Car modified NK cells

Abstract

The present invention relates to an isolated natural killer (NK) cell, wherein a KLRC1 gene is replaced by a sequence encoding an anti-CD19 chimeric antigen receptor (CAR). The invention also relates to a production method of the NK cell, to its medical use and to a nucleic acid molecule for insertion into an NK cell.

Description

[0001] CAR modified NK cells

[0002] This application claims the right of priority of European Patent Applications EP24212340.4 EP filed 12 November 2024, and EP24221987.1 filed 20 December 2024, both of which are incorporated by reference herein.

[0003] Field

[0004] The present invention relates to an isolated natural killer (NK) cell, wherein a KLRC1 gene is replaced by a sequence encoding an anti-CD19 chimeric antigen receptor (CAR). The invention also relates to a production method of the NK cell, to its medical use and to a nucleic acid molecule for insertion into an NK cell.

[0005]

[0006] Cellular immunotherapy has already had a practice-changing impact on the treatment of difficult-to-treat advanced hematological cancers and research to improve the available therapies is constantly ongoing. The focus of early cancer immunotherapy research was on conventional T cells, which is reflected by the fact that all approved cellular cancer immunotherapies to date are T cell based. However, other cell types like natural killer (NK) cells are attracting ever more interest and are being studied extensively. In contrast to T cells, NK cells can be used far more readily in an allogenic setting, which opens up the possibility for off-the-shelf products. This would allow banking, rendering the therapy faster to reach the patient and the manufacturing more standardized, easier and cheaper.

[0007] NK cells, as part of the innate immune system, have a T cell receptor-independent mechanism of killing virus- infected or transformed cells. Whether an NK cell gets activated is regulated by the expression and ligation of various germline-encoded activating and inhibitory receptors. Overexpression of ligands for inhibitory receptors is a mechanism by which cancer cells avoid killing by NK cells, and the NKG2A-HLA-E axis has been identified as an outstanding negative regulator of NK cell activity in cancer. HLA-E is a non-classical HLA class I molecule and binds epitopes derived from signal peptides from other, classical HI_A class I allotypes. HI_A-E is expressed at low levels on most tissues but is upregulated on a wide variety of tumor cells and primary human tumors including lymphoma. NKG2A is a C-type lectin that is specific for HLA-E. It forms a heterodimer with CD94 and contains two immunoreceptor tyrosine-based inhibitory motifs (ITIM) in the cytoplasmic domain, which are involved in the transduction of the inhibitory signal. NKG2A is expressed on about 40-50% of peripheral blood NK cells and on a subset of CD8+T cells.

[0008] HI.A-E is upregulated by IFN-y, which is secreted in the tumor microenvironment (TME) of solid tumors. Similarly, NKG2A is upregulated on tumor-infiltrating NK cells and T cells, making HLA-E-NKG2A-mediated inhibition of cytotoxic lymphocytes an immune evasion mechanism of tumors.

[0009] Thus, disrupting the NKG2A-HLA-E axis is a promising strategy for unleashing NK cells. Different ways of blocking NKG2A have been explored, including a protein-retention system, the anti-NKG2A monoclonal antibody monalizumab, or genomic knock-out of the NKG2A encoding gene KLRC1, which all showed to effectively increase the cytotoxicity of NK cells. In this study, we aim to overcome the NKG2A-HI_A-E mediated inhibition of CAR NK cells by generating anti-CD19 CAR NK cells harboring a genetic knock-out (KO) of KLRC1, the gene encoding for NKG2A. We explored the activity of NKG2A edited CAR NK cells generated by two methods, namely by CRISPR-Cas9 mediated knock-out of NKG2A followed by retroviral CAR transduction, and by site-specific knock-in of the CAR construct into the KLRC1 locus with CAR expression under endogenous gene regulation. We compared the cytotoxicity of NKG2A edited CAR NK cells and non-edited CAR NK cells against HI_A-E+tumor cell lines in both short-term and long-term cytotoxicity assays.

