Antigen-specific T cells by gene editing of CD3 epsilon

Abstract

The present invention relates to a nucleic acid construct for targeting and in-frame integration of a binding domain into the endogenous CD3 epsilon gene of a human cell. The present invention further relates to genetically modified human T cells comprising an exogenous nucleic acid sequence region encoding an antigen-binding domain integrated in-frame into the endogenous CD3 epsilon gene. In a preferred embodiment, the T cells are regulatory T cells (Tregs). The present invention further relates to genetically modified T cells used as a medicament for preventing and / or treating unwanted (pathogenic) immune responses, preferably before and / or after allogeneic transplantation or autoimmune diseases, more preferably for preventing and / or treating graft-versus-host disease (GVHD). The present invention also relates to a method for preparing therapeutic genetically modified T cells, comprising repeated stimulation via the TCR / CD3 complex and CD28 coreceptor, wherein the therapeutic genetically modified regulatory T cells exhibit essentially unchanged expansion (±50%) in culture compared to unmodified T cells.

Description

[Technical Field]

[0001] The present invention relates to the fields of biology and medicine, in particular to the genetic modification of therapeutic cell products.

[0002] The present invention relates to nucleic acid constructs that allow targeted in-frame integration of a binding domain into the endogenous CD3 epsilon gene to generate a gene fusion and thus an engineered functional CD3-T cell receptor (TCR) complex comprising an exogenous binding domain, such as an antibody or antigen-binding fragment thereof, fused to the endogenous CD3 epsilon protein.

[0003] Furthermore, the present invention relates to a nucleic acid construct comprising two nucleic acid sequence regions, wherein a first nucleic acid sequence region encodes an antigen-binding domain and a second nucleic acid sequence region encodes a targeting sequence configured to integrate the construct in frame with the endogenous CD3 epsilon gene of a human cell.

[0004] In a further aspect, the present invention relates to genetically modified human T cells comprising an exogenous nucleic acid sequence region encoding an antigen-binding domain integrated in-frame into an endogenous CD3 epsilon gene. Furthermore, the present invention relates to genetically modified human T cells that express the CD3 epsilon protein as an in-frame fusion protein comprising the antigen-binding domain and the endogenous CD3 epsilon protein, wherein T cell receptor (TCR) function is maintained in the presence of the in-frame fusion protein. The modified T cells are preferably regulatory T cells (Tregs). Furthermore, the present invention relates to a polypeptide expressed from the genetically modified T cells as a fusion protein comprising the antigen-binding domain and the endogenous CD3 epsilon protein.

[0005] Furthermore, the present invention relates to a pharmaceutical composition for therapeutic intervention in human diseases, comprising genetically modified T cells, preferably Tregs, and a pharmaceutically acceptable carrier. Furthermore, the present invention relates to corresponding medical uses and therapeutic methods in the treatment and / or prevention of pathologies such as unwanted (pathogenic) immune responses, including unwanted (pathogenic) immune responses after allogeneic transplantation, and for the prevention and / or treatment of graft-versus-host disease (GvHD).

[0006] In a further aspect, the present invention relates to a method for preparing therapeutic genetically modified T cells, preferably Treg cells, comprising repeatedly stimulating genetically modified T cells expressing an altered functional CD3-TCR complex with an in-frame fusion protein comprising an exogenous antigen-binding domain and an endogenous CD3 epsilon protein, wherein said therapeutic genetically modified T cells, preferably genetically modified Treg cells, exhibit essentially unchanged expansion kinetics in culture (±50%) compared to unmodified T cells.

[0007] Furthermore, the present invention relates to oligonucleotides, such as gRNAs and primers, suitable for in-frame integration into the endogenous CD3 epsilon gene or amplification of the nucleic acid constructs according to the present invention. [Background technology]

[0008] Adoptive transfer of immune cells is an immunotherapeutic treatment with great potential for the treatment of immune-mediated diseases and cancer. In particular, the adoptive transfer of T cells, such as regulatory T cells (Tregs), has attracted considerable interest. Treg cells are immunosuppressive lymphocytes that prevent autoimmune diseases, regulate and terminate excessive immune responses, ensure tissue regeneration, and control bacterial homeostasis at mucosal surfaces. Furthermore, clinical data from patients undergoing solid organ transplantation or hematopoietic stem cell transplantation suggest that a higher frequency of activated Treg cells after transplantation is associated with better graft survival and a lower incidence of graft-versus-host disease (GvHD). Congenital Treg cell dysfunction is associated with severe autoimmune diseases. Therefore, adoptive transfer of Treg cells is an emerging modality for personalized immunosuppression with many potential applications.

[0009] Adoptive transfer of ex vivo expanded polyclonal Treg cells has been clinically tested as a potential treatment for inducing tolerance to solid organ transplants (Non-Patent Document 1), resolving chronic GvHD (Non-Patent Document 2), and ameliorating autoimmune diseases such as type 1 diabetes (Non-Patent Document 3) or inflammatory bowel disease (Non-Patent Document 4). The transfer of antigen-specific Treg cells has shown high efficacy in most preclinical models of the aforementioned diseases due to their excellent homing and tissue-specific activation (Non-Patent Document 5). Clinical studies on kidney transplant patients have demonstrated that ex vivo expansion and transfer of autologous Treg cells allows for the reduction of drug-based immunosuppression without increasing graft rejection. In summary, this study demonstrates that Treg cell therapy is safe, does not increase infections, and can effectively replace drug-based immunosuppression (Non-Patent Document 1). Further studies have demonstrated that adoptive transfer of donor Treg cells after allogeneic stem cell transplantation in refractory chronic GVHD can lead to remarkable clinical remission (Non-Patent Document 2).

[0010] However, the low abundance of antigen-specific Treg cells in peripheral blood has led to manufacturing concerns, slowing the adoption of antigen-specific Treg cells in clinical settings (Non-Patent Document 6). To date, Treg therapy has primarily been performed using polyclonal Treg cells. Here, Treg cells are isolated from the donor's peripheral blood along with other mononuclear cells and enriched via conventional Treg markers. Treg cells are then expanded by polyclonal stimulation via their T cell receptor / CD3 complex and additional costimulation using beads equipped with anti-CD3 and anti-CD28 antibodies. Furthermore, high doses of IL-2 and the mTOR inhibitor rapamycin are added to the culture medium as growth factors. However, in polyclonal products, only a low percentage of Treg cells exhibit the desired antigen specificity and are alloreactive, i.e., capable of recognizing the kidney of an allogeneic organ donor and thus exerting a protective effect on the graft.

[0011] An alternative approach to producing antigen-specific Treg cell products is genetic engineering of cells. Polyclonal Treg cells can be made specific for a single target tissue through overexpression of transgenic T cell receptors (TCRs) or chimeric antigen receptors (CARs). CARs, in particular, are a clinically relevant strategy. Chimeric antigen receptors (CARs) are tools that direct Treg cells to specific tissues or cell types.

[0012] CARs are synthetic fusion genes that typically combine the antigen-specific portion of a monoclonal antibody (called a single-chain variable fragment [scFv]) with an intracellular signaling domain that triggers T cell activation, usually via CD3 zeta. The poor performance of "first-generation" CARs prompted the incorporation of additional costimulatory signaling domains, such as 4-1BB or CD28, to enhance T cell function. Although the CAR-mediated immune synapse remains inefficient compared to the T cell receptor (TCR), a number of second-generation CARs have produced dramatic clinical success in the treatment of B cell malignancies. In Treg cells, CD28 is the preferred costimulatory domain in second-generation CARs (Non-Patent Document 7). An alternative strategy to generate antigen-specific T cell products from conventional T cells is to lentivirally transduce T cells with so-called T cell receptor fusion constructs (TruCs), which encode fusion proteins in which the antigen-binding domain is fused to the CD3 delta, gamma, or epsilon subunit and which, upon expression, form an integral part of the CD3 / TCR complex (Non-Patent Document 8).

[0013] However, retroviral gene transfer predominates in the production of CAR-redirected T cells and TruC T cells. Retroviral production is costly, time-consuming, and requires sophisticated safety measures in the clinical stage. Non-viral approaches using transposase technologies such as PiggyBac or Sleeping Beauty represent more affordable alternatives for gene transfer. However, these have not been adopted for Treg cells, likely due to the toxicity of plasmid transfection. Furthermore, two recent cases of CAR-derived lymphoma in studies using hyperactive PiggyBac-modified T cells (Non-Patent Document 9, Non-Patent Document 10) are evidence of the potential risk of mutagenesis when using transposase technologies, which may result from random insertion of multiple transgene copies, gene dysregulation by exogenous promoters, or genetic scarring due to transgene hopping of transposable elements (Non-Patent Document 11).

[0014] CRISPR-Cas gene editing is an efficient alternative that allows site-specific gene transfer in Treg cells (Non-Patent Document 12), thereby reducing the risk of insertional mutagenesis. Virus-free knock-in using synthetic dsDNA and CRISPR-Cas9 RNP electroporation has been shown to enable efficient reprogramming of T cells, such as Treg cells, including the transfer of therapeutic TCRs and CARs (Non-Patent Document 12, Non-Patent Document 13, Non-Patent Document 14, Non-Patent Document 15). In studies using this approach, transgenic antigen receptors are typically integrated into the TCR alpha constant gene (TRAC) because this leads to TCR replacement with a low risk of alloreactivity and physiological control of the transgene, which has been associated with improved fitness of CAR T cells in leukemia models (Non-Patent Document 16, Non-Patent Document 17). Alternatively, it has been shown that endogenous CD3 zeta chain (CD247) can be used to express CARs in Tregs as well as T cells (Patent Document 1).

[0015] However, both TRAC-KI and CD247-KI approaches result in CAR-Treg cells that no longer have a TCR on their surface because the TCR is replaced by the CAR. This makes cell expansion by repeated stimulation with anti-CD3 / 28 beads difficult, if not impossible. Therefore, the absence of endogenous TCR-CD3 receptors results in the lack of stimulatory capacity of the cells and the irreproducible low yield of antigen-specific Treg cells, preventing their clinical and large-scale application.

[0016] Patent Document 2 describes the first human CD4 + T cells and CD8 +They disclose the insertion of a heterologous CD19 scFv into the CD3 epsilon locus of T cells to generate a fusion protein of the scFv and the CD3 epsilon protein, whereby the scFv is located extracellularly at the N-terminus of the CD3 epsilon molecule while maintaining the TCR alpha chain. However, they do not disclose such modification of Treg cells.

[0017] Patent Document 3 discloses inserting an scFv sequence that binds to an anti-tumor antigen into the N-terminus of the human CD3 epsilon locus of immune cells such as T cells, thereby producing T cells that express the fusion protein, but does not disclose such modification of Treg cells.

[0018] Non-Patent Document 18 discloses a method for generating Treg cells expressing an N-terminal fusion protein of FVIII scFv and CD3 epsilon by retroviral transduction of a nucleic acid construct. However, this method requires the use of a viral vector that involves safety concerns, high complexity, and costly handling.

[0019] Given the medical need and great therapeutic potential of adoptive T cell therapy, particularly Treg cell therapy, for the prevention and / or treatment of unwanted pathogenic immune responses, simple and reproducible means of producing antigen-specific T cells in sufficient numbers are urgently needed to enable improved clinical use of such cells. Furthermore, there is a need for efficient, affordable, and particularly safe techniques for obtaining antigen-specific T cells that do not incur the risks known in prior art techniques, such as mutagenesis. [Prior art documents] [Patent documents]

[0020] [Patent Document 1] International Publication No. 2022 / 136551 [Patent Document 2] International Publication No. 2022 / 098787 [Patent Document 3] International Publication No. 2021 / 022327

Non-licensed literature

[0021]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

[0022] In view of the prior art, the technical problem underlying the present invention is to provide improved or alternative means for immunotherapy of unwanted (pathogenic) immune responses and cancerous diseases. A further object of the present invention is to provide a novel strategy for immunotherapy using genetically engineered therapeutic T cells.

[0023] In particular, one of the problems underlying the present invention is to provide a means for generating therapeutic modified T cells having antigen-specific receptors that can be expanded ex vivo and overcome the disadvantages of the prior art. More specifically, one of the problems underlying the present invention is to provide a means for generating antigen-specific modified Tregs that allow reliable expansion ex vivo. Another problem to be solved is to provide a means for increasing the production capacity of genetically modified antigen-specific Tregs. Another problem underlying the present invention is to provide a gene editing strategy for reprogramming T cells against specific antigens by genetically modifying the CD3 / TCR complex. [Means for solving the problem]

[0024] These problems are solved by the features of the independent claims. Preferred embodiments of the invention are provided by the dependent claims.

[0025] In one embodiment, the present invention provides a nucleic acid construct comprising: a first nucleic acid sequence region encoding an antigen-binding domain; a second nucleic acid sequence region comprising a targeting sequence configured to integrate the construct in frame with the endogenous CD3 epsilon gene of a human cell; The present invention relates to a nucleic acid construct comprising:

[0026] Surprisingly, the nucleic acid construct of the present invention allows for the easy and precise in-frame integration of a transgene in the form of a sequence encoding an antigen-binding domain into the endogenous CD3 epsilon gene of a T cell. This provides a novel, non-viral gene editing strategy for reprogramming T cells to specific antigens while (i) preserving the functionality of the CD3 / TCR receptor complex and (ii) maintaining T cell fitness. Furthermore, in-frame integration of the nucleic acid construct allows for the minimization of transgenes, which is beneficial for non-viral gene editing methods that typically exhibit limited gene payload capacity. This non-viral in-frame integration avoids the use of viral vectors and the associated safety concerns, complexity, and costly handling.

[0027] In contrast, conventional methods for obtaining antigen-specific T cells typically involve introducing a chimeric antigen receptor (CAR) to replace the cellular CD3 / TCR complex. However, because the CD3 / TCR complex is essential for effective ex vivo expansion of T cells using conventional methods, such as repeated stimulation with antibody-coated beads, impaired CD3 / TCR complex function prevents the effective expansion of these cells, thus resulting in high costs and low efficacy of T cell immunotherapy. Given the low abundance of antigen-specific T cells typically obtained from patients' peripheral blood, in-frame integration using the nucleic acid constructs of the present invention allows both simple, non-viral generation of antigen-specific cells and effective ex vivo expansion of these modified cells through repeated stimulation. This allows for the production of large numbers of antigen-specific cells, thereby improving the efficacy of immunotherapy using such cells while further reducing costs and making these cells widely applicable in various clinical settings.

[0028] Furthermore, the nucleic acid constructs of the present invention can be easily modified by those skilled in the art, particularly with respect to the sequence region encoding the antigen-binding domain, so that T cells specific to a particular antigen can be obtained, and the construct and the resulting modified T cells will be specific for a particular antigen and therefore applicable to a wide variety of indications, such as undesired (pathogenic) immune responses, autoimmune diseases, or cancerous diseases.

[0029] In one embodiment, the targeting sequence is configured to integrate the construct in frame with an exon of the endogenous CD3 epsilon gene.

[0030] In some embodiments, the exon of the endogenous CD3 epsilon gene into which the nucleic acid construct is inserted may be exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or exon 8, preferably exon 1, exon 2, exon 3, exon 4, exon 5, or exon 6, more preferably exon 3 or exon 6, more preferably exon 3.

[0031] In one embodiment, the targeting sequence is configured to integrate the construct in-frame into exon 3 or exon 6 of the endogenous CD3 epsilon gene.

[0032] Surprisingly, the nucleic acid construct of the present invention allows for direct in-frame integration of an antigen-binding site into an exon of the endogenous CD3 epsilon gene without resulting in loss of function of the endogenous CD3 epsilon gene upon expression. This is advantageous for obtaining an antigen-specific, functional T cell fusion protein (also called receptor fusion construct (TruC)) containing a functional CD3 / TCR complex and an antigen-binding domain. Furthermore, in-frame integration into an exon of the endogenous CD3 epsilon gene allows for control of the expression of the TRuC construct by the endogenous promoter, thereby ensuring the maintenance of functionality and fitness of the resulting genetically modified T cells. Surprisingly, exons 3 and 6 in particular resulted in efficient expression of TRuC in T cells.

[0033] In an embodiment of the invention, the target site in the CD3 epsilon gene into which the first sequence region is integrated is located downstream of the sequence encoding the CD3 epsilon signal peptide and upstream of the endogenous CD3 epsilon sequence (generating an N-terminal fusion after removal of the signal peptide).

[0034] The generation of an N-terminal fusion advantageously generates a fusion protein comprising an antigen-binding domain and an endogenous CD3 epsilon protein, which is functional with respect to both the CD3 epsilon protein and the antigen-binding domain. This N-terminal fusion protein is advantageously able to form an integral part of the endogenous CD3 / TCR complex of a T cell, thereby generating antigen-specific T cells that can be efficiently expanded ex vivo.

[0035] In some cases, sequence regions upstream of the integrated sequence region encoding the antigen binding domain are transcribed along with the integrated sequence region, and therefore, in some embodiments, means are provided to isolate any sequence upstream of the antigen binding domain during or after translation.

[0036] In some embodiments, a nucleic acid sequence region encoding a protein separation site, such as a self-cleaving peptide, a protease cleavage site, or an internal ribosome entry site (IRES), preferably a self-cleaving peptide, is located upstream of the integrated sequence region encoding the antigen-binding domain. The protein separation site is upstream of the sequence region encoding the antigen-binding domain and is translated together with that sequence, allowing separation of any potentially dysfunctional or unwanted sequences in the in-frame fusion peptide.

[0037] Thus, in one embodiment, the antigen binding domain coding region is separated from the endogenous gene and / or mRNA sequence segment by a sequence encoding a self-cleaving peptide, a proteolytic cleavage site, and / or an IRES site.

[0038] In one embodiment, the polypeptide cleavage site is selected from the group consisting of P2A, T2A, E2A, and F2A.

[0039] In some embodiments, the self-cleaving peptide is preferably a 2A peptide, more preferably selected from the group consisting of a foot-and-mouth disease virus (FMDV) 2A peptide, an equine rhinitis A virus (ERAV) 2A peptide, a Thosea asigna virus (TaV) 2A peptide, a porcine teschovirus-1 (PTV-I) 2A peptide, a tylovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.

