferation marker Ki-67, exhaustion markers PD-1, LAG-3, TIM-3, IFN-γ, and additional phenotypic markers in human primary T cells transduced to express a switchable CAR (UniCAR or RevCAR). Human primary T cells from four healthy donors were transduced via lentiviral vector-mediated gene transfer to express a switchable CAR (UniCAR = UC02 or RevCAR = RC01). After harvesting, the T cells were stained for the proliferation marker Ki-67 and the exhaustion markers PD-1, LAG-3, TIM-3, IFN-γ, and additional phenotypic markers. Since there was a 2pA site and GFP behind the CAR cDNA, GFP expression in CD3+ cells was used as a surrogate for CAR positivity. T cells transduced with RC01 showed a significant reduction in the expression of the exhaustion markers PD-1 and LAG-3 and a numerical reduction in intracellular IFN-γ expression.

[0267] Figure 5 shows a comparison of the differentiated phenotypes after generation of human primary T cells transduced to express a switchable CAR (UniCAR or RevCAR). Human primary T cells obtained from healthy donors were transduced via lentiviral vector-mediated gene transfer to express a switchable CAR (UniCAR02 = UC02 or RevCAR01 = RC01). After harvesting on day 14, the T cells were stained for the cell surface markers CD4, CD45RO, CD197, and CD28. Cells were gated as GFP+ and CD4+ (A, C, E, G, I) or CD4- (B, D, F, H, J) respectively, and the percentage of the effector / memory population was determined based on the expression of CD45RO, CD197, and CD28. T cells transduced with RC01 showed enrichment of the undifferentiated memory population (SCM: stem cell memory, CM: central memory), and a reduction in the proportion of the more differentiated populations (TM: transitional memory, EM: effector memory, LE: late effector). Thus, RevCAR-T cells showed a less differentiated phenotype after generation.

[0268] Design of a Targeting Module for a Kit According to the Present Invention The targeting module R-TM123 is a soluble recombinant fusion protein having two antibody-derived binding domains. One selectively binds to the target antigen CD123, and the other recognizes the RCE or tag presented on the RevCAR-expressing cells (epitope E5B9 of the human La protein). Thus, R-TM123 functions as a cross-linking module between the Allo-RevCAR-T and the target cancer cells expressing CD123 (Figure 2). The targeting module further includes an 8x-histidine tag at the C-terminus for detection and purification purposes.

[0269] Cytotoxicity Assay The efficacy of the anti-CD123 TM in inducing the elimination of tumor cells by switchable CAR-T cells was tested in a flow cytometry-based cytotoxicity assay using the AML cell lines MV4-11 or OCI-AML3 (Figure 6). Human primary T cells obtained from the same healthy donor were transduced via lentiviral vector-mediated gene transfer to express a switchable CAR (Auto RevCAR-T), or further edited via CRISPR / Cas-mediated knockout of the TRAC locus (Allo RevCAR-T). The efficacy of the TM was evaluated in a flow cytometry-based cytotoxicity assay. The switchable CAR-T cells were incubated with the target cells at an E:T ratio of 2:1 for 48 hours in the presence of various TM concentrations. The target cells were quantified, and the cell numbers of each sample were normalized to the control sample seeded with only tumor cells to calculate lysis. The data were fitted to a four-parameter model with a variable slope of a sigmoid curve. The calculated EC50 values can be interpreted as representative values of the efficacy of the TM against these tumor cells.

[0270] Figure 6 shows the lysis of CD123-expressing AML cell lines MV4-11 or OCI-AML3 in the presence of human primary T cells expressing a switchable CAR (Auto RevCAR-T) or further edited via CRISPR / Cas-mediated knockout of the TRAC locus (Allo RevCAR-T) and different concentrations of the anti-CD123 targeting module (R-TM123). The modified T cells were incubated with the CD123-expressing AML cell lines MV4-11 or OCI-AML3 in the presence of different R-TM123 concentrations (i.e., antibody concentrations). OCI-AML3 cells were pre-stained with the cell tracer eFluor670. The number of surviving eFlour670-positive AML cells was determined by flow cytometry after 48 hours. Lysis was calculated by normalizing the number of surviving AML cells to a control of only surviving AML cells. The derived dose-response curves, EC50 values, and Hill slopes are shown. Non-specific lysis was determined in the absence of R-TM123. The respective EC50 values of Auto RevCAR-T cells and Allo RevCAR-T cells in this short-term assay were comparable. Thus, the lysis activity of RevCAR-T cells was not affected by TRAC editing. Furthermore, non-specific lysis against MV4-11 cells was suppressed by TRAC editing in Allo RevCAR-T cells.

[0271] For the effectiveness of adoptive immunotherapy involving CAR-T, it is necessary for the transferred T cells to survive in vivo. This is particularly interesting for switchable CAR-T platforms such as the RevCAR platform, as long-term survival enables reactivation after discontinuation of soluble adapter administration. A certain combination of surface markers has been associated with an excellent clinical T cell phenotype, and the combination enriched in clinical T cell products enhances anti-tumor efficacy and overall clinical performance (Mahnke et al., 2013).

[0272] Figure 7 shows the phenotypic analysis of RevCAR-T products made using a clinical-scale GMP-compliant process. Accordingly, samples of the formulated and cryopreserved final drug product were thawed and stained for the surface markers CD4, CD28, CD45RA, CD45RO, and CD197 (CCR7). Human T cell subsets were classified according to Mahnke and colleagues (Mahnke et al., 2013). Tcm: Central memory T cells CD28+CD197+, Ttm: Transitional memory T cells CD28+CD197-, Tem: Effector memory T cells CD28-CD197-. Selective enrichment of CD28 and C-C chemokine receptor type 7 (CCR7, CD197) double-positive T cells that can be classified as central memory T cells (Tcm) is seen. In addition to the predominantly Tcm phenotype seen in the product, transitional memory T cells (Ttm) and memory effector T cells (Tem) were detected in much smaller amounts in the RevCAR product (Figure 7). Most T cells (>90%) co-express the two markers CD45RO and CD45RA on the surface, which is typical of T cells in the process of cell division (LaSalle and Hafler, 1991). Cell products containing a greater fraction of CD28-positive and / or CCR7-positive T cells have been shown to correlate with objective tumor responses in non-human primate models and patients (reviewed in Busch et al., 2016; Gattinoni et al., 2017).

