MODIFIED HUMAN T CELL WITH A REDUCED HLA-Ia REPERTOIRE AND METHOD OF PRODUCTION

Abstract

The present invention provides a method of producing a modified human T cell for cell therapy of a given recipient from a T cell obtained from a given donor, wherein the donor has at least one HLA-la allele that does not match an HLA-la allele of the recipient, wherein the method comprises inhibiting a cell surface expression of at least one HLA-la allele of the T cell obtained from the donor that does not match the H LA-la allele of the recipient, wherein the modified human T cell expresses beta-2-microglobulin on the cell surface. The present invention also provides a modified human T cell for cell therapy of a given recipient, wherein an expression of at least one HLA-la allele is inhibited, wherein said at least one allele of a class HLA-la does not match an HLA-allele of the recipient, wherein the expression of beta-2-microglobulin has not been modified.

Description

[0001] MODIFIED HUMAN T CELL WITH A REDUCED HLA-la REPERTOIRE AND METHOD OF PRODUCTION

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims the benefit of priority of European Patent Application EP 24170951 .8 filed on 18 April 2024, the content of which is hereby incorporated by reference in its entirety for all purposes.

[0004] TECHNICAL FIELD

[0005] The present invention provides a method of producing a modified human T cell for cell therapy of a given recipient from a T cell obtained from a given donor, wherein the donor has at least one HLA-la allele that does not match an HLA-la allele of the recipient, wherein the method comprises inhibiting a cell surface expression of at least one HLA-la allele of the T cell obtained from the donor that does not match the HLA- la allele of the recipient, wherein the modified human T cell expresses beta-2- microglobulin on the cell surface, and wherein the human T cell from the donor is preferably a primary T cell, comprising inhibiting a cell surface expression of at least one HLA-la allele of an isolated human T cell, wherein the modified human T cell expresses beta-2-microglobulin on the cell surface, and wherein the isolated human T cell is preferably an isolated human primary T cell. The present invention further provides a method of generating a human T cell population for allogeneic cellular therapy, wherein the method comprises, in case a donor has at least one HLA-la allele that does not match an HLA-la allele of a recipient, genetically modifying the T cell population to inhibit cell surface expression of at least one HLA-la allele of the isolated T cell population of the donor, wherein the modified human T cell expresses beta-2- microglobulin on the cell surface, and a modified human T cell obtainable or obtained by the methods. The present invention also provides a modified human T cell for allogeneic cell therapy of a given recipient, wherein an expression of at least one HLA- la allele is inhibited, wherein said at least one HLA-la allele does not match an HLA- allele of the recipient, wherein the expression of beta-2-microglobulin has not been modified, and wherein the modified human T cell is preferably a modified human primary T cell. The invention further provides the modified human T cell for use in an adoptive cell therapy or in a cellular therapy of a human subject in need thereof by allogenic T cell transfer, wherein those therapies could be a treatment of cancer, an autoimmune disease, or an infectious disease. The present invention further provides a pharmaceutical composition comprising the modified human T cell.

[0006] BACKGROUND

[0007] The adoptive transfer of T cells is a powerful treatment option for cancer, infections, and autoimmune diseases. However, polymorphic human leukocyte antigens (HLA) of donor T cells and recipient need to be matched in order to prevent rejection of transferred T cells.

[0008] Generating autologous T cell products or donor registries is time-, labor- and costintensive. Therefore, several approaches have been proposed to generate “universal” or at least broadly applicable allogeneic donor cells. This encompassed knocking out (KO) HLA class I and II through targeting of beta-2-microglobulin (Abbr.: |32M) (Gene: B2M) and the transcription factor CIITA, respectively.

[0009] While KO of HLA prevents rejection through allogeneic T cells, NK cells can recognize such cells through “missing self” and promote their lysis. To reverse the KO of HLA-E, a fusion construct of HLA-E and beta-2-microglobulin was knocked in a T cell with KO of beta-2-microglobulin. These multiple genomic alterations prevent these cells from lysis through NK cells. However, such additional editing steps complicate the generation process of cellular products that are intended for clinical application.

[0010] Therefore, allogenic modified human T cells are needed which are not rejected by T cells or NK cells of the recipient.

[0011] SUMMARY OF INVENTION

[0012] This object is accomplished, inter alia, by the modified human T cells and the method for producing modified human T cells having the features of the independent claims. According to a first aspect, the invention provides a method of producing a modified human T cell for cell therapy of a given recipient from a T cell obtained from a given donor, wherein the donor has at least one HLA-la allele that does not match an HLA- la allele of the recipient, wherein the method comprises inhibiting a cell surface expression of at least one HLA-la allele of the T cell obtained from the donor that does not match the HLA-la allele of the recipient, wherein the modified human T cell expresses beta-2-microglobulin on the cell surface, and wherein the human T cell from the donor is preferably a primary T cell.

[0013] According to a second aspect, the invention provides a method of generating a human T cell population for allogeneic cellular therapy, wherein the method comprises, in case a donor has at least one HLA-la allele that does not match an HLA-la allele of a recipient, genetically modifying the T cell population to inhibit cell surface expression of at least one HLA-la allele of the isolated T cell population of the donor, wherein the modified human T cell expresses beta-2-microglobulin on the cell surface.

[0014] According to a third aspect, the invention provides a modified human primary T cell obtainable or obtained by the method mentioned above.

[0015] According to a fourth aspect, the invention provides a modified human T cell for allogeneic cell therapy of a given recipient, wherein an expression of at least one HLA- la allele is inhibited, wherein said at least one HLA-la allele does not match an HLA-la allele of the recipient, wherein the expression of beta-2-microglobulin has not been modified, and wherein the modified human T cell is preferably a modified human primary T cell.

[0016] According to a fifth aspect, the invention provides a pharmaceutical composition comprising the modified human T cell of the third or fourth aspect or the modified T cell obtained by the method of the first or second aspect for use in therapy.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Generation of HLA KO primary human T cells with reduced allogeneic recognition. a, Flow-cytometric analysis of b2rrr-T cells (gated on living lymphocytes) from B2M gRNA edited (left) and unedited (right) peripheral blood mononuclear cells of a healthy donor, b, Discordance plot as generated by ICE analysis of a b2m-edited sample (blue) in comparison to mock edited cells (grey, control). Discordance refers to the extent of disagreement between the wild type and edited sample at each base within a defined inference window (black line), c, Percentage of successful HLA class I KO as detected by flow cytometry (FACS) and in ICE analysis after sequencing (DNA) (n=6 technical replicates), d, Percentage of CD69+CD137+CD8+effector cells after 48h of co-culture with indicated target cells (n=3 technical replicates). Effector cells were peripheral mononuclear blood cells from donor A, co-cultured for 7 days together with PBMCs from donor B (allo priming). Target cells were autologous unedited cells from donor A (HLA I match), allogeneic B2M KO cells (no HLA I), and allogeneic unedited cells (HLA I mismatch). Target cells were sorted for CD3+and successful KO if applicable.

[0018] Figure 2 Generation of HLA KO primary human T cells with reduced allogeneic recognition. a, Flow-cytometric analysis of CIITA KO T cells (gated on living CD8+ lymphocytes) from CIITA gRNA edited (left) and unedited (right) peripheral blood mononuclear cells of a healthy donor, b, Percentage of HLA class II KO as detected by flow cytometry (FACS) and by ICE analysis after sequencing (DNA) (n=2 or 4 technical replicates, respectively), c, Flow-cytometric analysis of B2M KO targets for mixed lymphocyte reaction (MLR) assay (gated on living lymphocytes) after electroporation with indicated gRNAs prior to cell sorting, d, as in c, now after cell sorting prior to MLR assay in case of B2M KO cells (WT cells were not sorted).

[0019] Figure 3 Intrinsic in vitro functionality of HLA class I and II deficient primary human T cells (I) a, Representative flow-cytometric intracellular cytokine staining of IFNy and TNFa in response to no antigen, increasing amounts of antigen or phorbol 12-myristate 13- acetate (PMA) and ionomycin; human peripheral blood mononuclear cells as effector cells underwent orthotopic TOR replacement with CMV NLV-specific TOR 6-2 (TOR KI) and were simultaneously edited with B2M gRNA (b2m KO) or CIITA gRNA (CIITA KO); K562 cells loaded with NLV-peptide pp65495-503 as antigen were used for stimulation, b, Quantification of data from a (n=2-3 technical replicates, mean with SD), x-axis showing logarithmic molar peptide concentration; neg, negative control.

[0020] Figure 4 Intrinsic in vitro functionality of HLA class I and II deficient primary human T cells (II) a, Killing of target cells (HepG2 cells pulsed with NLV-peptide PP65495-503) by HLA class II KO T cells as measured through changes in cell index over time (left panel) in an xCELLigence assay and quantified as the area under the curve (right panel) calculated over the entire time period shown; addition of effector cells indicated by dashed line; positive control of target cell lysis achieved through addition of detergent Triton-X; negative control (neg. Ctrl.) shows uninhibited target cell growth through absence of effector cells; mock edited effector cells were used as control to show non-specific effect on target cell growth through superseding; CD8+ T cells from a and b were used as effector cells and sorted for successful editing (CD8+hTCR’ mTRBC+b2rrr / Cl ITA- ); statistical testing by ordinary one-way ANOVA and Tukey’s multiple comparisons test), n=3 technical replicates, mean with SD; ns, not significant; **** P<0.0001. b, As in a with HLA class I KO T cells as investigated effector cells.

[0021] Figure 5 Generation of HLA KO primary human T cells with orthotopic T cell receptor replacement for subsequent functionality testing

[0022] Flow cytometric analysis of primary human T cells after CRISPR / Cas9 editing and cell sorting for CD8+hTCR’ mTRBC+pregated on CD8+(left) or CD8+hTCR’ mTRBC+(right) cells; all conditions received orthotopic T cell receptor replacement with TCR 6- 2 and either no additional editing (“TCR KI only”) or knocked out endogenous B2M (“B2M KO”) or CIITA (“CIITA KO”), unedited cells shown for comparison (“mock”).

[0023] Figure 6 NK cell recognition of HLA class I deficient primary human T cells a, Experimental setup for NK cell recognition assay, b, Representative flow cytometric analysis of CD107a+NK cells (percentage of living CD56+CD8’ lymphocytes) after 5h of co-incubation with indicated target T cells, K562 cells or PMA / lono; cells were sorted for successful editing, c, Quantification of data shown in b; n=3 technical replicates; statistical testing was done using an unpaired two-tailed t-test and results are only shown for comparison of WT and B2M KO T cells; mean with SD; ***, P< 0.001 . Figure 7 Intrinsic in vivo functionality of HLA class I deficient primary human T cells a, Experimental setup for in vivo transfer of antigen-specific HLA-edited primary human T cells; effector cells were administered i.p. into NSG / HHD HLA-A*02 mice after irradiation with 2 Gy; the next day the mice were infected i.p. with mCMV-NLV; CD8+ T cells used as effector cells all underwent orthotopic TOR replacement with TCR 6-2 (TCR KI) and were simultaneously edited with B2M gRNA (b2m KO) and sorted for successful editing, b, Representative flow cytometric analysis of b2m and transgenic TCR (mTRBC) expression (as percentage of hCD8+) of primary human CD45+ T cells (blue) recovered in the liver seven days after in vivo transfer of TCR KI cells into mice. All living murine cells (red) are included for comparison, c, Percentage of HLA class I deficient primary human T cells before in vivo transfer and after recovery in the liver; pooled data from two independent experiments (light blue or purple color barcode) with n=2 mice each; statistical testing by paired two-tailed t-test; ns, not significant.

[0024] Figure 8 Escape of HLA reduced primary human T cells from allogeneic recognition in vitro (I) a, Experimental scheme showing concept of HLA class I reduction through single HLA allele targeting, b, Characterization of 6-1 (A2) KO T cells (in which all HLA class I molecules were targeted except for HLA A2) compared to B2M KO T cells; left: HLA BC’ population pre-gated on living CD8+T cells, second from left: s2m expression of HLA BC’ and HLA BC+populations pre-gated on living CD8+T cells; second from right: HLA A2 and HLA A3 expression pre-gated on HLA BC’ CD8+living T cells; right: s2m expression of HLA BC- subsets as defined in dot plots second from right.

[0025] Figure 9 Escape of HLA reduced primary human T cells from allogeneic recognition in vitro (II) a, Percentage of CD137+CD8+effector cells after 48h of co-culture with indicated target cells. Effector cells were peripheral mononuclear blood cells from indicated donors, co-cultured for 7 days together with PBMCs from donor A (allo priming); donors only share an allele for HLA A2 or HLA A3 as indicated; target cells were T cells from donor A without (WT) or with HLA class I reduction, sorted for CD8+and HLA BC’ and A2’ A3’, A2+A3’ or A2’ A3+; HLA-reduced target cells with HLA BC’A2’A3+(left) or HLA BC’A2+A3’ phenotype respectively signify a synthesized HLA match; statistical testing was done using an ordinary one-way ANOVA and Tukey's multiple comparisons test and results are only shown for selected comparisons; n=2-3 technical replicates; ns, not significant; *, P< 0.05; **, P< 0.01 ; ***, P< 0.001 ; **** P< 0.0001. b, Quantification of CD107a+ NK cells (percentage of living CD56+CD8’ lymphocytes) from three different indicated donors after 5h of co-incubation with indicated target cells; n / a, not performed experimental conditions; statistical testing was done using an ordinary two- way ANOVA and Tukey’s multiple comparisons test and results are only shown for selected comparisons; n=2-3 technical replicates; ns, not significant; **, P< 0.01 ; ***, P< 0.001 ; **** P< 0.0001. c, Expression of HLA E for target populations in b as assessed by flow cytometry; n / a, not performed experimental conditions; statistical testing was done using an ordinary two-way ANOVA and Tukey’s multiple comparisons test and results are only shown for selected comparisons; n=2-3 technical replicates; ns, not significant; *, P< 0.05; **, P< 0.01 ; ***, P< 0.001 ; **** P< 0.0001.

