Gene-modified t regulatory cell

Abstract

The invention relates to a safe and feasible triple strategy based on i) a gene-modified T regulatory cell characterised by genetic silencing of CIITA (HLA class II); and one, or two of the HLA class I gene loci HLA-A, HLA-B, and HLA-C; ii) particular embodiments provide a cell modified to have resistance to a particular immunosuppressive drug by gene silencing of the respective target gene; iii) the partial stealth concept combined with immunosuppression facilitates donor recipient matching using AI tools.

Description

[0001] Gene-modified T Regulatory Cell

[0002] Field

[0003] The present invention relates to multiplex strategies to combat the immunogenicity of allogenic ‘off-the- shelf’ regulatory T cell products.

[0004] Background

[0005] The adoptive transfer of T cell products has developed into a promising immunotherapy with the prospect of a cure instead of lifelong treatment of severe chronic diseases. Several anti-cancer T cells, e.g. CD19-specific or BMCA-specific CAR-T cells, have already been approved as therapeutics, and hundreds of clinical trials with advanced approaches are currently underway.

[0006] So far, the autologous approach has dominated, i.e. the starting product for the cell products is obtained from the patient himself and enriched, modified and expanded in the GMP manufacturing process. The autologous product produced is then administered to the same patient. Similar approaches are performed for anti-infection memory / effector T cell products.

[0007] These processes are logistically challenging, reach their limits in heavily pre-treated patients and are very cost-intensive. For this reason, approaches for off-the-shelf allogeneic therapy using starting material from healthy individuals or iPSC are being developed. However, the (allo)immunogenicity of these products poses a challenge, at least in patients who are not severely immunocompromised (e.g. as in post-hematopoietic stem cell transplantation), as the allogeneic cell products are commonly rejected.

[0008] Regulatory T cells (Treg) are a completely different category of T cell products. Unlike the anti-cancer or anti-infection T cells mentioned above, they are not used for immune reconstitution, but for the treatment of undesired immune reactions, such as after transplantation, gene therapies or diseases such as autoimmunity, allergy or impaired regeneration. Treg are the natural ‘peacemakers’ of the cellular immune system, and can rebalance disturbed immune regulation through cell-contact mediated and secreted factors. Similar to effector T cells, at present mainly autologous Treg products are used in patients. Only severely immunocompromised patients, e.g. after haematopoietic stem cell therapy (HSCT), have been treated with allogeneic Treg to date. It was hoped that the inherent immunosuppressive properties of Tregs would be able to prevent / inhibit alloreactive responses against off-the-shelf Treg products in cell transfer recipients. However, in a clinically relevant humanized murine model, an off-the-shelf Treg product was associated with significant immunogenicity, leading to loss of function and survival of transferred cells (reference 8).

[0009] Therein, the inventors were able to show in a human immune system (HIS) mouse model that despite their inherent immunoregulatory properties, regulatory T cells (Treg) exhibit alloimmunogenicity similar to that of conventional T cells directed against tumours or infectious agents. In other words, they are recognized and eliminated by the host's human immune system, which makes a simple off-the-shelf use ineffective (ref 8; McCallion et al. 2023). The inventors were also able to demonstrate that use of “stealth” Treg by knock-out of HLA calls I and class II molecules (to prevent recognition by host's T cells) combined with knock-in of less polymorphic HLA-E molecule (to prevent rejection by host's NK cells) results in escape of off-the-shelf Treg from allorejection (loss of alloimmunogenicity) and functionally comparable data as seen for autologous ones (Ref. 8).

[0010] Off-the-shelf allogeneic Treg products have great potential for treating a wide array of inflammatory conditions, however Treg function is impaired in many (autoimmune) diseases, which at present limits the use of potentially immunogenic autologous preparations.

[0011] Off-the-shelf allogeneic T-cell therapy will have many advantages in terms of cost, logistical challenges, quality of the cell product and scalability compared to the most commonly used autologous approach. To achieve this, two important hurdles must be overcome:

[0012] Alloimmunogenicity of the off-the-shelf T cell product, which leads to its rejection by the host's immune system after triggering alloreactive (host-reactive) T-cell receptors of the product (host- versus-transplant reaction

[0013] Alloreactivity of the off-the-shelf T cell product to the host tissues (graft-versus-host reaction)

[0014] Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to reduce the immunogenicity of cellular T regulatory cell-based therapies. This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.

[0015] Summary of the Invention

[0016] The invention relates to a gene and cell therapy advanced medicinal therapeutic product (ATMP) - a T regulatory cell characterised by genetic deletion or genetic silencing, particularly a gene knock-down or gene knock-out of the Transactivator of the Major Histocompatibility Complex of Class II gene (CIITA) (HLA class II); and one, or two of the HLA class I gene loci HLA-A, HLA-B, and HLA-C. Partial, as opposed to complete knock-down of HLA the loci allows easier matching of donors and recipients, particularly using artificial intelligence (Al) tools.

[0017] In particular embodiments, the gene-edited T regulatory cells according to the invention are characterised by a second gene-modification conferring resistance to an immunosuppressive drug, particularly one selected from the group consisting of tacrolimus, cyclosporin, and steroids by genetic silencing / knock-out of the drug’s respective molecular target (Fig. 5).

[0018] The data shown in the Examples demonstrate that in order to evade immune recognition, no need exists for a total “stealth cap’’ (knock-out of all HLA cells l+ll), which is accompanied by significant inherent risk of a lack of immune surveillance of tumour formation or viral infection of these stealth cap cells (Table 1+2).

[0019] The data of the specification further show an effective combination of immunosuppressive therapy such as calcineurin inhibitors (to prevent rejection) with immunosuppressant-resistant Treg (to prevent harm for Treg regarding their survival and function) (Table 2). The combination of temporary immunosuppression with partial stealth (knock-out 2 of 3 HLA class I genes with or without knock-out of HLA-class II) is most powerful in preventing rejection of allogeneic Treg but keeping their survival and functionality.

[0020] Terms and definitions

[0021] General

[0022] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

[0023] The terms “comprising”, “having”, “containing”, and “including”, and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of’ or “consisting of.”

[0024] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0025] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X."

[0026] As used herein, including in the appended claims, the singular forms “a”, “or” and “the” include plural referents unless the context clearly dictates otherwise.

[0027] "And / or" where used herein is to be taken as specific recitation of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of 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).

[0028] Unless defined otherwise, 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). Standard techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et aL, Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

[0029] Any patent document cited herein shall be deemed incorporated by reference herein in its entirety.

[0030] General Molecular Biology: Nucleic Acid Sequences, Expression

[0031] The term gene refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. A polynucleotide sequence can be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.

[0032] The term transgene in the context of the present specification relates to a gene or genetic material that has been transferred from one organism to another. In the present context, the term may also refer to transfer of the natural or physiologically intact variant of a genetic sequence into tissue of a patient where it is missing. It may further refer to transfer of a natural encoded sequence the expression of which is driven by a promoter absent or silenced in the targeted tissue.

[0033] The term recombinant in the context of the present specification relates to a nucleic acid, which is the product of one or several steps of cloning, restriction and / or ligation and which is different from the naturally occurring nucleic acid. A recombinant virus particle comprises a recombinant nucleic acid.

