Genetically modified regulatory t cells

Abstract

The invention relates to a safe and feasible triple strategy based on: i) a genetically modified regulatory T cell characterized by a genetic silencing of CIITA (HLA class II) and one or two of the HLA class I loci HLA-A, HLA-B and HLA-C; ii) in some specific embodiments, a cell modified to be resistant to a specific immunosuppressive drug by silencing the corresponding target gene; iii) the partial stealth concept combined with immunosuppression facilitates the matching of donors and recipients using artificial intelligence tools.

Description

Technical Field

[0001] This invention relates to multiple strategies for combating the immunogenicity of allogeneic "off-the-shelf" regulatory T cell products. Background Technology

[0002] Adoptive transfer of T-cell products has emerged as a promising immunotherapy with the potential to cure serious chronic diseases, thus eliminating the need for lifelong treatment. Several anti-cancer T-cells, such as CD19-specific or BMCA-specific CAR-T cells, have been approved as therapeutic products, and hundreds of clinical trials employing advanced methodologies are currently underway.

[0003] To date, the autologous approach has been dominant, where the starting material for cell products is taken from the patient themselves and enriched, modified, and expanded during GMP production. The prepared autologous product is then administered to the same patient. A similar approach is used for anti-infection memory / effective T cell products.

[0004] These processes are logistically challenging, have limitations in patients with a history of extensive treatment, and are very costly. Therefore, off-the-shelf allogeneic therapies using starting materials from healthy individuals or iPSCs are being developed. However, the (allogeneic) immunogenicity of these products poses a challenge, at least for patients whose immune function is not severely impaired (e.g., those following hematopoietic stem cell transplantation), as allogeneic cell products are often rejected.

[0005] Regulatory T cells (Tregs) are a completely different class of T cell products. Unlike the aforementioned anti-cancer or anti-infection T cells, they are not used for immune reconstitution, but rather to treat unwanted immune responses, such as those occurring after transplantation, gene therapy, or in autoimmune, allergic, or aplastic diseases. Tregs are natural "peacemakers" of the cellular immune system, restoring balance to disordered immune regulation through cell-contact mediated factors and secreted factors. Similar to effector T cells, patients currently primarily use autologous Treg products. To date, allogeneic Treg therapy has only been received by patients with severely compromised immune function, such as those who have undergone hematopoietic stem cell therapy (HSCT). It was hoped that the inherent immunosuppressive properties of Tregs would prevent / suppress allogeneic reactions in cell transfer recipients to off-the-shelf Treg products. However, in clinically relevant humanized mouse models, off-the-shelf Treg products have shown significant immunogenicity, leading to loss of function and viability of metastatic cells (Reference 8).

[0006] The inventors demonstrated in a mouse model of the human immune system (HIS) that, despite their inherent immunomodulatory properties, regulatory T cells (Tregs) exhibit allogeneic immunogenicity similar to that of conventional T cells targeting tumors or infectious pathogens. In other words, they are recognized and eliminated by the host's human immune system, rendering them ineffective as off-the-shelf products (Reference 8; McCallion et al., 2023). The inventors also demonstrated that by knocking out HLA class I and II molecules (to prevent recognition by host T cells) and knocking in less polymorphic HLA-E molecules (to prevent rejection by host NK cells), using "reclusive" Tregs allows off-the-shelf Tregs to evade allogeneic rejection (losing allogeneic immunogenicity) and achieve functional data similar to autologous Tregs (Reference 8).

[0007] Off-the-shelf allogeneic Treg products have great potential in treating a variety of inflammatory conditions; however, Treg function is impaired in many (autoimmune) diseases, which currently limits the use of autologous preparations with potential immunogenicity.

[0008] Compared to the most commonly used autologous therapies, off-the-shelf allogeneic T-cell therapies will have many advantages in terms of cost, logistical challenges, cell product quality, and scalability. To achieve this, two significant hurdles must be overcome:

[0009] - The allogeneic immunogenicity of off-the-shelf T-cell products can lead to a rejection response (host-resistant transplantation response) of the host immune system after activation of the allogeneic reactive (host-responsive) T-cell receptor for the product.

[0010] - Allogeneic reactivity of off-the-shelf T-cell products to host tissues (graft-versus-host disease)

[0011] Based on the above-mentioned prior art, the object of the present invention is to provide means and methods for reducing the immunogenicity of regulatory T cell-based cell therapies. This object is achieved by the subject matter described in the independent claims of this specification, while other advantageous embodiments are described in the dependent claims, examples, drawings, and general description of this specification. Summary of the Invention

[0012] This invention relates to an advanced therapeutic product (ATMP) for gene and cell therapy—a regulatory T cell characterized by gene deletion or silencing, particularly gene knockdown or knockout, of one or two of the major histocompatibility complex transcription activator gene (CIITA) (HLA class II) and HLA class I loci HLA-A, HLA-B, and HLA-C. Compared to complete knockdown of HLA loci, partial knockdown of these loci allows for easier donor-recipient matching, especially when using artificial intelligence (AI) tools.

[0013] In some specific embodiments, the gene-edited regulatory T cells according to the present invention are characterized by a second gene modification that confers resistance to an immunosuppressive drug by silencing / knocking out the corresponding molecular target of the drug, particularly resistance to one drug selected from the group consisting of tacrolimus, cyclosporine, and steroids. Figure 5 ).

[0014] The data shown in the examples indicate that complete "invisibility capping" (knockout of all HLA cells I+II) is not necessary to evade immune recognition; complete "invisibility capping" comes with significant inherent risks, namely the lack of immune surveillance when these occult cells develop tumors or become infected with viruses (Tables 1 and 2).

[0015] The data in this specification further demonstrate that the combination of immunosuppressive therapy (such as the use of calcineurin inhibitors to prevent rejection) with Tregs resistant to immunosuppressants (to prevent impaired survival and function of Tregs) is effective (Table 2).

[0016] Temporary immunosuppression combined with partial recessive (knockout of 2 out of 3 HLA class I genes, with or without HLA class II knockout) is most effective in preventing allogeneic Treg rejection while maintaining their survival and function.

[0017] Terms and Definitions

[0018] General

[0019] For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, singular terms will also include plural forms, and vice versa. If any of the following definitions conflict with any document incorporated herein by reference, the following definitions shall prevail.

[0020] The words “comprising,” “having,” “containing,” and “including,” as well as other similar forms and grammatical equivalents, used herein shall have the same meaning and shall be open-ended, meaning that one or more items following these words are not intended to be exhaustive or limited to the listed items. For example, an article “comprising” components A, B, and C may consist of components A, B, and C (i.e., containing only these components), or it may contain not only components A, B, and C, but also one or more other components. Therefore, it is intended herein that “comprising” and its similar forms and grammatical equivalents be understood to include disclosure of embodiments “substantially consisting of” or “consisting of”.

[0021] When a range of values ​​is given, it should be understood that, unless the context explicitly specifies otherwise, every intermediate value between the upper and lower limits of the range (accurate to one-tenth of the unit used for the lower limit), as well as any other explicitly listed value or intermediate value within the range, is included in this disclosure, except for the limits explicitly excluded from the range. When the range includes one or both endpoints, the range excluding any one or both included endpoints is also included in this disclosure.

[0022] The use of "about" in this article to refer to a value or parameter includes (and describes) the various variations of that value or parameter itself. For example, a description of "about X" includes a description of "X".

[0023] As used herein (including the appended claims), the singular forms “a,” “an,” and “the” include plural references to objects, unless the context clearly specifies otherwise.