[0026] Figure 10 HLA reduction through cocktails of HLA allele-specific gRNAs a, 50 most frequent HLA class I alleles of European Caucasian population in descending order, with HLA-A*02:01 shown in red (left panel); cumulative coverage of 50 most frequent HLA class I alleles (area under the curve in grey), dashed line indicates cumulative frequency of most common ten HLA class I alleles (right panel), data retrieved from Effenberger et al,. 2019. b, Flow-cytometry histograms showing expression of I3>2m, HLA BC, HLA A2 and HLA A3 of primary human CD8+T cells of an HLA-A*02+A*03+B*41+B*55+C*15+C*16+donor edited with HLA allele-specific gRNAs; editing was performed for individual HLA alleles (e.g. for HLA-A*03 in case of “A03 KO”), for all HLA alleles except one (e.g. for all except HLA-A*03 in case of “6-1 (A03) KO”), for all six alleles (“6x KO”), for B2M (“b2m KO”) or for no target (“mock”), c, d, Quantification of b2m geometric mean fluorescence intensity (MFI; c) or percentage of HLA BC- CD8 T cells (d) for data from b. e, Flow-cytometric analysis showing HLA A2 and HLA A3 expression of CD8+living T cells edited with indicated supposedly allele-specific guides, f, Bar graph showing quantification of data shown in (e), n=2 technical replicates.

[0027] Figure 11 Improved functionality of HLA reduced primary human T cells in the presence of HLA mismatched T and NK cells in vivo a, Experimental setup for in vivo transfer of HLA reduced aCD19-CAR T cells; effector cells from HLA-A*02+donor A (same donor as in Fig. 4) were administered i.v. one day after assessment of humanization of hu-CD34 NSG-SGM3 mice; humanization was performed with cord blood derived hCD34+cells from donor C, which were different from cells of donor A for every HLA class I allele except for HLA-A*02; every three days, blood samples were drawn and analyzed via flow cytometry; CAR T cells used as effector cells all received an aCD19-CAR knock-in into the endogenous TRAC gene locus and were simultaneously edited for all HLA class I alleles except for HLA*02, and were administered directly after electroporation without prior sorting, b, CD19+B cells (percentage of living hCD45+lymphocytes) on day 11 after administration of no cells or aCD19 CAR T cells with unedited or reduced HLA alleles into humanized mice, c, CD19+B cells shown as change in percentage relative to one day prior to administration of no cells or aCD19 CAR T cells with unedited or reduced HLA alleles into humanized mice, d, CD19+B cells (percentage of hCD45+living lymphocytes) recovered in indicated organs on day 14 after aCD19 CAR T cell administration with unedited or reduced HLA alleles into humanized mice; n=2-3 mice, e, Numbers of CD19+ B cells recovered in blood of humanized mice on day 7 after aCD19 CAR T cell administration with unedited or reduced HLA alleles, pooled data from two independent experiments (dot or diamond symbol barcode), normalized to mean of allo CAR in both experiments; statistical testing was done using an unpaired t-test; n=8 mice; *, P<0.05. Bar height indicates mean, error bars indicate SD (c-e).

[0028] Figure 12 Reconstitution of humanized mice

[0029] Reconstituted populations from humanized mice one day prior to injection as determined by flow cytometry; mCD45, murine CD45; hCD45, human CD45.

[0030] Figure 13 Frequency of homozygous HLA class I haplotypes in European Caucasian population

[0031] 50 most frequent homozygous HLA class I haplotypes of European Caucasian population in descending order shown in blue; cumulative coverage of 50 most frequent homozygous HLA class I haplotypes (area under the curve in grey), dashed line indicates cumulative frequency of most common 20 HLA class I haplotypes (right).

[0032] DETAILED DESCRIPTION OF THE INVENTION Nearly all human nucleated cells express on their cell surface a major histocompatibility complex I (MHC I) with which the cell presents protein fragments to CD8+T cells. The MHC I protein complex is built of a membrane bound heavy chain with 3 alpha subunits and a soluble beta-2-microglobulin, the 4thsubunit of MHC I. The term human MHC is used herein synonymously with the term human leukocyte antigen (HLA). The heavy chain of HLA I is encoded by HLA class la genes (canonical HLA) HLA-A, HLA-B, HLA-C, and HLA class lb genes (non-canonical HLA) HLA-E, HLA-F, HLA-G. Several different HLA gene alleles exist that create a polymorphic HLA gene expression product. A human being expresses physiologically six alleles of the HLA- la gene family.

[0033] In contrast, only antigen-presenting cells express a major histocompatibility complex II (MHC II) on their cell surface, with protein fragments presented to CD4+T cells. The MHC II protein complex is built of a cell surface expressed alpha unit, with two alpha subunits, and a cell surface-expressed beta unit, with two beta subunits. The MHC II proteins are encoded by the HLA class II genes HLA-DM, -DO, -DP, -DQ, -DR.

[0034] Just as beta-2-microglobulin (B2M) knock-out (KO) cells, HLA-reduced T cells circumvent allogeneic T-cell recognition in case of HLA mismatches. However, one major drawback of B2M KO cells, which are incapable of expressing HLA I complexes, is that they elicit NK cell-mediated recognition through “missing self”. The inventors of the present application have surprisingly shown that such NK cell-mediated recognition can be avoided in human T cells if the expression of HLA I molecules is only reduced by not completely abolished. While transgenic expression of HLA-E or HLA-G has been proposed to counteract NK-cell reactivity in B2M KO cells, the inventors of the present invention have recognized that HLA reduction without knockout of B2M entails the unique advantage of preserving cellular physiology as much as possible.

[0035] HLA-la reduction, according to the present disclosure, can be achieved by inhibiting the expression of at least one HLA-la allele. Reducing the number of HLA-la alleles reduces HLA mismatches, and cells from fewer donors can be applied to a greater part of the population. It is also a possibility to leave more HLA molecules intact. While more donors are then needed to cover substantial parts of a population, the probability of finding a suitable donor is still much enhanced. For example, calculations with donors that have naturally occurring HLA class I homozygotes for HLA-A, HLA-B, and HLA-C (i.e., donors that are homozygous on HLA-A, HLA-B as well as HLA-C) from German blood donor registries show that 20 donors with the most frequent homozygous HLA class I haplotypes could already cover about two-thirds of the German population (Figure 13). It is preferred that the donor is HLA-A homozygote.

[0036] However, inhibition of more than one HLA allele, such as 2, 3, 4, 5, or even 6 alleles, preferably of HLA la, will further reduce the probability of potential HLA mismatches between donors and recipients. As shown in the present disclosure, the HLA reduction to a single, matching HLA molecule represents the most extreme case for which a match in canonical HLA class I molecules is still achieved. To demonstrate how much this improves the likelihood of finding suitable cell donors, the inventors calculated that HLA reduction to the 10 most prevalent HLA class I molecules would already cover about 80% of the European Caucasian population (Figure 10a).

[0037] Inhibiting all canonical HLA alleles (i.e., HLA la alleles) is also a possible strategy for generating broadly applicable cells, thereby preserving only non-canonical HLA expression. The inventors showed in the Examples that T cells that are negative for HLA-A2, HLA-A3, and HLA-BC prevent both NK cell as well as allogeneic T cell activation just as HLA-reduced T cells do. Fittingly, T cells that are negative for HLA- A2, HLA-A3, and HLA-BC sustain HLA-E expression. However, as shown in Example 5, the inventors observed a correlation between HLA-E and canonical HLA class I expression levels, as HLA-A2+HLA-A3’ HLA-BC’ T cells have higher HLA-E expression levels than HLA-ABC’ T cells (Figure 9c). It is thus believed that robust inhibition of NK cell recognition appears more likely when at least one canonical HLA molecule is preserved. It is also believed that preserving at least one canonical HLA class I molecule represents a safeguard in case engineered T cells are infected or undergo malignant transformation.

[0038] In Example 4, T cells have been HLA-reduced to a single remaining HLA allele by editing 7 different genetic loci (5 HLA class I alleles and two TCR targets; including knock-in into one TCR locus). This underlines the feasibility of highly multiplexed engineering through CRISPR / Cas9. The advantage of such a one-pot approach is that repeated experiments and / or editing steps are not needed, resulting in a more vital modified T cell and a shorter ex vivo cultivation.

[0039] Moreover, as shown in Example 6, the inventors observed enhanced functionality (i.e. , elimination of endogenous B cells) with HLA-reduced compared to HLA-non-reduced allogeneic donor T cells in the presence of allogeneic endogenous T and NK cells. This shows the advantages of the present invention. Without wishing to be bound by theory, it is believed that this enhanced functionality might be due to enhanced T-cell maintenance and / or improved maintenance that is due to escape from T or NK-cell mediated rejection. As of now, the Examples indicate superior functionality of HLA- reduced allogeneic T cells in a preclinical in vivo system, which mimics the actual in vivo situation in patients.

[0040] In summary, the inventors showed herein that HLA reduction is a feasible strategy to circumvent both allogeneic T as well as NK cell-mediated rejection while simultaneously preserving T-cell physiology and canonical HLA class I expression. HLA reduction thereby extends the toolbox of cellular engineering for the therapy of tumor diseases, infections, and autoimmunity.

[0041] The HLA reduction is enabled by the inhibition of the expression of at least one HLA- la allele, thereby reducing the expressed overall HLA repertoire of a given T cell.

[0042] It has been surprisingly found and included in the present application that the reduction of the expression of HLA-la alleles, by inhibiting the expression of at least one HLA-la allele, not only inhibits T-cell recognition in case of HLA mismatches but also inhibits the “missing self” recognition of the modified T cells by natural killer (NK) cells. In this invention, and in contrast to documents of the prior art, the expression of the beta-2- microglobulin of the modified human T cell has not been modified. Accordingly, it is contemplated that the expression of the beta-2-microglobulin is, in general, unaltered compared to an unmodified human T cell. As mentioned before, the beta-2- microglobulin is part of the HLA (MHC) class I complex expressed on the cell surface. By leaving the beta-2-microglobulin expression intact, the modified human T cell could at least express genes of the HLA-lb class.

[0043] The aim of the invention is the use of T cells of a given donor in personalized medicine. Therefore, the expression of HLA-la alleles of the donor T cells that do not match with the HLA-la alleles of a recipient could be inhibited using the inventive method. Thereby, the expression of beta-2-microglobulin is not inhibited. Thus, it is not the aim of the invention to create a universal T cell but to produce a modified human T cell which HLA-la alleles have ideally a complete match with the HLA-la alleles of the individual recipient.

[0044] Thus, the invention is directed in a first aspect to a method of producing a modified human T cell for cell therapy of a given recipient from a T cell obtained from a given donor, wherein the donor has at least one HLA-la allele that does not match an HLA- la allele of the recipient, wherein the method comprises inhibiting a cell surface expression of at least one HLA-la allele of the T cell obtained from the donor that does not match the HLA-la allele of the recipient, wherein the modified human T cell expresses beta-2-microglobulin on the cell surface, and wherein the isolated human T cell is preferably an isolated human primary T cell. Generally, the T cell obtained from the donor can be any type of T cell, including a primary T cell or a T cell derived from an iPSC. Primary T cells are however preferred. Also, primary NK cells or induced pluripotent stem cell (iPSC) derived NK or T cells could be used in the method.

[0045] In a second aspect, the invention is directed to a method of generating a human T cell population for allogeneic cellular therapy, wherein the method comprises, in case a donor has at least one HLA-la allele that does not match an HLA-la allele of a recipient, genetically modifying the T cell population to inhibit cell surface expression of at least one HLA-la allele of the isolated T cell population of the donor, wherein the modified human T cell expresses beta-2-microglobulin on the cell surface. Also, primary NK cells or induced pluripotent stem cell (iPSC) derived NK or T cells could be used in the method.

[0046] Regarding the second aspect, said donor may has at least one HLA-la allele that does not match an HLA-la allele of the recipient. Generally, the human T cell from the donor can be any type of T cell, including a primary T cell or a T cell derived from an iPSC. Preferably the human T cell from the donor is a primary T cell.

[0047] Regarding the first and the second aspect, it is preferred that the expression of one HLA-A allele and all HLA-B and HLA-C alleles of the T cell is inhibited and that the not inhibited HLA-A allele match with one HLA-A allele of the recipient. It is also preferred that the HLA-A alleles of the donor are homozygote and match with at leas tone HLA- A allele of the recipient and that (in this case) the expression of all HLA-B and HLA-C alleles is inhibited.