[0034] The terms gene expression or expression, or alternatively the term gene product, may refer to either of, or both of, the processes - and products thereof - of generation of nucleic acids (RNA) or the generation of a peptide or polypeptide, also referred to transcription and translation, respectively, or any of the intermediate processes that regulate the processing of genetic information to yield polypeptide products. The term gene expression may also be applied to the transcription and processing of a RNA gene product, for example a regulatory RNA or a structural (e.g. ribosomal) RNA. If an expressed polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Expression may be assayed both on the level of transcription and translation, in other words mRNA and / or protein product.

[0035] The terms “silence(ing) a gene”, which covers both “knock(ing) down a gene” and “knock(ing) out a gene” in the context of this specification relate techniques known in the art of synthetic biology:

[0036] Gene knock-down refers to the partial reduction of gene expression, typically at the mRNA level, resulting in decreased production of the corresponding protein. This is often achieved using RNA interference (RNAi) techniques, such as small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), which bind to the target mRNA and promote its degradation or inhibit its translation. Another method is antisense oligonucleotides, which hybridize to the mRNA and block translation. Gene knock-down is generally transient or reversible, making it useful for studying gene function without permanently altering the genome.

[0037] Gene knock-out, in contrast, involves the complete inactivation or deletion of a gene, leading to the total absence of the corresponding protein. This is typically achieved through genome-editing technologies such as CRISPR-Cas9, TALENs, or zinc finger nucleases (ZFNs), which introduce targeted double-strand breaks in DNA, resulting in frameshift mutations or deletions via error- prone repair mechanisms like non-homologous end joining. Knock-out models, particularly in mice, are widely used in research to investigate gene function and disease mechanisms by observing the effects of permanent gene loss.

[0038] Methods for modifying cells to treat diseases by regulating the expression of specific genes include, but are not necessarily limited to, the following methods:

[0039] RNA interference (RNAi): This method uses short interfering RNAs (siRNAs) or microRNAs (miRNAs) to target and degrade specific mRNA molecules, preventing the translation of the targeted gene into a protein. RNAi agents may be produced within the cell by transcription from a recombinant nucleic acid expression vector.

[0040] CRISPR-Cas9 gene editing or base editing: CRISPR-Cas9 and related systems derived from bacterial defence mechanisms can be programmed to target specific DNA sequences, allowing for the cutting of DNA at precise locations. This can be used to disrupt gene function or correct mutations.

[0041] Antisense oligonucleotides (ASOs): These are short, synthetic strands of DNA that bind to RNA transcripts and block their translation into proteins. They can also be designed to modulate RNA splicing.

[0042] Zinc finger nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs): These are engineered proteins that can be customized to bind specific DNA sequences, enabling targeted gene disruption.

[0043] Detailed Description of the Invention

[0044] T regulatory cell-based therapeutic products are known in the art, and have been demonstrated to allow minimal dosing of immunosuppressive therapy in transplant recipients (reference 10) and to reduce inflammatory responses to transplant complications arising from cytomegalovirus (CMV) infections in long term immunosuppressed patients (reference 13). Such cells are used to supress unwanted antiself, anti-allergen, or in some cases, anti-viral or anti-transplant immune responses in recipient. Accordingly, gene-modified T regulatory cells according to the invention display the canonical features of the Treg identity including a CD4+CD25+FOXP3+ phenotype, and no elevated Th1 cytokine production after polyclonal stimulation. The modifications described herein ensure efficient engraftment by modulating both HLA class I and II expression.

[0045] A first aspect of the invention relates to a gene-modified T regulatory cell characterised by genetic deletion or genetic silencing, particularly a knock-down, or gene knock-out of one, or two of the HLA class I gene loci HLA-A, HLA-B, and HLA-C. In other words, at least one of the HLA class I gene loci HLA-A, HLA-B, and HLA-C is expressed.

[0046] In certain embodiments, the Transactivator of the major histocompatibility complex of class II gene (CIITA) (HLA class II) is also silenced in the cell. In certain embodiments, the Transactivator of the major histocompatibility complex of class II gene (CIITA) (HLA class II) is not silenced in the cell, but the CIITA gene is expressed at or near wild type levels.

[0047] Preferably, ablation of HLA loci expression is achieved by targeted mutagenesis, gene-silencing, or gene-knockdown of one or at most two of the HLA-A gene, the HLA-B gene, and / or the HLA-C gene, and in addition, the CIITA gene. The resulting gene-modified T cell regulatory cell product expresses immunogenic HLA proteins from only 1 or at most 2 gene loci, resulting in 2 to 4 haplotypes, avoid the need for matching 12 haplotypes of HLA class I and II. The resulting cell product is more compatible with an Al-based haplotype matching tool configured to match a recipient with a product with a low potential for immunogenicity.

[0048] In certain embodiments, the gene-modified regulatory T cell is characterised by genetic silencing of the HLA-A and HLA-B gene loci, and HLA-C is expressed.

[0049] In certain embodiments, the gene-modified regulatory T cell is characterised by genetic silencing of the HLA-A gene locus, and HLA-B and HLA-C are expressed.

[0050] In certain embodiments, the gene-modified regulatory T cell is characterised by genetic silencing of the HLA-B gene locus, and HLA-A and HLA-C are expressed.

[0051] In particular embodiments, the gene-modified regulatory T cell is characterised by gene knock-down, or gene knock-out of the CIITA gene and both of the HLA-A and HLA-B gene loci.

[0052] In some embodiments, the gene-modified regulatory T cell is characterised by gene knock-down, or gene knock-out of the CIITA gene and both of the HLA-B and HLA-C gene loci.

[0053] In some embodiments, the gene-modified regulatory T cell is characterised by gene knock-down, or gene knock-out of the CIITA gene and both of the HLA-A and HLA-C gene loci.

[0054] In some embodiments, the gene-modified regulatory T cell is characterised by gene knock-down, or gene knock-out of the CIITA gene and the HLA-A gene loci, retaining expression of HLA-B and HLA-C.

[0055] In some embodiments, the gene-modified regulatory T cell is characterised by gene knock-down, or gene knock-out of the CIITA gene and the HLA-B gene loci, retaining expression of HLA-A and HLA-C.

[0056] In some embodiments, the gene-modified regulatory T cell is characterised by gene knock-down, or gene knock-out of the CIITA gene and the HLA-C gene loci, retaining expression of HLA-A and HLA-B.

[0057] In particular embodiments, the CIITA gene is silenced using a CRISPR-Cas9-derived adenine base editor (ABE) according to the method disclosed in references 8 and 15.

[0058] In particular embodiments, the gene-edited regulatory T cell product retains expression of an HLA loci that is very commonly expressed in the population, for example, HLA-A*01 , or HLA-A*02.