[0273] Dose-dependent activation of Allo-RevCAR-T by R-TM123 In the experiments described in the following section, clinical-scale Allo-RevCAR-T was used. CD25 (IL-2 receptor α, IL-2Rα), the high-affinity receptor for IL-2, is expressed on human T cells and can be detected on the cell surface upon stimulation of the endogenous TCR complex (Kmieciak et al., 2009). IL-2Rα regulates the proliferative response of T cells and is an indicator of the degree of TCR stimulation (Shatrova et al., 2016).

[0274] Stimulation of RevCAR via R-TM123 according to SEQ ID NO: 21 is similar to activation by the endogenous TCR, except that the artificial receptor activation signal from the immunoreceptor tyrosine-based activation motif (ITAM) of the CD3ζ moiety is accompanied by a simultaneous co-stimulation signal from the CD28 signaling chain (Cartellieri et al., 2016), and upregulation of CD25 can be monitored after this activation.

[0275] Figure 8 shows the surface expression of CD25 in Allo-RevCAR-T during R-TM123-mediated activation. Allo-RevCAR-T batches were co-cultured with the AML cell lines MOLM-13, MV4-11, and OCI-AML3 expressing CD123 for 48 hours in the presence of various concentrations of R-TM123. Cell samples were prepared and the surface expression of CD2, RevCAR, CD4, CD8, and CD25 was analyzed by flow cytometry. All samples were pre-gated for CD2+ / RevCAR+ cells. The frequency of CD25+ Allo-RevCAR-T cells is shown separately for CD4+ cells and CD8+ cells. Technical triplicates obtained from the co-cultures were pooled and staining data from four clinical-scale batches made from healthy donor material are shown.

[0276] Thus, the surface expression of CD25 in Allo-RevCAR-T in response to R-TM123-mediated stimulation in the presence of target cells expressing CD123 was determined. The frequency of RevCAR-T expressing CD25 is dependent on the dose of R-TM123 (Figure 8). Both CD4+ RevCAR-expressing T cells and CD8+ RevCAR-expressing T cells are activated when Allo-RevCAR-T crosslinks to target cells via R-TM123 (Figure 8). The degree of response dependent on the dose of R-TM123 after 48 hours is similar in both subpopulations (Figure 8 and Table 2).

[0277] [Table 2]

[0278] Cytotoxic response of R-TM123 against CD123 leukemia cell lines in a dose-dependent manner To evaluate dose-dependent target cell lysis, four clinical-scale batches of Allo-RevCAR-T were used in a cytotoxicity assay. Increasing concentrations of R-TM123 were used to analyze the cytotoxic response against three AML cell lines for all four Allo-RevCAR-T batches. The R-TM123 dose-response curves for MOLM-13, OCI-AML3, and MV4-11 are shown in Figure 9.

[0279] Figure 9 shows specific lysis of the AML cell line MOLM-13 by Allo-RevCAR-T redirected by R-TM123. The AML cell lines MOLM-13, MV4-11, and OCI-AML3 expressing CD123 were labeled with the cell tracer eFluor670 and then co-cultured at a 1:1 effector-to-target (E:T) ratio with 2×10 5 Allo-RevCAR-T cells from four clinical-scale batches. After 48 hours of co-culture, the number of viable target cells was determined by cytometry, and specific lysis was determined. The mean ± SD values of technical triplicates and the derived dose-response curves for four clinical-scale batches of Allo-RevCAR-T from independent donors are shown. Data points were fitted with a four-parameter non-linear regression in GraphPad9, and the half-maximal doses of R-TM123 are shown in Table 3.

[0280] The MOLM-13 cell line is derived from the peripheral blood of a patient with relapsed acute monocytic leukemia transformed from myelodysplastic syndrome (FAB M5a) (Matsuo et al., 1997). The OCI-AML3 cell line was established from a patient with AML (FAB M4) and has an NPM1 mutation (type A) and abnormal cytoplasmic translocation of nucleophosmin, an immunocytological feature of AML with mutated NPM1 (Quentmeier et al., 2005). Furthermore, it also has an R882C type DNMT3A mutation (Tiacci et al., 2012). Therefore, both cell lines are major AML subtypes that will be included in future clinical studies. The MV4-11 cell line originally derived from pediatric acute monocytic leukemia and has also been described to express CD123 (Mani et al., 2018).

[0281] Allo-RevCAR-T cells induced lysis of target cells in all of these cell lines, and the lysis occurred in a strictly R-TM123-dependent manner. Maximum half-maximal lysis (EC 50 ) in the single-digit picomolar range was seen in all of the three target cell lines (Table 3). All four clinical-scale batches of Allo-RevCAR-T showed similar maximum half-maximal lysis and reached the upper plateau (i.e., 100% target cell lysis) at approximately 1 nM in all AML cell lines.

[0282]

Table 3

[0283] R-TM123 dose-dependent cytokine release by Allo-RevCAR-T redirected against leukemia cell lines expressing CD123 Upon TCR engagement, T cells are activated and release large amounts of cytokines. Cytokines can have effector, stimulatory, regulatory, chemotactic, and inflammatory functions. Similarly, CAR-engineered T cells release cytokines when stimulated via their artificial receptors (Rossi et al., 2018).