[0048] The following explanations and embodiments are valid for the first and second embodiment, unless the wording explicit directs otherwise.

[0049] The term “match” in the context of HLA alleles is defined herein as a consensus between the given HLA-la allele of the donor and the given HLA-la allele of the recipient. For example, if the donor has the HLA-la allele of the donor is the HLA-A*02- 01 allele, then a match is given if the recipient has also the HLA-A*02-01 allele.

[0050] Vice versa, the term “mismatch” is defined herein as a non-consensus between the given HLA-la allele of the donor and the recipient, e.g. if the donor has an HLA-A*02- 01 allele and the recipient has another HLA-A allele which is not HLA-A+02-01 .

[0051] The at least one HLA-la allele is selected from the HLA-la gene family comprising HLA- A, HLA-B, and HLA-C. At least one HLA-la allele, in this context, can, e.g., mean 1 , 2, 3, 4, 5, or 6 HLA-la allele(s).

[0052] By expression inhibition of at least one HLA-la allele, the HLA repertoire of the modified human T cell is reduced. Furthermore, beta-2-microglobulin is still expressed by the modified human T cell on their cell surface. Therefore, the modified human T cell could present the MHC I complex on their cell surface comprising the not expression inhibited HLA class la gene products, the HLA-lb gene products, and beta-2-microglobulin to circumvent HLA-mismatch and recognition by NK cells of the recipient. As human T cell, preferably a primary human T cell is used. As mentioned before, the present disclosure contemplates that the expression of selected HLA-la alleles is reduced, which stands in contrast to the prior art, where beta- 2-microglobulin knock-out results in the loss of expression of all HLA-la and HLA-lb alleles. Although HLA-lb expression in such B2M-knock-out cells can theoretically be restored by knocking-in, e.g., an artificial fusion protein comprising an HLA-E allele fused to the beta-2-microglobulin. The inventors of the present application have recognized that the selective inhibition of HLA-la alleles is better suited for preserving cellular physiology. Thereby, the intervention on the genetic repertoire of a given human T cell is reduced by the methods of the present disclosure, giving a more vital modified human T cell.

[0053] As part of the inventive methods the HLA-la alleles of the donor may be determined before inhibiting the cell surface expression of the least one HLA-la allele of the T cell obtained from the donor. Furthermore, the HLA-la alleles of the recipient could be determined and afterwards the HLA-la alleles of the recipient may be compared with the HLA-la alleles of the donor for identifying HLA-la alleles which match and / or not match between the donor and the recipient, before inhibiting the cell surface expression of the least one HLA-la allele of the T cell obtained from the donor.

[0054] Thus, the method of producing a modified human T cell for cell therapy or generating a human T cell population for allogeneic cellular therapy may comprise a) a determination of the HLA-la alleles of the T cell of the donor and the T cell of the recipient, b) (optional) a comparison of the HLA-la alleles of the T cells of the donor and the T cells of the recipient for identifying HLA-la alleles which do not match between the donor and the recipient, b) the genetic modification of an isolated T cell of the donor by inhibiting the cell surface expression of the at least one HLA-la allele, which does not match between the donor and the recipient.

[0055] The donor could be selected from a group of potential donors, wherein the selection is based on the fitness of T cells for cell therapy. Optionally the group of potential donors comprises at least one individual potential donor that has a larger number of HLA-la alleles that matches the recipient than said given donor selected from said group. Thus, the fitness of the T cells of the donor plays a pivotal role for producing the modified human T cell. Such a cell could withstand cellular problems due to the genetic modifications and may be able to multiply in a higher rate in the organism of the recipient compared to an impaired cell. The fitness of the given T cell may be that the T cell is not exhausted, meaning that a fit T cell may not express PD-1 , TIM3 or LAG3. Also, the T cell should not be in a cell cycle arrest. Also, T cells are found to be fit which are e.g. CD8+CD45RO CD27+cells. Furthermore, it is advisable to use young replicative active T cells. The fitness of T cells and suitable markers are described and reviewed in Gett et al., 2003 and Mehta et al., 2021 , which are incorporated by reference here.

[0056] The human T cell from the donor used in this method may be an activated human T cell.

[0057] The inventors also surprisingly found that by inhibiting the expression of two, three, four, and / or five alleles of the HLA-la genes, the so-modified human T cells are still not recognized due to HLA mismatches or by NK cells. This is also true for modified human primary T cells in which the expression of all alleles of the HLA-la genes is inhibited.

[0058] It is, therefore, possible to use the inventive methods to inhibit the cell surface expression of one HLA-la allele in the human T cell of the donor, which do not match between the donor and the recipient. Thus, the modified human T cell expresses five HLA-la alleles on its cell surface.

[0059] It is also possible to inhibit the cell surface expression of two HLA-la alleles in the human T cell of the donor, which do not match between the donor and the recipient. Thus, the modified human T cell expresses four HLA-la alleles on their cell surface.

[0060] Using the inventive methods also the cell surface expression of three HLA-la alleles in the human T cell of the donor could be inhibited, which do not match between the donor and the recipient. Thus, the modified human T cell expresses three HLA-la alleles on their cell surface. The cell surface expression of four HLA-la alleles in the human T cell of the donor could be inhibited using the inventive methods, which do not match between the donor and the recipient. Thus, the modified human T cell expresses two HLA-la alleles on their cell surface. It is preferred that the cell surface expression of all HLA-B and HLA- C alleles is inhibited in the case that the donor is HLA-A homozygote.

[0061] Furthermore, using the inventive methods, the cell surface expression of five HLA-la alleles of the human T cell of the donor could be inhibited, which do not match between the donor and the recipient. Such modified human T cell may therefore express only one class HLA-la allele on their cell surface. It is preferred that the cell surface expression of one HLA-A allele and all of the HLA-B and HLA-C is inhibited

[0062] It is preferred, that the expression of all HLA-la alleles of the T cell of the donor is inhibited in the modified human T cell, which do not match with the HLA-la alleles of the recipient. Thus, the modified human T cell only express those HLA-la alleles which match between the donor and the recipient or, in the case that no HLA-la allele match with the respective HLA-la allele of the recipient, the modified human T cell may express no HLA-la allele.

[0063] In some modified human T cells, only one HLA-la allele, preferably one HLA-A allele, is expressed, and that this one HLA-A allele match with the one of the HLA-A alleles of the recipient. Here, the only HLA-la allele that is expressed on the cell surface of the modified human T cell can generally be an HLA-A, HLA-B, or HLA-C allele. It is especially preferred that the expression of one HLA-A allele and the expression of all HLA-B alleles and HLA-C alleles is inhibited.

[0064] In some modified human T cells, only the HLA-A allele is expressed, wherein the HLA- A allele can be any HLA-A allele known to the skilled person. In some modified human T cells, the only expressed HLA-la allele may be an allele selected from the group consisting of HLA-A*02:01 and HLA-A*03:01.

[0065] It is further preferred, that the HLA-A alleles of the T cell donor are homozygote and that the HLA-A alleles match with one of the HLA-A alleles of the recipient. In this case, the expression of all HLA-B and HLA-C alleles is inhibited. According to the methods of the disclosure, the modified T cell expresses HLA-E, HLA- F, and / or HLA-G on their cell surface.

[0066] The term “inhibition” as used herein may relate to any form of modification that results in a reduction of expression of a certain gene product. The reduction of expression may be at least 50%, at least 60%, at least 70%, at least 80%, preferably at least 90% or at least 95% or at least 99%, and most preferably 100% compared to the not modified human T cell. Hence, the inhibition of HLA-la allele expression on the cell surface may be achieved by any suitable method known to the skilled person. One exemplary method is the knock-out of the respective HLA-la allele, the genetic modification of the donor's T cell. This knock-out could be achieved using, for example, gene-editing or base-editing. Base-editing has the advantage that only point mutations are generated, and no double-strand breaks are generated. Thus, the risk of unwanted translocations of parts of the DNA is reduced. Preferably, the knock-out of the respective HLA-la allele(s) by either gene-editing or base-editing can be achieved by using CRISPR / Cas technology.

[0067] The methods of the disclosure also contemplate that it is also possible to inhibit the expression of a TRAC gene locus and / or a TRBC gene locus in addition to at least one HLA-la allele. Such a modification preferably results in a T cell that does not express its native TCR. It is further contemplated that one can introduce a gene encoding an antigen-specific receptor to the modified T cell. Examples of such antigen-specific receptors are a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In cases where a TRAC and / or TRBC gene locus is to be knocked out and a gene encoding an antigen-specific receptor is to be introduced, the gene encoding the antigen-specific receptor may be knocked into said TRAC and / or TRBC gene locus. Several methods for knocking in or integration of the antigen-specific receptor are known to the skilled person. One example may be the use of the CRISPR / Cas technology.

[0068] It is to be understood that with these methods, the gene or cell surface expression of the beta-2-microglobulin is not modified. Hence, the method of the invention does not include a step of modifying the gene encoding beta-2-microglobulin. The methods disclosure also contemplates that genetic engineering of multiple genes can be conducted in a single step. It is possible to inhibit the cell surface expression of at least one HLA-la allele, e.g., 1 , 2, 3, 4, 5, or 6 alleles in one single step. Said single step may further comprise inhibiting the expression of a TRAC gene locus and / or a TRBC gene locus and optionally the integration of the antigen-specific receptor, e.g., by knock-in into one of the TRAC, TRBC, or HLA gene loci. Hence, it is preferred that genetic engineering steps be conducted in a single step, i.e., in a one-pot reaction. This will reduce the time needed to manipulate the T cell and thus reduce the stress for the cell, resulting in a more vital modified human T cell.

[0069] In addition to inhibiting the cell surface expression of one or more HLA-la allele(s) and optionally knocking in or integrating the antigen-specific receptor, optionally, an expression of one or more genes of HLA class II could also be inhibited in the isolated human T cell. This could be accomplished by, e.g., knocking out a gene CIITA, which codes for a transactivator essential for the expression of the HLA class II genes. The inhibition of HLA class II gene expression could be done in the same step as the inhibition of HLA-la expression mentioned before.

[0070] The present disclosure also contemplates, that in these methods, in contrast to methods of the prior art, a step of culturing the modified human T cells after the inhibition of cell surface expression of the at least one HLA-la allele and / or a step of purification after the inhibition step can be omitted. Accordingly, it is contemplated that the methods disclosed herein may comprise no step of culturing the modified human T cells after the inhibition of cell surface expression of the at least one HLA-la allele and / or no step of purification after the inhibition step. Thus, the modified human T cell may be ready for administration directly after expression inhibition and need no timeconsuming culture and / or purification step after the inhibition step. This results in minimizing the “hands-on” time with the human T cell, reducing the contamination risks of the T cells, and allowing a rapid administration of the modified human T cell after expression inhibition.

[0071] The inventive methods may be conducted within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after T cell isolation. It is also possible to conduct the method in a time shorter than 1 day, e.g., within 3 hours, 4 hours, 5 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, or 23 hours after T cell isolation. The duration of the methods could be between about 3 hours to about 1 day, between about 3 hours to about 2 days, between about 3 hours to about 3 days, between about 3 hours to about 4 days, between about 3 hours to about 5 days, between about 3 hours to about 6 days, between about 3 hours to about 7 days. This is possible, in particular in the case when the step of culturing the modified human T cells after the inhibition of cell surface expression of the at least one HLA-la allele and / or a step of purification after the inhibition step is omitted. The inventive methods may also be conducted within one week, two weeks, three weeks, or four weeks after isolation of the human T cell after T cell isolation. Thus, the isolated human T cell is shorter cultivated ex-vivo compared to other methods and is therefore believed to be more vital. Furthermore, the risk of contamination during cultivation or purification is reduced. An additional advantage of the short method duration is that a patient could get the modified human T cells in a short time and thus on-site in a hospital or doctor’s office. This reduces the costs and administrative efforts of manufacturing a cellular therapy product significantly and thus allows making cellular therapy available for any patient for whom cellular therapy is indicated as medically beneficial.

[0072] In a third aspect, the invention is directed to a modified human T cell obtainable or obtained by a method described herein. Generally, the T cell can be any type of T cell, including a primary T cell or a T cell derived from an iPSC. Primary T cells are however preferred. Also, a primary NK cell or an induced pluripotent stem cell (iPSC) derived NK or T cell could be obtained or is obtainable by a method described herein. The description and examples regarding to the first and second aspect are incorporated herein by reference.

[0073] As explained above, in a fourth aspect the invention is directed to a modified human T cell for allogeneic cell therapy of a given recipient, wherein an expression of at least one HLA-la allele is inhibited, wherein said at least one HLA-la allele does not match an HLA-allele of the recipient. It is contemplated that the expression of beta-2- microglobulin has not been modified. Hence, beta-2-microglobulin is expressed by the T cell. Generally, the T cell can be any type of T cell, including a primary T cell or a T cell derived from an iPSC. It is further preferred that the modified human T cell is a modified human primary T cell. At least one, in this context can, e.g., mean 1 , 2, 3, 4, 5, or 6. Also, a primary NK cell or an induced pluripotent stem cell (iPSC) derived NK or T cell could be used instead of a T cell.