[0059] Treatment with an immunosuppression drug is required to prevent organ rejection in transplant recipients, and can also aid engraftment of cell therapy products such as gene-modified T cells expressing CAR or Tg TCR. Conventional immunosuppressive drug treatment includes three classes of immunosuppressants: the proliferation inhibitor mycophenolic acid (MPA), which also inhibits pro- inflammatory cytokine production, induces lymphocyte apoptosis and decreases homing of T cells. Calcineurin inhibitors (CNIs; e.g. tacrolimus and cyclosporine, also termed cyclosporine A) potently reduce T cell activation, maturation, and cytokine secretion. Glucocorticoids blunt cytokine production by T cells and provoke lymphocyte apoptosis. Preformed allo-antigen-reactive effector T cells are particularly difficult to control in transplant recipients. CNIs are currently the most effective treatment for sustained suppression of these allo-antigen-reactive cells. Therefore, solutions are required to maintain control of allograft-reactive T cells while overcoming suppression of protective antiviral T cell responses.

[0060] Corticosteroids are essential immunosuppressive agents in organ transplantation, used during induction therapy, maintenance, and treatment of acute rejection. The most important corticosteroids in this context are prednisone (or its active form prednisolone) and methylprednisolone, with dexamethasone playing a more limited role. Prednisone is commonly used for long-term oral maintenance due to its effective suppression of T-cell-mediated immune responses, while high-dose intravenous methylprednisolone is often administered during the perioperative period or in acute rejection episodes for rapid immunosuppression. These corticosteroids are typically not used as monotherapy but rather as part of a combination regimen alongside calcineurin inhibitors such as tacrolimus or cyclosporine, and antimetabolites like mycophenolate mofetil or azathioprine, to achieve synergistic immunosuppressive effects and reduce the risk of organ rejection.

[0061] In particular embodiments, the gene-modified regulatory T cell having ablation of CIITA and 1 or 2 HLA loci gene products, is resistant to an immunosuppressive drug. Gene silencing approaches conferring resistance to immunosuppressive drugs are known in the art to aid engraftment of cell therapy products in combination with immunosuppression, particularly following transplants. Examples include resistance to glucocorticoid treatment (such as dexamethasone) by disrupting expression of the glucocorticoid receptor (reference 14), or resistance to FKB12 conferring resistance to tacrolimus and / or cyclosporin.

[0062] In certain embodiments, the gene-modified regulatory T cell is resistant to tacrolimus.

[0063] In certain embodiments, the gene-modified regulatory T cell is characterized by gene knock-down, or gene knock-out of FKP12. In certain embodiments, the gene-modified regulatory T cells has been obtained by means of a vector-free clustered regularly interspaced short palindromic repeats (CRISPR)- Cas9-based protocol as described by Amini et al 2021 (13) is used to target and knock out the gene for the adaptor protein FK506-binding protein 12 (FKBP12), required for the immunosuppressive function of tacrolimus. This can be achieved, for example, by zinc-finger proteins, or by transient delivery of ribonucleoprotein complexes into CMV-specific T cells by electroporation.

[0064] In certain embodiments, the gene-modified regulatory T cell is resistant to cyclosporin.

[0065] In certain embodiments, the gene-modified regulatory T cell is characterized by gene knock-down, or gene knock-out of calcineurin. In other embodiments, the gene-modified cell is insensitive to tacrolimus by means of expression of a transgene encoding a tacrolimus-insensitive modified variant of the calcineurin protein.

[0066] In certain embodiments, the gene-modified regulatory T cell is resistant to an mTOR inhibitor. In certain embodiments, the gene-modified regulatory T cell is characterised by gene silencing, mutagenesis or genetic knock-down of a gene encoding a steroid receptor. In particular embodiments the gene encoding a steroid receptor is the glucocorticoid receptor.

[0067] Corticosteroids, particularly prednisone, methylprednisolone, and occasionally budesonide, are widely used in immunosuppression following organ transplantation. They are employed as part of induction therapy, maintenance immunosuppression, and for treating acute rejection episodes. These agents exert their effects by inhibiting pro-inflammatory cytokines and suppressing T-cell activation via genomic and non-genomic mechanisms. Prednisone, methylprednisolone, and other glucocorticoids exert their effects primarily 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.

[0068] In certain embodiments, the gene-modified regulatory T cell is characterized by gene knock-down, or gene knock-out of calcineurin and FKB12.

[0069] FKBP-12 (FK506-binding protein 12) is constitutively expressed in normal human cells and is widely distributed across tissues. FKBP-12 binds to tacrolimus (FK506) to form a complex that inhibits calcineurin, a calcium-dependent serine / threonine phosphatase critical for T-cell activation.

[0070] Calcineurin is a calcium- and calmodulin-dependent serine / threonine protein phosphatase that plays a central role in cellular signaling. Calcineurin is activated by increased intracellular calcium levels, binding to calmodulin. It regulates calcium-dependent processes in various tissues, including the immune system, nervous system, and cardiovascular system. Calcineurin dephosphorylates members of the Nuclear Factor of Activated T-cells (NFAT) family, enabling their translocation to the nucleus. This activates transcription of genes critical for T-cell activation, such as interleukin-2 (IL-2), which is essential for immune responses.

[0071] Calcineurin inhibitors (CNIs), such as cyclosporin A (CsA) and tacrolimus (FK506), bind to intracellular proteins (cyclophilins or FKBP-12) to form complexes that inhibit calcineurin's phosphatase activity. This prevents NFAT dephosphorylation and nuclear translocation.

[0072] Means to achieve gene silencing, mutation, or knockout are known in the art by which the HLA loci and / or drug target proteins can by modulated in a gene-modified T regulatory cell according to the invention. These include retroviral expression, RNA interference, and CRISPR-based strategies. In particular embodiments, a vector-free gene editing approach is used, targeting for example, the CIITA, HLA loci and / or immunosuppressive drug target such as the FKBP12 gene to make low immunogenic, drug-resistant T cells. Efficient gene editing may be achieved by transfection with ribonucleoprotein (RNP) complexes consisting of CRISPR-associated protein 9 (Cas9) and respective single guide RNA (sgRNA) (see reference 13 for protocol). In particular embodiments, an RNP nucleofection step is used.

[0073] In certain embodiments, the gene-modified regulatory T cell does not comprise a transgene encoding the HLA-E alpha chain (Uniprot P13747), or variants thereof. In other embodiments the gene-modified regulatory T cell does not comprise a transgene encoding the extracellular domain of HLA-E, or a variant polypeptide more that 95% similar to the extracellular domain of HLA-E. In certain embodiments, the cell is characterized by wildtype expression of the gene encoding human beta-2-microglobulin.

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

[0075] In other embodiments, the gene-modified regulatory T cell expresses from a transgenic expression cassette a chimeric antigen receptor (CAR). In some embodiments the T regulatory cell according to the invention expresses a chimeric antigen receptor protein comprising the following structural components: a. a signal peptide, b. a target specific recognition domain, particularly wherein the target is selected from a tumour- associated surface antigen, a lineage-specific antigen, a tissue-specific surface antigen, or a virus-specific surface antigen, c. an effector domain comprising a transmembrane region and one or more intracellular signalling domains, particularly a CD3 zeta signalling domain, and d. a linker region, connecting domain (b) and domain (c).

[0076] In other embodiments, the gene-modified regulatory T cell expresses from a transgenic expression cassette a transgenic T-cell receptor (tgTCR). In some embodiments, the modified T cell expresses a transgenic T cell receptor (TgTCR).