[0024] As used herein, “and / or” should be understood to explicitly state each of the two specified features or components, regardless of whether they coexist with the other. Therefore, the term “and / or” as used in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Molecular, genetic and biochemical methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th Edition (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Ausubel et al., Concise Laboratory Protocols for Molecular Biology (2002), 5th Edition, John Wiley & Sons, Inc.) and chemical methods employ standard techniques.

[0026] Any patent documents cited in this document shall be deemed to be incorporated herein in their entirety by reference.

[0027] General Molecular Biology: Nucleic Acid Sequences and Expression

[0028] The term "gene" refers to a polynucleotide containing at least one open reading frame (ORF) that, upon transcription and translation, encodes a specific polypeptide or protein. Polynucleotide sequences can be used to identify larger segments or the full-length coding sequence of a gene associated with it. Methods for isolating larger sequence segments are known to those skilled in the art.

[0029] In this specification, the term "transgenic" refers to a gene or genetic material transferred from one organism to another. In this context, the term can also refer to the transfer of a naturally occurring or physiologically intact variant of a genetic sequence into a patient tissue lacking that variant. It can also refer to the transfer of a naturally occurring coding sequence whose expression is driven by a promoter that is absent or silenced in the target tissue.

[0030] In this specification, the term "recombinant" refers to a nucleic acid that is different from naturally occurring nucleic acids, produced through one or more cloning, restriction enzyme digestion, and / or ligation steps. Recombinant viral particles contain recombinant nucleic acids.

[0031] The term gene expression, or gene product, can refer to either or both of the processes of generating nucleic acids (RNA) and generating peptides or polypeptides, and their products; these processes are also referred to as transcription and translation, respectively; or it can refer to any intermediate process that regulates the processing of genetic information to produce a polypeptide product. The term gene expression can also be used for the transcription and processing of RNA gene products, such as regulatory RNA or structural RNA (e.g., ribosomal RNA). If the expressed polynucleotide is derived from genomic DNA, expression may involve splicing of mRNA in eukaryotic cells. Expression can be detected at the transcriptional and translational levels; in other words, at the mRNA and / or protein product level.

[0032] In this specification, the term "silent gene" encompasses both "knockdown gene" and "gene knockout," referring to techniques known in the field of synthetic biology:

[0033] Gene knockdown refers to the partial reduction of gene expression, typically at the mRNA level, resulting in a decrease in the production of the corresponding protein. This is often achieved using RNA interference (RNAi) techniques, such as small interfering RNA (siRNA) or short hairpin RNA (shRNA), which bind to target mRNA, promoting its degradation or inhibiting its translation. Another approach is to use antisense oligonucleotides, which can hybridize with mRNA and block translation. Gene knockdown is usually temporary or reversible, and therefore can be used to study gene function without permanently altering the genome.

[0034] In contrast, gene knockout involves the complete inactivation or deletion of a gene, resulting in the complete absence of the corresponding protein. This is typically achieved through genome editing technologies such as CRISPR-Cas9, TALEN, or zinc finger nucleases (ZFNs), which introduce targeted double-strand breaks into the DNA and cause frameshift mutations or deletions through error-prone repair mechanisms such as non-homologous end joining. Gene knockout models, particularly mouse gene knockout models, are widely used in research to study gene function and disease mechanisms by observing the effects of permanent gene deletion.

[0035] Methods of modifying cells by regulating the expression of specific genes to treat diseases include, but are not limited to, the following:

[0036] - RNA interference (RNAi): This method uses small interfering RNA (siRNA) or microRNA (miRNA) to target and degrade specific mRNA molecules, thereby preventing the target gene from being translated into protein. RNAi reagents can be produced intracellularly through transcription using recombinant nucleic acid expression vectors.

[0037] - CRISPR-Cas9 gene editing, or base editing: CRISPR-Cas9 and related systems derived from bacterial defense mechanisms can be programmed to target specific DNA sequences, thereby cutting DNA at precise locations. This can be used to disrupt gene function or correct mutations.

[0038] - Antisense oligonucleotides (ASOs): These are short synthetic DNA strands that bind to RNA transcripts, preventing RNA from being translated into proteins. They can also be engineered to regulate RNA splicing.

[0039] - Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs): These are engineered proteins that can be customized to bind to specific DNA sequences, thereby achieving targeted gene destruction.

[0040] Detailed description of the invention

[0041] Therapeutic products based on regulatory T cells are known in the art and have been shown to enable transplant recipients to receive minimal doses of immunosuppressive therapy (Reference 10) and reduce inflammatory responses to transplant complications caused by cytomegalovirus (CMV) infection in patients on long-term immunosuppression (Reference 13). These cells are used to suppress unwanted anti-autoimmune and anti-allergen immune responses in recipients, or in some cases, to suppress antiviral or anti-graft immune responses. Therefore, the genetically modified regulatory T cells according to the present invention exhibit typical characteristics of Tregs, including the CD4+CD25+FOXP3+ phenotype, and do not produce elevated Th1 cytokines upon polyclonal stimulation. The modifications described herein ensure effective engraftment by regulating the expression of HLA classes I and II.

[0042] The first aspect of the invention relates to a genetically modified regulatory T cell, characterized by gene deletion or silencing, particularly gene knockdown or gene elimination, at one or two of the HLA class I loci HLA-A, HLA-B, and HLA-C. In other words, at least one of the HLA class I loci HLA-A, HLA-B, and HLA-C is expressed.

[0043] In some implementations, the major histocompatibility complex transcription activator gene class II (CIITA) (HLA class II) is also silenced in the cell.

[0044] In some implementations, the major histocompatibility complex transcription activator gene (CIITA) (HLA class II) is not silenced in the cells, but is expressed at wild-type or near-wild-type levels.

[0045] Preferably, the elimination of HLA locus expression is achieved through targeted mutagenesis, gene silencing, or gene knockdown of one or at most two of the HLA-A, HLA-B, and / or HLA-C genes, as well as an additional CIITA gene. The resulting genetically modified regulatory T cell product expresses only immunogenic HLA proteins from one or at most two loci, generating two to four haplotypes, thus avoiding the need to match the 12 haplotypes of HLA classes I and II. The resulting cell product is more compatible with AI-based haplotype matching tools configured to match recipients with products of lower immunogenicity potential.

[0046] In some implementations, the genetically modified regulatory T cells are characterized by gene silencing at HLA-A and HLA-B loci, and HLA-C expression.

[0047] In some implementations, the genetically modified regulatory T cells are characterized by gene silencing at the HLA-A locus, while HLA-B and HLA-C are expressed.

[0048] In some implementations, the genetically modified regulatory T cells are characterized by gene silencing at the HLA-B locus, while HLA-A and HLA-C are expressed.

[0049] In some specific embodiments, the genetically modified regulatory T cells are characterized by gene knockdown or gene deletion of the CIITA gene and the HLA-A and HLA-B loci.

[0050] In some implementations, the genetically modified regulatory T cells are characterized by gene knockdown or gene deletion of the CIITA gene and the HLA-B and HLA-C loci.

[0051] In some implementations, the genetically modified regulatory T cells are characterized by gene knockdown or gene deletion of the CIITA gene and the HLA-A and HLA-C loci.

[0052] In some implementations, the genetically modified regulatory T cells are characterized by gene knockdown or gene deletion of the CIITA gene and the HLA-A locus, while preserving the expression of HLA-B and HLA-C.

[0053] In some implementations, the genetically modified regulatory T cells are characterized by gene knockdown or gene deletion of the CIITA gene and the HLA-B locus, while preserving the expression of HLA-A and HLA-C.