[0074] It is an aim of the invention in the context of personalized medicine to provide a modified human T cell for allogeneic cell therapy of a given recipient and therefore, the HLA-la alleles of the T cell should be match with the HLA-la alleles of the recipient.

[0075] It is preferred that the expression of one HLA-A allele and all HLA-B and HLA-C alleles of the T cell is inhibited and that the not inhibited HLA-A allele match with one HLA-A allele of the recipient. It is also preferred that the HLA-A alleles of the donor are homozygote and match with at leas tone HLA-A allele of the recipient and that (in this case) the expression of all HLA-B and HLA-C alleles is inhibited.

[0076] The term “match” in the context of HLA alleles is defined herein as a consensus between the given HLA-la allele of the donor and the given HLA-la allele of the recipient. For example, if the donor has the HLA-la allele of the donor is the HLA-A*02- 01 allele, then a match is given if the recipient has also the HLA-A*02-01 allele.

[0077] Vice versa, the term “mismatch” is defined herein as a non-consensus between the given HLA-la allele of the donor and the recipient, e.g. if the donor has an HLA-A*02- 01 allele and the recipient has another HLA-A allele which is not HLA-A+02-01 .

[0078] The term “inhibition”, “inhibit”, or “inhibited” as used herein may relate to any form of modification that results in a reduction of expression of a certain gene product. The reduction of expression may be at least 50%, at least 60%, at least 70%, at least 80%, preferably at least 90%, or at least 95%, or at least 99%, and most preferably 100% compared to the not modified human T cell.

[0079] The description and examples regarding to the first and second aspect are incorporated herein by reference. Hence, any feature disclosed in the context of the method of producing a T cell disclosed herein should also be regarded as being disclosed in the context of a modified T cell of the invention, where applicable.

[0080] The at least one HLA-la allele can be selected from the HLA-la gene family comprising HLA-A, HLA-B, and HLA-C. Thus, by expression inhibition of at least one HLA-la allele, the HLA repertoire of the modified human T cell is reduced. Furthermore, beta-2-microglobulin is still expressed by the modified human T cell on their cell surface. Therefore, the modified human T cell could present the MHC I complex on their cell surface comprising the not expression inhibited HLA class la gene products, the HLA-lb gene products, and beta- 2-microglobulin to circumvent recognition by NK cells of the recipient.

[0081] In this invention and also in the fourth aspect of the invention, only the expression of selected HLA-la alleles is inhibited and not, as shown in the prior art, the expression of all HLA-la and HLA-lb alleles by knocking-out beta-2-microglobulin and afterwards knocking-in e.g. an HLA-E allele fused to beta-2-microglobulin. Thus, the intervention on the genetic repertoire of a given human T cell is reduced giving a more vital modified human T cell.

[0082] In the modified human T cell, it is possible that the expression of two alleles of the class HLA-la is inhibited, which do not match with the recipient. This means that the expression of four alleles of the class HLA-la is not inhibited.

[0083] In the modified human T cell, it is further possible that the expression of three alleles of the class HLA-la is inhibited, which do not match with the recipient. This means that the expression of three alleles of the class HLA-la is not inhibited.

[0084] In the modified human T cell, it is further possible that the expression of four alleles of the class HLA-la is inhibited, which do not match with the recipient. This means that the expression of two alleles of the class HLA-la is not inhibited.

[0085] In the modified human T cell, it is further possible that the expression of five alleles of the class HLA-la is inhibited, which do not match with the recipient. Thus, the modified human T cell expresses only one class HLA-la allele. It is especially preferred that the HLA-la allele expressed by the modified human T cell is an HLA-A allele which match with the HLA-A allele of the recipient. Thus, the expression of 2, 3, 4, or 5 HLA-la alleles, which do not match with the recipient, could be inhibited in the modified human T cell. It is preferred, that the expression of all HLA-la alleles of the T cell of the donor is inhibited in the modified human T cell, which do not match with the HLA-la alleles of the recipient. Thus, the modified human T cell only express those HLA-la alleles which match between the donor and the recipient or, in the case that no HLA-la allele match with the respective HLA-la allele of the recipient, the modified human T cell may express no HLA-la allele.

[0086] It is within the scope of the invention that only one HLA-la allele is expressed. Generally, said only one HLA-la allele can be an HLA-A, HLA-B, or HLA-C allele. Such class HLA-la allele is preferably the HLA-A allele. Said HLA-A allele can be any HLA- A allele known to the skilled person. Further preferred is the expression of only both HLA-A alleles in the case that the donor is homozygote in these alleles, which gives the same result as mentioned before.

[0087] The modified human T cell may only express an HLA-la gene allele selected from the group consisting of HLA-A*02:01 and HLA-A*03:01 of the HLA-la class.

[0088] Since only the expression of HLA-la alleles is concerned by the inventive modification, the modified human T cell still expresses beta-2-microglobulin, which allows for the expression of HLA-E, HLA-F, and / or HLA-G. Accordingly, the T cell disclosed herein may express HLA-E, HLA-F, and / or HLA-G. Expression of any one of HLA-E, HLA-F, and / or HLA-G can for example be assessed by using antibodies directed to HLA-E, HLA-F or HLA-G e.g. in a FACS analysis, e.g. as essentially described in Example 5. Thus, the present disclosure contemplates a modified human T cell, in which the expression of HLA-lb has not been modified.

[0089] The present disclosure also contemplates that expression of a TRAC gene locus and / or a TRBC gene locus in the modified T cell may be inhibited. Such a modification preferably results in a T cell that does not express its native TCR.

[0090] It is further within the scope of the invention that, optionally, an antigen-specific receptor may be introduced to the modified human T cell. For example, said antigenspecific receptor may be knocked-in or integrated into a human gen locus, such as in a TRAC gen locus or a TRBC gene locus of the modified human T cell. Such an antigen-specific receptor could be a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0091] In addition to the inhibition of the expression of at least one HLA-la allele and the optional knock-in or integration of the antigen-specific receptor, an expression of one or more genes of an HLA class II may also be inhibited in the modified human T cell. This could be accomplished by e.g. genetic modifications of one or more HLA-II gene(s) or by using specific siRNA. Furthermore, the expression of all HLA-II genes could be inhibited by knocking out a gene encoding a transactivator, e.g. CIITA, which is essential for expression of the HLA class II genes.

[0092] The inventive modified human T cell may have an ex vivo duration of not more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after T cell isolation. It is also possible that the ex vivo duration of the modified human T cell is shorter than 1 day, e.g. within 3 hours, 4 hours, 5 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, or 23 hours after T cell isolation. The ex vivo duration of the modified human T cell could be between about 3 hours to about 1 day, between about 3 hours to about 2 days, between about 3 hours to about 3 days, between about 3 hours to about 4 days, between about 3 hours to about 5 days, between about 3 hours to about 6 days, between about 1 day to about 7 days. This is possible, in particular in the case when the modified human T cells are not further cultivated or purified after modification. The inventive modified human T cell may also have an ex vivo duration of not more than one week, two weeks, three weeks, or four weeks. Thus, the human T cell is shorter cultivated ex-vivo, compared to other methods, and therefore may be more vital. Furthermore, the risk of contamination during cultivation or the like is reduced. An additional advantage of the short ex vivo duration is that a patient could get the modified human T cells in a short time and thus on-site in a hospital or doctor’s office. This reduces the costs and administrative efforts of manufacturing a cellular therapy product significantly and thus allows cellular therapy to be made available for any patient for whom cellular therapy is indicated as medically beneficial.

[0093] The modified human T cell described above and / or the modified human T cell obtainable or obtained by the methods described above could further be used in an adoptive cell therapy. The adoptive cell therapy could be a treatment of cancer, an autoimmune disease, or an infectious disease. This includes the use of the modified human T cell in an adoptive cell therapy.

[0094] The modified human T cell described above and / or the modified human T cell obtainable or obtained by the methods described above could also be used in an cellular therapy of a human subject in need thereof by an allogeneic T cell transfer. This cellular therapy may be a treatment of cancer, an autoimmune disease, or an infectious disease. This includes the use of the modified human T cell in a cellular therapy.

[0095] The invention relates in a fifth aspect to a pharmaceutical composition comprising the modified human T cell of the fourth aspect and / or the modified human T cell obtainable or obtained by the methods described above according to the third aspect. The pharmaceutical composition may comprise further pharmaceutical suitable ingredients, such as a pharmaceutically acceptable carrier, a pharmaceutical solution and / or excipient. Further pharmaceutical suitable ingredients are known by the skilled person. The pharmaceutical composition may be used as a treatment of cancer, an autoimmune disease, or an infectious disease.

[0096] The invention is also directed to a use of a human T cell obtained from a given donor for the manufacture of a modified human T cell for allogeneic cell therapy comprising conducting the methods of the first and second aspect. This modified human T cell may be used for the treatment of cancer, an autoimmune disease or an infectious disease. In alternatives the cell therapy mentioned before may be an adoptive cell therapy or an allogenic T cell transfer.

[0097] The invention is further directed to a use of the modified human T cell of the third and / or fourth aspect in an adoptive cell therapy. The adoptive cell therapy may be for use in a treatment of cancer, an autoimmune disease, or an infectious disease.

[0098] The invention also relates to a use of the modified human T cell of the third and / or fourth aspect for a cellular therapy of a human subject in need thereof by allogeneic T cell transfer. The cellular therapy may be for use in a treatment of cancer, an autoimmune disease, or an infectious disease

[0099] Furthermore, the invention relates to a method of treating a recipient in need of an allogeneic cellular therapy, comprising generating a modified human T cell population from a T cell population of a donor by inhibiting the cell surface expression of at least one HLA-la allele that does not match with an HLA-la allele of the recipient, wherein the modified human T cell expresses beta-2-microglobulin on the cell surface, and wherein the human T cell from the donor is preferably a primary T cell, the method further comprises administering to the recipient an effective amount of the modified human T cell population.

[0100] The description and examples regarding to all aspects above are incorporated herein by reference.

[0101] The method of treating may comprise the steps of a) isolation of T cells from the donor and the recipient. b) determination of the HLA-la alleles of the T cell of the donor and the T cell of the recipient, c) (optional) comparison of the HLA-la alleles of the T cells of the donor and the T cells of the recipient for identifying HLA-la alleles which do not match between the donor and the recipient, d) (optional) isolation of T cells from the donor, e) genetic modification of the T cells of the donor by inhibiting the cell surface expression of the at least one HLA-la allele, which does not match between the donor and the recipient, wherein optional the genetic modification takes place in one step, f) administration of the modified T cells to the recipient, wherein optional no culturing of the modified T cells after the inhibition step and / or no purification after the inhibition step takes place.

[0102] In the case that the HLA-la alleles of donor and recipient are known before, the method of treating may comprise the steps of a) (optional) comparison of the HLA-la alleles of the T cells of the donor and the T cells of the recipient for identifying HLA-la alleles which do not match between the donor and the recipient, b) isolation of T cells from the donor, c) genetic modification of the T cells of the donor by inhibiting the cell surface expression of the at least one HLA-la allele, which does not match between the donor and the recipient, wherein optional the genetic modification takes place in one step, d) administration of the modified T cells to the recipient, wherein optional no culturing of the modified T cells after the inhibition step and / or no purification after the inhibition step takes place.

[0103] The method of treating may further comprise the selection of the donor according to the fitness of T cells according to the first and second aspect mentioned above.

[0104] The T cell of the donor used in the method of treating may be an activated T cell.

[0105] The method of treating may further comprise inhibiting the expression of two, three, four, five or six HLA-la alleles of the T cell of the donor, wherein the inhibited HLA-la alleles do not match with the HLA-la alleles of the recipient. Preferably the expression of one HLA-A allele and all HLA-B alleles and HLA-C alleles is inhibited, whereby the expressed HLA-A allele match with at least one HLA-A allele of the recipient. It is further preferred that the expression of all HLA-B and HLA-C alleles is inhibited and that the HLA-A alleles are homozygote and that the HLA-A alleles match with at least one HLA-A allele of the recipient.

[0106] The method of treating may further comprise inhibiting the expression of all HLA-la alleles of the T cell of the donor, which do not match with the HLA-la alleles of the recipient, in the modified human T cell. If all HLA-la alleles of the donor T cell do not match with the HLA-la alleles of the recipient, then the method of treating includes the inhibition of all six HLA-la alleles of the donor T cell.

[0107] The method of treating comprise that the modified T cell expresses HLA-E, HLA-F, and / or HLA-G. The method of treating comprises further no modification of the expression of HLA-lb or any HLA-lb allele. The method of treating comprises the possible genetic modification of the T cell by knocking out one or more HLA-la allele(s) according to the first and second aspect.

[0108] The method of treating comprises also the optional modification of an antigen specific receptor according to the first and second aspect.

[0109] The method of treating may additional comprises inhibiting the expression of HLA class II as further described in the first and second aspect.

[0110] Within the method of treating the modified human T cell is ready for administration directly after inhibition of the cell surface expression of at least one HLA-la allele. Thus, it is a one-pot reaction. This is further described in the first, second and fourth aspect above and incorporated by reference herein.