[0284] To characterize the cytokine release capacity of Allo-RevCAR-T, a co-culture assay was used. For this purpose, Allo-RevCAR-T derived from the same four clinical scale batches was used for specific target cell lysis, and the T cell activation studies described in the previous section were analyzed. Cells were thawed and co-cultured with MOLM-13 AML cells in the presence of R-TM123 for 48 hours, and effector cytokines released into the cell culture supernatant were quantified using the MACSPlex Cytotoxic T / NK Cell kit (Miltenyi, Germany).

[0285] Qualitatively, very similar cytokine release profiles showing a large amount of effector cytokine release (granzyme B and perforin) as well as an increase in inflammatory cytokines such as GM-CSF, IFN-γ, TNF-α and IL-2 were seen in different Allo-RevCAR-T clinical scale batches (Figure 10). The anti-inflammatory and regulatory cytokine IL-4 was detected in only two Allo-RevCAR-T batches. Quantitatively, it was revealed that the absolute amounts of individual cytokines were donor- or product-dependent. Furthermore, the maximum half-maximal cytokine release (EC 50 of R-TM123) was determined from sigmoid dose-response curves via non-linear regression (see Table 4). The EC 50 values can be used as a direct indicator of the dynamics of cytokine release and can be compared with other effector functions such as target cell lysis or T cell activation. Usually, the maximum half-maximal cytokine release was significantly different between cytokines rather than between Allo-RevCAR-T cell products. The effector cytokines granzyme B and perforin were already secreted at low R-TM123 concentrations and correlated with target cell lysis (2 - 6 pM in lysis, whereas the EC 50It should be noted that (4 - 12 pM). Inflammatory cytokines showed maximum half - cytokine secretion at much higher R - TM123 doses. For example, GM - CSF was about 35 - 40 pM, IFN - γ was about 20 - 25 pM, or TNF - α was about 50 - 80 pM. In conclusion, the clinical - scale product of Allo - RevCAR - T shows cytokine release typical of T cells. The release varies in amount between cytokines and between T - cell donors or products. Cytokine release correlates with other effector functions of the tested Allo - RevCAR - T (i.e., activation and target - cell lysis), but shifts to higher R - TM123 doses.

[0286] Figure 10 shows the release of cytokines and effector molecules by Allo - RevCAR - T redirected against the AML cell line MOLM - 13 by R - TM123.

[0287] Allo - RevCAR - T, derived from 4 healthy donors and manufactured by a clinical - scale process, was incubated with the AML cell line MOLM - 13 expressing CD123 at the indicated concentrations of R - TM123 and at a 1:1 effector - to - target cell ratio. After 48 hours, T - cell co - culture supernatants were harvested, technical replicates were pooled, and analyzed using a flow - cytometry - based multiplex assay (MACSPlex Cytotoxic T / NK Cell kit; Miltenyi, Germany). Each dose - response curve is shown. Sigmoid data points were fit using 4 - parameter logistic regression in GraphPad Prism 9 to determine the maximum half - cytokine release (EC 50 50) (summarized in Table 4).

[0288]

Table 4

[0289] R - TM123 dose - dependent lysis of primary AML cells by Allo - RevCAR - T The ability of clinical-scale Allo-RevCAR-T batches to lyse primary patient AML material was analyzed in a flow-based cytotoxicity assay. The cytotoxicity assay was performed on primary AML cells (AML1, AML3, AML4, AML5) from four AML patients, each combined with three Allo-RevCAR-T batches (i.e., a total of 12 primary AML / Allo-RevCAR-T pairs). On the day the assay was set up, patient-derived AML cells were thawed, washed, and characterized via flow cytometry for the expression of markers CD45, CD14, HLA-DR, CD33, CD34, and to confirm the expression of target CD123 (data not shown). The Allo-RevCAR-T batches were co-cultured with primary AML cells at an effector-to-target ratio of 1:2 in the presence of R-TM123. After 48 hours of co-culture, the number of viable AML cells was determined via flow cytometry staining (Figure 11).

[0290] Figure 11 shows the cytotoxic response of Allo-RevCAR-T redirected by R-TM123 against primary leukemia cells from AML patients. Allo-RevCAR-T, derived from healthy donors and manufactured in a clinical-scale process, was thawed and co-cultured with 1.25 - 1.5×10 4 primary AML cells at an effector-to-target ratio of 1:2 in the indicated R-TM123 concentration range. IMDM supplemented with 5% FBS, 5 μM β-mercaptoethanol, 1% penicillin / streptomycin, 100 ng / mL stem cell factor (SCF), 10 ng / mL IL-3, 10 ng / mL thrombopoietin (TPO), and 10 ng / mL Fms-related tyrosine kinase 3 ligand (FLT-3L) was used as the culture medium. After 48 hours, the number of viable AML cells was determined by flow cytometry. A four-parameter non-linear fit of log(agonist) vs. response with variable slope was applied to calculate the dose-response curve using GraphPad prism 9. The calculated EC50 values for target cell lysis are summarized in Table 5.

[0291]

Table 5

[0292] Intravenous administration of a single dose of R-TM123 in mice The pharmacokinetic profile of R-TM123 was investigated in vivo in NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ immunodeficient mouse model (hereinafter referred to as NSG). NSG mice were injected intravenously (IV) with a single dose of 1000 or 3000 ng / g of the R-TM123 pharmaceutical from the confirmation experiment. Blood was collected by retro-orbital puncture at 15, 30, 60, 120, 180, 210, 255, 270, 300 and 360 minutes after injection. After plasma was isolated, the concentration of R-TM123 was measured using a specific sandwich ELISA assay with CD123-Fc as the capture agent and an anti-poly His tag monoclonal antibody conjugated to horseradish peroxidase (HRP) for detecting the bound R-TM123. Capture with CD123-Fc indicates the integrity of the anti-CD123 domain, while detection of the poly His tag indicates the presence of the second scFv specific for RCE, since this is located at the N-terminus of the poly His tag. Quantification of the test samples was performed by comparing them to the titration of the R-TM123 standard. The detection limit of the sandwich ELISA assay was determined to be 0.6 ng / mL of assay concentration.