[0040] In an embodiment of the invention, the targeting sequence of the second sequence region is configured to be integrated into the host genome by gene editing techniques, preferably CRISPR-Cas, zinc finger nuclease (ZFN), integrase, site-specific recombinase, meganuclease, homing endonuclease, or TALEN, more preferably CRISPR-Cas12a from Acidaminococcus sp. BV3L6 or CRISPR-Cas9 from Streptococcus pyogenes.

[0041] In an embodiment of the invention, the targeting sequence comprises a sequence identical to a recognition site of the endogenous DNA sequence into which integration is intended, preferably a recognition site selected from the group consisting of a homology arm, a guide RNA target site, a restriction enzyme recognition site, a ZFN recognition site, a ZFN cleavage site, a TALEN DNA binding site, a recombinase recognition site, an integrase site, and / or a homing nuclease recognition site.

[0042] In certain embodiments, the targeting sequence, also referred to as a "homology arm," comprises at least one sequence that is complementary to an endogenous genomic sequence of the target cell. In one embodiment, the targeting sequence of the second sequence region configured for integration into the host genome comprises a homology arm that is complementary to the endogenous integration site described above, preferably complementary to a site in the endogenous CD3 epsilon gene.

[0043] In some embodiments, the nucleic acid construct comprises, in 5' to 3' order, a left homology arm, a nucleic acid sequence encoding an antigen-binding domain, and a right homology arm. In certain embodiments, the nucleic acid construct may further comprise one or more linkers between the nucleic acid sequence encoding the antigen-binding domain and the left homology arm and / or the right homology arm, and / or within the nucleic acid sequence encoding the antigen-binding domain. In some embodiments, the nucleic acid construct may optionally comprise a nucleic acid sequence encoding a self-cleaving peptide.

[0044] In one embodiment, the nucleic acid construct encodes an antigen-binding domain, which comprises: a left homology arm, preferably according to SEQ ID NO: 11 or SEQ ID NO: 19, or a sequence having at least 80% sequence identity to SEQ ID NO: 11 or SEQ ID NO: 19; a sequence region according to SEQ ID NO: 13 and SEQ ID NO: 15 encoding an antigen-binding domain of a CAR that specifically binds to an antigen, preferably HLA, more preferably HLA-2, linked to a linker according to SEQ ID NO: 14, or a sequence having at least 80% sequence identity to SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; a right homology arm, preferably a right homology arm according to SEQ ID NO: 17 or SEQ ID NO: 28, or a sequence having at least 80% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 28; Includes.

[0045] In some embodiments, the nucleic acid construct encodes an antigen-binding domain, which further comprises: a sequence region encoding a self-cleavage site, preferably according to SEQ ID NO: 20, or a sequence having at least 80% sequence identity to SEQ ID NO: 20; Includes.

[0046] In one embodiment, gene editing techniques are used to create a double-strand break at a target site, preferably in the CD3 epsilon gene, and the nucleic acid construct is integrated into the target site by homologous sequence-dependent repair at the site of the double-strand break. A non-viral vector is preferably used to transfer the nucleic acid construct encoding the antigen-binding domain fragment into cells. In one embodiment, the delivery method is non-viral, preferably electroporation.

[0047] In one embodiment, the present invention provides a means for inserting a transgene into a T cell using homology-directed repair (HDR). In one embodiment, a CRISPR-Cas ribonucleoprotein (Cas) complexed with a guide RNA (gRNA) is introduced into the T cell along with a nucleic acid construct. In a preferred embodiment, the CRISPR-Cas is CRISPR-Cas9 or CRISPR-Cas12a, preferably CRISPR-Cas12a.

[0048] Compared to retroviral gene transfer, the non-viral method of the present invention represents a significant improvement over conventional techniques, particularly with regard to GMP-compliant production of the necessary starting materials. The production and purification of viral vectors according to GMP standards is complex and very costly. In contrast, GMP-certified gene editing tools, such as gRNAs and recombinant CRISPR-Cas proteins, are readily available and inexpensive.

[0049] Further genome editing via HDR using CRISPR-Cas technology is advantageous for specifically and efficiently inserting large transgenes into target cells without the need for viral vectors. It is advantageous to maintain the viability and function of transfected cells and enable a high yield of genetically modified cells for clinical use. Because gene transfer is sequence-specific, this method also provides targeted and safe integration of nucleic acid constructs, resulting in a reduced risk of mutagenesis. In comparison, in viral gene transfer or transposon technology, DNA molecules are randomly integrated into the genome. Random insertion of DNA fragments poses the risk of mutagenesis, such as the occurrence of malignant transformation.

[0050] In one embodiment, T cells are preferably co-electroporated with a gRNA targeting the CD3 epsilon gene. The gRNA sequence is important for targeted gene disruption. The gRNAs disclosed herein (see Table 1) direct specific cleavage, thereby providing beneficial effects for targeted gene disruption. Targeted disruption of the CD3 epsilon gene leads to correct integration of the antigen-binding domain via gene fusion, resulting in efficient translation of a functional in-frame fusion protein comprising the antigen-binding domain and the endogenous CD3 epsilon protein. Such a functional in-frame fusion protein is advantageous because it is efficiently transported and anchored to the plasma membrane, allowing functional presentation of the antigen-binding domain on the cell surface while maintaining the function of the CD3 / TCR complex. Such specific guidance of correct integration is important for the accurate synthesis of the in-frame fusion protein and its functionality, such as surface presentation and antigen specificity, and is an advantageous feature of the guide RNAs disclosed herein over prior art guide RNAs.

[0051] Thus, the present invention provides gRNA sequences that target the CD3 epsilon gene that can be applied to T cells and obtain antigen-specific T cells with preserved CD3 / TCR complex function that can be expanded ex vivo and used for immunotherapy.

[0052] In an embodiment of the invention, the antigen binding domain is in the form of an antibody or an antigen binding fragment thereof.

[0053] In a preferred embodiment, the antigen-binding domain is an antigen-binding fragment, preferably a single-chain fragment (scFv), a single variable fragment (ssFv), a single-domain antibody (such as a VHH fragment), a Fab fragment, a F(ab')2 fragment, a fragment produced by a Fab expression library, an anti-idiotypic antibody, or an epitope-binding fragment, or a combination thereof, preferably a single-chain fragment (scFv).

[0054] The use of antigen-binding fragment domains such as scFV fragments has proven particularly advantageous because these fragments are small, can be easily modified to target a variety of antigens, and can be encoded by small nucleic acid fragments, making them easy to integrate into the endogenous CD3 epsilon gene of T cells using non-viral methods.

[0055] In one embodiment, the invention provides an in-frame fusion protein comprising an antigen binding domain as described herein and an endogenous CD3 epsilon protein, wherein the antigen binding domain is A heavy chain complementarity determining region 1 (H-CDR1) having at least 80% sequence identity to SEQ ID NO: 52(51) (GVTLSDY); a heavy chain complementarity determining region 2 (H-CDR2) having at least 80% sequence identity to SEQ ID NO: 52 (RNDGSD); a heavy chain complementarity determining region 3 (H-CDR3) having at least 80% sequence identity to SEQ ID NO: 53 (NGESGPLDYWYFDL); and a variable heavy chain (VH) comprising a light chain complementarity determining region 1 (L-CDR1) having at least 80% sequence identity to SEQ ID NO: 54 (QASQDISNYLN); a light chain complementarity determining region 2 (L-CDR2) having at least 66% sequence identity to SEQ ID NO: 55 (DASNLET); a light chain complementarity determining region 3 (L-CDR3) having at least 80% sequence identity to SEQ ID NO: 56 (QQYSSFPLT); a variable light chain (VL) comprising The present invention relates to an in-frame fusion protein comprising:

[0056] In one embodiment, the invention relates to an in-frame fusion protein comprising an antigen binding domain as described herein and an endogenous CD3 epsilon protein, wherein the fusion protein comprises a VH domain comprising the CDR sequences of SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, and a VL domain comprising the CDR sequences of SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56.

[0057] In some embodiments, sequence variants with 80% or greater sequence identity to the specific antigen-binding sequence of SEQ ID NO: 47 maintain HLA-A2 binding with essentially the same or similar functional properties as the VH and VL domains having the specific sequences of SEQ ID NO: 48 and SEQ ID NO: 50. That is, the HLA-A2 binding is essentially the same or similar in terms of affinity, specificity, and / or epitope binding mode.

[0058] In one embodiment of the invention, the antigen binding domain is specific for an autoantigen, an alloantigen, or a cancer antigen.

[0059] In one embodiment, the antigen binding domain is specific for an autoantigen or an alloantigen.

[0060] In some embodiments, the autoantigen or alloantigen is an HLA protein, preferably an HLA protein selected from the group of HLA proteins including HLA-A, HLA-B, HLA-DR, and HLA-associated antigens such as Mic A or Mic B. In preferred embodiments, the autoantigen or alloantigen is selected from HLA-A2, HLA-B7, HLA-DR, and HLA-associated antigens such as Mic A or Mic B.

[0061] In one embodiment, the antigen binding domain is specific for an HLA protein, preferably HLA-A2.

[0062] In one embodiment, the antigen binding domain is specific for a cancer antigen, wherein the cancer antigen is selected from the group of ErbB proteins such as EGFR and HER2 / neu and B lymphocyte antigens such as CD 19. In a preferred embodiment, the cancer antigen is selected from HER2 / neu or CD19.

[0063] In a further aspect, the present invention relates to genetically modified human T cells comprising an exogenous nucleic acid sequence region encoding an antigen-binding domain integrated in-frame into the endogenous CD3 epsilon gene.

[0064] In an embodiment of the invention, the genetically modified human T cells are human regulatory T cells (Tregs).

[0065] In some embodiments, the present invention relates to genetically modified human T cells, wherein the CD3 epsilon protein is expressed as an in-frame fusion protein comprising the antigen binding domain and the endogenous CD3 epsilon protein.

[0066] In some embodiments, the present invention relates to genetically modified human regulatory T cells (Tregs) in which the CD3 epsilon protein is expressed as an in-frame fusion protein comprising the antigen-binding domain and the endogenous CD3 epsilon protein.

[0067] In some embodiments, the present invention relates to genetically modified human T cells in which the function of the T cell receptor (TCR) complex is maintained in the presence of an in-frame fusion protein comprising an antigen-binding domain and an endogenous CD3 epsilon protein.

[0068] In some embodiments, the present invention relates to genetically modified human regulatory T cells (Tregs), in which the function of the T cell receptor (TCR) complex is maintained in the presence of an in-frame fusion protein comprising an antigen-binding domain and an endogenous CD3 epsilon protein.

[0069] Advantageously, the genetically modified T cells of the present invention exhibit maintenance of CD3 / TCR receptor complex functionality, cell fitness, and antigen specificity. Retention of functional CD3 / TCR complex receptors (receptor function) maintains the stimulatory properties of genetically modified antigen-specific T cells, and therefore allows them to be efficiently expanded ex vivo to a similar extent as unmodified T cells with functional CD3 / TCR complexes. In contrast, in conventionally modified T cells, such as CAR-T cells, the CD3 / TCR complex is replaced by a CAR, resulting in impaired function and stimulatory properties, limiting effective expansion and limiting the clinical applicability of these cells.

[0070] Furthermore, the genetically modified T cells of the present invention can be easily modified with respect to the antigen specificity of the expressed in-frame fusion protein, thereby obtaining T cells specific for a particular antigen for use in preventing and / or treating unwanted (pathogenic) immune responses, autoimmune diseases, or cancerous diseases associated with that particular antigen.

[0071] Furthermore, genetically modified T cells advantageously exhibit comparable functionality to unmodified T cells in terms of signal transduction, expression of activation markers, and traditional T cell growth suppression assays.

[0072] In some embodiments, the present invention relates to genetically modified human T cells, wherein the fusion protein is expressed under the control of the endogenous CD3 epsilon promoter.

[0073] In some embodiments, the present invention relates to genetically modified human regulatory T cells (Tregs), wherein the fusion protein is expressed under the control of the endogenous CD3 epsilon promoter.

[0074] An endogenous promoter is endogenously controlled and / or induced, for example, by a cellular signal from a T cell. A particular advantage of the present invention is the use of endogenous promoter expression. Control of the expression of the integrated nucleic acid construct by an endogenous promoter ensures that the functionality and fitness of the resulting genetically modified T cells are maintained. Furthermore, omitting an exogenous promoter can reduce the risk of mutagenesis due to gene dysregulation via regulatory elements. Furthermore, due to the absence of an exogenous promoter, the nucleic acid construct of the present invention is smaller and provides particularly efficient transfer and integration of nucleic acid sequences via non-viral methods.

[0075] In some embodiments, the present invention relates to genetically modified human T cells, wherein an exogenous nucleic acid sequence region encoding a recombinant antigen-binding domain is integrated in-frame within an exon of the endogenous CD3 epsilon gene.

[0076] In some embodiments, the present invention relates to genetically modified human regulatory T cells (Tregs), in which an exogenous nucleic acid sequence region encoding a recombinant antigen-binding domain is integrated in-frame within an exon of the endogenous CD3 epsilon gene.

[0077] In a further embodiment, the exogenous nucleic acid sequence region encoding the antigen binding domain is integrated in-frame within exon 3 or exon 6 of the endogenous CD3 epsilon gene.

[0078] In some embodiments, the present invention relates to genetically modified T cells, wherein the target site in the CD3 epsilon gene into which the first sequence region is integrated is located downstream of the sequence encoding the CD3 epsilon signal peptide and upstream of the endogenous CD3 epsilon sequence (generating an N-terminal fusion after removal of the signal peptide).

[0079] In some embodiments, the present invention relates to genetically modified regulatory T cells (Tregs), wherein the target site in the CD3 epsilon gene into which the first sequence region is integrated is located downstream of the sequence encoding the CD3 epsilon signal peptide and upstream of the endogenous CD3 epsilon sequence (generating an N-terminal fusion after removal of the signal peptide).

[0080] In some embodiments, the present invention relates to genetically modified human T cells, wherein the recombinant antigen-binding domain binds to an autoantigen, a transplant alloantigen, or a cancer antigen.

[0081] In some embodiments, the present invention relates to genetically modified human regulatory T cells (Tregs), wherein the recombinant antigen-binding domain binds to an autoantigen, a transplant alloantigen, or a cancer antigen.

[0082] In one embodiment, the antigen binding domain binds to an autoantigen or an alloantigen, wherein the autoantigen or alloantigen is selected from the group of HLA proteins including HLA-A, HLA-B, HLA-C, HLA-E, HLA-DR, HLA-DQ, and HLA-associated antigens such as Mic A or Mic B. In a preferred embodiment, the autoantigen or alloantigen is selected from the group of HLA-associated antigens such as HLA-A2, HLA-B7, HLA-B27, HLA-DR, and Mic A or Mic B.

[0083] In one embodiment, the antigen binding domain binds to an HLA protein, preferably HLA-A2.

[0084] In one embodiment, the antigen binding domain binds to a cancer antigen, wherein the cancer antigen is selected from the group of ErbB proteins such as EGFR and HER2 / neu and B lymphocyte antigens such as CD 19. In a preferred embodiment, the cancer antigen is selected from HER2 / neu or CD19.

[0085] In a further aspect, the present invention relates to genetically modified T cells for use as a medicament for the prevention and / or treatment of unwanted (pathogenic) immune responses, preferably unwanted (pathogenic) immune responses before and / or after allogeneic transplantation, more preferably for the prevention and / or treatment of graft-versus-host disease (GvHD).

[0086] In a further aspect, the present invention relates to genetically modified regulatory T cells (Tregs) as described herein for use as a medicament for the prevention and / or treatment of unwanted (pathogenic) immune responses, preferably unwanted (pathogenic) immune responses before and / or after allogeneic transplantation, more preferably for the prevention and / or treatment of graft-versus-host disease (GvHD).

[0087] In a further aspect, the present invention relates to the genetically modified T cells described herein for use as a medicament for the prevention and / or treatment of cancerous diseases.

[0088] In a further aspect, the present invention relates to genetically modified regulatory T cells (Tregs) as described herein for use as a medicament for the prevention and / or treatment of autoimmune diseases.

[0089] In a further aspect, the present invention relates to a polypeptide expressed from the genetically modified cells described herein as a fusion protein comprising a recombinant antigen-binding domain and an endogenous CD3 epsilon protein.

[0090] In a further aspect, the invention relates to a pharmaceutical composition comprising a genetically modified cell as described herein and a pharmaceutically acceptable carrier.

[0091] In a further aspect, the present invention relates to a method for preparing therapeutic genetically modified T cells as described herein, comprising repeated stimulation of genetically modified regulatory T cells via the TCR / CD3 complex and the CD28 coreceptor, wherein said therapeutic genetically modified regulatory T cells exhibit essentially unchanged expansion kinetics (±50%) in culture compared to unmodified T cells.

[0092] In a further aspect, the present invention relates to a method of preparing therapeutic genetically modified regulatory T cells (Tregs) as described herein, comprising repeated stimulation of genetically modified regulatory T cells via the TCR / CD3 complex and the CD28 coreceptor, wherein the therapeutic genetically modified regulatory T cells exhibit essentially unchanged expansion kinetics in culture (±50%, ±40%, ±30%, ±20%, ±10%, or ±5%) compared to unmodified T cells.

[0093] The term unaltered expansion kinetics in culture refers to beneficial properties of the modified cells during culture and repeated stimulation. The modified cells of the invention have no worse expansion kinetics in culture compared to cells without modification (thus, the cells of the invention represent an improvement over alternative cells lacking a TCR; see Comparative Example 1 below). In some embodiments, expansion kinetics refers to the growth rate and / or maintenance of Treg properties of the cells during culture. The necessary properties are described in more detail in the Examples below.

[0094] Given the generally low abundance of expandable, unmodified, antigen-specific T cells in peripheral blood and the lack of expandability of conventionally engineered antigen-specific T cells, such as CAR-T cells, it is particularly advantageous that genetically modified T cells can be efficiently expanded by repeated stimulation via the TCR / CD3 complex and CD28 coreceptor. This allows for efficient expansion to high cell numbers and high reproducibility while maintaining the fitness of the genetically modified T cells upon repeated stimulation. Sufficient, reproducible expansion of antigen-specific T cells is a key factor in enabling the clinical application of the cells and further reducing the cost of such applications.