[0111] The method of treating does not comprise a step of culturing after the inhibition step and / or a step of purification after the inhibition step as further described in the first, second and fourth aspect above and incorporated by reference herein.

[0112] The method of treating may include the step of obtaining T cells from a donor.

[0113] The method of treating may also include the step of providing T cells from a donor.

[0114] Within the method of treating, the T cell has an ex vivo duration of not more than four weeks according to the first, second and fourth aspect above and incorporated by reference herein.

[0115] The use of primary NK cells or induced pluripotent stem cell (iPSC) derived NK or T cells instead of T cells in the methods mentioned above and instead of the modified human T cell is within the scope of the application.

[0116] It is noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0117] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0118] The term "and / or", wherever used herein, includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0119] The terms “less than” or in turn “more than” does not include the concrete number.

[0120] For example, “less than 20” means less than the number indicated. Similarly, “more than” or “greater than” means more than or greater than the indicated number, e.g. “more than 80 %” means more than or greater than the indicated number of 80 %.

[0121] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes, when used herein, with the term “having”. When used herein, “consisting of' excludes any element, step, or ingredient not specified.

[0122] The term “including” means “including but not limited to”, “Including” and “including but not limited to” are used interchangeably.

[0123] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments, only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. All publications cited throughout the text of this specification (including all patents, patent applications, scientific publications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[0124] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

[0125] The invention is further characterized by the following items:

[0126] 1 . A method of producing a modified human T cell for cell therapy of a given recipient from a T cell obtained from a given donor, wherein the donor has at least one HLA-la allele that does not match an HLA-la allele of the recipient, wherein the method comprises inhibiting a cell surface expression of at least one HLA-la allele of the T cell obtained from the donor that does not match the HLA-la allele of the recipient, wherein the modified human T cell expresses beta-2-microglobulin on the cell surface, and wherein the human T cell from the donor is preferably a primary T cell, optionally wherein a primary NK cell or an induced pluripotent stem cell (iPSC) derived NK or T cell is used instead of the T cell.

[0127] 2. A method of generating a human T cell population for allogeneic cellular therapy, wherein the method comprises, in case a donor has at least one HLA-la allele that does not match an HLA-la allele of a recipient, genetically modifying the T cell population to inhibit cell surface expression of at least one HLA-la allele of the isolated T cell population of the donor, wherein the modified human T cell expresses beta-2- microglobulin on the cell surface, optionally wherein a primary NK cell population or an induced pluripotent stem cell (iPSC) derived NK or T cell population is used instead of the T cell population. 3. The method of item 2, wherein the donor has at least one HLA-la allele that does not match an HLA-la allele of the recipient, and wherein the human T cell from the donor is preferably a primary T cell.

[0128] 4. The method of any one of the preceding items, wherein the method comprises a) a determination of the HLA-la alleles of the T cell of the donor and the T cell of the recipient, b) (optional) a comparison of the HLA-la alleles of the T cells of the donor and the T cells of the recipient for identifying HLA-la alleles which do not match between the donor and the recipient, c) the genetic modification of an isolated T cell of the donor by inhibiting the cell surface expression of the at least one HLA-la allele, which does not match between the donor and the recipient.

[0129] 5. The method of any one of the preceding items, wherein the given donor has been selected from a group of potential donors, wherein the selection is based on the fitness of T cells for cell therapy.

[0130] 6. The method of item 5, wherein the group of potential donors comprises at least one individual potential donor that has a larger number of HLA-la alleles that matches the recipient than said given donor selected from said group.

[0131] 7. The method of any one of the preceding items, wherein the T cell of the donor is an activated T cell.

[0132] 8. The method of any one of the preceding items, wherein the method comprises inhibiting the expression of two HLA-la alleles, which do not match with the HLA-la alleles of the recipient.

[0133] 9. The method of any one of the preceding items, wherein the method comprises inhibiting the expression of three HLA-la alleles, which do not match with the HLA-la alleles of the recipient. 10. The method of any one of the preceding items, wherein the method comprises inhibiting the expression of four HLA-la alleles, which do not match with the HLA-la alleles of the recipient.

[0134] 11 . The method of any one of the preceding claims, wherein the method comprises inhibiting the expression of all HLA-B and HLA-C alleles, wherein the HLA-A alleles of the donor are homozygote and wherein the HLA-A alleles of the donor match at least one HLA-A allele of the recipient.

[0135] 12. The method of any one of the preceding items, wherein the method comprises inhibiting the expression of five HLA-la alleles, which do not match with the HLA-la alleles of the recipient.

[0136] 13. The method of any one of the preceding claims, wherein the method comprises inhibiting the expression of one HLA-A allele and all HLA-B and HLA-C alleles, wherein the expressed HLA-A allele match with at least one of the HLA-A alleles of the recipient.

[0137] 14. The method of any one of the preceding items, wherein the method comprises inhibiting the expression of all HLA-la alleles of the T cell of the donor, which do not match with the HLA-la alleles of the recipient, in the modified human T cell.

[0138] 15. The method of any one of items 1 to 7 or 11 to 12, wherein the modified human T cell expresses only one class HLA-la allele.

[0139] 16. The method of item 15, wherein the HLA-la allele is an HLA-A allele.

[0140] 17. The method of any one of items 1 to 7 or 11 to 16, wherein the modified human T cell only expresses the allele selected from the group consisting of HLA-A*02:01 and HLA-A*03:01.

[0141] 18. The method of any one of items 1 to 17, wherein the modified T cell expresses HLA-E, HLA-F, and / or HLA-G. 19. The method of any one of the preceding items, wherein the method comprises inhibiting the cell surface expression of at least one HLA-la allele by a knock-out of the respective allele.

[0142] 20. The method of item 19, wherein the method for the knock-out is gene-editing or base-editing, preferably by using CRISPR / Cas technology.

[0143] 21. The method of any one of the preceding items, wherein additional an antigenspecific receptor is knocked-in or integrated in a TRAC gene locus or a TRBC gene locus.

[0144] 22. The method of item 21 , wherein the antigen-specific receptor is a TCR.

[0145] 23. The method of item 21 , wherein the antigen-specific receptor is a CAR.

[0146] 24. The method of any one of items 21 to 23, wherein the method comprises using gene-editing for the knock-out and the knock-in, preferably by using CRISPR / Cas technology for gene-editing.

[0147] 25. The method of any one of the preceding items, wherein the method does not include modifying the gene expression of beta-2-microglobulin.

[0148] 26. The method of any one of the preceding items, the method comprises conducting the inhibition of cell surface expression of at least one HLA-la allele and optionally knock-in or integration of the antigen-specific receptor in a single step.

[0149] 27. The method of any one of the preceding items, wherein the method comprises additional inhibiting the cell surface expression of HLA class II.

[0150] 28. The method of any one of the preceding items, wherein the modified human T cell is ready for administration directly after inhibition of the cell surface expression of at least one HLA-la allele. 29. The method of any one of items 1 to 26, wherein the method does not comprise a step of culturing after the inhibition step and / or a step of purification after the inhibition step.

[0151] 30. The method of any one of the preceding items, wherein the method comprises conducting the method within four weeks after isolation of the human T cell.

[0152] 31 . A modified human T cell obtainable or obtained by a method of any one of items 1 to 30, optionally wherein a primary NK cell or an induced pluripotent stem cell (iPSC) derived NK or T cell is used instead of the T cell.

[0153] 32. A modified human T cell for allogeneic cell therapy of a given recipient, wherein an expression of at least one HLA-la allele is inhibited, wherein said at least one HLA-la allele does not match an HLA-la allele of the recipient, wherein the expression of beta- 2-microglobulin has not been modified, and wherein the modified human T cell is preferably a modified human primary T cell, optionally wherein a primary NK cell or an induced pluripotent stem cell (iPSC) derived NK or T cell is used instead of the T cell.

[0154] 33. The modified human T cell of item 32, wherein the expression of two HLA-la alleles, which do not match with the HLA-la alleles of the recipient, is inhibited.

[0155] 34. The modified human T cell of any one of items 32 or 33, wherein the expression of three HLA-la alleles, which do not match with the HLA-la alleles of the recipient, is inhibited.

[0156] 35. The modified human T cell of any one of items 32 to 34, wherein the expression of four HLA-la alleles, which do not match with the HLA-la alleles of the recipient, is inhibited.

[0157] 36. The modified human T cell of any one of claim 32, wherein the expression of all HLA-B and HLA-C alleles is inhibited, wherein the HLA-A alleles of the donor are homozygote and wherein the HLA-A alleles of the donor match at least one HLA-A allele of the recipient. 37. The modified human T cell of any one of items 32 to 36, wherein the expression of five HLA-la alleles, which do not match with the HLA-la alleles of the recipient, is inhibited.

[0158] 38. The modified human T cell of claim 32, wherein the expression of one HLA-A allele and all HLA-B and HLA-C alleles is inhibited, and wherein the expressed HLA-A allele match with at least one of the HLA-A alleles of the recipient.

[0159] 39. The modified human T cell of any one of items 32 to 38, wherein the expression of all HLA-la alleles that do not match the HLA-la alleles of the recipient are inhibited.

[0160] 40. The modified human T cell of any one of items 32 to 39, wherein the T cell expresses only one class HLA-la allele.

[0161] 41 . The modified human T cell of item 40, wherein the HLA-la allele is an HLA-A allele.

[0162] 42. The modified human T cell of any one of items 32 or 37 to 41 , wherein the modified human T cell only expresses the HLA-la allele selected from the group consisting of HLA-A*02:01 and HLA-A*03:01 of the HLA-la class.

[0163] 43. The modified human T cell of any one of items 32 to 42, wherein the modified T cell expresses HLA-E, HLA-F, and / or HLA-G.

[0164] 44. The modified human T cell of any one of items 32 to 43, wherein the expression of HLA-lb has not been modified.

[0165] 45. The modified human T cell of any one of items 32 to 44, wherein the T cell expresses beta-2-microglobulin.

[0166] 46. The modified human T cell of any one of items 32 to 45, wherein an antigen-specific receptor is knocked-in or integrated in a TRAC gene locus or a TRBC gene locus.

[0167] 47. The modified human T cell of item 46, wherein the antigen-specific receptor is a TCR. 48. The modified human T cell of item 46, wherein the antigen-specific receptor is a CAR.

[0168] 49. The modified human T cell of any one of items 32 to 48, wherein additional expression of HLA class II is inhibited.

[0169] 50. The modified human T cell of any one of items 32 to 49, wherein the T cell has an ex vivo duration of not more than four weeks.

[0170] 51. The modified human T cell of any one items 31 to 50 for use in an adoptive cell therapy.

[0171] 52. The modified human T cell of item 51 , wherein the adoptive cell therapy is a treatment of cancer, an autoimmune disease, or an infectious disease.

[0172] 53. The modified human T cell of any one of items 31 to 50 for use in a cellular therapy of a human subject in need thereof by allogeneic T cell transfer.

[0173] 54. The modified human T cell of item 53, wherein the cellular therapy is a treatment of cancer, an autoimmune disease, or an infectious disease.

[0174] 55. A pharmaceutical composition comprising the modified human T cell of any one of items 31 to 50 or the modified T cell obtained by the method of any one of items 1 to 30 for use in therapy.

[0175] 56. The pharmaceutical composition of item 55, wherein the therapy is a treatment of cancer, an autoimmune disease, or an infectious disease.

[0176] 57. A use of a human T cell obtained from a given donor for the manufacture of a modified human T cell for allogeneic cell therapy comprising conducting the method of any one of items 1 to 30, optionally wherein a primary NK cell or an induced pluripotent stem cell (iPSC) derived NK or T cell is used instead of the T cell. 58. The use of item 57, wherein the modified human T cell is for the treatment of cancer, an autoimmune disease or an infectious disease.

[0177] 59. A use of the modified human T cell of items 31 to 50 in an adoptive cell therapy, optionally wherein a primary NK cell or an induced pluripotent stem cell (iPSC) derived NK or T cell is used instead of the T cell.

[0178] 60. The use of item 59, wherein the adoptive cell therapy is for use in a treatment of cancer, an autoimmune disease, or an infectious disease.

[0179] 61 . A use of the modified human T cell of items 31 to 50 for a cellular therapy of a human subject in need thereof by allogeneic T cell transfer, optionally wherein a primary NK cell or an induced pluripotent stem cell (iPSC) derived NK or T cell is used instead of the T cell.

[0180] 62. The use of item 61 , wherein the cellular therapy is for use in a treatment of cancer, an autoimmune disease, or an infectious disease.

[0181] 63. A method of treating a recipient in need of an allogeneic cellular therapy, comprising generating a modified human T cell population from a T cell population of a donor by inhibiting the cell surface expression of at least one HLA-la allele that does not match with an HLA-la allele of the recipient, wherein the modified human T cell expresses beta-2-microglobulin on the cell surface, and wherein the human T cell from the donor is preferably a primary T cell, the method further comprises administering to the recipient an effective amount of the modified human T cell population, optionally wherein a primary NK cell population or an induced pluripotent stem cell (iPSC) derived NK or T cell population is used instead of the T cell population.