[0293] To determine the plasma half-life, non-compartmental analysis (NCA) and two-compartment analysis (2CA) of plasma data after intravenous bolus injection were performed using PkSolver 2.0 (Zhang et al., 2010). The data of 2CA were further weighted via W = 1 / C obs 2 to fit the final disappearance phase T 1 / 2Focused on -β. The graphic output of the experiment conducted is shown in Fig. 12, and the summary of important pharmacokinetic data is shown in Table 6.

[0294] Fig. 12 shows the in vivo pharmacokinetics of R - TM123 in NSG mice. Graphic output of non - compartmental analysis (NCA) and two - compartment analysis (2CA) of R - TM123 concentration in peripheral blood plasma obtained with the R - TM123 pharmaceutical. Experimental mice (n = 5) were injected IV with 1000 or 3000 ng of R - TM123 per gram of body weight. Peripheral blood samples were collected by retro - orbital puncture at 15, 30, 60, 120, 180, 210, 255, 270, 300, and 360 minutes after the IV bolus injection. The R - TM123 concentration determined by ELISA was analyzed using PkSolver 2.0 (Zhang et al., 2010) NCA and 2CA. The mean ± SD values of samples obtained from 5 individual mice are shown per data point.

[0295] The area - under - the - curve values (AUC) were determined to be 255603.5 (NCA) and 220241.7 ng / mL×min (2CA) for the dose of 1000 ng per gram of body weight, and 644179.4 (NCA) and 555559.9 ng / mL×min (2CA) for the dose of 3000 ng per gram of body weight, respectively, for R - TM123. The calculated elimination half - life (T 1 / 2 ) was 36.5 minutes (NCA) and 38.0 minutes (2CA) for the dose of 1000 ng per gram of body weight, and 47.8 minutes (NCA) and 45.3 minutes (2CA) for the dose of 3000 ng per gram of body weight, respectively, for R - TM123. The obtained T 1 / 2 is consistent with the values reported in the literature for similar scFv constructs (Hutt et al., 2012), and as expected, the plasma concentration of R - TM123 was highest 15 minutes after intravenous injection (T max ), i.e., the earliest time point measured. The maximum plasma concentration (C max) was detected 15 minutes after injection and was determined to be doses of 4682.6 and 11522.7 ng / mL at 1000 ng per gram of body weight or 3000 ng per gram of body weight, respectively. The short half-life observed, as was done with bispecific T cell engagers with comparable short half-lives (Chichili et al., 2015; Hijazi et al., 2018), supports the delivery of R-TM123 by continuous infusion.

[0296]

Table 6

[0297] The results obtained at 255, 270, 300 and 360 minutes at 1000 ng per gram of body weight or at 360 minutes at 3000 ng per gram of body weight were below the lower limit of quantification (LLoQ) of 0.67 - 1.42 ng / mL and were thus excluded from further analysis. In the analysis, the NCA model and 2CA model of plasma data after intravenous bolus injection were applied using PkSolver 2.0 (Zhang et al., 2010). The observed differences between the two doses are most likely due to technical variations in sample collection and / or ELISA performance, especially at lower R-TM123 concentrations. Due to the overall lower plasma levels of R-TM123, the LLoQ was reached significantly earlier at a dose of 1000 ng per gram of body weight (210 minutes) than at a dose of 3000 ng per gram of body weight (300 minutes). The plasma half-life is likely underestimated due to the smaller number of valid measurement points obtained in the final elimination phase. Therefore, the estimated plasma half-lives of the 3000 ng per gram of body weight dose, 47.8 minutes (NCA model) and 45.3 minutes (2CA model), calculated from six measurement points above the LLoQ in the final phase, reflect a more robust dataset.

[0298] In vivo efficacy of Allo-RevCAR-T in different CDX AML models The in vivo efficacy of Allo-RevCAR-T redirected by R-TM123 was confirmed in an extramedullary lesion AML-CDX model using a fluorescence-based readout. In this model, MOLM-13 cells expressing mCherry were subcutaneously injected into the flanks of NSG mice on day 0, either alone or in combination with Allo-RevCAR-T cells. R-TM123 was administered once daily for 5 days in 4 cycles at the indicated dose per gram of body weight around the tumor (Figure 13). A sustained anti-tumor response against MOLM-13 AML cells was observed based on fluorescence in vivo imaging.

[0299] Figure 13 shows the elimination of leukemia by Allo-RevCAR-T redirected against extramedullary lesions of AML by R-TM123. NSG mice were injected subcutaneously with 1×10 6 MV4-11 cells expressing mCherry, either alone or in combination with 5×10 5 Allo-RevCAR-T cells from a clinical-scale experiment, and tumor growth was monitored by optical imaging. Subsequently, the mice were injected with R-TM123 (at the indicated dose per gram of body weight, daily, around the tumor) in 4 cycles of 5 days, separated by a 2-day off period. The percentage of tumor signal was referenced to the first measurement (geometric mean, t0) of each group. Statistical significance was evaluated by two-way analysis of variance (ANOVA) using Dunnett's multiple comparison test, and as a result, in all treatment groups, the P-value was less than 0.05 compared to the group administered Allo-RevCAR-T cells and tumor cells without R-TM123.