[0095] All features described herein can be used to define any other embodiment or aspect of the invention, for example, features used to describe genetically modified T cells can be used to describe a nucleic acid construct, a polypeptide, a pharmaceutical composition, or a method of preparing therapeutic genetically modified T cells, and vice versa. Similarly, features used to describe a method of the invention can be used to describe a cell, construct, polypeptide, or composition, and vice versa.

[0096] Detailed Description of the Invention The present invention provides a strategy for immune cell therapy using gene editing methods that preferably incorporate one or more antigen binding domains under the control of the endogenous CD3 epsilon promoter, allowing cell-specific and endogenously regulated expression in therapeutic T cells. Various aspects and embodiments of the present invention, as well as their advantages over the prior art, have been described above.

[0097] "T cells," also called "T lymphocytes," are immune cells that belong to the lymphocyte group. T cells can be thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, cytokine-induced killer cells (CIK cells), or activated T lymphocytes. T cells originate from the bone marrow and migrate via the bloodstream to the thymus, where they develop T cell receptors (TCRs) and undergo positive and negative selection, during which cells that display high affinity for endogenous proteins are lysed. T cells are classified as T helper (Th, CD4 + T cells can be T helper (Th) cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, or T cells. T cells can be cytotoxic T cells (CTLs, CD8 + T cells), CD4 + CD8 + The T cells may be T cells, CD4 CD8 T cells, or regulatory T cells (reg), or any other T cell subset, such as cytokine-induced killer (CIK) cells, which are typically CD3-positive and CD56-positive major histocompatibility complex (MHC)-unrestricted natural killer (NK)-like T lymphocytes. The T cells may be naive T cells, effector T cells, memory T cells, effector memory T cells (effector storage), central memory T cells (central storage), or memory stem T cells. The T cells may be cord blood cells. The T cells may be peripheral lymphocytes. The T cells may be derived and expanded from peripheral mononuclear blood cells (PBMNC). The T cells may be autologous to the individual to whom they are administered. The T cells may be allogeneic to the individual to whom they are administered.

[0098] Regulatory T cells (Tregs), typically CD4+, play a key role in preventing autoimmune diseases by maintaining tolerance to self-recognized antigens, also called "autoantigens," regulating and terminating excessive immune responses, ensuring tissue regeneration, and controlling bacterial homeostasis at mucosal surfaces. +Treg cells are immunosuppressive T cells. Activation of Treg cells is antigen-specific. Upon activation, Treg cells secrete various cytokines, including, but not limited to, interleukin 10 (IL-10), transforming growth factor β (TGF-β), and interleukin 35 (IL-35). This results in, for example, increased activation levels of naive T cells, inhibition of the pro-inflammatory function of antigen-presenting cells (APCs), and downregulation of the induction and proliferation of T effector cells. In the circulation, peripheral blood CD4 + 2%–5% of T cells represent Treg cells, characterized by classical markers such as high expression of the transcription factor forkhead box protein P3 (Foxp3) and the high-affinity interleukin-2 (IL-2) receptor chain CD25, and low expression of the interleukin-7 (IL-7) receptor alpha chain CD127.

[0099] A "T cell receptor" (TCR) is a protein complex found on the surface of T cells, responsible for recognizing and binding fragments of antigens bound to major histocompatibility complex (MHC) molecules on the surface of antigen-specific receptors (APCs), for example. TCRs are heterodimers containing TCR alpha and TCR beta protein chains, or TCR gamma and TCR delta protein chains. T cells with TCRs composed of TCR alpha and TCR beta protein chains account for approximately 95% of the T cell population in peripheral blood. Each subunit of the heterodimer consists of a constant domain (C) and a variable domain (V), a transmembrane domain, and a short C-terminal cytoplasmic region. The N-terminus of the chain belonging to the C domain penetrates the cell membrane and reaches the cytoplasmic space, anchoring the receptor. The two subunits are linked extracellularly by disulfide bridges in the constant regions. The "TCR complex," also referred to as the "TCR / CD3 complex," is formed by the TCR and CD3, which contains the CD3 gamma, delta, epsilon, and zeta subunits. Upon binding to antigens presented by MHC II on the surface of antigen-binding cells (antigen / MHC II complexes) or MHC I on any other cell type (antigen / MHC I complexes), TCR-mediated signals are transmitted across the cell membrane by the CD3 chains zeta, delta, epsilon, and gamma, resulting in T cell activation. All CD3 chains contain immunoreceptor tyrosine-based activation motifs (ITAMs) in their cytoplasmic domains. The genes encoding the epsilon, gamma, and delta polypeptides are located within the same cluster on chromosome 11.

[0100] As used herein, the terms "polynucleotide," "nucleic acid," "nucleic acid molecule," or "nucleic acid construct" refer to a polymeric form of nucleotides (nt) of any length, where the nucleotides can be deoxyribonucleotides (DNA) or ribonucleotides (RNA), or analogs thereof. These terms include, but are not limited to, DNA, messenger RNA (mRNA), RNA, genomic RNA (gRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), small interfering RNA (siRNA), single guide RNA (sgRNA), piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), positive-strand RNA (RNA(+)), negative-strand RNA (RNA(-)), genomic DNA (gDNA), complementary DNA (cDNA), recombinant polynucleotides, branched polynucleotides, plasmids, nucleic acid probes, and primers. Polynucleotides include single-stranded and double-stranded polynucleotides. Preferably, polynucleotides of the invention include polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein, where typically the variant retains at least one biological activity of the reference sequence.

[0101] As used herein, "sequence identity," "identity," "sequence homology," or "homology" in the context of two nucleic acid sequences refers to a specified percentage of residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window, as measured by a sequence comparison algorithm or visual inspection.

[0102] As used herein, "percent sequence identity," "sequence with % identity," "percent sequence homology," or "sequence with % homology" to a particular "reference sequence" (e.g., SEQ ID NO: 1) refers to the percentage of nucleotides or amino acids in a particular sequence that are identical to the nucleotides or amino acids of the reference sequence, as determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or amino acid sequence within the comparison window may contain additions or deletions (i.e., gaps) relative to the reference sequence (which does not contain additions or deletions) upon optimal alignment of the two or more sequences. This percentage is calculated by determining the number of positions in two or more sequences where identical nucleic acid bases or amino acid residues occur to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein, where typically the polypeptide variant retains at least one biological activity of the reference polypeptide.

[0103] Any suitable sequence alignment method for determining percent sequence identity may be used and is known to those skilled in the art. The determination of percent identity between any two or more sequences can be achieved using published mathematical algorithms. Computer software implementations of these mathematical algorithms include, but are not limited to, the ClustalW algorithm (VNTI software, InforMax Inc.), ALIGN (version 2.0), GAP (Genetics Computer Group software, currently available through Accelrys at http: / / www.accelrys.com), BESTFIT, BLAST™, FASTA, and TFASTA in the Wisconsin Genetics software package (version 8) (available from Genetics Computer Group (GCG), Madison, Wisconsin, USA), and the multiple sequence alignment MUSCLE (European Bioinformatics Institute at EMBL (EMBL-EBI), Cambridgeshire, UK). Alignments using these programs can be performed using default parameters. Software for performing BLAST™ analyses is publicly available from the National Center for Biotechnology Information. A nucleic acid sequence of interest can be used to search sequence databases. Algorithms for database searches are typically based on BLAST software (Altschul et al., 1990). In some embodiments, the percent homology or identity can be determined along the entire length of the nucleic acid.

[0104] Those skilled in the art will appreciate that, as a result of the degeneracy of the genetic code, there are numerous nucleotide sequences that encode the in-frame fusion proteins described herein. Some of these polynucleotides bear minimal homology or sequence identity to the nucleotide sequence of any native gene. Nevertheless, variations in codon usage make polynucleotides specifically contemplated by the present invention. Deletions, substitutions, and other changes in the sequences that fall within the described sequence identities are also encompassed by the present invention.

[0105] Modifications of protein sequences that may occur through substitutions are also within the scope of the present invention. Substitutions, as defined herein, are modifications made to the amino acid sequence of a protein, whereby one or more amino acids are replaced with the same number of (different) amino acids, resulting in a protein containing an amino acid sequence different from that of the original protein. Preferably, substitutions can be made that do not significantly alter the function of the protein. Like additions, substitutions can be natural or artificial. It is well known in the art that amino acid substitutions can be made without significantly altering the function of a protein. This is particularly true when the modification involves "conservative" amino acid substitutions, in which one amino acid is replaced with another amino acid of similar properties. Such "conserved" amino acids can be natural or synthetic amino acids that, due to their size, charge, polarity, and conformation, can be substituted without significantly affecting the structure and function of the protein. In many cases, many amino acids can be substituted with conservative amino acids without adversely affecting the function of the protein.

[0106] Generally, the nonpolar amino acids Gly, Ala, Val, Iie (Ile), and Leu; the nonpolar aromatic amino acids Phe, Trp, and Tyr; the neutral polar amino acids Ser, Thr, Cys, Gin (Gln), Asn, and Met; the positively charged amino acids Lys, Arg, and His; and the negatively charged amino acids Asp and Glu represent conservative amino acid groups. This list is not exhaustive. For example, it is well known that Ala, Gly, Ser, and sometimes Cys can be substituted for each other even though they belong to different groups.

[0107] Substitutional variants involve removing at least one amino acid residue in an antibody molecule or antibody fragment and inserting a different residue in its place. The sites of greatest interest for substitutional mutagenesis include hypervariable regions, although framework modifications are also contemplated. If such substitutions result in altered biological activity, more drastic changes, designated "exemplary substitutions" in the table immediately below or further described below for amino acid classes, can be introduced and the products screened.

[0108] Potential amino acid substitutions: [Table A] Original residue: Preferred conservative substitutions Examples of exemplary substitutions Norleucine

[0109] Substantial alterations in the biological properties of antibodies or antibody fragments are achieved by selecting substitutions that differ significantly in their effect on (a) the structure of the polypeptide backbone in the region of the substitution, e.g., as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining side chain bulk.

[0110] Conservative amino acid substitution is not limited to naturally occurring amino acids, but also includes synthetic amino acids.Commonly used synthetic amino acids are omega amino acids of various chain lengths, and neutral non-polar analogues cyclohexylalanine, neutral non-polar analogues citrulline and methionine sulfoxide, aromatic neutral analogues phenylglycine, negatively charged analogues cysteic acid, and positively charged amino acid analogues ornithine.Similar to naturally occurring amino acids, this list is not exhaustive, but merely exemplifies the substitutions that are well known in the art.

[0111] An "antigen (Ag)" refers to a compound, composition, or substance capable of binding to an antibody. An antigen that stimulates an "immune response" or "immune reaction," such as the production of antibodies or a T-cell response, in an animal or human subject is also called an "immunogen." Antigens and / or immunogens typically comprise protein, lipid, and / or carbohydrate structures. Antigens typically have several substructures, called "determinants" or "epitopes," which are regions of the antigen to which binding factors bind. Thus, an epitope may be formed from a continuous structure, such as an amino acid sequence, or from a discontinuous structure, such as two or more amino acid sequences juxtaposed by tertiary folding of a protein. Thus, an epitope may be formed in close proximity in two different proteins, or as a complex in a protein and another immunogen.

[0112] The term "self-antigen" refers to an endogenous substance or structure that is not normally recognized by T cells due to their selection in the thymus, which gives them the ability to distinguish between physiological endogenous structures and pathological and / or foreign structures. If self-antigens are not recognized by T cells as healthy endogenous structures, they may trigger "unwanted immune responses," such as "autoimmune responses" due to pathological T-cell activation and antibody production, which may manifest as autoimmune diseases. Autoantigens involved in the manifestation of autoimmune diseases include, but are not limited to, IgA immunoglobulin in IgA nephropathy, the Fc portion of IgG and cyclic citrullinated peptide (CCP) in rheumatoid arthritis, thyroid-stimulating hormone (TSH) receptor and thyroid peroxidase (microsomes) in Graves' disease, acetylcholine receptor (AChR) and muscle-specific kinase (MUSK) in myasthenia gravis, insulin, pancreatic islet cell surface antigen, glutamic acid decarboxylase (mainly GAD65, but also GAD67), zinc transporter 8 and tyrosine phosphatase IA-2 in type 1 diabetes, thrombin, ribonucleoproteins, double-stranded DNA, snRNP core protein and histones in systemic lupus erythematosus, phospholipids in antiphospholipid syndrome, thyroglobulin and thyroid peroxidase (microsomes) in Hashimoto's thyroiditis, and nucleoporin 62, Sp100 nuclear antigen, and nucleoporin 210 in primary biliary cirrhosis. kDa, voltage-gated calcium channels (P / Q type) in Lambert-Eaton myasthenic syndrome, type IV collagen in Goodpasture's syndrome, DNA topoisomerase in scleroderma, centromere protein A, centromere protein B, and centromere protein C in CREST syndrome, ribonucleoprotein in Sjogren's syndrome, and reticulin, endomysial, and tissue transglutaminase in celiac disease.Autoimmune diseases include, but are not limited to, rheumatoid arthritis, Graves' disease, myasthenia gravis, type 1 diabetes, systemic lupus erythematosus, antiphospholipid syndrome, Hashimoto's thyroiditis, primary biliary cirrhosis, Lambert-Eaton myasthenic syndrome, Goodpasture's syndrome, Sjogren's syndrome, celiac disease, multiple sclerosis, ulcerative colitis, pemphigus vulgaris, polymyositis, scleroderma, systemic vasculitis, alopecia areata, autoimmune liver disease (AILD), autoimmune hepatitis, primary sclerosing cholangitis, and autoimmune These include chronic hemolytic anemia, Ord's thyroiditis, bullous pemphigoid, dermatomyositis, eosinophilic granulomatosis with polyangiitis (EGPA), Guillain-Barré syndrome, suppressed hemophilia, immune thrombocytopenia, Lambert-Eaton syndrome, Adamantiades-Behçet's disease, narcolepsy, primary chronic polyarthritis, polychondritis, psoriasis, Schönlein-Henoch purpura, rheumatic fever, giant cell arteritis (RZA), SAPHO syndrome, stiff-man syndrome, type A gastritis, vitiligo, and Wegener's granulomatosis.

[0113] Autoantigens are also involved in graft-versus-host reaction (GVHR), also known as graft-versus-host disease (GVHD), an undesirable systemic cytotoxic immune response caused by transplanted or transfused immune cells against the host organism. Transplanted T cells recognize the recipient's autoantigens as foreign and trigger a cellular immune response in the recipient organism. This results in the production of specific cytotoxic T cells and antibodies against the host. In addition, a response from the innate immune system is suspected. Furthermore, immunocompromised hosts may develop severe diseases such as hepatosplenomegaly, lymphoid organ atrophy, diarrhea, skin lesions, and cachexia. In chronic graft-versus-host reaction, both alloreactive and autoreactive immunological processes occur. Impaired peripheral immune tolerance is associated with a deficiency of Treg cells. This results in chronic inflammatory responses and secondary fibrosis. GVHD plays a particularly important role in allogeneic stem cell transplantation and also occurs rarely after transfusion in patients with severe congenital or acquired immunodeficiencies.

[0114] An "alloantigen" is an antigen produced by an individual that elicits an immune response in individuals of the same biological species. Typical alloantigens include surface antigens on red blood cells and lymphocytes and on allogeneic grafts. Alloantigens are potential triggers of "unwanted immune responses," which can lead to blood group incompatibility and graft rejection. The most important group of alloantigens are human leukocyte antigens (HLA), also known as major histocompatibility complex (MHC) molecules. HLA are glycoproteins anchored to cell membranes and classified as immunoglobulins. HLA is classified into class I MHC molecules and class II MHC molecules. Class I includes HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, and their subgroups. Class II includes HLA-DP, HLA-DQ, HLA-DR, and their subgroups. Class I MHC molecules are present as transmembrane glycoproteins on the surface of all nucleated cells. Class II MHC molecules are typically found on APCs, such as B cells, phagocytes (e.g., macrophages), and dendritic cells. Generally, both classes of HLA molecules function to bind peptides and present them on the cell surface to cells of the immune system. In particular, the HLA antigens HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, and HLA-DPB1 play important roles in allograft rejection. Certain HLA antigens are also involved in autoimmune diseases, including HLA-B27 in ankylosing spondylitis, reactive arthritis, and acute anterior uveitis, HLA-B47 in 21-hydroxylase deficiency, HLA-DR2 in systemic lupus erythematosus, HLA-DR3 in autoimmune hepatitis, primary Sjögren's syndrome, type 1 diabetes, and systemic lupus erythematosus, HLA-DR4 in rheumatoid arthritis and type 1 diabetes, and HLA-DQ2 and HLA-DQ8 in celiac disease.Alloantigens may be classified as major alloantigens, such as MHC mismatches, but minor mismatches may also occur, for example, donor A has a mutation in protein X, and a fragment of the mutated protein X is presented via the matched HLA, which may recognize the mutated fragment as non-self, thereby eliciting an immune response in T cells.

[0115] The term "immune response" refers to the reaction of a subject's immune system to encountering an antigen. In this way, the immune system typically distinguishes between endogenous structures, such as autoantigens, and exogenous structures. An "undesirable immune response" refers to a reaction of a subject's immune system against an autoantigen or alloantigen, as specified above. The terms "pathogenic" and "pathogenic immune response" refer to a reaction of the immune system that is harmful to the body. Such reactions include, for example, the autoimmune reactions and autoimmune diseases listed under the term autoantigen, as well as GVHD or graft rejection due to a reaction against an alloantigen.

[0116] "Cancer antigens," "cancer-associated antigens," or "tumor antigens" are expressed on the surface of cancer cells, either in their entirety or as fragments (e.g., MHC / peptides). Tumor antigens are either specifically expressed on tumor cells and not found on non-pathogenic cells, or are aberrantly expressed, e.g., at least two-fold higher than the levels found on non-pathogenic cells. Antigens uniquely found on tumor cells are considered foreign to the immune system, and their presence may cause immune cells to attack transformed tumor cells. Tumor antigens refer to antigens commonly found in certain hyperproliferative diseases. In one embodiment, the hyperproliferative disease antigens of the present invention are primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, and adenocarcinomas such as bladder cancer, prostate cancer, ovarian cancer, and pancreatic cancer derived from cancers such as uterine cancer, cervical cancer, kidney cancer, and breast cancer. Cancer-associated antigens include, but are not limited to, CD19, APRIL / TNFSF13, Mic A / B, T cell-associated antigens: CD3, CD5, CD7, folate receptor alpha (FRα), ERBB2 (HER2 / neu), EphA2, IL-13Ra2, epidermal growth factor receptor (EGFR), mesothelin, TSHR, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, and TnAg, prostate-specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, interleukin-11 receptor a (IL-11Ra), PSCA, PRSS21, VEGFR2, Lewis Y, CD24, platelet-derived growth factor receptor beta (PDGFR-beta), SSEA-4, CD20, MUC1, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr- abl, tyrosinase, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mutThese include hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, and derivatives thereof, including both naturally occurring and non-naturally occurring post-translational modifications of polypeptides, such as glycosylation, ubiquitination, acetylation, phosphorylation, etc., and other modifications known in the art.