[0182] 64. The method of treating of item 63, comprising the steps of a) isolation of T cells from the donor and the recipient. b) determination of the HLA-la alleles of the T cell of the donor and the T cell of the recipient, c) (optional) comparison of the HLA-la alleles of the T cells of the donor and the T cells of the recipient for identifying HLA-la alleles which do not match between the donor and the recipient, d) (optional) isolation of T cells from the donor, e) genetic modification of the T cells of the donor by inhibiting the cell surface expression of the at least one HLA-la allele, which does not match between the donor and the recipient, wherein optional the genetic modification takes place in one step, f) administration of the modified T cells to the recipient, wherein optional no culturing of the modified T cells after the inhibition step and / or no purification after the inhibition step takes place.

[0183] 65. The method of treating of item 63, comprising the steps of a) (optional) comparison of the HLA-la alleles of the T cells of the donor and the T cells of the recipient for identifying HLA-la alleles which do not match between the donor and the recipient, b) isolation of T cells from the donor, c) genetic modification of the T cells of the donor by inhibiting the cell surface expression of the at least one HLA-la allele, which does not match between the donor and the recipient, wherein optional the genetic modification takes place in one step, d) administration of the modified T cells to the recipient, wherein optional no culturing of the modified T cells after the inhibition step and / or no purification after the inhibition step takes place, wherein the HLA-la alleles of donor and recipient are known before.

[0184] 66. The method of treating of any on of items 63 to 65, wherein the method of treating comprise the method according to any one of items 5 or 6.

[0185] 67. The method of treating of any one of items 63 to 66, wherein the T cell of the donor is an activated T cell.

[0186] 68. The method of any one of items 63 to 67, wherein the method of treating comprises inhibiting the expression of two, three, four, five or six HLA-la alleles of the T cell of the donor, wherein preferably the inhibited HLA-la alleles do not match with the HLA-la alleles of the recipient. 69. The method of any one of items 63 to 68, wherein the method of treating comprises inhibiting the expression of one HLA-A allele and all HLA-B and HLA-C of the T cell of the donor, wherein preferably the expressed HLA-A allele match with at least one of the HLA-A alleles of the recipient.

[0187] 70. The method of any one of items 63 to 68, wherein the method of treating comprises inhibiting the expression of all HLA-B and HLA-C of the T cell of the donor, wherein the HLA-A alleles of the donor are homozygote, wherein preferably the HLA-A alleles of the donor match with at least one of the HLA-A alleles of the recipient.

[0188] 71 . The method of treating of any one of items 63 to 70, wherein the method of treating comprises inhibiting the expression of all HLA-la alleles of the T cell of the donor, which do not match with the HLA-la alleles of the recipient, in the modified human T cell.

[0189] 72. The method of treating of any one of items 63 to 71 , wherein the modified T cell expresses HLA-E, HLA-F, and / or HLA-G.

[0190] 73. The method of treating of any one of items 63 to 72, wherein the method of treating does not comprise modifying the expression of HLA-lb.

[0191] 74. The method of treating of any one of items 63 to 73, wherein the method of treating comprises the method of any one of items 19 or 20.

[0192] 75. The method of treating of any one of items 63 to 74, wherein the method of treating comprises the method of any one of items 21 to 24.

[0193] 76. The method of treating of any one of items 63 to 75, wherein the method of treating additional comprises inhibiting the expression of HLA class II.

[0194] 77. The method of treating of any one of items 63 to 76, wherein the modified human T cell is ready for administration directly after inhibition of the cell surface expression of at least one HLA-la allele. 78. The method of treating of any one of items 63 to 77, wherein the method of treating does not comprise a step of culturing after the inhibition step and / or a step of purification after the inhibition step.

[0195] 79. The method of treating of any one of items 63 to 78, wherein the method includes the step of obtaining T cells from a donor.

[0196] 80. The method of treating of any one of 63 to 79, wherein the method includes the step of providing T cells from a donor.

[0197] 81 . The method of treating of any one of items 63 to 80, wherein the T cell has an ex vivo duration of not more than four weeks.

[0198] A better understanding of the present invention and its advantages will be had from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.

[0199] EXAMPLES

[0200] Material and Methods

[0201] T cells from peripheral blood mononuclear cells (PBMCs) and cell culture

[0202] T cells were cultured in RPMI 1640 (Gibco) supplemented with 10% FCS, 0.025% L- glutamine, 0.1 % HEPES, 0.001 % gentamycin and 0.002% streptomycin (hereafter RPMI) at 37 °C and 5% CO2 unless indicated otherwise. For resting of cells, medium was supplemented with 50 lll / ml Interleukin 2 (IL-2). For feeder-free expansion of cells, medium was supplemented with 180 lll / ml IL-2. Written informed consent was obtained from the donors, and use of the blood samples was approved according to national law by the local Institutional Review Board (Ethikkommission der Medizinischen Fakultat der Technischen Universitat Muenchen).

[0203] Cell culture of NK cells

[0204] For short-term culture of NK cells, cells were cultured in full medium supplemented with 100 lll / ml IL-2 at a density of 1 x 106 / ml at 37 °C and 5% CO2. Feeder cell culture

[0205] For feeder cell-based rapid expansion, allogeneic PBMCs were mitotically inactivated by irradiation with 35 Gy. Cells were washed two times with full medium. Cells were adjusted to a target to feeder ratio of 1 :5 and a total density of 1 x 106 / ml. Medium was supplemented with 180 lll / ml IL-2 and 1 pg / ml Phytohaemagglutinin (PHA). If the medium turned acidic, fresh full medium containing 50 lll / ml IL-2 was added. The feeder cell culture was renewed weekly with fresh irradiated allogeneic feeder cells.

[0206] CRISPR / Cas9 mediated knock-out and knock-in

[0207] Frozen PBMCs were thawed and rested overnight in RPMI + 50 lU / ml IL-2. Cells were activated two days prior to electroporation using 4.8 pg aCD3 / aCD28 Expamer (Juno Therapeutics), 300 lU / ml IL-2, 50 lU / ml IL-7 and 50 lU / ml IL-15 for 1 x 1 O6cells. Stimulus was removed by incubating in 1 mM D-Biotin (Sigma Aldrich) for 20 min at room temperature. Cells were electroporated (pulse code EH-110) with Cas9 RNP and 1 pg DNA template in P3 electroporation buffer (20 pl per 1 x 106T cells; Lonza) with a 4D Nucleofector X unit (Lonza). After electroporation, cells were cultured in RPMI containing 180 lU / ml IL-2.

[0208] Used Cas9 RNPs crRNA sequences for gRNAs were

[0209] SEQ ID NO: 1 : 5’-GGCCACGGAGCGAGACATCT-3’ for B2M,

[0210] SEQ ID NO: 2: 5’-GGTCCATCTGGTCATAGAAG-3’ for CIITA,

[0211] SEQ ID NO: 3: 5’-GGAGAATGACGAGTGGACCC-3’ for TRBC (targeting both TRBC1 and TRBC2),

[0212] SEQ ID NO: 4: 5’-AGAGTCTCTCAGCTGGTACA-3’ for TRAC,

[0213] SEQ ID NO: 5: 5’-ACCCAGTTCTCACTCCCATCGGG-3’ for HLA-A*02:01 , SEQ ID NO: 6: 5’-ACCCTGAGATGGGATAAGGAGGG-3’ for HLA-A*03:01 , SEQ ID NO: 7: 5’-ACTCCACGCACAGACCCTCCAGG-3’ for HLA-B*44:03, SEQ ID NO: 8: 5’-ACGCTGCAGCGCGCGGGTACCGG-3’ for HLA-B*51 :01 , SEQ ID NO: 9: 5’-AAAGTCACCAGTCACCCACCCGG-3’ for HLA-C*15:02, SEQ ID NO: 10: 5’-CACACCCTCCAGTGGATGTACGG-3’ for HLA-C*16:01. For delivering up to three guides simultaneously, 80 pM tracrRNA (IDT DNA) and 80 pM crRNA (IDT DNA) were mixed 1 :1 and incubated at 95°C for 5 min, then cooled on benchtop to room temperature. 6 pM high fidelity Cas9 (IDT DNA) was added slowly to gRNA solution to yield RNPs with 3 pM Cas9, 20 pM gRNA and 20 pM electroporation enhancer (IDT DNA). RNPs were incubated for 15 min at RT. 3 pl of RNPs were used for 1 x 106cells. For delivering more than three guides simultaneously, RNPs with a smaller volume were needed. 80 pM tracrRNA (IDT DNA) and 80 pM crRNA (IDT DNA) were mixed 1 :1 and incubated at 95°C for 5 min, then cooled on benchtop to room temperature. 20 pM high fidelity Cas9 (IDT DNA) was added slowly to gRNA solution to yield RNPs with 10 pM Cas9, 20 pM gRNA and 20 pM electroporation enhancer (IDT DNA). RNPs were incubated for 15 min at RT. 1 pl of RNPs were used for 1 x 106cells.

[0214] T-cell receptor replacement for functional assays

[0215] For functional assays the endogenous T-cell Receptor was replaced with either TCR 6-2 (HLA-A*02:01 restrictive; NLV-CMV-epitope) or with JCAR 21 (aCD19 CAR). Replacement took place via CRISPR / Cas9 mediated KO of TRAC and TRBC and use of Homologous Directed Repair (HDR) to insert construct into the endogenous TRAC locus.

[0216] HDR DNA template design and production

[0217] The DNA template for TCR 6-2 for orthotopic TCR replacement via CRISPR / Cas9- mediated HDR KI has been described before (Shultz et al., 2010). In short, the template had the following structure: 5’ homology arm (300-400 base pairs (bp)), P2A, TCR-[3 (including mTRBC with additional cysteine bridge (Effenberger et al., 2019)), T2A, TCR-a (including mTRBC with additional cysteine bridge), bovine growth hormone polyA signal (bGHpA) tail, 3’ homology arm (300-400 bp); synthesized by GeneArt (Thermo Fisher Scientific) and cloned into a vector with Kanamycin resistance for bacterial amplification. The DNA template of anti-human CD19 targeting CAR clone (JCAR021 ) for CRISPR / Cas9-mediated HDR KI was designed in silico, synthesized by Twist Bioscience, and cloned into a vector with Ampicillin resistance for bacterial amplification. The scFv sequence of JCAR021 was kindly provided by Juno Therapeutics - a Bristol Myers Squibb Company. The extracellular binding domain of the CAR construct was linked to a spacer domain consisting of a triple repetitive sequence of Strep-tag II (STII)19 and parts of the lgG4-Fc molecule; followed by a trans-membrane region originated from the CD28 chain; followed by intracellular signaling domains, CD3- and 4-1 BB. As a transduction marker, a truncated version of EGFR (EGFRt) could be linked by a viral T2A peptide to the aforementioned CAR construct (Lee et al., 1998). CAR construct HDR templates had the following structure: 5’ homology arm (300-400 base pairs (bp)), P2A, CAR construct, TGA Stop codon, bGHpA tail, 3’ homology arm (300-400 bp). Unless indicated otherwise, linearized double-stranded DNA PCR products were used for electroporation. Plasmid DNA was amplified by PCR and PCR products were purified by Ampure XP beads (Beckman Coulter) at a 1 :1 ratio.

[0218] Sequencing for KO confirmation

[0219] Genomic DNA was extracted using DNeasy Blood & Tissue Kit (QIAGEN) from PBMCs three days after CRISPR / Cas9 mediated editing. PCRs were performed for amplification of the targeted locus. Purified PCR products were sent for Sanger sequencing (Eurofins).

[0220] Antibody staining for flow cytometry

[0221] Cells were harvested and washed two times in cold FACS buffer (PBS containing 0.5% (w / v) BSA, pH = 7.45). For antibody staining, cells were resuspended in cold FACS buffer containing antibodies and incubated 20 min on ice in the dark. Samples were then washed thrice in cold FACS buffer, filtered through a nylon mesh and analyzed on a flow cytometer. Live / dead discrimination was done by using Propidiumiodide (Invitrogen).

[0222] Flow cytometric cell sorting

[0223] For sorting, staining was performed as described above under sterile conditions. Cells were sorted in 1 ml of sterile FCS. Finally, cells were pelleted and resuspended in full medium with or without feeder cells depending on cell numbers and the following experiment.

[0224] Intracellular cytokine staining

[0225] K562 cells bearing the correct HLA were irradiated (80 Gy) and pulsed with NLV- peptide pp65495-503 (10‘12M, 10’10M, 10’9M, 10’8M, 10’7M, 10’6M, 10’4M) overnight at 37 °C. T cells were then co-incubated with peptide pulsed K562 cells and 2 pl / ml GolgiPlug (BD Biosciences) in a 1 :1 ratio for 4 h at 37 °C. Positive control was stimulated with phorbol myristate acetate (25 ng / ml) and lonomycin (1 pg / ml). Surface staining for CD8 (FITC, Beckman Coulter), HLA-ABC (APC, Biolegend), HLA-DR (PB, Biolegend) and mTRBC (APCFire, Biolegend) was followed by intracellular staining after permeabilization using Cytofix / Cytoperm Kit (BD Biosciences) with IFNy (FITC, BD Pharmingen), TNFa (PE-Cyanine7, eBioscience). Live / dead discrimination was done by using Ethidium monoazide bromide (Invitrogen).