[0054] In some implementations, the genetically modified regulatory T cells are characterized by gene knockdown or gene deletion of the CIITA and HLA-C loci, while preserving the expression of HLA-A and HLA-B.

[0055] In some specific embodiments, the CIITA gene is silenced using a CRISPR-Cas9-derived adenine base editor (ABE) according to the methods disclosed in references 8 and 15.

[0056] In some specific implementations, the gene-edited regulatory T cell product retains the expression of HLA loci that are very commonly expressed in the population, such as HLA-A*01 or HLA-A*02.

[0057] Transplant recipients require immunosuppressive therapy to prevent organ rejection and to facilitate the implantation of cell therapy products such as gene-modified T cells expressing CARs or Tg TCRs. Traditional immunosuppressive therapy includes three classes of immunosuppressants: mycophenolic acid (MPA), a proliferation inhibitor that also inhibits the production of pro-inflammatory cytokines, induces lymphocyte apoptosis, and reduces T cell homing; calcineurin inhibitors (CNIs; such as tacrolimus and cyclosporine, also known as cyclosporine A) effectively reduce T cell activation, maturation, and cytokine secretion; and glucocorticoids inhibit T cell cytokine production and induce lymphocyte apoptosis. In transplant recipients, pre-existing allogeneic antigen-responsive effector T cells are particularly difficult to control. CNIs are currently the most effective treatment for sustainably suppressing these allogeneic antigen-responsive cells. Therefore, a solution is needed to overcome the suppression of protective antiviral T cell responses while maintaining control of allogeneic graft-responsive T cells.

[0058] Corticosteroids are essential immunosuppressants in organ transplantation, used for induction therapy, maintenance therapy, and treatment of acute rejection. In this context, the most important corticosteroids are prednisone (or its active form, prednisolone) and methylprednisolone, while dexamethasone has limited effectiveness. Prednisone is often used for long-term oral maintenance therapy because it effectively suppresses T-cell-mediated immune responses; high-dose intravenous methylprednisolone is commonly used perioperatively or during acute rejection to rapidly suppress the immune response. These corticosteroids are usually not used as monotherapy but rather as components of combination therapy with calcineurin inhibitors (such as tacrolimus or cyclosporine) and antimetabolites (such as mycophenolate mofetil or azathioprine) to achieve synergistic immunosuppression and reduce the risk of organ rejection.

[0059] In some specific embodiments, regulatory T cells with gene modifications that eliminate the gene products of CIITA and one or two HLA loci are resistant to immunosuppressive drugs. It is known in the art that gene silencing methods that confer resistance to immunosuppressive drugs to cells facilitate the implantation of cell therapy products when used in combination with immunosuppression, particularly post-transplantation. For example, resistance to glucocorticoid therapy (such as dexamethasone) can be induced by disrupting the expression of glucocorticoid receptors (Reference 14), or resistance to tacrolimus and / or cyclosporine can be conferred by resistance to FKB12.

[0060] In some implementations, the genetically modified regulatory T cells are resistant to tacrolimus.

[0061] In some embodiments, the genetically modified regulatory T cells are characterized by knockdown or deletion of the FKP12 gene. In some embodiments, the genetically modified regulatory T cells are obtained using a vector-free CRISPR-Cas9-based protocol described by Amini et al. (2021, 13) that targets and knocks out the gene for the adaptor protein FK506-binding protein 12 (FKBP12), which is essential for tacrolimus to exert its immunosuppressive function. This can be achieved, for example, via zinc finger proteins or by transiently delivering the ribonucleoprotein complex to CMV-specific T cells via electroporation.

[0062] In some implementations, the genetically modified regulatory T cells are resistant to cyclosporine.

[0063] In some embodiments, the genetically modified regulatory T cells are characterized by gene knockdown or gene knockout of calcineurin. In other embodiments, the genetically modified cells are made insensitive to tacrolimus by expressing a transgene encoding a calcineurin-modified variant of the calcineurin protein that is insensitive to tacrolimus.

[0064] In some implementations, the genetically modified regulatory T cells are resistant to mTOR inhibitors.

[0065] In some embodiments, the genetically modified regulatory T cells are characterized by gene silencing, mutagenesis, or knockdown of the gene encoding the steroid receptor. In some specific embodiments, the gene encoding the steroid receptor is the glucocorticoid receptor gene.

[0066] Corticosteroids, particularly prednisone, methylprednisolone, and occasionally budesonide, are widely used for immunosuppressive therapy after organ transplantation. They are used to induce therapy, maintain immunosuppression, and treat acute rejection. These drugs exert their effects through genomic and non-genomic mechanisms that inhibit pro-inflammatory cytokines and suppress T cell activation. Prednisone, methylprednisolone, and other glucocorticoids primarily function through the glucocorticoid receptor (GR), a ligand-dependent transcription factor that regulates gene expression to suppress immune responses, including cytokine production and T cell activation.

[0067] In some implementations, the genetically modified regulatory T cells are characterized by gene knockdown or gene knockout of calcineurin and FKB12.

[0068] FKBP-12 (FK506-binding protein 12) is constitutively expressed in normal human cells and widely distributed in various tissues. FKBP-12 binds to tacrolimus (FK506) to form a complex that inhibits calmolysin, a calcium-dependent serine / threonine phosphatase that is crucial for T cell activation.

[0069] Calcineurin is a calcium and calmodulin-dependent serine / threonine protein phosphatase that plays a central role in cell signaling. Calcineurin is activated when intracellular calcium levels rise and binds to calmodulin. It regulates calcium-dependent processes in various tissues, including the immune, nervous, and cardiovascular systems. Calcineurin dephosphorylates members of the nuclear factor-activating factor (NFAT) family of activated T cells, causing them to translocate into the nucleus. This activates the transcription of genes crucial for T cell activation, such as interleukin-2 (IL-2), which is essential for immune responses.

[0070] Calcineurin inhibitors (CNIs), such as cyclosporine A (CsA) and tacrolimus (FK506), bind to intracellular proteins (cyclophilin or FKBP-12) to form a complex, thereby inhibiting the phosphatase activity of calcineurin. This prevents NFAT dephosphorylation and nuclear translocation.

[0071] Techniques for gene silencing, mutation, or knockout are known in the art, through which HLA loci and / or drug target proteins in gene-modified regulatory T cells according to the present invention can be modulated. These methods include retroviral expression, RNA interference, and CRISPR-based strategies. In some specific embodiments, vector-free gene editing methods are employed to target, for example, CIITA, HLA loci, and / or immunosuppressive drug targets (such as the FKBP12 gene) to prepare T cells with low immunogenicity and drug resistance. Efficient gene editing can be achieved by transfecting a ribonucleoprotein (RNP) complex consisting of CRISPR-associated protein 9 (Cas9) and the corresponding single-guide RNA (sgRNA) (see reference 13 for a specific protocol). In some specific embodiments, an RNP nuclear transfection step is employed.

[0072] In some embodiments, the genetically modified regulatory T cells do not contain a transgene encoding the HLA-E α chain (UniprotP13747) or a variant thereof. In other embodiments, the genetically modified regulatory T cells do not contain a transgene encoding the HLA-E extracellular domain or a variant polypeptide that shares more than 95% similarity with the HLA-E extracellular domain.

[0073] In some embodiments, the cell is characterized by wild-type expression of the gene encoding human β2-microglobulin.

[0074] In some embodiments, the genetically modified regulatory T cells according to the invention comprise a T cell receptor characterized as an endogenous T cell receptor. Endogenous regulatory T cells can be enriched from patients according to known protocols (e.g., see references 10, 12) and genetically modified according to the invention to display only a subset of HLA molecules.