[0117] According to the present invention, any suitable autoantigen, alloantigen, or cancer antigen can be selected depending on the type of pathology for which the genetically modified T cells of the present invention are intended to be used. The genetically modified T cells of the present invention are preferably used in the prevention and / or treatment of unwanted (pathogenic) immune responses, such as unwanted immune responses before and / or after allogeneic transplantation and / or cancerous diseases. In a preferred embodiment, the genetically modified T cells are preferably used in the prevention and / or treatment of graft-versus-host disease (GVHD).

[0118] "Specifically binds" should be interpreted as an antigen-binding domain selectively binding to a specific antigen, and those skilled in the art will clearly recognize various experimental procedures that can be used to test binding and binding specificity. Methods for determining equilibrium association or dissociation constants are known in the art. In many antigen-binding domain-antigen interactions, some cross-reactivity or background binding may be unavoidable, but this does not diminish the "specificity" of the binding between the antigen-binding region and the antigen. By way of example, "specific binding" describes that, for example, an HLA-A2 antibody or antigen-binding fragment thereof (or a fusion protein comprising the same and the endogenous CD3 epsilon protein) binds to the HLA2 antigen with a binding affinity higher than background binding. The term "directed against" is also applicable when considering the term "specificity" in understanding the interaction of an antibody with an epitope.

[0119] The term "antigen-binding domain" refers to an antibody or antibody fragment that binds to an antigen, such as an HLA2 antigen. Thus, antibodies or antibody fragments of the present invention include, but are not limited to, polyclonal, monoclonal, bispecific, human, humanized, or chimeric antibodies, single-chain fragments (scFv), single variable fragments (ssFv), single-domain antibodies (such as VHH fragments from nanobodies), Fab fragments, F(ab')2 fragments, fragments produced by Fab expression libraries, anti-idiotypic antibodies, and epitope-binding fragments, or any combination thereof, provided they retain similar binding characteristics. Miniantibodies and multivalent antibodies, such as diabodies, triabodies, tetravalent antibodies, and peptabodies, may also be included in the present invention. The immunoglobulin molecules of the present invention may be of any class (i.e., IgG, IgE, IgM, IgD, and IgA) or subclass of immunoglobulin molecule. Thus, the term antibody, as used herein, also includes antibodies and antibody fragments produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. The antigen-binding domain, in some embodiments, can be the extracellular domain of a receptor, such as an endogenous receptor (e.g., to target CD137, CD137L can be added to CD3e to redirect it).

[0120] As used herein, "antibody" generally refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. When the term "antibody" is used, the term "antibody fragment" may also be considered a reference. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes: IgG, IgM, IgA, IgD, and IgE, respectively. The basic immunoglobulin (antibody) structural unit is known to comprise a tetramer or a dimer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (L) chain (approximately 25 kD) and one "heavy" (H) chain (approximately 50 kD-70 kD). The N-terminus of each chain defines a variable region of approximately 100-110 or more amino acids, which is primarily responsible for antigen recognition. The terms "variable light chain" and "variable heavy chain" refer to these variable regions of the light and heavy chains, respectively. The variable region of an antibody refers to either the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs within each chain are held in close proximity by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. Optionally, antibodies or immunological portions of antibodies can be chemically conjugated to or expressed as fusion proteins with other proteins.

[0121] A "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, where these domains are present in either orientation on a single polypeptide chain (e.g., VL-VH or VH-VL). Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, enabling the scFv to form the desired structure for antigen binding. In a preferred embodiment, the antigen-binding domain in the in-frame fusion protein of the present invention is an scFv antibody fragment, which can be a murine scFv, human scFv, or humanized scFv. Single-chain antibodies can be cloned from the V-region genes of a hybridoma specific for a desired target. The antigen-binding domain of the present invention and the in-frame fusion protein comprising it and the endogenous CD3 epsilon protein are intended to bind to mammalian, particularly human, protein targets. In a specific embodiment, the antigen-binding domain is a humanized scFv that binds to an autoantigen, an alloantigen, or a cancer antigen. In certain embodiments, the autoantigen or alloantigen is an HLA antigen, preferably HLA-A2. In further embodiments, the cancer antigen is an ErbB protein or a B lymphocyte antigen, preferably HER2 / neu or CD19.

[0122] In certain embodiments, the antigen-specific binding domain is a humanized scFv that binds to an HLA-A2 polypeptide. An example of a variable heavy chain suitable for constructing an in-frame fusion protein comprising an HLA-A2-specific antigen-binding domain contemplated herein includes, but is not limited to, the amino acid sequence set forth in SEQ ID NO: 47. An example of a variable light chain suitable for constructing an anti-an in-frame fusion protein comprising an HLA-A2-specific antigen-binding domain contemplated herein includes, but is not limited to, the amino acid sequence set forth in SEQ ID NO: 47.

[0123] The affinity of antigen-binding domains and in-frame fusion proteins according to the invention for a particular antigen can be readily determined using conventional techniques, for example by competitive ELISA (enzyme-linked immunosorbent assay), or by binding association, or by displacement assays using labeled ligands or using surface plasmon resonance devices such as Biacore.

[0124] "Gene" refers to a region of DNA that encodes a gene product, including regions that control the production of the gene product, whether or not these sequences are adjacent to the coding sequence and / or the transcribed sequence. Genes include, but are not limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0125] The term "exon" refers to the portion of a eukaryotic gene that is retained after splicing of the initially transcribed pre-mRNA. In contrast, introns are excised and degraded during splicing, resulting in the mature mRNA that is transcribed into a protein sequence. An exon of a protein-coding gene includes the open reading frame (ORF) as well as the 5' and 3' untranslated regions (UTR) from the terminal exon. An ORF is a region of DNA located between the start and stop codons (reading frame). In some embodiments, the nucleic acid construct of the present invention is integrated into an exon of an endogenous CD3 epsilon gene. In a preferred embodiment, the nucleic acid construct of the present invention is integrated into exon 3 or exon 6 of the endogenous CD3 epsilon gene.

[0126] Gene editing is a type of genetic engineering that uses engineered nucleases, or "molecular scissors," to insert, delete, or replace DNA within the genome of a living organism. These nucleases generate site-specific double-strand breaks (DSBs) at desired sites within the genome. The induced double-strand breaks are repaired by non-homologous end compound (HEJ) or homologous recombination (HR), thus resulting in the targeted mutation ("edit"). There are five examples of engineered nucleases used in gene editing: (a) meganucleases, (b) zinc finger nuclei (zinc finger nucleases), and (c) nucleotide sequences. (c) Transcription activator-like effector-based nucleases (TALENs), (d) Mega-TALENs, and (e) CRISPR-Cas systems can be used.

[0127] Methods for introducing exogenous molecules, e.g., nucleic acid sequences, into cells are well known to those skilled in the art and include lipid-mediated transfer (i.e., liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, biopolymer nanoparticles, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer. The terms "gene transfer," "nucleic acid transfer," "gene sequence transfer," and "transgene transfer" are used interchangeably. In some embodiments, nucleic acid constructs are preferably transferred into cells by methods such as non-viral vectors and electroporation.

[0128] "Gene expression" refers specifically to the conversion of information encoded by a gene into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein produced by translation of mRNA. Gene products also include RNAs modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins modified by processes such as methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and glycosylation.

[0129] As used herein, "endogenous" is used to specifically define gene expression that originates from a system such as a host, organism, tissue, or cell, while "exogenous" specifically defines a gene sequence that has been introduced into a cell from outside by one or more genetic, biochemical, or other methods.

[0130] The term "promoter" refers to a DNA sequence that initiates and controls transcription of downstream DNA by binding an enzyme that reads and transcribes the DNA. The resulting transcribed RNA may encode a protein. Promoters are preferably located near the transcription start site of a gene, upstream of the coding DNA, and typically before the start of the open reading frame (toward the 5' region of the sense strand).

[0131] In some embodiments, the nucleic acid construct is integrated into an endogenous CD3 epsilon gene. In some embodiments, the nucleic acid construct is integrated into an endogenous CD3 epsilon gene. In some embodiments, the nucleic acid construct is integrated into an endogenous CD3 epsilon gene. In some embodiments, the endogenous promoter is the promoter of the ...

[0132] "Sequence to be integrated," "integrating sequence," or "integrated nucleic acid construct" refers specifically to a nucleic acid construct comprising coding and / or non-coding elements that are inserted into the genome of a transfected cell, also referred to as the "host genome." In some embodiments, the edited chromosomal sequence may comprise an integrated sequence. The integrated sequence may encode an endogenous protein, an exogenous or heterologous protein, a wild-type protein, a modified protein, a fusion protein, etc. In an embodiment of the invention, the integrated sequence encodes an antigen-binding domain.

[0133] As used herein, an "integration site" or "fusion site" refers to a precise nucleic acid location within a genome where a double-strand break is generated and a nucleic acid construct is introduced into the genome by homologous sequence-dependent repair at the location of the double-strand break. Integration of an exogenous sequence may interrupt an endogenous sequence such that the exogenous sequence is under the control of an endogenous promoter. In some embodiments, the integrated exogenous sequence and / or the integrated exogenous sequence will be expressed together with the endogenous sequence. The integrated protein-coding sequence may be under the control of an endogenous promoter and / or fused in-frame with the endogenous protein-coding sequence. Additionally, the integrated sequence may function as a regulatory element. Thus, the integrated sequence may be endogenous or exogenous to the cell. The exogenous sequence may be a homolog of or related to the endogenous sequence. The exogenous sequence may be a human sequence. An animal or cell containing such an integrated sequence may be referred to as a "knock-in." In an embodiment of the present invention, the incorporated sequence may be a nucleic acid sequence encoding an antigen-binding domain.

[0134] As used herein, "upstream" and "downstream" are used in the context of the 5' to 3' direction of a nucleic acid strand, with upstream referring to nucleotides toward the 5' end of any given nucleic acid and downstream referring to nucleotides toward the 3' end of any given nucleic acid.

[0135] A fusion product is defined as "in-frame" if the open reading frame remains intact in the 3' region downstream of the nucleic acid integration site, regardless of the number of amino acids inserted or deleted at the fusion junction. A shift in the open reading frame of a protein-coding gene sequence falls under the category of "out-of-frame." According to the present invention, the nucleic acid construct encoding the antigen-binding domain is preferably integrated in-frame into the endogenous CD3 epsilon gene.

[0136] According to the present invention, a nucleic acid construct adapted to be integrated in frame with the endogenous CD3 epsilon gene of a human cell preferably comprises a first sequence encoding an antigen-binding domain, a second sequence encoding a target sequence such as a left homology arm and / or a right homology arm, one or more sequences encoding a linker, and optionally a sequence encoding a CD3 signal peptide, a protein cleavage site such as a self-cleaving peptide and / or an expression termination signal.

[0137] The terms "targeting sequence," "target sequence," or "target site" primarily refer to a chromosomal or extrachromosomal nucleic acid sequence that defines a portion of nucleic acid to which a binding molecule binds and / or cleaves when conditions for binding and / or cleavage are sufficient. In gene editing techniques that induce double-strand breaks, the target sequence includes a sequence identical to a recognition site in the endogenous DNA sequence intended for integration, preferably a recognition site selected from the group consisting of a homology arm, a guide RNA target site, a restriction enzyme recognition site, a ZFN recognition site, a ZFN cleavage site, a TALEN DNA binding site, a recombinase recognition site, an integrase site, and / or a homing nuclease recognition site. Techniques using each of these artificial nucleases are well known among experts. For example, when cells are edited using genome editing complexes (e.g., TALENs, CRISPR-Cas9 from Streptococcus pyogenes, CRISPR / Cas12a from Acidaminococcus species BV3L6, ZFNs, Mega-TALENs, or meganucleases) to introduce or knock out functional genes, double-strand breaks are induced at the site of modification by the nuclease (e.g., Cas9). Upon induction of the double-strand break, DNA repair proteins, such as phosphorylated (serl778) 53BP1 (p53BP1) or gH2AC, can accumulate at the double-strand break site, indicating a DNA damage response.

[0138] The term "signal sequence" refers to a nucleic acid sequence encoding a "signal peptide," also known as a "targeting signal." Signal peptides are short peptides, typically containing 16 to 30 amino acids, present at the N-terminus of most synthesized proteins. Proteins containing signal peptides typically include proteins present in certain cellular organelles (such as the endoplasmic reticulum, Golgi apparatus, or endosomes), proteins secreted from the cell, or proteins inserted into the cell membrane, such as the TCR / CD3 complex. The end of a signal peptide typically contains a stretch of amino acids that is recognized and cleaved by a signal peptidase, hence the term "cleavage site." Signal peptidases can be cleaved either during translocation or after translocation is complete, generating a free signal peptide and a mature protein.

[0139] As used herein, the term "protein separation site" includes a nucleic acid sequence element that causes separation of bicistronic or polycistronic expression of protein sequences to result in separately expressed proteins. Protein separation sites may refer to, but are not limited to, self-cleaving peptides, internal ribosome entry sites (IRES), and / or proteolytic cleavage sites. In preferred embodiments of the present invention, nucleic acid constructs may be designed to optionally include a protein separation site, preferably upstream of the nucleic acid sequence encoding the antigen-binding domain.

[0140] "Self-cleaving peptides" or "self-cleaving peptides" such as P2A can mediate ribosome skipping during protein translation in cells. The term "proteolytic cleavage site" refers to a recognition nucleic acid sequence for an intracellular proteolytic cleavage enzyme that breaks the peptide bond between amino acids in a protein.

[0141] In certain embodiments, the in-frame fusion proteins contemplated herein may contain linker residues, also referred to as "linkers," between the various domains, added for proper spacing and conformation of the molecule, e.g., a linker comprising an amino acid sequence connecting the VH and VL domains and providing a spacer function compatible with the interaction of the two partial binding domains, so that the resulting polypeptide retains the same specific binding affinity for the target molecule as an antibody comprising the same light chain variable region and heavy chain variable region. The in-frame fusion proteins contemplated herein may contain one, two, three, four, five, or more linkers. In certain embodiments, the linker is from about 1 to about 60 amino acids in length, from about 5 to about 55 amino acids, or from about 10 to about 50 amino acids in length, or any length therebetween, such as 2, 4, 5, 6, 8, 10, 12, 14, 15, 16, 18, 20, 22, 24, 25, 26, 28, 30, 32, 34, 35, 36, 38, 40, 42, 44, 45, 46, or 48 amino acids. Suitable linkers according to the present invention include glycine peptides, glycine-serine peptides such as polyglycine-serine (G4S), glycine-alanine peptides, alanine-serine peptides, and other flexible linkers known in the art, such as a Whitlow linker. Because glycine and glycine-serine peptides are relatively unstructured, they may be able to function as neutral tethers between domains of fusion proteins, such as the in-frame fusion proteins described herein. In certain embodiments, the antigen-binding domain of an in-frame fusion protein is followed by one or more "spacers" or "spacer polypeptides," which refer to regions that distance the antigen-binding domain from the effector cell surface, allowing for proper cell-cell contact, antigen binding, and activation.

[0142] "Expression termination signals" include any structural elements at the RNA and / or DNA levels that specify the end of transcription and / or translation and / or polyadenylation of heterologous nucleic acid transcripts. The termination of transcription is usually determined by a terminator, a nucleic acid sequence that defines the end of transcription (e.g., of a gene) and initiates the release of newly synthesized RNA. Terminators are located downstream of the transcribed gene and immediately precede a 3' regulatory element, such as a polyA signal. As used herein, the terms "polyA site," "polyA signal," or "polyA sequence" refer to a nucleic acid sequence that signals both the termination and polyadenylation of nascent RNA transcripts by RNA polymerase II. Polyadenylation sequences can increase mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thereby contributing to improved translation efficiency. Efficient polyadenylation of recombinant transcripts is preferred because transcripts without a polyA tail are unstable and rapidly degraded. A "stop codon" is also a sequence of three consecutive nucleotides (called a triplet) in a messenger RNA that signals the end of the protein translation process in the ribosome.

[0143] Polynucleotides, such as nucleic acid constructs of the present invention, can be prepared, manipulated, and / or expressed using any of a variety of established techniques known and available in the art. To express a desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector. Examples of vectors are plasmids, autonomously replicating sequences, and transposable elements. Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of categories of animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors are the pClneo vector (Promega) for expression in mammalian cells, and pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentiviral-mediated gene transfer and expression in mammalian cells. The "control elements" or "regulatory sequences" present in an expression vector are the untranslated regions of the vector (origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine-Dalgarno or Kozak sequences), introns, polyadenylation sequences, 5'-untranslated regions, and 3'-untranslated regions) that interact with host cell proteins to drive transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of appropriate transcription and translation elements, including ubiquitous and inducible promoters, can be used.

[0144] Methods for amplifying the nucleic acid constructs of the present invention are known to those skilled in the art and include polymerase chain reaction (PCR)-based methods, such as real-time PCR, quantitative real-time PCR (qPCR), quantitative real-time reverse transcription PCR (qRT-PCR), and digital PCR, using appropriate primers that hybridize to the nucleic acid construct.