[0300] Enhanced RevCAR transduction efficiency in Allo-RevCAR-T compared to the unedited RevCAR-T product T cells were isolated from healthy human donors via positive selection for CD4 and CD8 and cultured in 6-well GRex plates (Wilson Wolf, St. Paul, MN, USA). On the second day after polyclonal activation, transduction of Allo-RevCAR-T and unedited RevCAR-T was performed by adding virus supernatant containing RevCAR to the T cells at a multiplicity of infection (MOI) of 2. For gene editing of Allo-RevCAR-T, steps for knockout of TRAC, HLA-A, and CIITA were performed by adding LNPs containing Cas9 mRNA and the respective sgRNA before and after retroviral transduction. Genetically engineered T cells were expanded on the ninth day after activation, and surface expression of RevCAR was analyzed using flow cytometry. For this purpose, the monoclonal antibody anti-5B9-AF647, which recognizes the functional binding site of RevCAR, was used.

[0301] T cells transduced with RevCAR alone showed an average transduction efficiency of 36.7% ± 17.4%, while T cells further engineered with LNPs for knockout of TRAC, HLA-A, and CIITA showed a percentage of RevCAR-positive cells of 57.9% ± 16.5% (Figure 14). The results suggest that the enhancement of RevCAR transduction is mediated by editing of T cells with LNPs containing sgRNA / mRNA for gene modification.

[0302] Figure 14 shows the percentage of RevCAR-positive T cells in Allo-RevCAR-T compared to the unedited RevCAR-T (Auto-RevCAR-T) product. The data shown are the mean ± SD of RevCAR-T products from 15 independent donors. Statistical significance was calculated using a paired t-test. p < 0.0001 = **** is.