[0075] In other embodiments, genetically modified regulatory T cells express chimeric antigen receptors (CARs) from a transgenic expression cassette. In some embodiments, regulatory T cells according to the invention express chimeric antigen receptor proteins comprising the following structural components:

[0076] a. Signal peptide,

[0077] b. Target-specific recognition domain, wherein the target is selected from tumor-associated surface antigens, lineage-specific antigens, tissue-specific surface antigens, or virus-specific surface antigens.

[0078] c. Effector domains containing transmembrane regions and one or more intracellular signal transduction domains, particularly the CD3ζ signal transduction domain, and

[0079] d. The junction area connecting structural domain (b) and structural domain (c).

[0080] In other embodiments, the genetically modified regulatory T cells express the transgenic T cell receptor (tgTCR) from a transgenic expression cassette. In some embodiments, the modified T cells express the transgenic T cell receptor (TgTCR).

[0081] In some embodiments, the gene-edited regulatory T cells according to the invention are characterized by having a T-cell receptor (TCR) that can recognize targets selected from tissue-specific surface antigens, autoantigens, inflammatory molecules, allogeneic antigens, products derived from nucleic acid expression vectors, or transgenes expressed by gene therapy products.

[0082] In some embodiments, the gene-edited T cells are derived from peripheral blood cells. In some embodiments, the gene-edited T cells are derived from thymic T cells. In some embodiments, the gene-edited T cells are derived from induced pluripotent stem cells (iPSCs).

[0083] On the other hand, it relates to recombinant cells according to any of the above embodiments, used in subjects who simultaneously receive (or receive within one week before or one week after treatment) immunosuppressive drug treatment.

[0084] In some implementations, the patient has been diagnosed with an autoimmune disease, an allergic disease, or has received a bone marrow transplant, tissue transplant, or an immunogenic in vivo gene therapy product.

[0085] In some specific implementations, the genetically engineered cells are provided to patients who are simultaneously receiving treatment with another T-cell therapy product, and who may have developed or have developed an immune intolerance to that product (e.g., an HLA-mismatched cell therapy product).

[0086] In addition, a combination drug is provided comprising a genetically modified regulatory T cell and an immunosuppressive drug as described in any aspect of this document and embodiments.

[0087] In combination therapies or certain specific embodiments, the immunosuppressive treatment is selected from the group consisting of tacrolimus, cyclosporine, mTOR inhibitors, corticosteroids, kinase inhibitors, and / or monoclonal antibodies that deplete T cells. In some specific embodiments, the immunosuppressive treatment is tacrolimus and / or cyclosporine (cyclosporine A).

[0088] Another aspect of the invention is a gene-edited regulatory T cell according to the invention, or a combination drug comprising the regulatory T cell and an immunosuppressive drug, for use in patients diagnosed with an autoimmune disease.

[0089] Another aspect of the invention is a gene-edited regulatory T cell according to the invention, or a combination drug comprising the regulatory T cell and an immunosuppressive drug, for use in a patient diagnosed with an allergic disease.

[0090] Another aspect of the invention is a gene-edited regulatory T cell according to the invention, or a combination drug comprising the regulatory T cell and an immunosuppressive drug, for use in patients receiving bone marrow transplantation.

[0091] Implantation of cell therapies for treating cancer or viral infections, characterized by the expression of foreign substances such as CARs or Tg TCRs, may have efficacy limited by the immune response to anti-cell therapy. Another aspect of the invention is gene-edited regulatory T cells or combination drugs comprising said regulatory T cells and immunosuppressive drugs for use in patients receiving cell therapy.

[0092] Another aspect of the invention is the use of gene-edited regulatory T cells according to the invention, or a combination of said regulatory T cells and an immunosuppressive drug, for patients receiving tissue transplants. In some specific embodiments, gene-edited cells or combinations are provided for patients who have received kidney transplants.

[0093] The manufacturing method and treatment method according to the present invention

[0094] The present invention also covers an additional aspect, namely the use of the genetically modified cells described herein in a method for preparing a medicament for treating or preventing conditions requiring concurrent immunosuppressive therapy.

[0095] Similarly, the present invention covers a method for treating a patient diagnosed with a condition requiring concurrent immunosuppressive therapy, the method comprising administering a therapeutically effective amount of the genetically modified cells detailed herein to the patient in need.

[0096] This invention also includes the following items:

[0097] 1. A genetically modified regulatory T cell, characterized by gene deletion or silencing of the following genes, particularly gene knockdown or gene elimination:

[0098] -Class II major histocompatibility complex transcription activator genes (CIITA); and

[0099] - One or at most two of the HLA class I loci: HLA-A, HLA-B, and HLA-C.

[0100] 2. The gene-modified regulatory T cell according to claim 1, wherein the gene-modified regulatory T cell is characterized by gene knockdown or gene knockout of the CIITA gene and the HLA-A and HLA-B loci.

[0101] 3. The genetically modified regulatory T cell according to claim 1 or 2, wherein the genetically modified regulatory T cell is resistant to immunosuppressive drugs.

[0102] 4. Genetically modified regulatory T cells according to item 3, wherein the genetically modified regulatory T cells are resistant to tacrolimus and / or mTOR inhibitors.

[0103] 5. The gene-modified regulatory T cell according to any one of the preceding claims, wherein the gene-modified regulatory T cell is characterized by gene knockdown or gene knockout of the FK506 binding protein 12 gene (FKBP12).

[0104] 6. Regulatory T cells with gene modifications according to any one of items 3 to 5, wherein the immunosuppressive drug is cyclosporine.

[0105] 7. The recombinant regulatory T cell according to any one of the preceding claims, wherein the recombinant regulatory T cell is characterized by gene knockdown or gene knockout of calcineurin and / or genes encoding steroid receptors.

[0106] 8. The gene-modified regulatory T cell according to any one of the preceding claims, wherein the gene-modified regulatory T cell is characterized by gene knockdown or gene knockout of calcineurin and FKB12.

[0107] 9. A genetically modified regulatory T cell according to any of the preceding claims, wherein the genetically modified regulatory T cell does not contain a transgene encoding HLA-E.

[0108] 10. A regulatory T cell with gene modification according to any of the preceding claims, characterized in that the wild-type expression of the gene encoding human β2-microglobulin is present.

[0109] 11. A genetically modified regulatory T cell according to any one of the preceding claims, comprising a T cell receptor having the following characteristics:

[0110] -Endogenous T cell receptors,

[0111] - Chimeric antigen receptor (CAR), and / or

[0112] - Transgenic T-cell receptor (tgTCR).

[0113] 12. A regulatory T cell with a gene modified according to any of the preceding claims, wherein the T cell is derived from peripheral blood cells, thymic T cells or induced pluripotent stem cells (iPSCs).

[0114] 13. Genetically modified cells according to any one of items 1 to 12, for use in subjects who are simultaneously receiving (or receiving within one week before or one week after treatment) immunosuppressive drugs.

[0115] 14. Genetically modified T cells for use as described in paragraph 13, wherein the patient has been diagnosed with an autoimmune disease, an allergic disease, or is a recipient of a bone marrow transplant, cell therapy, or tissue transplant.

[0116] 15. A combination drug comprising a genetically modified regulatory T cell and an immunosuppressive agent according to any one of claims 1 to 14, wherein the immunosuppressive agent is selected from the group consisting of tacrolimus, cyclosporine, mTOR inhibitors, corticosteroids, kinase inhibitors, and / or T cell-depleting monoclonal antibodies.