[0145] The term "primer" refers to an oligonucleotide, whether naturally occurring, as in a purified restriction digest, or synthetically produced, that can act as an initiation point for synthesis when placed under conditions that induce synthesis of a primer extension product complementary to a nucleic acid strand, i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase, and at an appropriate temperature and pH. Thus, a primer can serve as an initiation point for a DNA replicating enzyme, such as DNA polymerase. A primer or oligonucleotide is preferably single-stranded to maximize amplification efficiency, but may alternatively be double-stranded. If double-stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligonucleotide, more preferably an oligodeoxyribonucleotide. The primer or oligonucleotide must be sufficiently long to prime the synthesis of an extension product in the presence of an inducing agent. The exact length of the primer will depend on many factors, including temperature, the source of the primer, and the intended use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, oligonucleotide primers typically contain 15 to 25 or more nucleotides, although fewer nucleotides may be used. In an embodiment of the present invention, primers according to SEQ ID NO: 43 to SEQ ID NO: 46 are used for the amplification of a nucleic acid construct according to the present invention encoding an antigen-binding domain integrated into the endogenous CD3 epsilon gene.

[0146] Hybridization is the process of establishing non-covalent, sequence-specific interactions between two or more complementary nucleic acid strands, resulting in a single hybrid called a duplex in the case of two strands, or a DNA double strand in the case of DNA. In the present invention, the terms "binding" or "annealing" may be used instead of hybridization. Hybrids can be dissociated by thermal denaturation, also known as melting. Here, heating a solution of the hybrid breaks the hydrogen bonds between the nucleic acid bases, and the two strands then separate. In the absence of external negative factors, the hybridization and melting processes can be repeated continuously and indefinitely. This forms the basis of PCR.

[0147] As used herein, the term "hybridization" refers to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (e.g., the strength of the association between nucleic acids) are affected by factors such as the degree of complementarity between nucleic acids, the stringency of the conditions involved, the Tm (melting temperature) of the formed hybrid, and the G:C ratio within the nucleic acids. As used herein, the term "stringency" refers to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents under which nucleic acid hybridization is performed. In an illustrative example, hybridization under "highly stringent conditions" may mean hybridizing in 5xSSPE and 50% formamide at 65°C and washing in 0.5xSSPE at 65°C. In another example, "highly stringent conditions" can mean hybridization at 55°C for 18 to 24 hours in a hybridization buffer consisting of 50% formamide (volume / volume), 10% dextran sulfate, 1x Denhardt's solution, 20 mM sodium phosphate (pH 6.5), 5x SSC, and 200 µg of salmon sperm DNA per ml of hybridization buffer, followed by four washes (5 minutes each) with 2x SSC, 1% SDS at room temperature, followed by a 15-minute wash with 0.1x SSC at 50°C to 55°C. In another example, conditions for high stringency hybridization are described in Sambrook et al., "Molecular Cloning: A Laboratory Manual," 3rd ed., Cold Spring Harbor Laboratory Press, (2001) (incorporated herein by reference). In some exemplary embodiments, hybridization occurs along the entire length of the nucleic acid. Highly stringent detection of hybridization in the context of the present invention indicates, for example, strong structural similarity or homology (e.g., nucleotide structure, base composition, arrangement, or order) to the nucleic acids provided herein.

[0148] The terms "peptide," "polypeptide," "polypeptide fragment," and "protein" are used interchangeably according to their conventional meaning, i.e., as sequences of amino acids, unless otherwise specified. Polypeptides are not limited to a particular length, e.g., they can include full-length protein sequences or fragments of full-length proteins, and can include post-translational modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications (both naturally occurring and non-naturally occurring) known in the art.

[0149] As used herein, the term "fusion protein" or "chimeric protein" includes proteins formed by joining two or more nucleic acid segments that originally encoded separate proteins, polypeptides, or protein fragments. A "fusion gene," "gene fusion," or "fused gene," according to the present invention, preferably refers to joined nucleic acid sequence segments in which at least one nucleic acid sequence is also an exogenous nucleic acid sequence and at least one other nucleic acid sequence is also an endogenous nucleic acid sequence. In preferred embodiments, the exogenous nucleic acid is integrated into and fused with an endogenous nucleic acid sequence, such as the endogenous CD3 epsilon gene. In some embodiments, the integrated and fused exogenous nucleic acid sequence may have several combined gene structures and / or polypeptide coding sequences separated by one or more protein separation sites. Translation of the fusion gene results in single or multiple polypeptides possessing functional properties derived from each of the original proteins or protein fragments. In further embodiments, the in-frame fusion proteins contemplated herein contain a signal (or leader) sequence at the N-terminus of the protein, which co- or post-translationally directs transport of the protein. Polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. Polypeptides contemplated herein specifically include the in-frame fusion proteins of the present disclosure, or sequences having deletions from, additions to, and / or substitutions of one or more amino acids of the in-frame fusion proteins disclosed herein.

[0150] As used herein, "isolated peptide" or "isolated polypeptide" and the like refer to the in vitro isolation and / or purification of a peptide or polypeptide molecule from its cellular environment and from association with other components of a cell, i.e., it is not significantly associated with in vivo materials. Similarly, an "isolated cell" refers to a cell obtained from an in vivo tissue or organ and that is substantially free of extracellular matrix.

[0151] In certain embodiments, the present invention contemplates T cells genetically modified to express in-frame fusion proteins contemplated herein for use in the prevention and / or treatment of unwanted (pathogenic) immune responses, such as unwanted (pathogenic) immune responses before and / or after allogeneic transplantation. In a preferred embodiment, the present invention contemplates T cells genetically modified to express in-frame fusion proteins contemplated herein for use in the prevention and / or treatment of graft-versus-host disease. In a further embodiment, the present invention contemplates T cells genetically modified to express in-frame fusion proteins contemplated herein for use in the prevention and / or treatment of cancerous diseases.

[0152] As used herein, the term "genetic manipulation" or "genetic modification" refers to the addition of additional genetic material in the form of DNA or RNA to the total genetic material in a cell. In contrast, an "unmodified" cell refers to a cell in which no additional genetic material has been added or introduced into the total genetic material of the cell. The genetic modification may or may not be selective at a specific location within the genome of the cell. In one embodiment, the genetic change is site-specific. In one embodiment, the genetic modification is not site-specific. The terms "genetically modified cells," "genetically modified immune cells," "modified cells," and "redirected cells" are used interchangeably. As used herein, the term "gene therapy" refers to the permanent or transient introduction of additional genetic material in the form of DNA or RNA into the total genetic material of a cell to restore, correct, or alter the expression of a gene or to express a polypeptide, e.g., an antigen-binding domain. In certain embodiments, the antigen binding domains contemplated herein are introduced into and expressed in T cells as in-frame fusion proteins comprising the antigen binding domain and the endogenous CD3 epsilon protein, thereby directing their specificity to a target antigen of interest, e.g., the HLA-A2 antigen.

[0153] The T cells of the present invention can be autologous / autologous ("autologous") or non-autologous ("non-autologous", e.g., allogeneic, syngeneic, or xenogeneic). As used herein, "autologous" refers to cells from the same subject and represents a preferred embodiment of the present invention. As used herein, "allogeneic" refers to cells of the same species that are genetically different from the cells of a comparison subject. As used herein, "allogeneic" refers to cells of a different subject that are genetically identical to the cells of a comparison subject. As used herein, "xenogeneic" refers to cells of a different species than the cells of a comparison subject. In preferred embodiments, the cells of the present invention are autologous or allogeneic.

[0154] In certain embodiments, a cell source is obtained from a subject prior to in vitro manipulation or genetic modification of immune effector cells as described herein. T cells can be obtained from a number of sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood drawn from a subject using any number of techniques known to those skilled in the art, such as sedimentation, e.g., FICOLL™ separation, antibody-conjugated bead-based methods, e.g., MACS™ separation (Miltenyi). In one embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, and B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing. Cells can be washed with PBS or another suitable solution lacking calcium, magnesium, and most, if not all, other divalent cations. As will be appreciated by those skilled in the art, the washing step can be accomplished by methods known to those skilled in the art, such as by using a semi-automated flow-through centrifuge, such as the Cobe 2991 cell processing device or the Baxter CytoMate. After washing, cells can be resuspended in a variety of biocompatible buffers or other physiological saline solutions, with or without buffers. In certain embodiments, undesirable components of the apheresis sample can be removed directly in the culture medium in which the cells are resuspended.

[0155] In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and removing monocytes, for example, by PERCOLL™ gradient centrifugation. Specific subpopulations of T cells can be further isolated by positive or negative selection techniques. One method used herein is negative magnetic immunoadhesion or flow cytometric cell sorting and / or cell selection using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells.

[0156] T cells may be genetically modified after isolation and optional selection using known methods, or T cells may be activated and expanded (or differentiated, in the case of progenitor cells) in vitro and then genetically modified. In certain embodiments, T cells, such as Tregs, are genetically modified with a nucleic acid construct (e.g., transfected with a nucleic acid construct and a pre-complexed Cas12a or Cas9 enzyme) comprising a sequence region encoding an antigen-binding domain as contemplated herein, and then expanded in vitro. In various embodiments, T cells can be activated and expanded before or after genetic modification to express the in-frame fusion protein using, for example, methods described in U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 2006 / 0121005. For example, the expansion of Treg cells requires repeated stimulation via the TCR / CD3 complex and additional CD28 costimulation using anti-CD3 and anti-CD28 antibodies on beads. High doses of IL-2 as a growth factor and rapamycin, an mTOR inhibitor, are also added to the culture medium. In one aspect, the present invention relates to a method for preparing genetically modified T cells, which comprises repeated stimulation via the TCR / CD3 complex, wherein the genetically modified T cells exhibit unchanged expansion kinetics (±50%) in culture compared to unmodified T cells. In one embodiment of the present invention, the genetically modified T cells can be expanded to an unchanged extent (±50%) compared to unmodified T cells by the above method.

[0157] In one embodiment, the present invention provides a method for storing genetically modified T cells, e.g., targeting the HLA-A2 antigen, comprising cryopreserving the T cells so that the cells remain viable after thawing. A portion of the expressing genetically modified T cells can be cryopreserved by methods known in the art to provide a permanent source of such cells for future administration to a subject, such as for preventing and / or treating an unwanted (pathogenic) immune response or cancerous disease. If desired, the cryopreserved transformed T cells can be thawed and grown and expanded to produce larger numbers of such cells.

[0158] A "pharmaceutical composition" refers to a composition formulated into a pharmaceutically or physiologically acceptable solution for administration to a cell or animal, alone or in combination with one or more other therapeutic modalities. It will also be understood that, if desired, the compositions of the present invention may be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharmaceutically active agents. There is virtually no limit to other components that may be included in the composition, provided that the additional agents do not adversely affect the ability of the composition to deliver its intended therapy.

[0159] The term "pharmaceutically acceptable" is used herein to refer to compounds, substances, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0160] As used herein, "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, additive, glidant, sweetener, diluent, preservative, dye / colorant, flavoring, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surface active agent, or emulsifier approved by the U.S. Food and Drug Administration as acceptable for use in humans or domestic animals.

[0161] Plasmids, nucleic acids, nucleic acids complexed with Cas enzymes, and / or other compositions, agents, drugs, biologics (proteins) can be incorporated into pharmaceutical compositions, e.g., pharmaceutically acceptable carriers or excipients. Such pharmaceutical compositions are particularly useful for administration and delivery to a subject in vivo or ex vivo.

[0162] The pharmaceutical compositions of the present invention comprising a genetically modified T cell population, such as genetically modified Treg cells, may contain a buffer solution, such as neutral buffered saline, phosphate buffered saline, or the like; a carbohydrate, such as glucose, mannose, sucrose, or dextran, mannitol; a protein; a polypeptide or an amino acid, such as glycine; an antioxidant; a chelating agent, such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The compositions of the present invention are preferably formulated for parenteral administration, for example, intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration.

[0163] Liquid pharmaceutical compositions (whether in the form of a solution, suspension, or the like) may contain one or more of the following: a sterile diluent, such as water for injection, saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides that can serve as a solvent or suspending medium, polyethylene glycol, glycerin, propylene glycol, or other solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates, and isotonic agents such as sodium chloride or dextrose. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials. Pharmaceutical compositions for injection are preferably sterile.

[0164] In certain embodiments, pharmaceutical compositions contemplated herein comprise a quantity of genetically modified T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. In certain embodiments, pharmaceutical compositions contemplated herein comprise a quantity of genetically modified T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0165] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or condition, and may include even a slight decrease in one or more measurable markers of the disease or condition being treated. Treatment may optionally include a reduction or amelioration of symptoms of the disease or condition, or a delay in the progression of the disease or condition. "Treatment" does not necessarily mean a complete eradication or cure of the disease or condition or its associated symptoms.

[0166] As used herein, "prevent" and similar terms, such as "prevented," "preventing," or "prophylactic," refer to an approach that prevents, inhibits, or reduces the likelihood of the onset or recurrence of a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevent" and similar terms also include reducing the intensity, impact, symptoms, and / or burden of a disease or condition before the onset or recurrence of the disease or condition.

[0167] In one embodiment, a method for preventing and / or treating an unwanted (pathogenic) immune response or cancerous disease in a subject comprises administering an effective amount, e.g., a therapeutically effective amount, of a composition comprising a genetically modified immune effector cell as contemplated herein. The amount and frequency of administration will depend on factors such as the condition of the patient and the type and severity of the patient's disease, although appropriate dosages may be determined through clinical trials.

[0168] Administration of the compositions contemplated herein can be carried out by any convenient method, including aerosol inhalation, injection, oral infusion, infusion, implant, or transplant. In a preferred embodiment, the compositions are administered parenterally. As used herein, the phrases "parenteral administration" and "parenterally administered" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumor, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0169] In certain embodiments, the compositions of the invention comprise an amount of genetically modified T cells as contemplated herein. As used herein, the term "amount" refers to an "effective amount" or "effective dose" of genetically modified therapeutic T cells to achieve a beneficial or desired prophylactic or therapeutic result, including a clinical result.

[0170] A "prophylactically effective amount" refers to an amount of genetically modified T cells effective to achieve a desired prophylactic result. Typically, a prophylactic dose is used in subjects prior to or at an early stage of an unwanted (pathogenic) immune response or cancerous disease, and therefore the prophylactically effective amount is less than a therapeutically effective amount, but this is not necessarily the case. The term prophylactic does not necessarily refer to the complete prevention or obviation of a particular condition, such as an unwanted (pathogenic) immune response or cancerous disease. The term prophylactic can also refer to reducing the risk of a particular condition occurring or its symptoms worsening.

[0171] A "therapeutically effective amount" of genetically modified T cells may vary depending on factors such as the condition, age, sex, and weight of an individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the genetically modified cells are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the exact amount of the composition of the present invention to be administered may be determined by a physician, taking into account individual differences in the age, weight, stage of disease, and condition of the patient (subject). Generally, pharmaceutical compositions comprising the genetically modified T cells described herein are administered in an amount of 10 per kg of body weight. 2 ~10 pieces 10 cells per kg of body weight, preferably 10 5 ~10 pieces 7 It can be stated that the composition may be administered in a dosage of 10 cells (including all integer values ​​within these ranges). The number of cells will depend on the intended end use of the composition and also on the type of cells contained therein. For the uses provided herein, the cells will generally be in a volume of 1 liter or less, and may be in a volume of 500 mL or less, or even 250 mL or 100 mL or less. Thus, a desirable cell density is typically 10 per ml. 6 More than 10 cells per mL, typically 10 7 >10 cells, typically 10 per mL 8 Clinically meaningful numbers of T cells can be distributed over multiple infusions, which cumulatively amount to 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 pieces, 10 10 pieces, 10 11 Pieces or 10 12The number of cells administered may be equal to or greater than 100 cells. In some embodiments of the invention, fewer cells may be administered, particularly since all infused cells will be directed to a specific target antigen. Compositions of genetically modified T cells may be administered multiple times at dosages within these ranges. Cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient receiving therapy.

[0172] Immunotherapy in the context of the present invention should be construed to include any therapeutic agent that utilizes the immune system to prevent and / or treat unwanted (pathogenic) immune responses, such as those before and / or after allogeneic transplantation and autoimmune diseases, as well as for the treatment of cancerous diseases. With regard to cancer, immunotherapy exploits the fact that cancer cells have subtle differences in molecules on their surface that can be recognized by the immune system. Immunotherapy includes, but is not limited to, cell therapy and antibody therapy. Cell therapy according to the present invention involves the administration of genetically modified T cells that contain and / or express an in-frame fusion protein comprising an antigen-binding domain and the endogenous CD3 epsilon protein, whereby the function of the TCR complex is maintained.

[0173] In certain embodiments, compositions contemplated herein comprise an effective amount of genetically modified T cells, alone or in combination with one or more therapeutic agents. Thus, genetically modified T cell compositions can be administered alone or in combination with other treatments known for a particular condition, such as immunomodulatory or cancer treatments, including immunotherapy, hormone therapy, photodynamic therapy, radiation therapy, chemotherapy, transplantation, etc. The compositions can also be administered in combination with antibiotics. Such therapeutic agents may be accepted in the art as standard treatments for certain conditions described herein, such as certain autoimmune or cancerous diseases. Exemplary contemplated therapeutic agents include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory drugs, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies, or other active and adjuvant agents.

[0174] Combination immunotherapy encompasses simultaneous, concomitant, or co-treatment, and involves administering genetically modified T cells in combination with immunotherapeutic agents, such as checkpoint inhibitors and / or immunostimulatory cytokines, whereby treatments may occur within minutes of each other, within the same hour, on the same day, within the same week, or within the same month. Combination medicaments containing one or more of the above genetically modified T cells and another immunotherapeutic agent can also be used to simultaneously administer various components in a single administration or dosage. "Medication" refers specifically to a drug containing immunotherapeutic or genetically modified cells used to cure, treat, or prevent disease.