[0077] In particular embodiments, the gene-edited regulatory T cell according to the invention is characterized by a T cell receptor (TCR) which recognizes a target selected from a tissue-specific surface antigen, an autoantigen, an inflammatory molecule, an alloantigen, a product derived from a nucleic acid expression vector, or a transgene expressed by a gene therapy product.

[0078] In certain embodiments, the gene-edited T cell is derived from a peripheral blood cell. In certain embodiments, the gene-edited T cell is derived from a thymic T cell. In certain embodiments, the gene- edited T cell is derived from an induced pluripotent stem cell (iPSC).

[0079] Another aspect relates to the recombinant cell according to any of the above embodiments, for use in a subject who is receiving concurrent treatment with (or has received in the week prior to treatment with, or week following) an immunosuppressive drug.

[0080] In particular embodiments, the patient has been diagnosed with an autoimmune condition, with an allergic condition, or is the recipient of a bone marrow transplant, or a tissue transplant or an immunogenic in vivo gene therapeutic product.

[0081] In particular embodiments, the gene-engineered cells is provided for use in a patient receiving concurrent treatment with an additional T cell therapy product, to which the patient is likely to, or has already developed immunological intolerance towards (for example, an H LA-mismatched cell therapy product). Also provided is combination medicament comprising a gene-modified T regulatory cell according to any one of the aspects and embodiments herein, and an immunosuppressive drug.

[0082] In particular embodiments or the combination medicament, the immunosuppressive drug treatment is selected from the group of tacrolimus, cyclosporin, an mTOR inhibitor, a corticosteroid, a kinase inhibitor, and / or a T-cell depleting monoclonal antibody. In particular embodiments, the immunosuppressive drug treatment is tacrolimus and / or cyclosporin (cyclosporine A).

[0083] Another aspect of the invention, is a gene-edited regulatory T cell according to the invention, or a combination medicament comprising said regulatory T cell and an immunosuppressive drug for use in a patient has been diagnosed with an autoimmune condition.

[0084] Another aspect of the invention, is a gene-edited regulatory T cell according to the invention, or a combination medicament comprising said regulatory T cell and an immunosuppressive drug for use in a patient has been diagnosed with an allergic disease.

[0085] Another aspect of the invention, is a gene-edited regulatory T cell according to the invention, or a combination medicament comprising said regulatory T cell and an immunosuppressive drug for use in a patient who is the recipient of a bone marrow transplant.

[0086] The engraftment of cell therapies used to treat cancer, or viral infections are characterised by expression of foreign material such as CAR, or Tg TCR, and their efficacy can be limited by anti-cell therapy reactions. Another aspect of the invention, is a gene-edited regulatory T cell, or a combination medicament comprising said regulatory T cell and an immunosuppressive drug for use in a patient who is the recipient of a cell therapy

[0087] Another aspect of the invention, is a gene-edited regulatory T cell according to the invention, or a combination medicament comprising said regulatory T cell and an immunosuppressive drug for use in a patient who is the recipient of a tissue transplant. In particular embodiments, the gene-edited cell or combination is provided for use in a patient who has received a kidney transplant.

[0088] Method of Manufacture and Method of Treatment according to the invention

[0089] The invention further encompasses, as an additional aspect, the use of a gene-modified cell as identified herein, for use in a method of manufacture of a medicament for the treatment or prevention of a condition requiring concurrent treatment an immunosuppressive drug.

[0090] Similarly, the invention encompasses methods of treatment of a patient having been diagnosed with a condition requiring the patient to receive concurrent treatment with an immunosuppressive drug, comprising administering to a patient in need thereof a therapeutically effective amount of the gene- modified cell as specified in detail herein.

[0091] The invention further encompasses the following items:

[0092] 1. A gene-modified T regulatory cell characterised by genetic deletion or genetic silencing, particularly a knock-down, or gene knock-out, of

[0093] - the transactivator of the major histocompatibility complex of class II gene (CIITA); and

[0094] - one, or at most two of the HLA class I gene loci HLA-A, HLA-B, and HLA-C. The gene-modified T regulatory cell according to item 1 , wherein the gene-modified regulatory T cell is characterised by gene knock-down, or gene knock-out, of the CIITA gene and of the HLA-A and HLA-B gene loci. The gene-modified T regulatory cell according to item 1 or 2, wherein the gene-modified regulatory T cell is resistant to an immunosuppressive drug. The gene-modified T regulatory cell according to item 3, wherein the gene-modified regulatory T cell is resistant to tacrolimus and / or an mTOR inhibitor. The gene-modified T regulatory cell according to any one of the preceding items, wherein the gene-modified regulatory T cell is characterized by gene knock-down, or gene knock-out of FK506-binding protein 12 gene (FKBP12). The gene-modified T regulatory cell according to any one of the items 3 to 5, wherein wherein the immunosuppressive drug is cyclosporin. The recombinant T regulatory cell according to any one of the preceding items, wherein the recombinant regulatory T cell is characterized by gene knock-down, or gene knock-out, of calcineurin, and / or a gene encoding a steroid receptor. The gene-modified T regulatory cell according to any one of the preceding items, wherein the gene-modified regulatory T cell is characterized by gene knock-down, or gene knock-out of calcineurin and FKB12. The gene-modified T regulatory cell according to any one of the preceding items, wherein the gene-modified regulatory T cell does not comprise a transgene encoding HLA-E. The gene-modified T regulatory cell according to any one of the preceding items, wherein the cell is characterized by wildtype expression of the gene encoding human beta-2-microglobulin. The gene-modified T regulatory cell according to any one of the preceding items, comprising a T cell receptor characterized as: an endogenous T-cell receptor, a chimeric antigen receptor (CAR), and / or a transgenic T-cell receptor (tgTCR). The gene-modified T regulatory cell according to any one of the preceding items, wherein the T cell is derived from a peripheral blood cell, a thymic T cell, or an induced pluripotent stem cell (iPSC). A gene-modified cell according to any of the items 1 to 12, for use in a subject who is receiving concurrent treatment with (or has received in the week prior to treatment with, or week following) an immunosuppressive drug. The gene-modified T cell for use according to item 13, wherein the patient has been diagnosed with an autoimmune condition, with an allergic condition, or is the recipient of a bone marrow transplant, a cell therapy, or a tissue transplant. A combination medicament comprising a gene-modified T regulatory cell according to any one of the items 1 to 14, and an immunosuppressive drug, particularly wherein the immunosuppressive drug is selected from the group of tacrolimus, cyclosporin, an mTOR inhibitor, a corticosteroid, a kinase inhibitor, and / or a T-cell depleting monoclonal antibody, more particularly wherein the immunosuppressive drug is tacrolimus and / or cyclosporin. Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

[0095] The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

[0096] Description of the Figures

[0097] Fig. 1 shows in vivo humanised mouse model system to prove the immunosuppressive capacity of human autologous and allogeneic Treg products (adapted from reference 8).

[0098] Fig. 2 shows human Treg product allogeneic to the PBMC (peripheral blood mononuclear cells) are less potent in preventing rejection of allogeneic third-party skin allograft (adapted from reference 8).

[0099] Fig. 3 shows only combination of HLA class I + II K.O. and HLA-E K.L hypo-immunogenic allogeneic Treg are comparably effective as autologous Treg (adapted from reference 5).