[0226] NK cell assay

[0227] One day prior to the analysis, PBMCs were isolated from fresh blood and sorted for CD3’ CD8’ CD56+cells. The next day, NK cells were co-incubated with T cells in a 1 :2 ratio for 5 h in the presence of CD107a PE (Biolegend) antibody. After one hour, 6 pg / ml GolgiStop (BD Biosciences) was added. Cells were then washed and additional antibody staining for flow cytometry was performed. xCELLigence killing assay

[0228] HepG2 cells were loaded with NLV-peptide pp65495-503 (10-6M) for 2 h at room temperature. 8 x 104peptide pulsed HepG2 cells were seeded per well onto an E-Plate (OLS) and placed in an xCELLigence™ RTCA System (ACEA Bio). T cells were added when curve hits saturation no less than 24 hours later. As a positive control, 100 pl of full medium containing 2 % Triton-X was added; as a negative control served HepG2 cells cultured alone. xCELLigence RTCA Software Pro (ACEA) and Prism8 (GraphPad) were used to analyze the data.

[0229] Mixed lymphocytes reaction (MLR) assay

[0230] Fresh blood was collected from two donors. PBMCs were isolated and half of the cells were mitotically inactivated through irradiation (35 Gy). Cells from the donor used for target cell generation were then co-cultured with irradiated cells from the same donor (auto priming) or from the second donor (allo priming) for 7 days with 10 lU / ml IL-2 at 37 °C. After priming of the effector cells, cells were labelled with the eBioscience™ Cell Proliferation Dye eFluorTM 450 Kit (Thermo Fisher) according to manufacturer’s protocol. Target cells were labelled with CFSE Cell Division Tracker Kit (Biolegend) according to manufacturer’s protocol. Target and effector cells were then co-cultured for 48 h. Cells were harvested, washed and additional antibody staining for flow cytometry was performed.

[0231] In vivo transfer in syngeneic infection mouse model

[0232] Before T cell transfer NSG-HLA-A2 / HHD mice (Jackson Laboratories) were irradiated with 2 Gy to create a niche for the transferred cells. CD8+TCR-transgenic T cells with and without B2M KO were then injected intraperitoneally. The endogenous TCR was orthotopical ly replaced with a CMV-specific TCR (TCR 6-2). The next day, mice were infected intraperitoneally with 5 x 103PFU mCMV-NLV (virus provided by Luka Cicin- Sain). On day 7 after infection, the mice were sacrificed and the liver was processed for flow cytometry analysis. Lymphocytes were isolated with Percoll (GE Healthcare) and red blood cell lysis was performed with ACT buffer (10% v / v 0.17 M Tris-HCI pH=7.5, 90% v / v 0.17 M NH4CI). Cells were stained with hCD8 PE (Invitrogen), mTRBC APCFire780 (Biolegend), [32m APC (Biolegend) and hTCR FITC (Biolegend) antibodies.

[0233] In vivo transfer in humanized mouse model

[0234] Female 4-week-old NSG-SGM3 mice (Jackson Laboratories) were humanized with human CD34+cells following irradiation with 1 Gy. Human immune system reconstitution took place over 12 weeks. Blood was then analyzed via flow cytometry to identify different immune cell populations. PBMCs used as effector cells all received an aCD19-CAR (JCAR 21 ) knock-in into the endogenous TRAC gene locus and were (in the case of HLA reduction) simultaneously edited for all HLA class I alleles except for HLA-A*02:01 . Finally, cells were administered directly after electroporation without prior sorting via the intravenous route into recipient mice.

[0235] Flow cytometry

[0236] Samples were acquired on a Cytoflex (S) flow cytometer (Beckman Coulter). Flow sorting was done on a MoFlo Astrios EQ (Beckman Coulter).

[0237] Data analysis

[0238] All flow cytometry data were analyzed with FlowJo v10 and GraphPad PRISM 9 software. Genomic KO was scored using Synthego’s ICE. xCELLigence RTCA Software Pro (ACEA) was used for analysis of xCELLigence assays. HLA population data

[0239] HLA data and frequencies used for calculation of Figure 13 (homozygous HLA haplotypes) were kindly provided by the Stem Cell Donor Registry of the German Red Cross Blood Donor Service North-East.

[0240] Experimental examples

[0241] Example 1 Generation of HLA KO primary human T cells with reduced allogeneic recognition

[0242] To replicate HLA KO as a strategy to circumvent T cell alloreactivity, the inventors knocked out the B2M or CIITA gene through specific gRNAs in primary human T cells using electroporation of CRISPR / Cas9 gRNA ribonucleoproteins (Schober ef al., 2019) (Figure 1 a-c; Figure 2 a-b).

[0243] The inventors next induced alloreactivity through co-incubation of effector T cells from one healthy donor for seven days with allogeneic target T cells from another healthy donor with complete HLA mismatch, followed by 48 hours of re-stimulation. This led to preferential activation of HLA mismatched target cells (Figure 1 d). Survival of allogeneic T cells was significantly rescued when [32M- T cells after B2M KO (Figure 2c-d) were used as target cells (Figure 1 d).

[0244] Example 2 Intrinsic in vitro and in vivo functionality of HLA class deficient primary human T cells

[0245] Given the potential of HLA KO to generate T cell products that can escape allogeneic T-cell recognition, the inventors next studied whether loss of HLA expression is associated with changes in T-cell functionality (Torikai et al., 2013; Gornalusse et al., 2017; Lee et al., 2020). The inventors equipped T cells with a TOR specific for the HLA-A*02:01 -restricted Cytomegalovirus (CMV) epitope pp65495-503 via orthotopic TCR replacement (OTR) (Schober et al., 2019; Muller et al., 2021 ). Simultaneous to TCR knock-in (TCR KI) into the endogenous TCR Alpha Constant (TRAC) gene locus and KO of TCR Beta Constant (TRBC), the inventors performed no additional editing (“TCR KI only”), knocked out endogenous B2M (“B2M KO”) or CIITA (“CIITA KO”), followed by purity sorting on CD8+hTCR’ mTRBC+cells (Figure 5). The inventors then coincubated these effector cells with peptide-loaded K562 cells and checked for release of IFNy and TNFa (Figure 3a). HLA- edited cells showed no change in their sensitivity of target-cell recognition compared to unedited cells (Figure 3b). The inventors also investigated killing of peptide-loaded HepG2 target cells using a live-cell imaging system. Again, neither CIITA KO (Figure 4c) nor B2M KO (Figure 4d) affected effector function of TCR-transgenic T cells.

[0246] Next, the inventors studied whether HLA editing would affect T-cell functionality in vivo. The inventors adoptively transferred TOR KI cells with or without B2M KO into irradiated NSG / HHD HLA-A*02:01 - transgenic recipient mice (Shultz et al., 2010) which were subsequently infected with human NLV peptide-expressing murine CMV (mCMV) (Muller et al., 2021 ) (Figure 7a). Human T cells could be recovered from livers on day 8 (Figure 7b). The frequency of [32M- T cells was completely preserved compared to the infusion product (Figure 7c). This indicates that [32M- T cells are neither positively nor negatively selected in vivo and confirms that HLA class I editing does not change intrinsic T-cell functionality.

[0247] Example 3 NK cell recognition of HLA class I deficient primary human T cells

[0248] NK cells may recognize HLA-negative cells through a “missing self” mechanism

[0249] (Bix et al., 1991 ; Liao et al., 1991 ). The inventors therefore co-incubated NK cells with unedited T cells or B2M KO T cells and analyzed NK cell activation (Figure 6a). Sort- purified B2M KO T cells induced NK cell activation significantly more than WT T cells, almost to the same degree as positive controls did (Figure 6b-c). These data confirm that HLA KO T cells are prone to rejection through NK cells.

[0250] Example 4 Generation of HLA class I reduced primary human T cells

[0251] Based on these findings, the inventors aimed to explore whether a reduction, rather than complete elimination, of the diversity of HLA molecules (Han et al., 2019; Xu et al., 2019) can be achieved in primary human T cells in a single editing event.

[0252] The inventors took T cells of an HLA-A*02:01+A*03:01+B*44:03+B*51 :01+C*15:02+0*16:01+donor (Figure 8a) and left these cells unedited, knocked out only one individual HLA allele at a time or individually knocked out all six HLA class I alleles, except for one. The sole preservation of a single HLA allele would allow coverage of 80% of the European Caucasian population if performed for the 10 most frequent individual HLA class I alleles (Figure 10a) (Effenberger et al., 2019). As additional controls, the inventors knocked out all six HLA class I alleles individually in one sample or knocked out B2M (Figure 10b). Flow cytometric antibody staining can be used to distinguish HLA BC and individual HLA-A*02 and HLA-A*03 proteins (Figure 10b-d). The inventors observed absence of [32m protein only upon B2M KO, but not in any of the other samples in which HLA alleles were edited individually. HLA BC- T cells were generated by B2M KO, but also observed in the two samples in which either HLA- A*02:01 or HLA-A*03:01 -specific gRNAs were left out of the editing cocktail while all gRNAs targeting HLA-B and HLA-C alleles were present. T cells lacking HLA-A*02:01 or HLA-A*03:01 were most effectively generated with editing cocktails containing both HLA-A-specific gRNAs, indicating some degree of cross-reactive editing. In fact, T cells lacking HLA-A*02:01 were more robustly induced by the HLA-A*03:01 -targeting gRNA and vice versa (Figure 10e).

[0253] Still, editing with individual gRNA cocktails yielded cell products with very specific HLA allele expression profiles. The number of expressed individual HLA alleles was thereby associated with distinct [32m protein expression levels (Figure 8b). Overall, these data indicate that targeting single HLA alleles with individual gRNAs can generate HLA- reduced T cells.

[0254] Example 5 Escape of HLA-reduced primary human T cells from allogeneic recognition in vitro

[0255] HLA-reduced T cells should not elicit alloreactive T cell and NK cell mediated recognition. To test this, the inventors repeated the MLR assay with sort-purified HLA- reduced target T cells (expressing HLA-A*02:01 or HLA-A*03:01 ) from donor A, who had a complete HLA mismatch with the three donors providing effector T cells, except for HLA-A*02:01 or HLA-A*03:01 respectively (Figure 9a). As controls, the inventors also used HLA mismatched target T cells that were not edited, HLA matched autologous target cells, as well as sort-purified cells lacking HLA BC and HLA-A*02:01 or HLA-A*03:01 or both. The HLA-A2’ HLA-A3+donor shows alloreactivity towards HLA reduced cells that are HLA-A2+HLA-A3’. Both HLA-A2+HLA-A3’ donors, on the other hand, do not show such alloreactivity towards HLA-reduced cells that are HLA- A2+HLA-A3-. The latter donors also do not show alloreactivity towards HLA-reduced cells that are HLA-A2’ HLA-A3+, but alloreactivity does not necessarily have to be consistently present against all individual HLA molecules in all donors (Figure 9a). Overall, HLA-reduced T cells seem to be largely protected from allogeneic T cell recognition. Importantly, HLA-reduced T cells also did not activate NK cells (Figure 9b). Next to T cells expressing only HLA-A*02:01 or HLA-A*03:01 , cells lacking HLA-A*02:01 , HLA- A*03:01 and HLA BC did not stimulate NK cells, protecting from missing-self recognition to a similar degree as HLA-E knock-in T cells did. The inventors hypothesize that protection of HLA-reduced cells from NK cell recognition could be due to preservation of non-canonical HLA expression. Indeed, cells lacking HLA-A*02:01 , HLA-A*03:01 and HLA BC could be stained for HLA E (Figure 9c), which is in line with preserved [32m protein expression upon 6xK0 (Figure 10b-c). In summary, HLA- reduced T cells escape both alloreactive T cell and NK cell mediated recognition in vitro.

[0256] Example 6 Improved functionality of HLA-reduced primary human T cells in the presence of HLA mismatched T and NK cells in vivo

[0257] Finally, the inventors aimed to investigate the functionality of HLA-reduced primary human T cells in vivo. Studying allogeneic T cell and NK cell-mediated recognition of human T cells in vivo is challenging. Usually, for in vivo testing of allogeneic rejection of human T cells, the individual cell products (T cells from one donor, HLA mismatched T cells from another donor, as well as NK cells) are actively injected into mice, often pre-primed before ex vivo, and then only monitored over short periods of time due to poor maintenance of transferred T cells. For this reason, the inventors humanized mice after transplantation with CD34 (hCD34)+hematopoietic stem and progenitor cells (HSPCs) that build up a human endogenous immune cell repertoire encompassing B, T and NK cells (Figure 12). In addition to this, recipient mice had a NSG-SGM3 background, which allows more rapid and more complete reconstitution of human immune cell lineages compared to conventional NSG mice (Norelli et al., 2018).