[0175] As used herein, a "cancerous disease" is a disease characterized by the uncontrolled growth of abnormal cells. Cancer refers to any type of cancerous growth or oncogenic process, metastatic tissue, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasion. Cancer cells can spread locally or to other parts of the body via the bloodstream or lymphatic system. Cancer cells that spread to other parts of the body are called "metastatic cells" or "metastatic tumor cells." As used herein, the terms "tumor" and "cancer" are used interchangeably, and for example, both terms include solid and liquid, e.g., systemic or circulating tumors, pre-malignant and malignant cancers and tumors. Examples of liquid cancers include acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (acute myeloid leukemia), and acute myeloid leukemia (acute myelogenous leukemia). Examples of solid tumors include, but are not limited to, myeloma, chronic myeloid leukemia (AML), chronic myeloid leukemia (CML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, and myeloma. Examples of solid tumors include malignant organ systems, including tumors of the liver, lung, breast, lymphatic system, gastrointestinal (e.g., colon), genitourinary (e.g., kidney, urothelial cell), prostate, and throat, such as sarcomas, adenocarcinomas, and carcinomas. Examples of breast cancers treatable with CTLA-1 include ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), invasive ductal carcinoma (IDC), and invasive ductal carcinoma (IDC). (Invasive ductal carcinoma includes tubular carcinoma, medullary carcinoma, mucinous carcinoma, papillary carcinoma, and cribriform carcinoma), invasive lobular carcinoma (ILC), inflammatory breast cancer, male breast cancer, Paget's disease of the nipple, phyllodes tumor of the breast, and recurrent and / or metastatic breast cancer. Adenocarcinomas include most malignant tumors, such as colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, and esophageal cancer. In some forms, the cancer is melanoma, e.g., advanced-stage melanoma. Metastatic lesions of cancer can also be treated or prevented with the methods and compositions of the present invention.Examples of other types of cancer that can be treated include bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, acute myeloid leukemia, chronic myeloid leukemia, acute phosphodiesterase II (PHX), and steroid hormone receptor agonists (SRH). These include chronic or acute leukemias, including leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphomas, bladder cancer, kidney or ureter cancer, renal pelvic carcinoma, central nervous system (CNS) neoplasms, primary CNS lymphomas, tumor angiogenesis, spinal axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, asbestos-induced T-cell lymphoma, including those associated with environmental cancers. Treatment of metastatic cancers, such as those expressing PD-L1 (Iwai et al. (2005) Int. Immunol. 17: 133-144), can be performed using the inhibitory molecules described in the present invention.

[0176] Medical indications related to unwanted (pathogenic) immune responses represent preferred embodiments of the medical use of the present invention in the treatment and / or prevention of unwanted (pathogenic) immune responses. Furthermore, medical indications related to cancerous diseases represent preferred embodiments of the medical use of the present invention in the treatment and / or prevention of cancerous diseases.

[0177] Table 1: Guide RNA targets for CD3 epsilon (sequence according to the invention) and TRAC (comparison example) Any of the sequences, any combination of sequences, or any marked subregion of any of the sequences set forth in the table below, SEQ ID NO: 1 to SEQ ID NO: 8 may represent an embodiment of the present invention and / or may be used in any of the aspects or embodiments of the invention described herein.

[0178] [Table 1] SEQ ID NO: Sequence number Sequence 5'-3': Sequence 5'-3' Name:Name Nuclease Target: target CD3E exon 3:CD3E exon 3 CD3E exon 6:CD3E exon 6 gRNA8:Cas9 #3 for CD3e exon 3: gRNA8:Cas9 #3 for CD3e exon 3 TRAC exon 1:TRAC exon 1

[0179] Table 2: Nucleic acid sequences for HDR donor templates for CD3 epsilon (sequences according to the invention) Any of the sequences, any combination of sequences, or any marked subregion of any of the sequences set forth in the table below, SEQ ID NO: 10 to SEQ ID NO: 28, may represent an embodiment of the present invention and / or may be used in any of the aspects or embodiments of the invention described herein.

[0180] SEQ ID NO: Sequence number Homology Directed DNA Repair (HDR) template Info HDR-Template Part: Information on the HDR template part Sequence: 5'-3' sequence HDRT HLA-A2 scFv into CD3e-exon3: HDRT HLA-A2 scFv into CD3e-exon3 Full Sequence: Full sequence Left Homology arm: Left homology arm Mys tag: Myc tag Hu anti-HLA-A2 VH(clone: ​​3PF12):Human anti-HLA-A2 VH(clone: ​​3PF12) Optimized (G4S) linker: Optimized (G4S)3 linker Hu anti-HLA-A2 VL(clone:3PF12):Human anti-HLA-A2 VL(clone:3PF12) Right homology arm: HDRT HLA-A2 scFv into CD3e-exon6: HDRT HLA-A2 scFv into CD3e-exon6 CD3 epsilon signal peptide Similar to SEQ ID NO 12 Similar to SEQ ID NO 13 Similar to SEQ ID NO 14 Similar to SEQ ID NO 15 CD3 epsilon 1-30aa: CD3 epsilon (amino acids 1-30) [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0181] Table 3: Nucleic acid sequences for HDR donor templates for TRAC (comparison sequences)

[0182] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] SEQ ID: Sequence number Homology directed DNA repair (HDR) template Info HDRHDR template Part:Template part information Sequence 5'-3': Sequence 5'-3' HDRT HLA-A2 CAR into TRAC: HDRT HLA-A2 CAR into TRAC Full sequence: Full sequence Left homology arm Similar to SEQ ID NO 20 Signal peptide Similar to SEQ ID NO 13 Hu anti-HLA-A2 VH(clone:3PF12):Human anti-HLA-A2 VH(clone:3PF12) Similar to SEQ ID NO 14 Optimized (G4S)3 linker: Optimized (G4S)3 linker Similar to SEQ ID NO 15 Human anti-HLA-A2 VL (clone: ​​3PF12) IgG4 hinge: IgG4 hinge CD28 transmembrane:CD28 transmembrane CD28 endodomain:CD28 endodomain CD28 zeta endodomain:CD3 zeta endodomain bgH terminator sequence: bgH terminator sequence Right homology arm:

[0183] Table 4: Primers for donor template amplification (sequences according to the invention) Any of the sequences, any combination of sequences, or any marked subregion of any of the sequences set forth in the table below, SEQ ID NO: 43 to SEQ ID NO: 46, may represent an embodiment of the present invention and / or may be used in any of the aspects or embodiments of the invention described herein.

[0184] [Table 4] SEQ ID NO: Sequence number Sequence 5'-3': Sequence 5'-3' Name:Name Comment:Remarks Primer for donor template amplification: Primer for donor template amplification

[0185] Table 5: Amino acid sequences of antigen-binding domains (sequences according to the invention) Any of the sequences, any combination of sequences, or any marked subregion of any of the sequences set forth in the table below, SEQ ID NO: 47 to SEQ ID NO: 56 may represent an embodiment of the present invention and / or may be used in any of the aspects or embodiments of the invention described herein.

[0186] [Table 5] SEQ ID NO: Sequence number Homology directed DNA repair (HDR) template part Info scFV Part: scFV part information Amino acid sequence: Hu anti-HLA-A2 scFV fragment (clone: ​​3PF12): Human anti-HLA-A2 scFV fragment (clone: ​​3PF12) Full Sequence: Full sequence Hu anti-HLA-A2 VH (clone 3PF12): Human anti-HLA-A2 VH (clone: ​​3PF12) Optimized(G4S)3 linker: Optimized (G4S)3 linker Hu anti-HLA-A2 scFV VL(clone:3PF12):Human anti-HLA-A2 VL(clone:3PF12) Hu anti-HLA-A2 scFV VH(clone:3PF12):Human anti-HLA-A2 VH(clone:3PF12) Hu anti-HLA-A2 scFV VL(clone:3PF12):Human anti-HLA-A2 VL(clone:3PF12)

[0187] drawing The present invention is further illustrated by the following drawings, which are not intended to limit the scope of the invention but rather represent preferred embodiments of aspects of the invention provided to more clearly illustrate the invention. [Brief explanation of the drawings]

[0188] [Figure 1-1] Figure 1 shows that TCR replaced the function of A2 CAR Tregs in vitro but suffered from poor expansion. [Figure 1-2] Same as above [Figure 2-1] FIG. 1 shows identification of a CD3 epsilon gene editing strategy to generate TruC+ T cells after integration of scFv cDNA. [Figure 2-2] Same as above [Figure 2-3] Same as above [Figure 3-1]Figure 1 shows that repeated stimulation preferentially expands CD3e-TruC+ Tregs over unedited Tregs or TRAC-substituted CAR Tregs. Schematic pathway for generation and expansion of CD3e-TruC+ and TRAC-CAR Treg cells. [Figure 3-2] Same as above [Figure 4-1] FIG. 1 shows that gene-edited and ex vivo expanded Tregs retain their classical phenotype and epigenetic identity. [Figure 4-2] Same as above [Figure 4-3] Same as above [Figure 5-1] FIG. 1 shows the activation profile of CD3e-TruC Treg cells compared to TRAC-CAR and WT Treg cells after antigen HLA-A2 specific stimulation. [Figure 5-2] Same as above [Figure 5-3] Same as above [Figure 5-4] Same as above [Figure 6] FIG. 10 shows that A2 CD3e-TruC+ Tregs functionally suppress the proliferation of autologous Tconv in vitro, regardless of polyclonal or HLA-A2 antigen-specific stimulation. [Figure 7] FIG. 1 shows purity check of polyclonal Treg selection. [Figure 8] FIG. 1 shows that CD3e-TruC Tregs specifically recognize the HLA-A2 antigen. [Figure 9] FIG. 1 shows that CD3e-TruC Tregs rapidly upregulate phosphorylation of TCR downstream molecules upon stimulation with antigens HLA-A2 and αCD3. [Figure 10] FIG. 1 shows purity check of gene-edited Treg selection. [Figure 11] FIG. 10 shows histogram plots of autologous Tconv proliferation without Tregs. [Figure 12]Figure 10 shows that alternative donors also demonstrate that A2 CD3e-TruC+ Tregs functionally suppress the expansion of autologous Tconv in vitro, regardless of polyclonal or HLA-A2 antigen-specific stimulation. [Figure 13] FIG. 1 shows that HLA-A2-specific TruC Tconvs and HER2-specific TruC Tconvs secrete inflammatory cytokines in an antigen-dependent manner. [Figure 14] FIG. 1 shows that CD3 epsilon-engineered CD19-TruC extends the lifespan of mice in a B-ALL xenograft model. DETAILED DESCRIPTION OF THE INVENTION

[0189] Figure 1: (A) Based on Watkins et al. (2000), this study used the moderate-affinity anti-human HLA-A2 scFv clone 3PF12. (B) HDR-mediated integration of the donor template into the TRAC locus. The donor template contains the 400-bp left homology arm (LHA), the self-cleaving peptide P2A, a signal peptide (SP), the heavy and light chains of the HLA-A2 scFv with a 3×G4S linker between them, an IgG1 hinge (used to detect CAR integration), the CD28 transmembrane domain (TD) and intracellular domain (ICD), a zeta chain, followed by a poly(A) tail, and a 400-bp right homology arm (RHA). (C) Dot plot of αFc A647 vs. CD3 PB staining for HLA-A2-specific TRAC-CAR Treg cells and untouched wild-type (WT) Treg cells. Gating was performed on live single lymphocytes. (D) Percentage of FOXP3 and CD25 double positive cells (left) and FOXP3 MFI (right) between TRAC-CAR and WT Treg cells. (E) Comparison of the percentage of inhibition between suppression assays performed with TRAC-CAR and WT Treg cells. (F) Irradiated K562.A2 +Fold change in expansion during 2 weeks of expansion after electroporation of TRAC-CAR Treg cells (1:1 ratio), or with 5 μg / ml A2 dimer coated on plates with either 1 μg / ml αCD28 or no αCD28, or no stimulation. WT Treg cells (1:1 ratio) plus Treg expansion bead stimulation served as control. Treg cells were stimulated every 2-3 days. N=3.

[0190] Figure 2: (A) Five gRNAs specifically targeting exon 3 and exon 6 of the CD3 epsilon locus were identified and used for knockout (KO) experiments in human conventional T cells. (B) Representative dot plots of CD3 PB against SSC-A. KO cells were CD3 negative (left). (C) Summary of KO efficacy for each gRNA, n=2 or 4 (right). (D) Donor template design and integration into the gRNA1 (left) and gRNA5 (right) cleavage sites. (E) Representative dot plots of myc A647 against CD3 PB. Successfully gene-edited cells were double-positive for CD3 and myc. (F) Comparison of HDR efficiency of donor templates integrated into the gRNA1 and gRNA5 cleavage sites. (G) Myc integrated into the gRNA1 and gRNA5 cleavage sites. + Overlaid histograms of cells. (H) CD3e-TruC integrated into the gRNA1 and gRNA5 cleavage sites. + MFI of cells. (I) CD3e-TruC integrated into the gRNA1 and gRNA5 cleavage sites 4 days after electroporation. + Relative expansion growth of cells (normalized to gRNA5).

[0191] Figure 3: (A) CD3e-TruC + Schematic pathway of Treg cell and TRAC-CAR Treg cell generation and expansion. (B) CD3e-TruC, indicated by myc and CD3 double positivity or HLA-A2 and myc double positivity. +(Left) and (Right) representative dot plots of TRAC-CAR Treg cells, as indicated by αFc positivity but CD3 negativity. (C) Fold change in total viable cell expansion between CD3e-TruC Treg cells and TRAC-CAR Treg cells compared to WT Treg cells. N=4. (D) Fold change in gene-edited cell expansion between CD3e-TruC Treg cells and TRAC-CAR Treg cells. N=4.

[0192] Figure 4: (A) Representative dot plots of FOXP3 versus CD25 between CD3e-TruC Treg cells, TRAC-CAR Treg cells, WT Treg cells, and WT Tconv cells. (B) Percentage of FOXP3 and CD25 double positive cells (left) and FOXP3 MFI (right) of CD3e-TruC Treg cells compared to TRAC-CAR Treg cells and WT Treg cells. n=4. (C) Expression levels of cytokines IFN-γ and TNF-α of CD3e-TruC Treg, TRAC-CAR Treg, and WT Treg after stimulation with PMA and ionomycin. WT Tconv was used as a high control. CD3e-TruC + Schematic pathway of Treg and TRAC-CAR Treg cell generation and expansion. (D) Treg-specific methylation analysis shows hypomethylation of all 10 CpGs in unedited Tregs (WT), CD3-TruC Tregs, and TRAC-replaced CAR Tregs, but not in conventional T cells (WT Tconv).

[0193] Figure 5: (A) Experimental setup for activation assay. (B) Representative offset histograms and summaries of each activation marker for CD3e-TruC and TRAC_CAR Treg cells for gradient concentrations of HLA-A2 stimuli coated on the plate. (C) Representative offset histograms (top) and summaries (bottom) of each activation marker for CD3e-TruC and TRAC_CAR Treg cells compared to WT Treg cells and WT Tconv (dotted line) as a negative control. N=2. (D) Fold change in MFI of activation markers for CD3e-TruC and TRAC_CAR Treg cells normalized to WT Treg cells. (E) Variance of activation marker MFI between two donors by comparing CD3e-TruC, TRAC_CAR, and WT Treg cells.

[0194] Figure 6: (A) Experimental setup for suppression assay. (B) Polyclonal beads and CD3-depleted HLA-A2 after stimulation with polyclonal beads alone. + After stimulation of cells (C), or CD3-depleted HLA-A2 + (D) CD4 Tregs during suppression assays performed with CD3e-TruC Treg cells, TRAC-CAR Treg cells, and WT Treg cells after stimulation with cells alone. + Tconv (left) and CD8 + Comparison of percentage of inhibition for Tconv (right).

[0195] Figure 7: (A) CD4 before sorting (top) and after two rounds of sorting with Tyto. + CD25 high CD127 - CCR7 + Representative dot plots of Treg cells, gated on single viable lymphocytes. Highly purified polyclonal Treg cells were used for HLA-A2-specific CD3e-TruC Treg cells and TRAC-CAR Treg cells. (B) CCR7 expression among single lymphocytes before and after Tyto sorting. + Percentage of Treg cells. n=8.

[0196] Figure 8: Representative dot plots of CD3e-TruC Tregs (top) and WT Tregs (bottom) showing that CD3e-TruC Tregs specifically recognize the HLA-A2 antigen.

[0197] Figure 9: (A) Experimental setup for phosphorylation assay. (B) Representative FACS plot showing the percentage of phosphorylated ERK among surviving cells after stimulation with 5 μg / ml HLA-A2. (C) Kinetics of phosphorylated ERK among surviving cells after stimulation with either 10 μg / ml αCD3 (left) or 5 μg / ml HLA-A2 (right).

[0198] Figure 10: (A) CD3 before sorting (top) and after sorting with Aria II. + Myc + CD3e-TruC Treg (left) and CD3-aFc + Representative dot plots of TRAC-CAR Treg (right) cells and single viable CD4 + Gating was on T lymphocytes. (B) Percentage of gene-edited Treg cells among single lymphocytes displayed for CD3e-TruC and TRAC-CAR cells.

[0199] Figure 11: Histogram plot of autologous Tconv proliferation in the absence of Tregs. CD4 + (left) and CD8 + Tconv cells (right) were co-cultured with aCD3 / CD28 beads at a 1:1 ratio (top) or with beads containing HLA-A2 + Co-cultured with CD3-depleted PBMCs at a 1:1:1 ratio (center) or with HLA-A2 + They were co-cultured with CD3-depleted PBMCs alone at a 1:1 ratio (bottom).

[0200] Figure 12: After stimulation with polyclonal beads alone (A), polyclonal beads and CD3-depleted HLA-A2 + After stimulation of cells (B), or CD3-depleted HLA-A2 +CD4 during suppression assays performed with CD3e-TruC Treg cells, TRAC-CAR Treg cells, and WT Treg cells after stimulation with cells alone (C). + Tconv (left) and CD8 + Comparison of the percentage of inhibition of Tconv (right).

[0201] Figure 13: HLA-A2-specific TruC Tconvs and HER2-specific TruC Tconvs secrete proinflammatory cytokines in an antigen- and TCR-dependent manner. Figure 13: HLA-A ... + :K562 overexpressing HLA-A2 cell line, Nalm-6 cell line, Her2 + Intracellular staining of inflammatory cytokines in HLA-A2-specific and HER2-specific TruC Tconvs in coculture with HLA-A2-specific and HER2-specific TruC Tconvs (ONCO-1 cell line) or in control conditions (no HLA-A2: K562 wild-type cell line, no HER2: DAOY cell line, no stimulation (no sti)). Brefeldin A (BFA) was added to prevent cytokine secretion. Six hours after the start of stimulation, cells were fixed, permeabilized, stained with fluorochrome-coupled antibodies, and visualized by flow cytometry analysis (Cytoflex LX, Beckman Coulter). The data show that different CD3 epsilon-engineered TruC Tconvs secrete similar amounts of inflammatory cytokines in both TruC antigen (HLA-A2 or HER2)-dependent and TCR-dependent manners, but not in control conditions.