[0117] More specifically, the immunosuppressants include tacrolimus and / or cyclosporine.

[0118] Wherever alternatives to a single separable feature are listed as "implementations" in this document, it should be understood that these alternatives can be freely combined to form the various specific embodiments of the present invention disclosed herein.

[0119] The following embodiments and figures further illustrate the present invention, from which further embodiments and advantages can be derived. These embodiments are intended to illustrate the invention, but do not limit its scope. Attached Figure Description

[0120] Figure 1 An in vivo humanized mouse model system is shown to demonstrate the immunosuppressive capabilities of human autologous and allogeneic Treg products (adapted from reference 8).

[0121] Figure 2 The study showed that human Treg products, which are allogeneic to PBMCs (peripheral blood mononuclear cells), are less effective in preventing rejection of third-party allogeneic skin grafts (adapted from reference 8).

[0122] Figure 3 It was shown that only low-immunogenic allogeneic Tregs combining HLA class I and II knockout (KO) with HLA-E knock-in (KI) have effects comparable to autologous Tregs (adapted from reference 5).

[0123] Figure 4 shows (A) partial allogeneic immunogenicity, (B) Treg donor / recipient matching can be achieved if reduced to only one or two polymorphic HLA genes, and (C) temporary combination use of tacrolimus does not target the off-the-shelf FKBP12 KOTreg.

[0124] Figure 5 An overview of the inventors’ novel triple strategy is shown, which is based on a feasible and safe “stealth” concept and enables the use of allogeneic Tregs through: i) partial knockdown + ii) low-dose immunosuppression and Tregs resistant to the corresponding drugs + iii) application of an AI-based matching tool. Example

[0125] Example 1:

[0126] The most important polymorphic structure distinguishing "you" from "me" is the human leukocyte antigen (HLA) system. The HLA complex is located on the short arm of chromosome 6. HLA genes follow Mendelian inheritance principles, encoding antigens that are co-dominantly expressed on the cell surface. In the absence of recombination events, HLA genes form tight physical linkages due to their proximity, and are usually inherited as a whole set from each parent. An HLA haplotype is a combination of linked HLA genes (HLA-A, -B, -C, -DR, -DQ, -DP) inherited from a single parental chromosome. HLA antigens are expressed on the surface of many cells, including T cells, and play important roles in self-recognition, triggering immune responses to antigenic stimuli, and coordinating cellular and humoral immunity. The HLA complex is known to be polygenic because it consists of many genes, which can be broadly classified into three classes: class I, class II, and class III. Polymorphism is another characteristic of HLA molecules. Polymorphism allows for multiple variations of antigens or alleles within a single gene. HLA class I and II antigens have the highest polymorphism among human structural genes, which means that the amino acids in any given HLA molecule vary slightly between individuals (Reference 1). There are more than 6,000 alleles known at the six HLA class I and II loci, theoretically allowing for billions of combinations.

[0127] In hematopoietic stem cell transplantation (HSCT), HLA matching of the recipient to a selected donor (from a list of thousands of donors) is standard practice. However, this is nearly impractical for the use of allogeneic T-cell products, as finding a suitable donor and subsequently producing the T-cell product separately offers no advantage over autologous methods. The goal is to obtain off-the-shelf products that can be repeatedly used for numerous patients as needed. Due to cost and logistical reasons, it is not feasible to produce and store thousands of allogeneic products to provide appropriately matched products when needed (or to discard them if they are never used and are subject to shelf-life limitations). Therefore, strategies to reduce allogeneic immunogenicity are being sought.

[0128] For transplantation, novel HLA matching tools have been developed that go beyond traditional serological or genotyping HLA typing. These new tools improve the feasibility of HLA matching by focusing on the detailed molecular interactions of the immune response during transplantation, while also enhancing the specificity and accuracy of the matching. Recent examples include HLA Twin, which focuses on molecular mismatches at the epitope level, using a comprehensive database to analyze HLA compatibility and predict the immunogenic potential of mismatches (Reference 5). Recently, studies have used machine learning methods to investigate clustering patterns of antibody responses to HLA class I antigens (Reference 6). PIRCHE-II is a tool that simulates the CD4+ T cell indirect allogeneic recognition pathway by calculating epitope load scores, aiming to improve the prediction of graft survival (Reference 7). However, for the reasons mentioned above, advanced HLA matching tools are difficult to apply to the selection of donor / recipient combinations in standard cell therapy without further manipulation.

[0129] The strategy of overcoming the risk of rejection of allogeneic cell products by the patient's own T cells through potent immunosuppression has been applied to organ transplantation and HSCT recipients who develop graft-versus-host disease, but it is not very suitable for T-cell therapy because this immunosuppression also inhibits the function and survival of metastatic T cells. One approach is to use genetic engineering to remove surface antigens (e.g., CD52) from cell products, making the cells resistant to antibodies targeting the corresponding antigens, such as anti-CD52 antibodies (alemumab). These antibodies can then be used to eliminate all T / B cells in the patient before cell administration; however, this significantly increases the risk of infection (References 2, 3). Other methods aim to produce "latent" cells, i.e., by genetically manipulating and knocking out the most important polymorphic immunogenic antigens (HLA class I + II). To prevent the NK cells and macrophages of the treated patient from recognizing these "latent" cells as dangerous cells and eliminating them due to "self-deficiency" signals, it is necessary to overexpress monomorphic / oligomorphic "self-signaling" molecules (e.g., HLA-E, HLA-G, or "don't eat me" molecules CD24 / CD47) (Reference 4). This requires complex genetic manipulation, is costly, and carries an inherent risk of genotoxicity.

[0130] Tregs are a new class of ATMPs whose goal is not to rebuild immune responses to fight cancer or infection, but rather to sustainably combat unwanted immune responses by reshaping immune balance. They are the immune system's natural peacemakers, possessing inert immunomodulatory / immunosuppressive capabilities.

[0131] Recent studies have demonstrated for the first time in preclinical model systems that mismatched allogeneic Treg products possess in vitro functionality and short-term in vivo implantation capability (<1 week) comparable to autologous Treg products. However, within 3 weeks, these products are challenged by allogeneic peripheral blood mononuclear cells (PBMCs) containing allogeneic reactive effector T cells, resulting in a reduction in their numbers and loss of immunosuppressive capacity in human skin xenograft model systems (Reference 8). Figure 1 , 2 ).

[0132] These data clearly demonstrate that HLA-mismatched allogeneic Tregs are also immunogenic and lose long-term efficacy due to rejection. The difference is even more pronounced if the number of adopted Treg cells is low (suboptimal).

[0133] Eliminating all CD8+ T cells in PBMCs almost restored the immunosuppressive efficacy of HLA-mismatched Tregs, while eliminating CD4+ T cells or CD56+ NK cells did not, suggesting that T cell-mediated killing of metastatic Treg products is the reason for the lower efficacy of allogeneic Tregs. However, this is not a truly safe treatment strategy. As mentioned above, HLA-matching strategies are difficult to achieve in clinical practice and have only been partially successful (Reference 8).