[0100] Fig. 4 shows (A) Partial Alloimmunogenicity , (B) Feasible Treg-Donor / -Recipient Matching if reduced to one or two polymorphic HLA genes only. (C) Temporary co-administration of tacrolimus does not target FKBP12 K.O. off-the-shelf Treg.

[0101] Fig. 5 shows an overview on the inventor’s novel triple strategy on feasible and safe “stealth” concept to allow use of allogeneic Treg by: i) partial knock-down + ii) low-dose immunosuppression and Teg resistant to the respective drug + iii) application of an Al-based matching tool.

[0102] Examples

[0103] Example 1:

[0104] The most dominant polymorphic structure differentiating “me from you” is the human leukocyte antigen (HLA) system. The HLA complex is located on the short arm of chromosome 6. The HLA genes follow the principles of Mendelian genetics and the encoded antigens are co-dominantly expressed on the cell surface. In the absence of a recombination event, HLA genes are normally inherited en bloc from each parent due to their close proximity resulting in their close physical linkage. The HLA haplotype is a combination of linked HLA genes (HLA-A, -B, -C, -DR, -DQ, -DP) transmitted on a single parental chromosome. HLA antigens are expressed on the surface of many cells, including T cells, and play a major role in self-recognition, evoking the immune response to an antigenic stimulus, and to the orchestration of cellular and humoral immunity. HLA complex is known to be polygenic as it is composed of many genes, which can divide broadly into three categories: Class I, Class II, and Class III. Polymorphism is another feature of the HLA molecule. Polymorphism allows the presence of multiple variations of antigens or alleles within one gene. The HLA class I and class II antigens have the most highly polymorphic structural genes found in humans, which allows amino acids in any given HLA molecule to vary slightly from one person to the next (reference 1). We know more than 6,000 alleles within the 6 HLA class I and II gene loci, allowing at least theoretically many billions of combinations. HLA matching recipients with a selected donor (from many thousands on the list), as it is routine for haematopoietic stem cell transplantation (HSCT), is hardly feasible for the use of allogeneic T-cell products, as finding a single suitable donor and the subsequent individual production of the T-cell products offers no advantage over the autologous approach. The aim is to have off-the-shelf products that can be called up several times for numerous patients if required. Since, for cost and logistic reasons, thousands of allogeneic products cannot be produced and stored so that they can be made available to a patient when required and suitably matched (or skipped if never needed and shelf life is superimposed), strategies are being pursued to reduce alloimmunogenicity.

[0105] For transplantation novel HLA matching tools have been developed that go beyond conventional serologic or genetic HLA-typing. Those novel HLA matching tools improve feasibility while enhancing specificity and accuracy of HLA matching by focusing on the detailed molecular interactions that contribute to immune responses in transplantation. Recently published examples include HLA Twin that focuses on molecular mismatching at the epitope level using a comprehensive database to analyze HLA compatibility and predict immunogenic potential of mismatches (ref.5). Recently, studies investigated the clustering patterns of antibody responses against HLA class I antigens applying machine learning approaches (reference 6). PIRCHE-II, a tool calculating an epitope load score to model the indirect pathway of allorecognition by CD4+ T cells, aimed at improving graft survival predictions (reference 7). Nevertheless, the applicability of advanced HLA matching tools for the selection of donor / recipient constellations for standard cell therapy approaches without further manipulation is very unlikely for the reasons mentioned above.

[0106] Strategies to overcome the risk of rejection of the allogeneic cell product by the patients' own T cells through powerful immunosuppression, as applied in organ transplantation and in case of graft-versus- host disease development also in HSCT recipients, are not very suitable for T cell therapy, as this immunosuppression also inhibits the function and survival of the transferred T cells. One approach is the deletion of a surface antigen (e.g. CD52) on the cell product using genetic engineering - this makes the cells resistant to the antibodies targeting the respective antigen, e.g. anti-CD52 antibody (alemtuzumab), which can then be used to deplete all T / B cells in the patient before cell administration - however, this leads to a greatly increased risk of infection (reference 2, 3). Other approaches are aimed at the production of "stealth" cells, i.e. the most important polymorphic immunogenic antigens (HLA class l+ll) are knocked-out by genetic manipulation. In order that NK cells and macrophages of the treated patients do not eliminate these “stealth” cells as dangerous due to the “missing self’-signal, mono- / oligomorphic "self-signalling” molecules must be overexpressed (e.g. HLA-E, HLA-G or the "do not eat me" molecules CD24 / CD47) (reference 4). This requires complex genetic manipulations with high costs and inherent genotoxicity risk.

[0107] Treg are a new ATMP category that do not aim to immune reconstitute the immune response for defending cancer or infection but to combat sustainably undesired immune reactions by reshaping immune balance. They represent natural peace makers of the immune system and have inert immunoregulatory / immunosuppressive capacity.

[0108] Very recent studies demonstrate in a preclinical model system for the first time that unmatched allogeneic Treg products show in vitro functionality and short-term engraftment in vivo (<1 week) comparable to autologous Treg products but are then attacked and reduced within 3 weeks by the allogeneic peripheral blood mononuclear cells (PBMC), containing alloreactive effector T cells and lost their immunosuppressive capacity in the human skin xenograft model system (reference 8, Fig. 1 ,2).

[0109] These data clearly demonstrate that allogeneic HLA-unmatched Treg are also immunogenic and lose long-term efficacy because of rejection). If lower (suboptimal) counts of Treg cells are adoptively transferred, the difference is even clearer.

[0110] Depletion of all CD8+ but not of CD4+ T cells or CD56+ NK cells within the PBMC almost rescued the immunosuppressive potency of HLA-unmatched Treg, suggesting T-cell mediated killing of the transferred Treg product as cause of the low performance of allogeneic Treg. However, this is not really an option for a safe therapeutic strategy. As mentioned above, HLA-matching strategy is hardly to realise for clinical practice and was also only partly successful (ref.8).

[0111] Therefore, as described for conventional T-cell products, the inventors designed “stealth” Treg by knocking-out polymorphic HLA class I and II molecules by CRISPR / Cas-mediated gene- / base-editing. To avoid the need for 12 gene edits (HLA class I: A, B, C and HLA class II: DR, DP, DQ each for both haplotypes), the approach specifically knocked out two key genes for the expression of HLA class I and class II by base editing - the monomorphic beta chain (B2-microglobulin, B2M) and the Transactivator of the major histocompatibility complex of class II (CIITA), respectively. As B2-microglobulin-edited T cells were shown to be targeted by NK cells via missing-self activation (reference 6), a gene editing strategy was established to introduce the NK-cell inhibitory receptor HLA-E into Tregs and knocking-in HLA-E to deliver a “self signal for preventing NK-mediated lysis. While HLA-E only partially protected from NK-cell lysis in vitro (reference 5), the combination of knock-out of HLA-class I and class II with parallel knock-in of HLA-E completely rescued the potency of HLA-unmatched allogeneic Treg in vivo (reference 8, Fig. 3).