[0202] Figure 14: CD3 epsilon-engineered CD19-TruC extends mouse lifespan in a B-ALL xenograft mouse model. Six- to eight-week-old NRG mice were administered 5 x 10 CD3 epsilon-engineered CD19-TruC by tail vein injection. 5 Four days later, 5 × 10 luciferase-labeled Nalm-6 cells (human B-ALL cell line) expressing CD19-specific scFv (FMC063) were injected. 5CD3 epsilon-engineered TruC Tconv (n=5) or wild-type, unedited Tconv (n=5) were administered intravenously. PBS was used as a vehicle control (n=3). Mice's health was monitored every other day for behavior, weight, and general appearance. Bioluminescence was measured weekly. Kaplan-Meier plots show the survival time of mice receiving different treatments and demonstrate that mice treated with CD3 epsilon-engineered CD19-specific TruC Tconv had a significantly prolonged survival time compared to controls. [Example]

[0203] The present invention is demonstrated through the examples disclosed herein. The examples provided represent embodiments of the present invention and are not intended to limit the scope of the present invention. These examples should be considered as providing non-limiting explanations and technical assistance for the practice of the present invention.

[0204] The nucleic acid constructs used in the Examples and Comparative Examples are disclosed in the above sequence tables (Tables 1 to 4).

[0205] Comparative Example 1: TCR-substituted A2 CAR Tregs (TRAC-CAR Tregs) were successfully generated via non-viral gene editing but suffered from poor expansion To generate an HLA-A2-specific CAR construct for Treg engineering, the antigen-binding domain of a second-generation CAR was replaced with an HLA-A2-specific scFv originally obtained from an allosensitized 57-year-old female patient (Figure 1A). The CAR was cloned into a homologous sequence-dependent repair template (HDRT) and inserted into the TRAC locus (Figure 1B), as previously described (Non-Patent Document 15). After isolating PBMCs from peripheral blood of an HLA-A2-negative healthy donor, Tregs were isolated from CD4 + , CD25 high, CD127 low, and CCR7 +Tregs were then activated using polyclonal stimulation and expanded in the presence of high-dose IL-2 and the mTOR inhibitor rapamycin. After 7–9 days of expansion, proliferating Tregs were harvested and electroporated with a precomplexed RNP containing a linear double-stranded (ds) DNA HDRT and a TRAC-specific guide RNA (gRNA) and recombinant Streptococcus pyogenes (Sp) Cas9 protein. The resulting TRAC-CAR + Tregs were predominantly CD3-negative (Figure 1C), retained classic markers of Treg identity, expressed high levels of CD25 and FOXP3 (Figure 1D), and secreted minimal amounts of proinflammatory cytokines, such as IFN-γ and TNF-α (Figure 1E). However, TRAC-CAR Tregs could not be efficiently expanded via CAR stimulation (Figure 1F). Re-stimulation every 2–3 days with plate-bound recombinant HLA-A2-IgG dimeric protein with or without CD28 mAb resulted in expansion ratios of 1.60–2.57, respectively. The addition of irradiated HLA-A2-overexpressing K562 cells improved expansion from 0.5-fold (unstimulated) to 5-fold. In contrast, polyclonally expanded CD4 Tregs were able to expand up to 500-fold within a similar expansion period (Figure 1F). Taken together, the suboptimal expansion of TRAC-CAR Tregs precludes the creation of a stable protocol on a clinical scale.

[0206] Example 1: TruC after integration of scFv cDNA + Identifying a CD3 epsilon gene editing strategy to generate T cells Homologous sequence-dependent repair by programmable nucleases relies on efficient placement of DNA double-strand breaks. Therefore, we first screened five potential gRNA candidates for the Acidaminococcus species CRISPR-Cas12a nuclease (AsCas12a) with no or minimal predicted off-targets for their ability to disrupt CD3e in exon 3 or exon 6 (Figure 2A). Synthetic gRNAs and recombinant AsCas12 Ultra (Zhang et al., 2021) were co-electroporated into polyclonally activated T cells, and CD3e expression was measured by flow cytometry. Four days after nucleofection, T cells treated with AsCas12a gRNAs #1 and #5 showed the highest knockout rates compared to other gRNAs within their respective exons (Figure 2B, Figure 2C). For each gRNA, we designed an HDRT with 400 bp of flanking homology arms to insert a myc tag for detection purposes, HLA-A2-scFv cDNA, and a linker to attach the antigen-binding domain to the CD3 epsilon protein (Figure 2D). After transfection of the gRNA and the respective dsDNA HDRT, the myc + We were able to detect TruC T cells (Figure 2E). + The knock-in rate, expression level, and total number of T cells were the highest (Figures 2F, 2G, 2H, and 2I).

[0207] Example 2: Repeated stimulation favors CD3e-TruC over TCR-negative Tregs + Tregs are preferentially expanded Using a non-viral gene editing protocol for Tregs, we generated HLA-A2-specific CD3e-TruC Tregs, TRAC-CAR Tregs, and wild-type (WT) unedited Tregs, followed by functional testing (Figure 3A). Gene editing of CD3e was higher in conventional T cells. CD3e-TruC in Tregs +The rate was comparable to or even higher than that of TRAC-CAR knock-in in the same donor (Figure 3B). + Tregs are mainly CD3 + Efficient engagement of TruC with HLA-A2 was further verified by staining with biotin-labeled recombinant HLA-A2 (Fig. 8). To examine signaling following TCR or antigen receptor engagement (Fig. 9A), flow cytometry analysis of phosphorylated extracellular signal-regulated kinase (ERK) was performed. Upon stimulation with plate-bound anti-CD3 mAb, CD3e-TruC exhibited time-dependent phosphorylation of ERK at a kinetics similar to that of WT Tregs (Fig. 9C, left). Stimulation with plate-bound HLA-A2-IgG protein resulted in ERK phosphorylation in CD3e-TruC but not in unedited Tregs (WT Tregs) (Fig. 9C, right).

[0208] During polyclonal expansion with anti-CD3 / 28 beads, CD3e-TruC Tregs expanded similarly to WT Tregs (Figure 3C). TRAC-CAR Tregs expanded at a significantly slower rate during anti-CD3 / 28 bead stimulation (Figure 3D). During expansion with repeated stimulation with anti-CD3 / 28 beads, TruC + / CD3 + Because Tregs expanded preferentially over TCR-negative Tregs (Figure 3B), the relative and absolute expansion of HLA-A2-specific Tregs was higher among CD3e-TruC Tregs compared with TRAC-CAR Tregs (Figure 3D). Thus, CD3e-TruC Tregs may be expanded more efficiently than TRAC-replaced CAR T cells.

[0209] Example 3: Gene-edited and in vitro expanded Tregs retained their classical phenotype and epigenetic identity After a total of 16 days of expansion, CD3e-TruC Tregs, TRAC-CAR Tregs, and WT Tregs all retained the typical Treg phenotype (Figures 4A and 4B). Furthermore, naive and gene-edited Tregs did not secrete Th1 cytokines, IFNγ, and TNFα, after polyclonal restimulation (Figure 4C). Analysis of the Treg-specific demethylated region (TDSR) in the Foxp3 locus confirmed that Tregs retained their epigenetic identity regardless of gene editing (Figure 4D). As expected, CD3e-TruC and TRAC-substituted CAR Tregs retained low TSDR methylation, similar to naive Tregs. In contrast, unedited conventional T cells from the same region were highly methylated (Figure 4D).

[0210] Example 4: Activation profile of CD3e-TruC Treg cells compared to TRAC-CAR and WT Treg cells after antigen HLA-A2-specific stimulation To compare the activation of TRAC-CAR Tregs or CD3e-TruC Tregs, we monitored the expression levels of various Treg activation markers in response to varying amounts of plate-bound HLA-A2-IgG protein (Figure 5A). Both CD3e-TruC Tregs and TRAC-CAR Tregs showed dose-dependent upregulation of ICOS, CD25, Foxp3, CD137, CD71, and LAP, and to a limited extent, CTLA4 and Helios (Figure 5B). CD3e-TruC Tregs showed stronger relative upregulation of ICOS, CD25, and CD137 compared to TRAC-CAR Tregs and their respective baseline expression without stimulation (Figure 5B). Notably, TRAC-CAR Tregs showed significantly higher baseline expression of CD25, CTLA-4, CD71, and CD137 compared to both CD3e-TruC Tregs and WT Tregs, potentially indicating antigen-independent signaling of the CAR (Figure 5C). In contrast, CD3e-TruC Tregs and WT Tregs share similar activation marker expression profiles at baseline (Figure 5D). Furthermore, TRAC-CAR Tregs showed higher donor variance in the MFI of LAP, CD25, CTLA-4, ICOS, CD71, and FOXP3 (Figure 5E).

[0211] Example 5: A2 CD3e-TruC + Tregs functionally suppress the proliferation of autologous T cells in vitro, regardless of polyclonal or HLA-A2 antigen-specific stimulation The primary purpose of Tregs is to suppress the unwanted proliferation of conventional T cells specific for autoantigens or alloantigens, which can be modeled in vitro. To compare the effects of various stimulatory measures on conventional responder T cells (Tresp) and Tregs, we used three different growth suppression assay configurations for comparing our Treg candidates (Figure 6A). The first assay (1) represents a standard growth suppression assay using various amounts of Tregs together with autologous T cells (Tresp) stimulated with anti-CD3 / 28 beads, but lacking HLA-A2-specific stimulation. In this assay, WT Tregs and CD3-TruC Tregs significantly outperformed TRAC-CAR Tregs (Figure 6B, Figure 12A). In the second assay (2), the addition of additional HLA-A2, which serves as a stimulator for the CAR / TruC receptor, was used. + CD3-depleted PBMCs were added, which partially rescued the suppressive effect of TRAC-CAR Tregs. However, CD3e-TruC Tregs and WT Tregs were not CD8+ Tregs in one of the two donors. + The third assay (3) showed a higher inhibitory potency against Tresp than against mismatched HLA-A2 + Aiming to model alloreactive T cell proliferation against CD3-depleted PBMCs, additional anti-CD3 / 28 beads were not included. As expected, the overall percentage of proliferating Tresp cells was much lower (Figure 11). Surprisingly, at lower Tresp:Treg ratios, higher proliferation suppression was observed in one donor (Figure 6D). HLA-A2 + The amount of CD3-depleted PBMCs from the donors was titrated against the total cell number, suggesting this may be an artifact. In one donor, CD3e-TruC Tregs exhibited the highest suppressive capacity at all Tresp:Treg ratios tested (Figure 6D). In the other donor, CD3e-TruC Tregs and TRAC-CAR Tregs suppressed CD8 +CD3e-TruC Tregs were superior to WT Tregs in suppressing Tresp (Figure 12C). Overall, CD3e-TruC Tregs exhibited high suppressive capacity in in vitro autologous and allogeneic proliferation suppression assays.

[0212] Methods and Materials: Polyclonal Treg cell isolation: All experiments were performed in accordance with the Declaration of Helsinki. Peripheral blood was obtained from healthy adults after obtaining informed consent (Charité Ethics Committee approval EA4 / 091 / 19). HLA-A2 phenotype was determined by flow cytometry using HLA-A2 conjugated with APC (BB7.2, Biolegend) and HLA-A2 conjugated with FITC (A28, Miltenyi). 120 mL of peripheral blood was obtained from an HLA-A2-negative donor, and PBMCs were isolated using standard density gradient centrifugation. CD4 + Cells were enriched by MACS technology and then stained with a cocktail of antibodies: CD4 VioBlue (REAQL103), CD25 APC (REAL128), CD127 PE-Vio770 (REAL102), CD45RA FITC (REAL164) (all from Miltenyi), and CCR7 PE (G043H7, Biolegend). Stained cells were resuspended in Tyto buffer (Miltenyi). CD4 + CD25 high CD127 low CCR7 +Treg cells were sorted in a sterile environment using a GMP-compliant Tyto sorter with a normal-speed cartridge (Miltenyi). After two rounds of sorting, sorted cells were manually counted using trypan blue (Gibco) in a hemocytometer under a light microscope (Zeiss). 100,000 cells were seeded per well in a 96-well U-bottom plate (Corning) containing 200 μL of Treg medium consisting of X-Vivo medium (Lonza), 10% FCS (Biochrom), 500 IU / mL recombinant human IL-2 (Miltenyi), and 10 μM rapamycin (Pfizer). The following morning, 400,000 Treg Expansion Beads (Miltenyi) were added to each well for initial stimulation (a 4:1 bead-to-cell ratio). The medium was replenished every 2–3 days. Cells were counted on day 5 and stimulated with Treg Expansion Beads at a 1:1 ratio for an additional 2 days prior to electroporation.

[0213] Screening of synthetic guide RNAs targeting the CD3 epsilon locus: In this study, five guide RNAs (gRNAs) were screened based on the criteria of fewer than 5–10 CFD off-targets calculated by CRISPR scan (Moreno-Mateos et al., 2015) for Cas12a and COSMID (Cradick et al., 2014) for Cas9 gRNA. Among them, three gRNAs targeted exon 3 of CD3e and two gRNAs targeted exon 6 of CD3e. These gRNAs were synthesized by IDT and their target sequences (SEQ ID NO: 9). Each gRNA was reconstituted in nuclease-free TE buffer to a stock concentration of 100 μM. Each gRNA combined with cas12a to form ribonucleoproteins (RNPs), which were then electroporated into conventional T cells (stimulated with 1 μg / mL plate-bound αCD3 / αCD28 for 2 days; see Kath et al. (2020) for further details). Knockout (KO) efficiency was confirmed on day 4 after electroporation by staining cells with CD3 PacBlue (UCHT1, Biolegend) and DAPI (Thermo Fisher) and then acquiring them on a CytoFLEX LX (Beckman Coulter). Mock cells were used as a staining control.

[0214] Donor template HDRT for the generation of TRAC-CAR and CD3e-TruC: Three donor templates were used in this study. One was for integration into exon 1 of the TRAC locus, and the other two were for integration into exons 3 and 6 of the CD3e locus, respectively. A moderate-affinity anti-HLA-A2 single-chain variable fragment (scFv, clone 3PF12) (REF) was used, whose mRNA sequence is available in Genebank (accession numbers AF163307 and AF163308). Each donor template was designed in Snapgene (Dotmatics) and synthesized by IDT as gBlocks gene fragments (double-stranded DNA fragments, 1600 bp to 2800 bp in length). The gBlocks were cloned into the plasmid PUC19 vector backbone using multiple-fragment In-Fusion cloning according to the manufacturer's recommendations (Clontech, Takara). Plasmid transformation, colony PCR, and plasmid purification and validation were previously described (Non-Patent Document 15). HA-flanked donor templates were amplified by PCR from validated plasmids using KAPA HiFi HotStart 2x Readymix (Roche) with a 500 μL reaction volume. The primers used can be found in the sequence table (SEQ ID NOs: 43 to 46). PCR products were purified and concentrated using paramagnetic beads (AMPure XP, Beckman Coulter Genomics). The concentration of HDRT was determined using a NanoDrop 1000 spectrophotometer (ThermoFisher) or Qubit (ThermoFisher) and adjusted to a concentration of 1 μg / μL in nuclease-free water and stored at -20°C until use.

[0215] Electroporation: After 7-9 days of stimulation of Treg cells, beads were removed by a separator (MACSiMAG™, Miltenyi), and bead-free Treg cells were counted, washed twice in PBS (Gibco), and collected at 1 × 10 6The cell pellet was resuspended in 20 μL of P3 buffer (Lonza). Electroporation was performed in a 16-well nucleocuvette™ strip using program EH-115 in a 4D-Nucleofector device (Lonza). The formulation of the RNP and HDRT mixture was previously described (Nguyen et al., 2020). 0.5 μg of HDRT was used per electroporation. To rescue the cells, 90 μL of prewarmed Treg medium was added to each well immediately after electroporation. After incubation in a 37°C incubator for 10 minutes, the cells were transferred equally to two wells of a 96-well U-bottom plate containing 150 μL of prewarmed Treg medium. The following morning, the cells were stimulated with Treg Expansion Beads at a 1:1 ratio.

[0216] Off-target identification by CASTseq: Off-target analysis of gRNAs was performed as previously described (Turchiano et al., 2021). CD3e KO using gRNA1 and mock Tconv cells were generated from two biological replicates and expanded for 2 weeks. Up to 10 million cells were washed in PBS, pelleted, and stored short-term at -20°C. KO efficiency on protein levels was determined by flow cytometry prior to cell pelleting. Genomic DNA was isolated using the Quick-DNA Miniprep Plus Kit (Zymo Research).

[0217] Expansion of CD3e-TruC Treg cells and TRAC-CAR Treg cells: First, TRAC-CAR Treg cells were electroporated and distributed evenly across four wells in a 96-well flat-bottom plate. They were incubated with 1) 2 μg / mL plate-bound HLA-A2 dimer:IgG fusion protein (BD) alone, or 2) plate-bound 1 μg / mL αCD28 (Biolegend) together with 3) gamma-irradiated (30 Gy) HLA-A2. +The cells were stimulated at a 1:1 ratio with the K562 cell line (a gift from Fatih Noyan, MHH). Plate coating with antigen and / or antibody was prepared the day before and kept in the refrigerator until use. No stimulation was included as a negative control. Uncontacted Treg cells stimulated with beads were included as a high control. Stimulation and medium replenishment were repeated every 2–3 days. CD3e-TruC Treg cells and TRAC-CAR Treg cells were then expanded using two methods: 1) expansion with Treg expansion beads at a 1:1 ratio every 2–3 days, or 2) plate-bound HLA-A2 dimer:IgG fusion protein at 2 μg / mL and αCD28 at 1 μg / mL. Cells were expanded 2 weeks after electroporation. The beads were removed and the cells were left overnight before assay preparation and reading.

[0218] Confirmation of gene editing efficiency by flow cytometry: 100,000–200,000 Treg cells were cultured overnight without beads. TRAC-CAR Treg cells were stained with CD3 PB and αFc A647 (Jackson Immuno Research Labs). CD3e-TruC Treg cells were stained with CD3 PB and αMyc A647 (Cell Signaling). CD3e-TruC cells were also first stained with 5 μg / mL HLA-A2-biotin (conjugated by the lab manager from DRFZ) and then with streptavidin A555 (Invitrogen) and αMyc A647. Uncontacted wild-type Treg cells were used as a control. Cells were resuspended in PBS containing DAPI and then acquired using a CytoFLEX LX. Gene editing efficiency of Treg cells was confirmed separately on days 4 and 12 after electroporation.