[0134] Therefore, as described in conventional T-cell products, the inventors designed "stealthy" Tregs by knocking out polymorphic HLA class I and II molecules through CRISPR / Cas-mediated gene / base editing. To avoid 12 gene edits (HLA class I: A, B, C; HLA class II: DR, DP, DQ, both haplotypes need to be edited), this method specifically knocked out two key genes required for HLA class I and II expression through base editing: the singlet β chain (β2-microglobulin, B2M) and the gene for major histocompatibility complex transcription activator (CIITA) in class II. Since it has been demonstrated that T cells with β2-microglobulin edits become targets of NK cells through a "self-deletion" activation mechanism (Reference 6), a gene editing strategy was developed to introduce the NK cell inhibitory receptor HLA-E into the Treg, providing a "self" signal by knocking in HLA-E to prevent NK cell-mediated lysis. Although HLA-E can only partially protect cells from NK cell-mediated lysis in vitro (Reference 5), HLA class I and II knockout combined with simultaneous HLA-E knock-in completely restores the potency of HLA-mismatched allogeneic Tregs in vivo (Reference 8). Figure 3 ).

[0135] Since obtaining a low-immunogenicity "recessive" Treg requires multiple gene modifications (two knockouts and one knock-in), the inventors performed transfections for both knock-in and knockout separately. For the latter, i.e., step 2, the inventors used a base editing technique with a catalytically modified Cas9 variant to reduce the risk of translocation caused by multiple gene editing (Reference 8).

[0136] Due to factors such as logistics, cost, effectiveness, and safety, different physical Treg products will significantly improve the broad feasibility of Treg therapy in clinical practice.

[0137] Contrary to the assumption that Tregs may have immune exemptions, a recent study (reference 8) shows that Tregs do indeed have allogeneic immunogenicity, which limits their use as off-the-shelf products that do not match HLA.

[0138] Using almost entirely conventional or advanced HLA typing (as used in HSCT or kidney transplantation) to select Treg donors and Treg recipients is practically not feasible.

[0139] Only by knocking out HLA-I (β2M) and HLA-II (CIITA) and knocking in HLA-E, producing "latent" Tregs, has shown efficacy comparable to autologous Tregs in clinically relevant humanized mouse models.

[0140] Considering the need for additional molecular modifications to Treg products to redirect their specificity (e.g., CAR expression), improve survival / adaptability (release growth factors, resist inflammation-induced reprogramming), and achieve targeted tissue adaptation, the aforementioned "stealthy" strategy is not well-suited for safely and effectively converting them into off-the-shelf clinical Treg products. Furthermore, completely "stealthy" Tregs cannot be recognized and controlled when infected by viruses or transformed into malignant cells, thus transferring completely "stealthy" Tregs carries a high risk.

[0141] Therefore, this invention provides a multi-pronged strategy to address immunogenicity issues in the development of allogeneic "off-the-shelf" regulatory T cell (Treg) products and provides a significantly safer cell therapy product.

[0142] The purpose of this method is twofold: firstly, to avoid additional knock-in (HLA-E or analogues) to replenish the lost autosignal during traditional β2-microglobulin knockout, as gene insertion (knock-in) particularly increases the risk of genotoxicity; and secondly, to preserve the presentation of viral or tumor antigens in the manipulated T-cell product, which also offers a safety advantage. To this end, a multi-concept based on three pillars has been developed:

[0143] 1) Partial (allogeneic) low immunogenicity:

[0144] Allogeneic immunogenicity is partially reduced by knocking out CIITA (HLA class II) and only knocking out one or two of the three HLA class I loci (e.g., knocking out HLA-A, -B, or -C alone, or a combination of knocking out HLA-A / -B, HLA-A / -C, or HLA-B / -C). Figure 4A ).

[0145] 2) Feasible Treg donor / recipient matching:

[0146] Reducing the expression of HLA class I polymorphic molecules (HLA-A, -B, -C) to only one or two instead of three allows for smarter and more feasible matching of Treg cell donors and recipients using HLA homozygous donors and / or existing or new genotyping procedures. Figure 4B ).

[0147] 3) Temporary combined use of immunosuppressants:

[0148] To overcome the residual trace allogeneic immunogenicity after the application of pillars 1 and / or 2, a temporary immunosuppressive agent should be used after cell administration to suppress any possible allogeneic reactions against the residual immunogenic structures.

[0149] Immunosuppressive drugs can be used alone or in combination: tacrolimus or its derivatives, cyclosporine or its derivatives, mTOR inhibitors, corticosteroids, kinase inhibitors, and monoclonal antibodies that target specific molecules common to Treg cells and regular T cells.

[0150] Because these immunosuppressants inhibit the survival and function of Tregs, "off-the-shelf" Tregs resistant to these immunosuppressants are used through additional stable or transient genetic modifications. Such modifications include, but are not limited to, knockout, silencing, or mutagenesis of drug target molecules, including but not limited to FKBP12 (resistance to tacrolimus and mTOR inhibitors), calcineurin (resistance to cyclosporine and its derivatives), and / or steroid receptor genes (…). Figure 4C ).

[0151] This system is applicable to any Treg product used as an allotropic, marketed therapeutic product, such as:

[0152] - First-generation polyclonal Treg products (polyclonal endogenous T cell receptor library)

[0153] - Next-generation Treg products that achieve specific retargeting through the expression of chimeric antigen receptors (CARs).

[0154] - Next-generation Treg products that achieve specific retargeting through the expression of transgenic T cell receptor (tgTCR).

[0155] - Next-generation Treg products that improve survival, function, implantation, and homing traits through genetic manipulation.

[0156] - Next-generation Treg products that improve survival, function, implantation, and homing properties through RNA technology manipulation.

[0157] - In-stock Tregs can be generated from peripheral blood (Reference 7), thymus (Reference 8), or iPSCs (Reference 9).

[0158] - Any combination of the above possible methods

[0159] Compared to the traditional "invisible" concept, the three-pillar concept has the following advantages:

[0160] • Reduce the risk of genotoxicity by avoiding the knock-in of self-signals (such as HLA-E) to prevent NK cell activation, a knock-in step that is necessary when using conventional β2-microglobulin knockout (pillar 1).

[0161] • By preserving antigen processing and presentation in the transferred Treg product, immune surveillance and control are maintained, thus providing a safety advantage in terms of malignant transformation or viral infection (Pillar 1).

[0162] • By reducing the differences in polymorphism and multigene HLA systems, we can improve the feasibility of matching patients awaiting Treg therapy using relatively well-matched off-the-shelf allogeneic Treg products from cell banks (Pillar 2).

[0163] • Temporary immunosuppression can be implemented (using tacrolimus and / or steroids or other commonly used immunosuppressants) to block the immune response against residual immunogenicity after molecular editing and matching, without affecting the survival and function of off-the-shelf Treg products (Pillar 3).

[0164] Example 2:

[0165] The problem with the complete knockout strategy currently used by many research groups is that the immune control over these "hidden" cells is completely lost; this poses a safety concern in the event of malignant transformation or infection, because these "hidden" cells cannot present tumor or viral antigens / peptides.

[0166] The following experiment confirms this:

[0167] Recessive Treg cells lose control over viral infections, but "partially recessive" Treg cells with residual HLA-I do not.

[0168] As a proof-of-concept (PoC) experiment to test immune surveillance capabilities, we pulsed "recessive" Tregs (HLA class I knockout (β2MG knockout) and HLA class II knockout (CIITA knockout), with or without HLA-E co-expression) with a cytomegalovirus (CMV)-derived key peptide library and compared their responses with those of "partially recessive" Tregs (only one HLA class I knockout, with or without HLA class II knockout) and unmodified Tregs (Table 1).

[0169] Table 1 shows that cloaked Tregs lose their ability to control viral infection, while "partially cloaked" Tregs do not lose this ability.