[0303] Cited non-patent literature Bae S, Park J, Kim JS (2014) Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases, Bioinformatics 30, 1473-1475. Brudno JN, Kochenderfer JN (2016) Toxicities of chimeric antigen receptor T cells: recognition and management. Blood 127, 3321-3330. Busch DH, Fraessle SP, Sommermeyer D, Buchholz VR, Riddell SR (2016) Role of memory T cell subsets for adoptive immunotherapy. Semin Immunol 28, 28-34. Cameron P, Fuller CK, Donohoue PD, Jones BN, Thompson MS, Carter MM, Gradia S, Vidal B, Garner E, Slorach EM, Lau E, Banh LM, Lied AM, Edwards LS, Settle AH, Capurso D, Llaca V, Deschamps S, Cigan M, Young JK, May AP (2017) Mapping the genomic landscape of CRISPR-Cas9 cleavage. Nat Methods 14, 600-606. Cartellieri M, Bachmann M, Feldmann A, Bippes C, Stamova S, Wehner R, Temme A, Schmitz M (2010) Chimeric Antigen Receptor-Engineered T Cells for Immunotherapy of Cancer J. Biomed. Biotechnol. Article ID 956304, doi: 10.1155 / 2010 / 956304. Cartellieri M, Feldmann A, Koristka S, Arndt C, Loff S, Ehninger A, von Bonin M, Bejestani EP, Ehninger G, Bachmann MP (2016) Switching CAR T cells on and off: a novel modular platform for retargeting of T cells to AML blasts. Blood cancer J. 6 (8), e458. Chichili GR, Huang L, Li H, Burke S, He L, Tang Q, Jin L, Gorlatov S, Ciccarone V, Chen F, Koenig S, Shannon M, Alderson R, Moore PA, Johnson S, Bonvini E (2015) A CD3xCD123 bispecific DART for redirecting host T cells to myelogenous leukemia: preclinical activity and safety in nonhuman primates. Sci Transl Med, 7(289), 289ra282. Ellis GI, Sheppard NC, Riley JL (2021) Genetic engineering of T cells for immunotherapy. Nature Reviews Genetics 22(7) 427-447. Fuerst D, Neuchel C, Tsamadou C, Schrezenmeier H, Mytilineos J (2019) HLA Matching in Unrelated Stem Cell Transplantation up to Date. Transfus Med Hemother 46(5):326-336. Gattinoni L, Speiser DE, Lichterfeld M, Bonini C (2017) T memory stem cells in health and disease. Nature Medicine 23, 18-27. Giannoukos G, Ciulla DM, Marco E, Abdulkerim HS, Barrera LA, Bothmer A, Dhanapal V, Gloskowski SW, Jayaram H, Maeder ML, Skor MN, Wang T, Myer VE, Wilson CJ (2018) UDiTaS TM , a genome editing detection method for indels and genome rearrangements. BMC Genomics 19, 212. Guedan S, Madar A, Casado-Medrano V, Shaw CE, Wing A, Liu F, Young RM, June CH, Posey AD (2020) Single residue in CD28-costimulated CAR T cells limits long-term persistence and antitumor durability. J Clin Invest. 133215. Hijazi Y, Klinger M, Kratzer A, Wu B, Baeuerle PA, Kufer P, Wolf A, Nagorsen D, Zhu M (2018) Pharmacokinetic and Pharmacodynamic Relationship of Blinatumomab in Patients with Non-Hodgkin Lymphoma. Curr Clin Pharmacol, 13(1), 55-64. Hutt M, Farber-Schwarz A, Unverdorben F, Richter F, and Kontermann RE (2012) Plasma Half-life Extension of Small Recombinant Antibodies by Fusion to Immunoglobulin-binding Domains. J Biol Chem. 287(7): 4462-4469. Kmieciak M, Gowda M, Graham L, Godder K, Bear HD, Marincola FM, Manjili MH (2009) Human T cells express CD25 and Foxp3 upon activation and exhibit effector / memory phenotypes without any regulatory / suppressor function. J Transl Med, 7, 89. LaSalle JM, Hafler DA (1991) The coexpression of CD45RA and CD45RO isoforms on T cells during the S / G2 / M stages of cell cycle. Cellular immunology 138, 197-206. Lek M, Karczewski KJ, Minikel EV, Samocha KE, Banks E, Fennell T, O’Donnell-Luria AH, Ware JS, Hill AJ, Cummings BB, Tukiainen T, Birnbaum DP, Kosmicki JA, Duncan LE, Estrada K, Zhao F, Zou J, Pierce-Hoffman E, Berghout J, Cooper DN, Deflaux N, DePristo M, Do R, Flannick J et al. (2016) Analysis of protein-coding genetic variation in 60,706 humans. Nature 536, 285-291. Liu D, Zhao J, Song Y (2019) Engineering switchable and programmable universal CARs for CAR T therapy. J Hematol Oncol 12, 69. Mahnke YD, Brodie TM, Sallusto F, Roederer M, Lugli E (2013) The who’s who of T-cell differentiation: human memory T-cell subsets. Eur J Immunol 43, 2797-2809. Mani R, Goswami S, Gopalakrishnan B, Ramaswamy R, Wasmuth R, Tran M, Mo X, Gordon A, Bucci D, Lucas DM, Mims A, Brooks C, Dorrance A, Walker A, Blum W, Byrd JC, Lozanski G, Vasu S, Muthusamy N (2018) The interleukin-3 receptor CD123 targeted SL-401 mediates potent cytotoxic activity against CD34(+)CD123(+) cells from acute myeloid leukemia / myelodysplastic syndrome patients and healthy donors. Haematologica, 103(8), 1288-1297. Matsuo Y, MacLeod RA, Uphoff CC, Drexler HG, Nishizaki C, Katayama Y, Kimura G, Fujii N, Omoto E, Harada M, Orita K (1997) Two acute monocytic leukemia (AML-M5a) cell lines (MOLM-13 and MOLM-14) with interclonal phenotypic heterogeneity showing MLL-AF9 fusion resulting from an occult chromosome insertion, ins(11;9)(q23;p22p23). Leukemia, 11(9), 1469-1477. Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA (2010) Case Report of a Serious Adverse Event Following the Administration of T Cells Transduced With a Chimeric Antigen Receptor Recognizing ERBB2. Mol. Ther. 18, 843-851. Neelapu SS, Munoz J, Locke FL, Miklos DB, Brown R, McDevitt JT, Mardiros A, Demirhan E, Konto C, Tees MT (2020) First-in-human data of ALLO-501 and ALLO-647 in relapsed / refractory large B-cell or follicular lymphoma (R / R LBCL / FL): ALPHA study. Journal of Clinical Oncology, 38: 8002-8002. Quentmeier H, Martelli MP, Dirks WG, Bolli N, Liso A, Macleod RA, Nicoletti I, Mannucci R, Pucciarini A, Bigerna B, Martelli MF, Mecucci C, Drexler HG, Falini B (2005) Cell line OCI / AML3 bears exon-12 NPM gene mutation-A and cytoplasmic expression of nucleophosmin. Leukemia, 19(10), 1760-1767. Pinello L, Canver MC, Hoban MD, Orkin SH, Kohn DB, Bauer DE, Yuan GC (2016) Analyzing CRISPR genome-editing experiments with CRISPResso. Nat Biotechnol 34, 695-697. Reinke AW, Grant RA, Keating AE (2010) A Synthetic Coiled-Coil Interactome Provides Heterospecific Modules for Molecular Engineering. JACS 132, 6025-6031. Qasim W, Zhan H, Samarasinghe S, Adams S, Amrolia P, Stafford S, Butler K, Rivat C, Wright G, Somana K, Ghorashian S, Pinner D, Ahsan G, Gilmour K, Lucchini G, Inglott S, Mifsud W, Chiesa R, Peggs KS, Chan L, Farzeneh F, Thrasher AJ, Vora A, Pule M, Veys P (2017) Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells. T cells. Sci Transl Med. 9(374), eaaj2013. Rossi J, Paczkowski P, Shen YW, Morse K, Flynn B, Kaiser A, Ng C, Gallatin K, Cain T, Fan R, Mackay S, Heath JR, Rosenberg SA, Kochenderfer JN, Zhou J, Bot A (2018) Preinfusion polyfunctional anti-CD19 chimeric antigen receptor T cells are associated with clinical outcomes in NHL. Blood 132(8), 804-814. Shatrova AN, Mityushova EV, Vassilieva IO, Aksenov ND, Zenin VV, Nikolsky NN, Marakhova, II (2016). Time-Dependent Regulation of IL-2R α-Chain (CD25) Expression by TCR Signal Strength and IL-2-Induced STAT5 Signaling in Activated Human Blood T Lymphocytes. PLoS One, 11(12), e0167215. Sotillo E, Barrett DM, Black K., Bagashev A, Oldridge D, Wu G, Sussman R, Lanauze C, Ruella M, Gazzara MR, Martinez NM, Harrington CT, Chung EY, Perazzelli J, Hofmann TJ, Maude SL, Raman P, Barrera A, Gill S, Lacey SF, Melenhorst JJ, Allman D, Jacoby E, Fry T, Mackall C, Barash Y, Lynch KW, Maris JM, Grupp SA, Thomas-Tikhonenko A (2015) Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy. Cancer Discov. 5, 1282-1295. Tiacci E, Spanhol-Rosseto A, Martelli MP, Pasqualucci L, Quentmeier H, Grossmann V, Drexler HG, Falini B (2012) The NPM1 wild-type OCI-AML2 and the NPM1-mutated OCI-AML3 cell lines carry DNMT3A mutations. Leukemia, 26(3), 554-557. Titov A, Petukhov A, Staliarova A, Motorin D, Bulatov E, Shuvalov O, Soond SM, Piacentini M, Melino G, Zaritskey A, Barlev NA (2018) The biological basis and clinical symptoms of CAR-T therapy-associated toxicites. Cell Death Dis 9, 897. Weber EW, Parker KR, Sotillo E, Lynn RC, Anbunathan H, Lattin J, Good Z, Belk JA, Daniel B, Klysz D, Malipatlolla M, Xu P, Bashti M, Heitzeneder S, Labanieh L, Vandris P, Majzner RG, Qi Y, Sandor K, Chen LC, Prabhu S, Gentles AJ, Wandless TJ, Satpathy AT, Chang HY, Mackall CL (2021) Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling. Science 372 (6537), eaba1786. Zhang Y (2021) A Novel Strategy for Off-the-shelf T Cell Therapies Evading Host T Cell and NK Cell Rejection. Oral presentation. European Society of Gene and Cell Therapy Annual Congress. Zhang Y, Huo M, Zhou J, Xie S (2010) PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. Comput Methods Programs Biomed. 99(3), 306-14.