[0219] Purification of gene-edited Treg cells using the Aria II sorter: Expanded CD3e-TruC Treg cells and TRAC-CAR Treg cells were removed from culture. After removing the beads, the cells were stained. CD3e-TruC Treg cells were purified using the Aria II sorter (BD) with an 85 μm nozzle size, and NearIR-CD3 + CD4 + Myc + TRAC-CAR Treg cells were sorted as NearIR-CD3 + CD4 + αFc + The cells were sorted as Treg medium. The sorted cells were collected in a 15 mL Falcon tube (pre-coated with 5 mL of FCS and then filled with 3 mL of Treg medium). Purity was checked after sorting. The sorted cells were centrifuged and resuspended in Treg medium at a concentration of 0.5 million cells per mL. Treg expansion beads were added to the culture at a 1:1 ratio and expanded.

[0220] Phenotypic characterization of gene-edited Treg cells: On day 9 of expansion, 1 million gene-edited and uncontacted Treg cells were removed from the culture and the beads were removed. Tconv cells were used as a negative control. One-third of the cells were extracellularly stained with the fixable dye Aqua (Invitrogen), followed by fixation and permeabilization using Transcription Factor Buffer Set (BD). Intracellular staining was performed with αFc A647 for TRAC-CAR Treg cells and αMyc A647 for CD3e-TruC, followed by two washes with permeabilization buffer. Cells were then stained with an antibody cocktail of CD3 PB, CD4 PE (Beckman Coulter), CD25 PC7 (Beckman Coulter), and FOXP3 FITC (BD). To examine the cytokine profile of gene-edited Treg cells, the other two-thirds of the cells were stimulated with 10 ng / mL PMA and 2.5 μg / mL ionomycin (Sigma-Aldrich) for 6 hours. No stimulation was included as a negative control. After 1 hour of co-culture, brefeldin A (Sigma-Aldrich) was added at a concentration of 10 μg / mL. After 6 hours of stimulation, cells were harvested and stained as above, except using TNFα A700 (Biolegend), IFNγ APC-eF780 (Invitrogen), and IL-2 PE-Cy7 (Biolegend) instead of CD25 and FOXP3 antibodies. Stained cells were acquired using a Cytoflex LX. Between 300,000 and 1 million Treg cells were flash-frozen and stored in liquid nitrogen until genomic DNA isolation using established methods.

[0221] ERK phosphorylation after stimulation: Beads were removed from the culture. Purified CD3e-TruC Treg cells, TRAC-CAR Treg cells, and WT Treg cells were cultured overnight in bead-free X-Vivo medium supplemented with 10% FCS and 1% penicillin / streptomycin without IL-2 or rapamycin, followed by assay. 48-well plates were coated with either 5 μg / mL HLA-A2:IgG dimer (BD) or 10 μg / mL αCD3 antibody (Invitrogen) in a 4°C refrigerator the day before. The next day, cells were first harvested, counted, and stained with fixable live / dead dye UV. 500,000 cells were seeded into each well on ice. After brief centrifugation, the plates were immediately incubated at 37°C in a thermomixer (Eppendorf). After the designated time intervals (0, 2, 5, 10, 15, and 30 min), 200 μL of prewarmed 4% fixation buffer (Biolegend) was added, gently pipetted, and incubated at 37°C for 15 min to stop the stimulation. The cells were washed, and the supernatant was discarded. 200 μL of True-Phos perm buffer (pre-chilled at -20°C) was added to each well and incubated at -20°C for ≥60 min. The cells were washed twice with PBS and then intracellularly stained with anti-ERK1 / 2 Phospho(Thr202 / Tyr204) FITC (Biolegend) for 30 min at 4°C. The cells were washed twice with PBS and acquired using a Cytoflex LX.

[0222] Activation profile of CD3e-TruC Treg cells versus TRAC-CAR Treg cells: As above, cells were incubated overnight without IL-2 and rapamycin. The day before, HLA-A2:IgG dimer (BD) was coated onto a 96-well flat-bottom plate in a 4°C refrigerator at a serial dilution of 10 μg / mL → 5 μg / mL → 2.5 μg / mL → 1 μg / mL, with or without 1 μg / mL αCD28. CD3e-TruC Treg cells, TRAC-CAR Treg cells, and WT Treg cells were resuspended in X-Vivo medium supplemented with 10% FCS and 100 IU / mL IL-2. 400,000 cells were seeded into each well and stimulated for one day. A well without stimulation was included as a negative control. Tconv cells were included as a parallel control. One day after stimulation, cells were harvested and split into two wells for activation antibody staining in two panels. The antibodies tested here included CD25 PC7 (Beckman), CD71 BV786 (BD Bioscience), FOXP3 FITC (BD Pharmingen), ICOS (CD278) BV650, LAP PE, CTLA-4 (CD152) BV421, CD137 PE, CD154 BV421, and Helios Percp-Cy5.5 (all Biolegend). Helios and FOXP3 were stained intracellularly. Stained cells were immediately acquired using a Cytoflex LX.

[0223] Suppression assay: Treg cells were incubated overnight without IL-2 or rapamycin, as described above. Autologous Tconv cells were enriched from PBMCs as responder cells (Tresp). Tresp cells were labeled with 1 μM CFSE for 10 minutes at 37°C. The labeled cells were washed twice with 5 mL of FCS, incubated at 37°C for 1 hour, and then seeded at 50,000 cells per well. Treg cells were seeded at Tresp:Treg ratios ranging from 1:1 to 1:1 / 8. Stimulation was performed with human Treg suppression inspector (Miltenyi) beads at a 1:1 ratio of total cells seeded per well or CD3-reduced PBMCs (A2) from an HLA-A2-positive donor. + Cells) were pre-labeled with CTFraRed and Tresp + Tregs and A2 + A2 in a 1:1 ratio with cells or beads + Cells were seeded together with Treg cells. Each condition had technical triplicates. Wells contained either stimuli with Tresp but no Treg cells as a positive control for proliferation. Wells contained Tresp alone as a negative control for proliferation. All cells were resuspended in X-vivo medium supplemented with 10% FCS and 1% penicillin / streptavidin (strep). After 5 days of incubation, cells were harvested, stained with CD4 PE (Beckman) and CD8 PE-Cy7 (BD Pharmingen), and finally resuspended in PBS containing DAPI and immediately acquired with a Cytoflex LX. The % suppressive capacity of Treg cells = (% of split Tresp alone - % of split Tresp treated with Tregs) / % of split Tresp alone × 100. [Explanation of symbols]

[0224] Drawing translation Figure 1 57-year-old female allo-sensitized Chr.14 TRAC TRAC on chromosome 14 gRNA cut site gRNA cut site exon 1 (G4S)3 linker (G4S)3 linker IgG1 hinge IgG1 hinge zeta polyA Zeta PolyA HLA-A2 specific TRAC-CAR HLA-A2 specific TRAC-CAR Untouched polyclonal WT Tregs MFI of FOXP3 % among viable cells Expansion fold change Irr.K562-A2 + Irradiated K562-A2 cells + cell A2 dimer + αCD28 sti A2 dimer + αCD28 stimulation A2 dimer sti A2 dimer stimulation w / o sti No stimulation days post E' days after electroporation Figure 2 KO check KO confirmation mock % KO efficacy Chr.11 CD3ε CD3ε on chromosome 11 exon 3 gRNA cut site gRNA cut site (G4S)3 linker (G4S)3 linker exon 6 Normalized To Mode Normalized to mode MFI of eTRuC + eTRuC+ MFI Relative eTRuC + expansion relative eTRuC + Expansion and proliferation of Figure 3 A2 donor KI check KI confirmation debead rest overnight In vitro assays, e.g., phenotype profile, cytokine profile Expansion count Expansion fold of total cells (normalized to d4) days post E' days after electroporation Expansion fold of proportion of edited cells (normalized to d4) Expansion fold of edited cells (normalized to d4) Figure 4 0.3mio cells (300,000 cells) PMA / Iono sti PMA / Ionomycin stimulation + BrefA + Brefeldin A Harvest cells sf stain - fix - perm - ic stain -> cytoflex Surface staining - fixation - permeabilization - intracellular staining → cytoflex % among viable cells Avg Average Donor A donor B Figure 5 Debead cells overnight Debead and leave cells overnight Activation Ab stain cytoflex readout cytoflex readout A2 dimer A2 dimer conc. concentration Fold change of MFI Fold change of % % of CD137 among live % of Helios among live Fold change of MFI (normalized to WT) Variance of MFI between donors Figure 6 Debead cells overnight Debead and leave cells overnight Stimuli Stimuli setup assays harvest cells -> stain cytoflex readout cytoflex readout ASSAY1 Assay 1 ASSAY2 Assay 2 ASSAY3 Assay 3 % Suppression of proliferation Ratio Tresp:Treg Ratio Tresp:Treg Figure 7 Pre-sort Post-2nd sort After the second sorting % CCR7 + Treg cells among single lymphocytes CCR7 + % of Treg cells Figure 8 A2 biotin-Streptavidin A555 Figure 9 0-30mins 0-30min debead sorted Treg cells A2 dimer of αCD3 sti A2 dimer or αCD3 stimulation phosphor stain phosphor stain cytoflex readout cytoflex readout 0 min 0 min 2 min 5 min 10 min 15 min 30 min αCD3 sti αCD3 stimulation A2 dimer sti A2 dimer stimulation % pERK among viable cells Figure 10 Pre-sort Post-sort % gene edited Treg cells among single lymphocytes Figure 11 Count ASSAY1 Assay 1 ASSAY2 Assay 2 ASSAY3 Assay 3 Figure 12 % Suppression of proliferation ASSAY1 Assay 1 ASSAY2 Assay 2 ASSAY3 Assay 3 Ratio Tresp:Treg Ratio Tresp:Treg Figure 13 Stimulation with αCD3-mAb or target cells Add 5 μg / ml BrefA Add 5 μg / ml of brefeldin A FACS ic stain FACS intracellular staining Flow cytometry HER2-specific scFv HER2-specific scFv % total cytokines CD4 + T cells CD4 + T cells αCD3 sti αCD3 stimulation no sti no stimulation CD8 + T cells CD8 + T cells mock Figure 14 Probability of Survival Blue: CD3e-TruC Blue: CD3e-TruC Black: Tconv Black: Tconv Grey: untreated

[0225] References 1. Roemhild, A. et al. Regulatory T cells for minimising immune suppression in kidney transplantation: phase I / IIa clinical trial. BMJ 371, (2020). 2. Landwehr-Kenzel, S. et al. Adoptive transfer of ex vivo expanded regulatory T cells improves immune cell engraftment and therapy-refractory chronic GvHD. Mol Ther 30, 2298-2314 (2022). 3. Marek-Trzonkowska, N. et al. Therapy of type 1 diabetes with CD4(+)CD25(high)CD127-regulatory T cells prolongs survival of pancreatic islets - results of one year follow-up. Clin Immunol 153, 23-30 (2014). 4. Voskens, C. et al. Autologous regulatory T-cell transfer in refractory ulcerative colitis with concomitant primary sclerosing cholangitis. Gut gutjnl-2022-327075 (2022) doi:10.1136 / gutjnl-2022-327075. 5. Amini, L. et al. Super-Treg: Toward a New Era of Adoptive Treg Therapy Enabled by Genetic Modifications. Frontiers in Immunology 11, 3868 (2021). 6. Landwehr-Kenzel, S. et al. Novel GMP-compatible protocol employing an allogeneic B cell bank for clonal expansion of allospecific natural regulatory T cells. Am J Transplant 14, 594-606 (2014). 7. Dawson, N. A. J. et al. Functional effects of chimeric antigen receptor co-receptor signaling domains in human regulatory T cells. Sci Transl Med 12, (2020). 8. Micklethwaite, K. P. et al. Investigation of product-derived lymphoma following infusion of piggyBac-modified CD19 chimeric antigen receptor T cells. Blood 138, 1391-1405 (2021). 9. Bishop, D. C. et al. Development of CAR T-cell lymphoma in 2 of 10 patients effectively treated with piggyBac-modified CD19 CAR T cells. Blood 138, 1504-1509 (2021). 10. Wilson, M. H. & Gottschalk, S. Expect the unexpected: piggyBac and lymphoma. Blood 138, 1379-1380 (2021). 11. Roth, T. L. et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 559, 405-409 (2018). 12. Schober, K. et al. Orthotopic replacement of T-cell receptor α- and β-chains with preservation of near-physiological T-cell function. Nat Biomed Eng 3, 974-984 (2019). 13. Muller, Y. D. et al. Precision Engineering of an Anti-HLA-A2 Chimeric Antigen Receptor in Regulatory T Cells for Transplant Immune Tolerance. Frontiers in Immunology 12, 3717 (2021). 14. Kath, J. et al. Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells. Mol Ther Methods Clin Dev 25, 311-330 (2022). 15. MacLeod, D. T. et al. Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells. Molecular Therapy 25, 949-961 (2017). 16. Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR / Cas9 enhances tumour rejection. Nature 543, 113-117 (2017). 17. Zhang, L. et al. AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines. Nat Commun 12, 3908 (2021). 18. Moreno-Mateos, M. A. et al. CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat. Methods 12, 982-988 (2015). 19. Cradick, T. J., Qiu, P., Lee, C. M., Fine, E. J. & Bao, G. COSMID: A Web-based Tool for Identifying and Validating CRISPR / Cas Off-target Sites. Mol Ther Nucleic Acids 3, e214 (2014). 20. Nguyen, D. N. et al. Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency. Nat Biotechnol 38, 44-49 (2020). 21. Turchiano, G. et al. Quantitative evaluation of chromosomal rearrangements in gene-edited human stem cells by CAST-Seq. Cell Stem Cell 28, 1136-1147.e5 (2021). 22. Baeuerle, P. A., et al. Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response. Nature communications 10(1), 1-12 (2019). 23. Watkins, N. A. et al. The isolation and characterisation of human monoclonal HLA-A2 antibodies from an immune V gene phage display library. Tissue Antigens 55(3):219-28 (2000).

Claims

1. A nucleic acid construct comprising: a first nucleic acid sequence region encoding an antigen-binding domain; a second nucleic acid sequence region comprising a targeting sequence configured to integrate said construct in frame with the endogenous CD3 epsilon gene of a human cell; A nucleic acid construct comprising:

2. 2. The nucleic acid construct of claim 1, wherein the targeting sequence is configured to integrate the construct in frame into exon 3 or exon 6 of the endogenous CD3 epsilon gene, and wherein preferably the target site of the CD3 epsilon gene into which the first sequence region is integrated is located downstream of the sequence encoding the CD3 epsilon signal peptide and upstream of the endogenous CD3 epsilon sequence.

3. the targeting sequence of the second sequence region is configured to be integrated into the host genome by gene editing techniques, preferably CRISPR-Cas, zinc finger nuclease (ZFN), integrase, site-specific recombinase, meganuclease, homing endonuclease or TALEN, more preferably CRISPR-Cas12a from Acidaminococcus sp. BV3L6 or CRISPR-Cas9 from Streptococcus pyogenes; and / or 3. The nucleic acid construct of claim 1, wherein the targeting sequence comprises a sequence identical to a recognition site of the endogenous DNA sequence into which integration is intended, preferably a recognition site selected from the group consisting of a homology arm, a guide RNA target site, a restriction enzyme recognition site, a ZFN recognition site, a ZFN cleavage site, a TALEN DNA binding site, a recombinase recognition site, an integrase site, and / or a homing nuclease recognition site.

4. 4. The nucleic acid construct of claim 1, wherein the antigen-binding domain is in the form of an antibody or an antigen-binding fragment thereof.

5. The nucleic acid construct according to any one of claims 1 to 4, wherein the antigen-binding domain is specific for an HLA protein, preferably HLA-A2.

6. A genetically modified human T cell comprising an exogenous nucleic acid sequence region encoding an antigen-binding domain integrated in-frame within the endogenous CD3 epsilon gene.

7. The genetically modified human T cells of claim 6, wherein the genetically modified human T cells are regulatory T cells (Tregs).

8. 8. The genetically modified T cell of claim 6 or 7, wherein the CD3 epsilon protein is expressed as an in-frame fusion protein comprising the antigen-binding domain and an endogenous CD3 epsilon protein.

9. 9. The genetically modified T cell of any one of claims 6 to 8, wherein the function of the T cell receptor (TCR) complex is maintained in the presence of an in-frame fusion protein comprising the antigen-binding domain and an endogenous CD3 epsilon protein.

10. 10. The genetically modified T cell of claim 6, wherein the fusion protein is expressed under the control of the endogenous CD3 epsilon promoter.

11. 11. The genetically modified T cell of claim 6, wherein the exogenous nucleic acid sequence region encoding the antigen-binding domain is integrated in-frame within exon 3 or exon 6 of the endogenous CD3 epsilon gene.

12. 12. The genetically modified T cell of any one of claims 6 to 11, wherein the target site of the CD3 epsilon gene into which said first sequence region is integrated is located downstream of the sequence encoding the CD3 epsilon signal peptide and upstream of the endogenous CD3 epsilon sequence (generating an N-terminal fusion after removal of said signal peptide).

13. The genetically modified T cell of any one of claims 6 to 12, wherein the antigen-binding domain binds to an HLA protein, preferably HLA-A2.

14. 14. The genetically modified T cell according to any one of claims 6 to 13, for use as a medicament for preventing and / or treating an unwanted (pathogenic) immune response, preferably an unwanted (pathogenic) immune response before and / or after allogeneic transplantation or an autoimmune disease, more preferably for preventing and / or treating graft-versus-host disease (GVHD).

15. A pharmaceutical composition comprising the genetically modified cell of any one of claims 6 to 13 and a pharmaceutically acceptable carrier.

16. 14. A method for preparing therapeutic genetically modified T cells, comprising repeatedly stimulating the genetically modified T cells of any one of claims 6 to 13 via the TCR / CD3 complex, wherein the therapeutic genetically modified T cells exhibit essentially unchanged expansion kinetics (±50%) in culture compared to unmodified T cells.

17. 17. The method of claim 16, wherein the genetically modified human T cells are regulatory T cells (Tregs).

Images