[0258] To probe the functionality of HLA-reduced primary human T cells, the inventors reduced HLA diversity so that HLA-A*02:01 would be the sole matching HLA allele compared to the donor that was used for recipient mouse humanization (Figure 11 a). Simultaneously, the inventors knocked-in (KI) an anti-CD19 CAR into the endogenous TRAC locus. In contrast to our previous in vivo analysis, the inventors here chose an anti-CD19 CAR as an antigen-specific receptor since the CAR would recognize the reconstituted human B cells in a continuous manner, and thereby renders the transgenic T cells a good target of allogeneic recognition through reconstituted T cells and NK cells for a period of at least one to two weeks. To maximize engraftment of allogeneic HLA-reduced donor T cells, the inventors directly transferred them after editing without further in vitro culture or purity sorting. As a control, the inventors transferred either allogeneic CAR KI T cells without HLA reduction or no cells. The inventors observed a mild decrease in CD19+B cells when allogeneic CAR T cells were infused (Figure 11 b-c). This elimination of B cells was, however, enhanced when allogeneic CAR T cells had been additionally HLA-reduced. HLA-reduced allogeneic CAR T cells consistently led to more effective B cell elimination also in the bone marrow and spleen at the end point analysis (Figure 11d) as well as in a second independent experiment (Figure 11e). In summary, HLA-reduced allogeneic CAR T cells show improved functionality in the presence of HLA mismatched T and NK cells in a humanized in vivo model of chronic antigen exposure.

[0259] REFERENCES

[0260] Bix M, Liao N-S, Zijlstra M, Loring J, Jaenisch R, Raulet D. Rejection of class I MHC- deficient haemopoietic cells by irradiated MHC-matched mice. Nature. 1991 ; 349(6307): 329-331.

[0261] Effenberger M, Stengl A, Schober K, Gerget M, Kampick M, Muller TR, Schumacher D, Helma J, Leonhardt H, Busch DH. A Double Tag for Universal Generation of Versatile Peptide-MHC Multimers. J Immunol. 2019;202(7):2164-2171 .

[0262] Gett AV, Sallusto F, Lanzavecchia A, Geginat J. T cell fitness determined by signal strength. Nat Immunol. 2003 Apr;4(4):355-60.

[0263] Gornalusse GG, Hirata RK, Funk SE, Riolobos L, Lopes VS, Manske G, Prunkard D, Colunga AG, Hanafi LA, Clegg DO, Turtle C, Russell DW. HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells. Nat Biotechnol. 2017;35(8):765-772.

[0264] Han X, Wang M, Duan S, Franco PJ, Kenty JH, Hedrick P, Xia Y, Allen A, Ferreira LMR, Strominger JL, Melton DA, Meissner TB, Cowan CA. Generation of hypoimmunogenic human pluripotent stem cells. Proc Natl Acad Sci. 2019; 116(21 ): 10441 -10446.

[0265] Lee J, Sheen JH, Lim O, Lee Y, Ryu J, Shin D, Kim YY, Kim M. Abrogation of HLA surface expression using CRISPR / Cas9 genome editing: a step toward universal T cell therapy. Sci Rep. 2020; 10(1 ): 1-10.

[0266] Lee N, Goodlett DR, Ishitani A, Marquardt H, Geraghty DE. HLA-E Surface Expression Depends on Binding of TAP-Dependent Peptides Derived from Certain HLA Class I Signal Sequences. J Immunol. 1998; 160(10):4951 -4960. Liao N-S, Bix M, Zijlstra M, Jaenisch R, Raulet D. MHC Class I Deficiency: Susceptibility to Natural Killer (NK) Cells and Impaired NK Activity. Science. 1991 ;253(5016): 199-202.

[0267] Mehta PH, Fiorenza S, Koldej RM, Jaworowski A, Ritchie DS, Quinn KM. T Cell Fitness and Autologous CAR T Cell Therapy in Haematologic Malignancy. Front Immunol. 2021 Nov 25; 12:780442.

[0268] Muller TR, Jarosch S, Hammel M, Leube J, Grassmann S, Bernard B, Effenberger M, Andra I, Chaudhry MZ, Kauferle T, Malo A, Cicin-Sain L, Steinberger P, Feuchtinger T, Protzer U, Schumann K, Neuenhahn M, Schober K, Busch DH. Targeted T cell receptor gene editing provides predictable T cell product function for immunotherapy. Cell Reports Med. 2021 ;2(8): 100374.

[0269] Norelli M, Camisa B, Barbiera G, Falcone L, Purevdorj A, Genua M, Sanvito F, Ponzoni

[0270] M, Doglioni C, Cristofori P, Traversari C, Bordignon C, Ciceri F, Ostuni R, Bonini C, Casucci M, Bondanza A. Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells. Nat Med. 2018 Jun;24(6):739-748.

[0271] Schober K, Muller TR, Gdkmen F, Grassmann S, Effenberger M, Poltorak M, Stemberger C, Schumann K, Roth TL, Marson A, Busch DH. Orthotopic replacement of T-cell receptor a- and [3- chains with preservation of near-physiological T-cell function. Nat Biomed Eng. 2019;3(12):974-984.

[0272] Shultz LD, Saito Y, Najima Y, Tanaka S, Ochi T, Tomizawa M, Doi T, Sone A, Suzuki

[0273] N, Fujiwara H, Yasukawa M, Ishikawa F. Generation of functional human T-cell subsets with H LA-restricted immune responses in HLA class I expressing NOD / SCID / IL2ry null humanized mice. Proc Natl Acad Sci. 2010; 107(29): 13022- 13027.

[0274] Torikai H, Reik A, Soldner F, Warren EH, Yuen C, Zhou Y, Crossland DL, Huis H, Littman N, Zhang Z, Tykodi SS, Kebriaei P, Lee DA, Miller JC, Rebar EJ, Holmes MC, Jaenisch R, Champlin RE, Gregory PD, Cooper LJ. Toward eliminating HLA class I expression to generate universal cells from allogeneic donors. Blood. 2013; 122(8): 1341 -1349.

[0275] Xu H, Wang B, Ono M, Kagita A, Fujii K, Sasakawa N, lleda T, Gee P, Nishikawa M, Nomura M, Kitaoka F, Takahashi T, Okita K, Yoshida Y, Kaneko S, Hotta A. Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility. Cell Stem Cell. 2019;24(4):566-578.e7.

Claims

CLAIMSWhat is claimed is :1 . A method of producing a modified human T cell for cell therapy of a given recipient from a T cell obtained from a given donor, wherein the donor has at least one HLA-la allele that does not match an HLA-la allele of the recipient, wherein the method comprises inhibiting a cell surface expression of at least one HLA-la allele of the T cell obtained from the donor that does not match the HLA-la allele of the recipient, wherein the modified human T cell expresses beta-2-microglobulin on the cell surface, and wherein the human T cell from the donor is preferably a primary T cell, and wherein optionally the T cell of the donor is an activated T cell, and wherein optionally the method comprises additional inhibiting the cell surface expression of HLA class II, and wherein optionally the method does not include modifying the gene expression of beta-2-microglobulin, and wherein optionally wherein the modified T cell expresses HLA-E, HLA-F, and / or HLA-G, and wherein optionally the method comprises inhibiting the cell surface expression of at least one HLA-la allele by a knock-out of the respective allele, and wherein optionally the method for the knock-out is gene-editing or base-editing, preferably by using CRISPR / Cas technology.

2. A method of generating a human T cell population for allogeneic cellular therapy, wherein the method comprises, in case a donor has at least one HLA-la allele that does not match an HLA-la allele of a recipient, genetically modifying the T cell population to inhibit cell surface expression of at least one HLA-la allele of the isolated T cell population of the donor, wherein the modified human T cell expresses beta-2-microglobulin on the cell surface, and wherein optionally the donor has at least one HLA-la allele that does not match an HLA-la allele of the recipient, and wherein the human T cell from the donor is preferably a primary T cell, and wherein optionally the T cell of the donor is an activated T cell, and wherein optionally the method comprises additional inhibiting the cell surface expression of HLA class II, and wherein optionally the method does not include modifying the gene expression of beta-2-microglobulin, and wherein optionally wherein the modified T cell expresses HLA-E, HLA-F, and / or HLA-G, and whereinoptionally the method comprises inhibiting the cell surface expression of at least one HLA-la allele by a knock-out of the respective allele, and wherein optionally the method for the knock-out is gene-editing or base-editing, preferably by using CRISPR / Cas technology.

3. The method of any one of the preceding claims, wherein the method comprises a) a determination of the HLA-la alleles of the T cell of the donor and the T cell of the recipient, b) (optional) a comparison of the HLA-la alleles of the T cells of the donor and the T cells of the recipient for identifying HLA-la alleles which do not match between the donor and the recipient, c) the genetic modification of an isolated T cell of the donor by inhibiting the cell surface expression of the at least one HLA-la allele, which does not match between the donor and the recipient.

4. The method of any one of the preceding claims, wherein the given donor has been selected from a group of potential donors, wherein the selection is based on the fitness of T cells for cell therapy, and wherein optionally the group of potential donors comprises at least one individual potential donor that has a larger number of HLA-la alleles that matches the recipient than said given donor selected from said group.

5. The method of any one of the preceding claims, wherein the method comprises inhibiting the expression of two, three, four, five or all HLA-la alleles, which do not match with the HLA-la alleles of the recipient, and wherein optionally the modified human T cell expresses only one class HLA-la allele, and wherein optionally the HLA-la allele is a HLA-A allele, and wherein optionally the modified human T cell only expresses the allele selected from the group consisting of HLA- A*02:01 and HLA-A*03:01.

6. The method of any one of the preceding claims, wherein the method comprises inhibiting the expression of all HLA-B and HLA-C alleles, wherein the HLA-A alleles of the donor are homozygote and wherein the HLA-A alleles of the donor match at least one HLA-A allele of the recipient or wherein the method comprisesinhibiting the expression of one HLA-A allele and all HLA-B and HLA-C alleles, wherein the expressed HLA-A allele match with at least one of the HLA-A alleles of the recipient.

7. The method of any one of the preceding claims, wherein additional an antigenspecific receptor is knocked-in or integrated in a TRAC gene locus or a TRBC gene locus, and wherein optionally the antigen-specific receptor is a TCR or a CAR, and wherein optionally the method comprises using gene-editing for the knock-out and the knock-in, preferably by using CRISPR / Cas technology for gene-editing.

8. The method of any one of the preceding claims, the method comprises conducting the inhibition of cell surface expression of at least one HLA-la allele and optionally knock-in or integration of the antigen-specific receptor in a single step.

9. The method of any one of the preceding claims, wherein the modified human T cell is ready for administration directly after inhibition of the cell surface expression of at least one HLA-la allele, wherein optionally the method does not comprise a step of culturing after the inhibition step and / or a step of purification after the inhibition step, and wherein optionally the method comprises conducting the method within four weeks after isolation of the human T cell.

10. A modified human T cell obtainable or obtained by a method of any one of claims 1 to 9.

11. A modified human T cell for allogeneic cell therapy of a given recipient, wherein an expression of at least one HLA-la allele is inhibited, wherein said at least one HLA-la allele does not match an HLA-la allele of the recipient, wherein the expression of beta-2-microglobulin has not been modified, and wherein the modified human T cell is preferably a modified human primary T cell, and wherein optionally the modified T cell expresses HLA-E, HLA-F, and / or HLA-G, and wherein optionally additional expression of HLA class II is inhibited, and wherein optionally the T cell has an ex vivo duration of not more than four weeks.

12. The modified human T cell of claim 11 , wherein the expression of two, three, four, five, or all HLA-la alleles, which do not match with the HLA-la alleles of the recipient, is inhibited; and wherein optionally the T cell expresses only one class HLA-la allele, and wherein optionally the HLA-la allele is an HLA-A allele, and wherein optionally the modified human T cell only expresses the HLA-la allele selected from the group consisting of HLA-A*02:01 and HLA-A*03:01 of the HLA- la class..

13. The modified human T cell of claims 11 or 12, wherein the expression of all HLA- B and HLA-C alleles is inhibited, wherein the HLA-A alleles of the donor are homozygote and wherein the HLA-A alleles of the donor match at least one HLA- A allele of the recipient or wherein the expression of one HLA-A allele and all HLA-B and HLA-C alleles is inhibited, and wherein the expressed HLA-A allele match with at least one of the HLA-A alleles of the recipient..

14. The modified human T cell of any one of claims 11 to 13, wherein an antigenspecific receptor is knocked-in or integrated in a TRAC gene locus or a TRBC gene locus, wherein optionally the antigen-specific receptor is a TCR or a CAR.

15. The modified human T cell of any one of claims 10 to 14 for use in an adoptive cell therapy, wherein optionally the adoptive cell therapy is a treatment of cancer, an autoimmune disease, or an infectious disease.

16. A pharmaceutical composition comprising the modified human T cell of any one of claims 10 to 14 or the modified T cell obtained by the method of any one of claims 1 to 9 for use in therapy, wherein optionally the therapy is a treatment of cancer, an autoimmune disease, or an infectious disease.

17. A use of a human T cell obtained from a given donor for the manufacture of a modified human T cell for allogeneic cell therapy comprising conducting the method of any one of claims 1 to 9, wherein optionally the modified human T cell is for the treatment of cancer, an autoimmune disease or an infectious disease.

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