[0112] As multiple gene modifications (two times knock-out, one times knock-in) to achieve hypoimmunogenic “stealth” Tregs, the inventors performed separate transfection for knock-in and knock-out. For the latter 2ndstep, the inventors employed a base editing technology using a catalytically modified Cas9 variant to reduce the risk of translocations by multiple gene edits (reference 8).

[0113] Allogeneic off-the-shelf Treg products will significantly improve the broad feasibility of Treg therapy in clinical practice for reasons of logistics, cost, effectiveness and safety.

[0114] In contrast to the assumption that Treg could be immune-privileged, a recent study (ref. 8) showed that Tregs do possess alloimmunogenicity, which limits their use as HLA-unmatched off-the-shelf products.

[0115] The selection of Treg donors and Treg recipients through almost complete conventional or advanced HLA matching as applied in HSCT or kidney transplantation is practically not feasible.

[0116] Only the combination of knock-out of HLA-class I (H2M) and HLA-class II (CIITA) and knock-in of HLA- E generates “stealth” Treg that show comparable efficacy to autologous Treg in a clinically relevant humanised mouse model.

[0117] Considering that additional molecular modifications of Treg products are also required to redirect specificity (e.g. CAR expression), improve survival / fitness (release of growth factors, resistance to reprogramming by inflammation), achieve targeted tissue adaptation, etc., the above-mentioned "stealth" strategy is less suitable for safe and effective translation into off-the-shelf clinical Treg products. Moreover, the complete “stealth” Treg would not be recognized and controlled in case of viral infection or transformation to a malign cell - so the transfer of complete “stealth” Treg is associated with a high risk profile.

[0118] The invention thus provides a multiplex strategy to combat the immunogenicity problem in the development of allogeneic 'off-the-shelf' regulatory T cell (Treg) products and provided a significantly safer cell therapy product.

[0119] The aim of the approach is, on the one hand, to avoid an additional knock-in (HLA-E or similar) for the self-signal that is omitted in the traditional procedure of B2-microglobulin knock-out, as gene insertions (knock-in) in particular increase the risk of genotoxicity, and, on the other hand, to maintain the presentation of viral or tumour antigens in the manipulated T-cell products, which also has a safety advantage. The multiplex concept, which is based on three pillars, was developed for this purpose:

[0120] 1) Partial (Allo)hypoimmunogenicity:

[0121] Partial reduction of alloimmunogenicity through knock-out of CIITA (HLA class II) and only one or two of the three HLA class I gene loci (e.g. each HLA-A, -B, -C alone, or a combination of HLA-A / -B, HLA- A / -C, HLA-B / -C (Fig.4A)

[0122] 2) Feasible Treg-Donor / -Recipient Matching:

[0123] The reduction to the expression of only one or two instead of three HLA-class I polymorphic molecule groups (HLA-A, -B, -C) enables a smarter and feasible matching of Treg cell donors and recipients by using HLA-homozygous donors and / or available or novel typing procedures (Fig.4B)

[0124] 3) Temporary co-administration of immunosuppression:

[0125] To overcome residual minimal allo-immunogenicity after applying pillar 1 and / or 2, temporary immunosuppression after cell application should be used to suppress a possible allo-response against residual immunogenic structures.

[0126] As immunosuppressive drugs are possible each alone or in combination: tacrolimus or derivates, cyclosporin or derivates, mTOR inhibitors, corticosteroids, kinase inhibitors, monoclonal antibodies targeting specific molecules shared by Treg and conventional T cells.

[0127] As those immunosuppressants inhibit the survival and function of the Treg, off-the-shelf' Treg are used that are resistant to these immunosuppressants through additional stable or transient genetic modification such as - but not limited to - knock-out, silencing or mutagenesis of drug target molecules, such as - but not limited to - FKBP12 (resistance to tacrolimus and mTOR inhibitors), calcineurin (resistance to cyclosporin and derivatives), and / or steroid receptor genes (Fig.4C).

[0128] The system is applicable for any Treg product used as allogeneic off-the-shelf therapeutics, such as:

[0129] 1stgeneration polyclonal Treg products (polyclonal endogenous T-cell receptor repertoire)

[0130] Next generation Treg products with redirected specificity by expression of chimeric antigen receptors (CARs) Next generation Treg products with redirected specificity by expression of transgenic T-cell receptors (tgTCRs)

[0131] Next-generation Treg products genetically manipulated to express improved survival, function, engraftment, homing properties

[0132] Next-generation Treg products manipulated by RNA technologies to express improved survival, function, engraftment, homing properties

[0133] The off-the-shelf Treg can be generated from peripheral blood (ref. 7), thymus (ref. 8), or iPSC (ref. 9)

[0134] Any combinations of the opportunities described above

[0135] The advantages of the three-pillar concept compared to conventional “stealth” concepts are:

[0136] • Reduction of the risk of genotoxicity by avoiding the need for knock-in of a self-signal (e.g. HLA- E) that is necessary in case of conventional 1 2-microglobulin knock-out in order to prevent NK cell activation (pillar 1)

[0137] • Safety advantage regarding malignant transformation or viral infection by maintenance of immunological monitoring and control through retained antigen processing and presentation in transferred Treg products (pillar 1)

[0138] • Increased feasibility of matching patients to be treated with Treg with relatively well-matched off-the-shelf allogeneic Treg products from the cell bank by reducing differences in the polymorphic and polygenic HLA system (pillar 2)

[0139] • Possibility of temporary immunosuppression (with common immunosuppressive agents, such as tacrolimus and / or steroids or others) to block an immune response against residual immunogenicity despite molecular editing and matching without affecting the survival and function of off-the-shelf Treg products (pillar 3)

[0140] Example 2:

[0141] The problem of a total knock-out strategy, an approach currently followed by many groups, is the total loss of immune control of those “stealth” cells - a safety issue in case of malign transformation or infection as no tumor or viral antigens / peptids can be presented by those “stealth” cells.

[0142] This is confirmed by the following experiments:

[0143] Loss of control of viral infections in stealth Treg but not in “partial stealth” Treg with residual HLA-I

[0144] As proof of concept (PoC) experiment for testing immune surveillance capacity, we pulsed the “stealth” Treg (knock-out of HLA- class I (KO of B2MG) and HLA-class II (KO of CIITA) with or without coexpression of HLA-E with cytomegalovirus (CMV)-derived key peptide pools and compared the response to “partial stealth” (one HLA class I KO only with or without HLA class II KO) and unmodified Treg (Table 1).