[0304] List of reference symbols 1 First domain, tag-binding domain or tag 2 Second domain, extracellular hinge and transmembrane domain 3 Third domain, signaling domain 4 Optional fourth domain, short peptide linker

Claims

**Claim 1** i. Reduced or eliminated surface expression of the endogenous T cell receptor alpha chain due to genetic modification in the T cell receptor alpha chain gene, ii. Reduced or eliminated surface expression of HLA-A compared to unmodified T cells due to genetic modification in the HLA-A gene, iii. Reduced or eliminated surface expression of HLA class II due to genetic modification in the CIITA gene, and iv. An inverse universal chimeric antigen cell surface receptor comprising: - A tag, - An extracellular hinge and transmembrane domain, and - A signal transduction domain A manipulated human T cell comprising the same. **Claim 2** The manipulated human T cell according to claim 1, wherein the tag is a peptide epitope tag. **Claim 3** The manipulated human T cell according to claim 2, wherein the peptide epitope tag is a myc tag, His tag, short linear peptide sequence derived from yeast transcription factor GCN4, leucine zipper sequence, or short linear peptide sequence derived from a human nuclear protein, preferably a short linear peptide sequence derived from human La protein. **Claim 4** The manipulated human T cell according to any one of claims 1 to 3, wherein the extracellular hinge and transmembrane domain are selected from the hinge and transmembrane domain of the human CD28 molecule, CD8a chain NK cell receptor, or a portion of the constant region of an antibody, mutants thereof, and combinations thereof. **Claim 5** The manipulated human T cell according to any one of claims 1 to 4, wherein the signal transduction domain is selected from the cytoplasmic regions of CD28, CD137 (4-1BB), CD134 (OX40), CD278 (ICOS); DAP10 and CD27, programmed cell death-1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), cytoplasmic region of the CD3 chain, DAP12, CD122 (interleukin-2 receptor β), CD132 (interleukin-2 receptor γ), CD127 (interleukin-7 receptor α), CD360 (interleukin-21 receptor), activating Fc receptor, mutants thereof, and combinations thereof. **Claim 6** The manipulated human T cell according to any one of claims 1 to 5, which is homozygous for HLA-B and homozygous for the HLA-C genotype. **Claim 7** A pharmaceutical composition comprising the engineered human T cells according to any one of claims 1 to 6.

8. The pharmaceutical composition according to claim 7, for use in a method for stimulating a chimeric antigen receptor-mediated immune response in a mammal, preferably for use in the treatment of cancer, infectious diseases or autoimmune diseases.

9. a) Engineered human T cells comprising: i. Reduced or eliminated surface expression of the endogenous T cell receptor alpha chain due to genetic modification in the gene of the T cell receptor alpha chain, ii. Reduced or eliminated surface expression of HLA-A compared to unmodified T cells due to genetic modification in the HLA-A gene, iii. Reduced or eliminated surface expression of HLA class II due to genetic modification in the CIITA gene, and iv. An inverse universal chimeric antigen cell surface receptor comprising: - A tag, - An extracellular hinge and transmembrane domain, and - A signal transduction domain, and b) A targeting module comprising a tag-binding domain and at least one target cell-binding domain, or a nucleic acid, vector or cell encoding said targeting module, A kit comprising, wherein the tag-binding domain of the targeting module binds to the tag of the inverse universal chimeric antigen cell surface receptor.

10. The kit according to claim 9, wherein the at least one target cell binding domain binds to a surface antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6, CD52, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD90, CD99, CD123, CD133, CD135, CD150, CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366 and CD371, cytokine receptors, CXCR4, c-Met, mesothelin, members of the epidermal growth factor receptor family and mutants thereof, members of the tumor necrosis factor receptor superfamily, claudin, ephrin, ephrin receptor, fucosyltransferase, prostate specific antigen, fetal antigen, members of the vascular endothelial growth factor family, EpCAM, AFP, members of the intercellular adhesion molecule family, C-type lectin, integrin, members of the mucin protein family, FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, ligands of the NKG2D receptor, members of the epithelial glycoprotein family, disialoganglioside, glypican, G protein-coupled receptor, human papillomavirus protein, cancer testis antigen, fibroblast activation protein, members of the carbonic anhydrase family, members of the carbohydrate antigen family, Notch ligand, MCSP, glycoprotein A33, guanylate cyclase 2C and tumor-specific glycans, and is an antibody, antibody fragment, protein, peptide or low molecular weight organic ligand.

11. further comprising at least one further targeting module or at least one further nucleic acid, vector or cell encoding a further targeting module, wherein the at least one further targeting module comprises at least one target cell binding domain and a tag binding domain. The at least one target cell binding domain binds to a surface antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6, CD52, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD90, CD99, CD133, CD135, CD150, CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366 and CD371, cytokine receptors, CXCR4, c-Met, mesothelin, members of the epidermal growth factor receptor family and mutants thereof, members of the tumor necrosis factor receptor superfamily, claudin, ephrin, ephrin receptor, fucosyltransferase, prostate specific antigen, fetal antigen, members of the vascular endothelial growth factor family, EpCAM, AFP, members of the intercellular adhesion molecule family, C-type lectin, integrin, members of the mucin protein family, FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, ligands of the NKG2D receptor, members of the epithelial glycoprotein family, disialoganglioside, glypican, G protein-coupled receptor, human papillomavirus protein, cancer testis antigen, fibroblast activation protein, members of the carbonic anhydrase family, members of the carbohydrate antigen family, Notch ligand, MCSP, glycoprotein A33, guanylate cyclase 2C and tumor-specific glycans, an antibody, antibody fragment, protein, peptide or low molecular weight organic ligand, the kit according to claim 9 or 10.