[0170]

[0171] Explanation: CMV-specific conventional T cells were enriched according to previously described methods (Brestrich et al., Am J Transplant. 2009 Jul;9(7):1679-84.; Bunde et al., J Exp Med.). Briefly, T cells were stimulated with an overlapping peptide library covering CMVIE-1 / pp65 protein, and IFNg-secreting T cells were isolated after overnight stimulation. After resting for 2–3 days, T cells were restimulated with a population of Treg cells pulsed with the CMVIE-1 / pp65 peptide library. Intracellular IFNg expression was determined by flow cytometry and expressed as a percentage of all viable responsive T cells selected by gating.

[0172] In summary, the proof-of-concept experiment demonstrated that "recessive" Tregs lacking HLA classes I and II, regardless of whether they co-express HLA-E, cannot present viral peptides. In contrast, "partially recessive" Tregs with residual HLA class I expression (e.g., HLA-A and HLA-C) can be controlled by conventional virus-specific CD8+ T cells; if they express HLA class II, they can also be controlled by virus-specific CD4+ T cells.

[0173] Therefore, Module 1 of our allogeneic Treg strategy is based on low immunogenic (partially recessive) Tregs that express only 1-2 HLA loci by knocking out only the B locus (HLA-B- / -) (producing HLA-A+C+B-Tregs) or knocking out only the A locus (producing HLA-B+C+A-Tregs), with or without additional HLA class II knockout (targeting or not targeting CIITA).

[0174] Partially recessive Treg cells with residual HLA-I still exhibit some allogeneic immunogenicity.

[0175] Allogeneic “partially clonic” (one HLA class I KO, with or without HLA class II KO) Treg cells retain their immune surveillance capabilities (see above), but may still be recognized by the host’s immune system, leading to reduced survival and function, as they can express up to 4 HLA class I mismatches and up to 6 HLA class II mismatches. We have confirmed this using an in vitro human cell co-culture model (see Table 2).

[0176] - The accompanying immunosuppression can overcome the allogeneic immunogenicity of Tregs.

[0177] Therefore, we added a second module to our allogeneic Treg approach: combining off-the-shelf Treg therapy with transient immunosuppression. To maintain Treg survival and function in the presence of immunosuppression, we used Tregs resistant to immunosuppressive drugs, obtained by knocking out the intracellular target protein of immunosuppressants. As a proof of concept, we used Tregs that had FKBP-12 (the binding molecule of the immunosuppressant tacrolimus) knocked out through gene editing, making these Tregs resistant to tacrolimus, one of the most potent immunosuppressants (TregTacRes). Our data showed that the combination of low immunogenicity (partially latent) and transient tacrolimus exposure was advantageous when using TregTacRes (Table 2).

[0178] Table 2. Temporary tacrolimus exposure eliminates allogeneic immunogenicity of Treg cells.

[0179]

[0180] Design: One million enriched and expanded Tregs (unmodified, partially recessive, and partially recessive + TacRes) were co-cultured with one million autologous or unmatched allogeneic conventional T cells (Tconv) in an IL-2-containing G-Rex culture apparatus for 14 days. Tacrolimus (10 pg / ml) was added to partial cultures from day 0 to day 7. The Treg / Tconv ratio was determined by flow cytometry.

[0181] In summary, the data indicate that conventional T cells (Tconv) reduce the number of surviving unmodified allogeneic Treg cells in co-culture. The reduction in Treg cell numbers was less pronounced with the addition of tacrolimus during the first week of co-culture (due to the inhibition of Tconv), but did not reach a level comparable to the autologous control group. This partial recovery was solely due to the strong inhibitory effect of tacrolimus on the survival and function of Tregs in autologous co-culture (Tac+). Although the clearance of low-immunogenic "partially latent" Tregs expressing or not expressing HLA class II (R+ / ++ or R+, respectively) was lower than that of unmodified Tregs (R++), the addition of tacrolimus did not improve survival data due to its direct inhibitory effect (Tac+).

[0182] In contrast, TregTacRes (FKBP12- / - knockout FK-binding protein 12) combined with low immunogenicity and tacrolimus resistance can evade allogeneic conventional T cell immune clearance in the presence of tacrolimus and simultaneously exhibit resistance to the inhibitory effects of tacrolimus. The presence or absence of HLA class II antigens has only a minor effect.

[0183] Notably, tacrolimus-resistant but HLA-expressing Tregs (TregTacRes) showed only a moderate effect (50% loss upon tacrolimus addition), indicating that Tconv recovered from immunosuppression after tacrolimus withdrawal (day 8) and partially eliminated fully immunogenic Tregs within week 2 of culture. No rejection was observed if tacrolimus persisted in the culture system for 14 days (not shown).

[0184] These data demonstrate the advantages of our novel approach, which combines drug resistance with low immunogenicity through gene editing, enabling off-the-shelf allogeneic Treg therapies. However, in vivo, using only one module (drug resistance or low immunogenicity) may be sufficient to achieve therapeutic effects.

[0185] References cited:

[0186] 1)Genetics, Human Major Histocompatibility Complex (MHC). GizemTumer, Brittany Simpson, Tiffany K. Roberts. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan. 2023 Aug 14. PMID:30855806

[0187] 2)Base-Edited CAR7 T Cells for Relapsed T-Cell Acute LymphoblasticLeukemia.

[0188] Chiesa R, Georgiadis C, Syed F, Zhan H, Etuk A, Gkazi SA, Preece R,Ottaviano G, Braybrook T, Chu J, Kubat A, Adams S, Thomas R, Gilmour K, O'Connor D, Vora A, Qasim W; Base-Edited CAR T Group. N Engl J Med. 2023 Sep 7;389(10):899-910. doi: 10.1056 / NEJMoa2300709. Epub 2023 Jun 14. PMID: 37314354

[0189] 3)Multiplex Genome-Edited T-cell Manufacturing Platform for "Off-the-Shelf" Adoptive T-cell Immunotherapies. Poirot L, Philip B, Schiffer-ManniouiC, Le Clerre D, Chion-Sotinel I, Derniame S, Potrel P, Bas C, Lemaire L,Galetto R, Lebuhotel C, Eyquem J, Cheung GW, Duclert A, Gouble A, Arnould S,Peggs K, Pule M, Scharenberg AM, Smith J. Cancer Res. 2015 Sep 15;75(18):3853-64. doi: 10.1158 / 0008-5472.CAN-14-3321. Epub 2015 Jul 16. PMID: 26183927

[0190] 4)New hope for tumor immunotherapy: the macrophage-related “do noteat me” signaling pathway. Han Deng, Guan Wang, Shengyan Zhao, Yiran Tao,Zhixiong Zhang, Jinliang Yang, and Yi Lei. Front Pharmacol. 2023; 14:1228962. Published online 2023 Jul 6. doi: 10.3389 / fphar.2023.1228962

[0191] 5)Class II HLA epitope matching—A strategy to minimize de novodonor-specific antibody development and improve outcomes. C Wiebe 1, DPochinco, T D Blydt-Hansen, J Ho, P E Birk, M Karpinski, A Goldberg, L JStorsley, I W Gibson, D N Rush, P W Nickerson (2013). American Journal ofTransplantation, Dec;13(12):3114-22. doi: 10.1111 / ajt.12478. Epub 2013 Oct 25

[0192] 6)Hidden Patterns of Anti-HLA Class I Alloreactivity Revealed ThroughMachine Learning. Angeliki G. Vittoraki, Asimina Fylaktou, Katerina Tarassi,Zafeiris Tsinaris , Alexandra Siorenta, George Ch. Petasis, DemetrisGerogiannis, Claudia Lehmann, Maryvonnick Carmagnat, Ilias Doxiadis, Aliki G.Iniotaki and Ioannis Theodorou (2021) Front. Immunol. 12:670956. doi:10.3389 / fimmu.2021.670956.