[0145] Table 1 Loss of control of viral infections in stealth Teg but not “partial stealth” Treg

[0146] Treg population Treg pulsed overnight % (+ / -SEM) intracellular IFN-y + with CMV-IE protein I pp65 CMV-specific conventional T protein-spanning peptide pool cells by flow cytometry CD8+ subset CD4+ subset unmodified Treg no 0.2 % (0.2) 0.05 % (0.05) unmodified Treg yes 26.8 % (2.5) 13.5 % (2.0)

[0147] “stealth” HLA-E- Treg no 0.05 % (0.03) 0.01 % (0.01)

[0148] “stealth” HLA-E- Treg yes 0.1 % (0.1) 0.01 % (0.01)

[0149] “stealth” HLA-E+ Treg no 0.1 % (0.1) 0.01 % (0.01)

[0150] “stealth” HLA-E+ Treg yes 0,4 % (0.2) 0.01 % (0.01)

[0151] Partial “stealth” Treg no 0.1 % (0.06) 0.01 % (0.01)

[0152] (HLA-A+, C+, B- class II -)

[0153] Partial “stealth Treg yes 18.5 % (2.0) 0.02 % (0.01)

[0154] (HLA-A+, C+, B- class II -)

[0155] Partial “stealth” Treg no 0.1 % (0.06) 0.02 % (0.01)

[0156] (HLA-A+, C+, B- class II +)

[0157] Partial “stealth Treg yes 21.4 % (2.3) 11.1 % (2.4)

[0158] (HLA-A+, C+, B- class II +)

[0159] Explanation: CMV-specific conventional T cells were enriched as shown before (Brestrich et al. Am J Transplant. 2009 Jul;9(7): 1679-84.) Bunde et al. JExp Med. 2005 Apr 4;201(7):1031-6.). Briefly, Tcells were stimulated by IE-1 / pp65 CMV protein-spanning overlapping peptide pools and IFNg secreting T cells were isolated after overnight stimulation. After 2-3 days of resting, T cells were re-stimulated with Treg populations pulsed with CMV IE- 1 / pp65 peptide pools. Intracellular IFNg expression was measured by flow cytometry in % of all gated living responder T cells.

[0160] In summary, we could demonstrate in the PoC experiment that “stealth” Treg with missing HLA-class I and class II cannot present viral peptides independent on the co-expression of HLA-E. In contrast, “partial stealth” Treg with residual HLA class I expression (e.g. HLA-A and -C) are controllable by conventional virus-specific CD8+ T cells and in case of expression of HLA class II, also by virus-specific CD4+ T cells.

[0161] Therefore, module 1 of our allo-Treg strategy is based on hypoimmunogenic (partial stealth) Treg expressing only 1-2 HLA gene loci by knock-out of the B-locus (HLA-B - / -), only (resulting in HLA-A+C+ B- Treg) or A-locus only (resulting in HLA-B+C+A- Treg) with or without additional knock-out of HLA- class II (targeting or non-targeting CIITA).

[0162] “Partial stealth” Treg with residual HLA-I show still some alloimmunogenicity

[0163] Allogeneic “partial stealth” (one HLA class I KO with / without HLA class II KO) Treg cells keep their immune surveillance capability (see above) but might be still recognized by the host's immune system resulting in reduced survival and functionality as they can express up to 4 HLA class I mismatches and up to 6 HLA-class II mismatches. We could confirm this using an in vitro human co-culture model (see Tab. 2). Concomitant immunosuppression overcomes alloimmunogenity of Treg

[0164] Therefore, we add a second module to our allo-Treg approach, the combination of off-the-shelf Treg therapy with temporary immunosuppression. To maintain the survival and functionality of the Treg in presence of immunosuppression, we used immunosuppressive drug-resistant Treg induced by knockout of the intracellular target protein of the immunosuppressants. As PoC we used gene-edited FKBP- 12 (binding molecule of the immunosuppressant tacrolimus) - knockout Treg that are resistant to one of the most powerful immunosuppressants, tacrolimus (TregTacRes). Our data demonstrate the advantage of a combination of hypoimmunogenicity (partial stealth) and temporary tacrolimus exposition if TregTacRes are used (Tab2).

[0165] Table 2 Alloimmunogenicity of Treg cells are abolished by temporary tacrolimus exposition

[0166] (R+ / ++ intermediate to strong allorejection of Treg; R- no allorejection,

[0167] Tac+ inhibition by tacrolimus, Tac- no inhibition)

[0168] Treg population Conventional Ratio Treg / Tconv (+ / -SEM)

[0169] T cells d14 - Tac d14 +Tac

[0170] Non-modified Treg:

[0171] Treg donorl Tconv donor 1 0.8 (0.1) R- 0.4 (0.2) R-Tac+

[0172] Treg donor 2 Tconv donor 2 0.9 (0.1) R- 0.4 (0.1) R-Tac+

[0173] Treg donor 2 T conv donor 1 0.1 (0.2) R++ 0.25 (0.2) R+Tac+

[0174] Treg donor 1 T conv donor 2 0.2 (0.1) R++ 0.3 (0.2) R+Tac+

[0175] Partial stealth Treg (HLA-A+C+B-, class II-)

[0176] Partial stealth Treg donor 2 0.4 (0.2) R+ 0.5 (0.2) R-Tac+

[0177] Partial stealth Treg donor 1 Tconv donor 2 0.45 (0.1) R+ 0.55 (0.2) R-Tac+

[0178] Partial stealth TregTacRes donor 2 Tconv donor 1 0.4 (0.2) R+ 0.9 (0.2) R-Tac=

[0179] Partial stealth TregTacRes donor 1 Tconv donor 2 0.5 (0.1) R+ 0.9 (0.1) R-Tac=

[0180] Partial stealth Treg (HLA-A+C+class 11+)

[0181] Partial stealth Treg donor 2 0.3 (0.2) R+ / ++ 0.5 (0.2) R-Tac+

[0182] Partial stealth Treg donor 1 Tconv donor 2 0.3 (0.1) R+ / ++ 0.5 (0.2) R-Tac+

[0183] Partial stealth TregTacRes donor 2 Tconv donor 1 0.3 (0.2) R+ / ++ 0.85 (0.2) R-Tac-

[0184] Partial stealth TregTacRes donor 1 Tconv donor 2 0.3 (0.1) R+ / ++ 0.8 (0.2) R-Tac=

[0185] Design: 1 Mill enriched and expanded Treg (non-modified vs. partial stealth vs. partial stealth + TacRes) were co-cultured with 1 Mill autologous or mismatched allogeneic conventional T cells (Tconv) for 14 days in G-Rex cultures devices in presence of IL-2. To a part of the cultures, tacrolimus ( 10 pg / ml) was added to the culture from d0-d7. The Treg / Tconv ratio was measured by flow cytometric analysis.

[0186] In summary, the data demonstrate that conventional T cells (Tconv) reduce the number of surviving non-modified allogeneic Treg cells in the co-culture. If Tacrolimus was added for the 1stweek of coculture, the reduction of Treg counts was less pronounced (because of inhibition of Tconv), however, did not achieve comparable number as in autologous controls. This partial recovery is due only to the strong inhibitory effects of tacrolimus on survival and function of Treg in autologous co-cultures (Tac+). Although hypoimmunogenic “partial stealth” Treg with or without HLA-class II expression showed a reduced elimination (R+ / ++ or R+, respectively)) compared to unmodified Treg (R++), tacrolimus addition did not improve the survival data because of its direct inhibitory effects (Tac+)

[0187] In contrast, the combination hypoimmunogenic “partial stealth” TregTacRes (knock-out of FK-binding protein 12 - FKBP12 - / -) were able to escape immune elimination by allogeneic conventional T cells in presence of tacrolimus and showed resistance to the inhibitory effects of tacrolimus as well. The presence or absence of HLA-class II had only minor impact.