12. The kit according to any one of claims 9 to 11, wherein the engineered human T cell and / or the targeting module is in the form of a pharmaceutical composition.

13. For use in a method for stimulating a chimeric antigen receptor-mediated immune response in a mammal, preferably for use in the treatment of cancer, infectious disease or autoimmune disease, the kit according to any one of claims 9 to 12.

14. a) A CRISPR system comprising the following: - A nucleic acid targeting a region of the endogenous T cell receptor alpha chain (TRAC) gene, - A nucleic acid targeting a region of the endogenous HLA-A gene, - A nucleic acid targeting a region of the endogenous CIITA gene, and b) A nucleic acid encoding a reverse universal chimeric antigen cell surface receptor or a vector containing the nucleic acid, comprising: - A tag, - An extracellular hinge and transmembrane domain, and - A signal transduction domain, and c) A targeting module comprising a tag-binding domain and at least one target cell-binding domain, or a nucleic acid, vector or cell encoding the targeting module, A kit comprising, wherein the tag-binding domain of the targeting module binds to the tag of the reverse universal chimeric antigen cell surface receptor.

15. The kit according to claim 14, wherein the CRISPR system is packaged in lipid nanoparticles and comprises mRNA of CRISPR-associated protein 9 and a nucleic acid targeting TRAC, HLA-A or CIITA.

16. The kit according to claim 15, wherein the nucleic acid targeting TRAC comprises or encodes SEQ ID NO: 1, and / or the nucleic acid targeting HLA-A comprises or encodes SEQ ID NO: 2, and / or the nucleic acid targeting CIITA comprises or encodes SEQ ID NO:

3.

17. A method for producing engineered human T cells expressing a reverse universal chimeric antigen cell surface receptor, comprising: (1) Providing human T cells, (2) Transfecting human T cells with a CRISPR system comprising a nucleic acid targeting a region of the endogenous T cell receptor alpha chain within the T cells, (3) Transfecting human T cells with a CRISPR system comprising a nucleic acid targeting a region of HLA-A within the T cells, and (4) Transfecting human T cells with a CRISPR system comprising a nucleic acid targeting a region of CIITA within the T cells to produce CRISPR-modified T cells, and (5) Transducing human T cells with a nucleic acid encoding a reverse universal chimeric antigen cell surface receptor or a vector containing the nucleic acid, comprising: - A tag, - An extracellular hinge and transmembrane domain, and ・A signal transduction domain A method comprising the same.

18. The method for producing an engineered human T cell expressing an inverse universal chimeric antigen cell surface receptor according to claim 17, wherein the T cell is homozygous for HLA-B and homozygous for HLA-C.

19. The method for producing an engineered human T cell expressing an inverse universal chimeric antigen cell surface receptor according to claim 18, further comprising matching the engineered human T cell to the patient for HLA-B alleles and HLA-C alleles.

20. The method for producing an engineered human T cell expressing an inverse universal chimeric antigen cell surface receptor according to any one of claims 17 to 19, wherein the CRISPR system comprises a lipid nucleic acid assembly composition comprising an endonuclease or an mRNA encoding the endonuclease and a target site-specific single guide RNA.

21. The method for producing an engineered human T cell expressing an inverse universal chimeric antigen cell surface receptor according to claim 20, wherein the endonuclease is CRISPR-associated enzyme 9.

22. The method for producing an engineered human T cell expressing an inverse universal chimeric antigen cell surface receptor according to claim 20 or 21, wherein the target site-specific single guide RNA in step (2) comprises a nucleotide sequence according to SEQ ID NO: 1, and / or the target site-specific single guide RNA in step (3) comprises a nucleotide sequence according to SEQ ID NO: 2, and / or the target site-specific single guide RNA in step (4) comprises a nucleotide sequence according to SEQ ID NO:

3.

23. The method for producing an engineered human T cell expressing an inverse universal chimeric antigen cell surface receptor according to any one of claims 20 to 22, wherein the lipid nucleic acid assembly composition comprises lipid nanoparticles.

24. The method for producing an engineered human T cell expressing an inverse universal chimeric antigen cell surface receptor according to claim 23, wherein the lipid nanoparticles are a mixture of cationic and / or ionizable lipids and helper lipids.

25. A method for producing engineered human T cells expressing an inverse universal chimeric antigen cell surface receptor according to claim 23 or 24, wherein the lipid nanoparticles are a mixture of an ionizable lipid, a helper lipid, and PEG-DMG.

26. The method for producing engineered human T cells expressing an inverse universal chimeric antigen cell surface receptor according to any one of claims 17 to 25, wherein the introduction of a nucleic acid or a vector containing the nucleic acid encoding the inverse universal chimeric antigen cell surface receptor according to step (5) is performed by adding retroviral vector particles, lentiviral vector particles, or adenoviral vector particles.

27. The following steps: c) administering to a mammal, preferably a human, having cancer, an infectious disease, or an autoimmune disease, an effective amount of a targeting module comprising a tag-binding domain and at least one target cell-binding domain, or a nucleic acid or pharmaceutical composition comprising the targeting module, and d) administering to a mammal an effective amount of the engineered human T cells according to any one of claims 1 to 6 or a pharmaceutical composition comprising the engineered human T cells, wherein the tag-binding domain of the targeting module binds to the tag of the inverse universal chimeric antigen cell surface receptor. A method for treating cancer, an infectious disease, or an autoimmune disease, comprising: The method wherein the targeting module is administered to the mammal before, simultaneously with, or after the administration of the engineered human T cells.

Images