[0193] 7)PIRCHE-II: an algorithm to predict indirectly recognizable HLAepitopes in solid organ transplantationGeneugelijk, K., & Spierings, E.(2020). Immunogenetics 72:119–129https: / / doi.org / 10.1007 / s00251-019-01140-x

[0194] 8)Matching or genetic engineering of HLA Class I and II facilitatessuccessful allogeneic ‘off-the-shelf’ regulatory T cell therapy.

[0195] Oliver McCallion, et al. bioRxiv https: / / doi.org / 10.1101 / 2023.08.06.551956.

[0196] 9)Targeted disruption of HLA genes via CRISPR-Cas9 generates IPSCswith

[0197] enhanced immune compatibility H. Xu, B. Wang, M. Ono, A. Kagita, K.Fujii, N. Sasakawa, T. Ueda, P. Gee, M. Nishikawa, M. Nomura, F. Kitaoka, T.Takahashi, K. Okita, Y. Yoshida, S. Kaneko, A. Hotta. Cell Stem Cell 24, 566-578.e567 (2019).

[0198] 10)Regulatory T cells for minimising immune suppression in kidneytransplantation: phase I / IIa clinical trial. Roemhild A, Otto NM, Moll G,Abou-El-Enein M, Kaiser D, Bold G, Schachtner T, Choi M, Oellinger R,Landwehr-Kenzel S, Juerchott K, Sawitzki B, Giesler C, Sefrin A, Beier C,Wagner DL, Schlickeiser S, Streitz M, Schmueck-Henneresse M, Amini L, StervboU, Babel N, Volk HD, Reinke P.

[0199] BMJ. 2020 Oct 21;371:m3734. doi: 10.1136 / bmj.m3734. PMID: 33087345

[0200] 11)A Novel GMP Protocol to Produce High-Quality Treg Cells from thePediatric Thymic Tissue to Be Employed as Cellular Therapy. Bernaldo-de-Quirós E, Cózar B, López-Esteban R, Clemente M, Gil-Jaurena JM, Pardo C, Pita A, Pérez-Caballero R, Camino M, Gil N, Fernández-Santos ME, Suarez S, Pion M,Martínez-Bonet M, Correa-Rocha R. Front Immunol. 2022 May 16;13:893576. doi:10.3389 / fimmu.2022.893576. eCollection 2022. PMID: 35651624

[0201] 12)A Serum- and Feeder-Free System to Generate CD4 and Regulatory TCells from Human iPSCs. Helen Fong, Matthew Mendel, John Jascur, Laeya Najmi,Ken Kim, Garrett Lew, Swetha Garimalla, Suruchi Schock, Jing Hu, AndresVillegas, Anthony Conway, Jason D. Fontenot, Simona Zompi. bioRxiv 2024. doi:https: / / doi.org / 10.1101 / 2023.07.01.547333

[0202] 13)Amini et al, Mol Ther 2021. 29(1): 32-46.

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[0204] 15)Gaudelli NM et al. Nat. Biotechnol. 38:892-900.

[0205] All scientific publications and patent documents cited in this specification are incorporated herein by reference.

Claims

1. A genetically modified regulatory T cell, characterized in that... The genes of one or at most two of the HLA class I loci HLA-A, HLA-B and HLA-C are silenced, wherein at least one of the HLA class I loci HLA-A, HLA-B and HLA-C is expressed.

2. The gene-modified regulatory T cell according to claim 1, wherein, The genetically modified regulatory T cells are characterized by gene silencing of the major histocompatibility complex transactivator gene (CIITA).

3. The gene-modified regulatory T cell according to claim 1, wherein, The genetically modified regulatory T cells are characterized by the expression of the major histocompatibility complex transcription activator gene (CIITA).

4. The genetically modified regulatory T cell according to any one of claims 1 to 3, wherein, The genetically modified regulatory T cells are characterized by gene silencing at the HLA-A and HLA-B loci.

5. The genetically modified regulatory T cell according to any one of claims 1 to 3, wherein, The genetically modified regulatory T cells are characterized by gene silencing at the HLA-A locus.

6. The genetically modified regulatory T cell according to any one of claims 1 to 3, wherein, The genetically modified regulatory T cells are characterized by gene silencing at the HLA-B locus.

7. The gene-modified regulatory T cell according to any one of the preceding claims, wherein, The genetically modified regulatory T cells are resistant to immunosuppressive drugs.

8. The gene-modified regulatory T cell according to any one of the preceding claims, wherein, The genetically modified regulatory T cells are resistant to tacrolimus and / or mTOR inhibitors.

9. The gene-modified regulatory T cell according to any one of the preceding claims, wherein, The genetically modified regulatory T cells are resistant to cyclosporine.

10. The gene-modified regulatory T cell according to any one of the preceding claims, wherein, The genetically modified regulatory T cells are characterized by gene silencing of the FK506-binding protein 12 gene (FKBP12).

11. The recombinant regulatory T cell according to any one of the preceding claims, wherein, The recombinant regulatory T cells are characterized by gene silencing of calcineurin.

12. The gene-modified regulatory T cell according to any one of the preceding claims, wherein, The genetically modified regulatory T cells are characterized by gene silencing of calcineurin and FKB12.

13. The recombinant regulatory T cell according to any of the preceding claims, wherein the recombinant regulatory T cell is characterized by gene silencing of the glucocorticoid receptor.

14. The gene-modified regulatory T cell according to any of the preceding claims, wherein gene silencing is achieved by gene knockdown.

15. The gene-modified regulatory T cell according to any one of the preceding claims, wherein, Gene silencing is achieved through gene deletion.

16. The genetically modified regulatory T cell according to any of the preceding claims, wherein the genetically modified regulatory T cell does not contain a transgene encoding HLA-E.

17. A genetically modified regulatory T cell according to any of the preceding claims, wherein the cell is characterized by wild-type expression of a gene encoding human β2-microglobulin.

18. The genetically modified regulatory T cell according to any of the preceding claims, comprising an endogenous T cell receptor.

19. The genetically modified regulatory T cell according to any one of the preceding claims, comprising a chimeric antigen receptor (CAR).

20. The genetically modified regulatory T cell according to any one of the preceding claims, comprising a transgenic T cell receptor (tgTCR).

21. The gene-modified regulatory T cell according to any one of the preceding claims, wherein, The T cells are derived from peripheral blood cells, thymic T cells, or induced pluripotent stem cells (iPSCs).

22. Genetically modified cells according to any one of claims 1 to 21, for use in subjects who simultaneously receive (or receive within one week before or one week after treatment) immunosuppressive drug therapy.

23. The gene-modified T cell for the use according to claim 22, wherein, The patient is receiving tacrolimus treatment.

24. The gene-modified T cell for the use according to claim 22, wherein, The patient is currently receiving treatment with sirolimus or everolimus.

25. The gene-modified T cell for the use according to claim 22, wherein, The patient is receiving cyclosporine treatment.

26. The gene-modified T cell for the use according to claim 22, wherein, The patient has been diagnosed with an autoimmune disease, an allergic disease, or is a recipient of a bone marrow transplant, cell therapy, or tissue transplant.

27. A combination drug comprising a genetically modified regulatory T cell and an immunosuppressive agent according to any one of claims 1 to 14, wherein the immunosuppressive agent is selected from tacrolimus, cyclosporine, mTOR inhibitors, corticosteroids, kinase inhibitors, and / or T cell-depleting monoclonal antibodies. More specifically, the immunosuppressive drugs included tacrolimus and / or cyclosporine.

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