[0188] Remarkably, Treg with resistance to tacrolimus (TregTacRes) but normal HLA expression showed only intermediate results (50% loss if tacrolimus was added) showing that after weaning of tacrolimus (day8) Tconv recover from immunosuppression and partly eliminate the fully immunogenic Treg within the second week of culture. If tacrolimus is kept in the culture for 14 days, no rejection is observed (not shown).

[0189] These data demonstrate advantage of our novel combinatory approach of drug-resistance + hypoimmunogenicity by gene-editing to allow an off-the-shelf allogeneic Treg therapy. In vivo, however, the use of only one module (drug-resistance or hypoimmunogenicity) might be sufficient for the therapeutic efficacy.

[0190] Cited references:

[0191] 1) Genetics, Human Major Histocompatibility Complex (MHC). Gizem Turner, Brittany Simpson, Tiffany K. Roberts. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan. 2023 Aug 14. PMID: 30855806

[0192] 2) Base-Edited CAR7 T Cells for Relapsed T-Cell Acute Lymphoblastic Leukemia.

[0193] 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 3) Multiplex Genome-Edited T-cell Manufacturing Platform for "Off-the-Shelf" Adoptive T-cell Immunotherapies. Poirot L, Philip B, Schiffer-Mannioui C, Le Clerre D, Chion-Sotinel I, Derniame S, Potrel P, Bas C, Lemaire L, Gaietto R, Lebuhotel C, Eyquem J, Cheung GW, Dueled 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

[0194] 4) New hope for tumor immunotherapy: the macrophage-related “do not eat 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

[0195] 5) Class II HLA epitope matching — A strategy to minimize de novo donor-specific antibody development and improve outcomes. C Wiebe 1 , D Pochinco, T D Blydt-Hansen, J Ho, P E Birk, M Karpinski, A Goldberg, L J Storsley, I W Gibson, D N Rush, P W Nickerson (2013). American Journal of Transplantation, Dec;13(12):3114-22. doi: 10.1111 / ajt.12478. Epub 2013 Oct 25

[0196] 6) Hidden Patterns of Anti-HLA Class I Alloreactivity Revealed Through Machine Learning. Angeliki G. Vittoraki, Asimina Fylaktou, Katerina Tarassi, Zafeiris Tsinaris , Alexandra Siorenta, George Ch. Petasis, Demetris Gerogiannis, Claudia Lehmann, Maryvonnick Carmagnat, llias Doxiadis, Aliki G. Iniotaki and loannis Theodorou (2021) Front. Immunol. 12:670956. doi: 10.3389 / fimmu.2021 .670956.

[0197] 7) PIRCHE-II: an algorithm to predict indirectly recognizable HLA epitopes in solid organ transplantationGeneugelijk, K., & Spierings, E. (2020). Immunogenetics 72:119— 129https: / / doi.org / 10.1007 / s00251 -019-01140-x

[0198] 8) Matching or genetic engineering of HLA Class I and II facilitates successful allogeneic ‘off-the- shelf’ regulatory T cell therapy.

[0199] Oliver McCallion, et al. bioRxiv https: / / doi.org / 10.1101 / 2023.08.06.551956.

[0200] 9) Targeted disruption of HLA genes via CRISPR-Cas9 generates IPSCs with 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).

[0201] 10) Regulatory T cells for minimising immune suppression in kidney transplantation: phase l / lla clinical trial. Roemhild A, Otto NM, Moll G, Abou-EI-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, Stervbo U, Babel N, Volk HD, Reinke P.

[0202] BMJ. 2020 Oct 21 ;371 :m3734. doi: 10.1136 / bmj.m3734. PMID: 33087345

[0203] 11) A Novel GMP Protocol to Produce High-Quality Treg Cells from the Pediatric Thymic Tissue to Be Employed as Cellular Therapy. Bernaldo-de-Quiros E, Cozar B, Lopez-Esteban R, Clemente M, Gil-Jaurena JM, Pardo C, Pita A, Perez-Caballero R, Camino M, Gil N, Fernandez-Santos ME, Suarez S, Pion M, Martinez-Bonet M, Correa-Rocha R. Front Immunol. 2022 May 16;13:893576. doi: 10.3389 / fimmu.2022.893576. eCollection 2022. PMID: 35651624 12) A Serum- and Feeder-Free System to Generate CD4 and Regulatory T Cells from Human iPSCs. Helen Fong, Matthew Mendel, John Jascur, Laeya Najmi, Ken Kim, Garrett Lew, Swetha Garimalla, Suruchi Schock, Jing Hu, Andres Villegas, Anthony Conway, Jason D. Fontenot, Simona Zompi. bioRxiv 2024. doi: https: / / doi.org / 10.1101 / 2023.07.01.547333 13) Amini et al, Mol Ther 2021 . 29(1): 32-46.

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[0206] All scientific publications and patent documents cited in the present specification are incorporated by reference herein.

Claims

Claims1 . A gene-modified T regulatory cell characterised by genetic silencing of one, or at most two of the HLA class I gene loci HLA-A, HLA-B, and HLA-C, wherein at least one of the HLA class I gene loci HLA-A, HLA-B, and HLA-C is expressed.

2. The gene-modified T regulatory cell according to claim 1 , wherein the gene-modified regulatory T cell is characterised by genetic silencing of the transactivator of the major histocompatibility complex of class II gene (CIITA).

3. The gene-modified T regulatory cell according to claim 1 , wherein the gene-modified regulatory T cell is characterised by expression of the transactivator of the major histocompatibility complex4. The gene-modified T regulatory cell according to any one of claims 1 to 3, wherein the gene- modified regulatory T cell is characterised by genetic silencing of the HLA-A and HLA-B gene loci.

5. The gene-modified T regulatory cell according to any one of claims 1 to 3, wherein the gene- modified regulatory T cell is characterised by genetic silencing of the HLA-A gene locus.

6. The gene-modified T regulatory cell according to any one of claims 1 to 3, wherein the gene- modified regulatory T cell is characterised by genetic silencing of the HLA-B gene locus.

7. The gene-modified T regulatory cell according to any one of the preceding claims, wherein the gene-modified regulatory T cell is resistant to an immunosuppressive drug.

8. The gene-modified T regulatory cell according to any one of the preceding claims, wherein the gene-modified regulatory T cell is resistant to tacrolimus and / or an mTOR inhibitor.

9. The gene-modified T regulatory cell according to any one of the preceding claims, wherein the gene-modified regulatory T cell is resistant to cyclosporin.

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

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

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

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

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

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

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

17. The gene-modified T regulatory cell according to any one of the preceding claims, wherein the cell is characterized by wildtype expression of the gene encoding human beta-2-microglobulin.

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

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

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

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

22. A gene-modified cell according to any of the claims 1 to 21 , for use in a subject who is receiving concurrent treatment with (or has received in the week prior to treatment with, or week following) an immunosuppressive drug.

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

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

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

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

27. A combination medicament comprising a gene-modified T regulatory cell according to any one of the claims 1 to 14, and an immunosuppressive drug, particularly wherein the immunosuppressive drug is selected from the group of tacrolimus, cyclosporin, an mTOR inhibitor, a corticosteroid, a kinase inhibitor, and / or a T-cell depleting monoclonal antibody, more particularly wherein the immunosuppressive drug is tacrolimus and / or cyclosporin.

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