[0010] Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to immune-cell treatment of cancer via engineered NK cells. This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.

[0011] Summary of the Invention

[0012] A first aspect of the invention relates to an isolated natural killer cell, wherein a KLRC1 gene is replaced by a sequence encoding an anti-CD19 chimeric antigen receptor (CAR).

[0013] A second aspect of the invention relates to an isolated nucleic acid molecule comprising a. a sequence encoding an anti-CD19 chimeric antigen receptor (CAR), wherein the CAR comprises:

[0014] i. an extracellular domain comprising a single chain variable region moiety (scFv) against CD19;

[0015] ii. a transmembrane domain comprising a transmembrane domain of CD28; and iii. an intracellular domain comprising an intracellular domain of CD28 and TCR-£; b. two flanking regions allowing for insertion of the nucleic acid molecule into a genetic locus of a KLRC1 gene.

[0016] A third aspect of the invention relates to an isolated natural killer cell of the first aspect or an isolated nucleic acid molecule of the second aspect for use in medicine. A fourth aspect of the invention relates to an isolated natural killer cell of the first aspect or an isolated nucleic acid molecule of the second aspect for use in treatment of cancer.

[0017] A fifth aspect of the invention relates to an isolated natural killer cell of the first aspect or an isolated nucleic acid molecule or a combination of the second aspect for use in treatment of an autoimmune disease.

[0018] A further aspect of the invention relates to a method for preparation of a natural killer cell as specified in the first aspect, the method comprising the steps:

[0019] a. providing a peripheral blood mononuclear cell (PBMC);

[0020] b. isolating an NKG2A+ natural killer (NK) cell and keeping the NK cell under cell culture conditions;

[0021] c. contacting the NKG2A+ NK cell with a vector comprising the isolated nucleic acid molecule as specified in the second aspect;

[0022] d. optionally, further knocking out FAS.

[0023] Terms and definitions

[0024] General

[0025] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

[0026] The terms “comprising”, “having”, “containing”, and “including”, and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of’ or “consisting of.”

[0027] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

[0028] As used herein, including in the appended claims, the singular forms “a”, “or” and “the” include plural referents unless the context clearly dictates otherwise.

[0029] "And / or" where used herein is to be taken as specific recitation of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

[0031] The term CD28 in the context of the present specification relates to Cluster of Differentiation 28 (Gene ID: 940, UniProt-ID: P10747).

[0032] The term TCR-^ in the context of the present specification relates to T-cell surface glycoprotein CD3 zeta chain (CD3Z) (Gene ID: 919, UniProt-ID: P20963).

[0033] The term KLRC1 in the context of the present specification relates to killer cell lectin like receptor C1 (Gene ID: 3821, UniProt-ID: P26715).

[0034] The term FAS in the context of the present specification relates to tumor necrosis factor receptor superfamily member 6 (Gene ID: 355, UniProt-ID: P25445).

[0035] The term PBS in the context of the present specification relates to Phosphate Buffered Saline: NaCI: 8.0 g / L; KCI: 0.2 g / L; Na2HPO4: 1.44 g / L KH2PO4: 0.24 g / L, pH 7.4.

[0036] Any patent document cited herein shall be deemed incorporated by reference herein in its entirety.

[0037] Sequences

[0038] Sequences similar or homologous (e.g., at least about 70% sequence identity) to the sequences disclosed herein are also part of the invention. In some embodiments, the sequence identity at the amino acid level can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. At the nucleic acid level, the sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively, substantial identity exists when the nucleic acid segments will hybridize under selective hybridization conditions (e.g., very high stringency hybridization conditions), to the complement of the strand. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.

[0039] In the context of the present specification, the terms sequence identity and percentage of sequence identity refer to a single quantitative parameter representing the result of a sequence comparison determined by comparing two aligned sequences position by position. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http: / / blast.ncbi.nlm.nih.gov / ).

[0040] One example for comparison of amino acid sequences is the BLASTP algorithm that uses the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 11, Extension 1; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match / Mismatch Scores: 1.-2; Gap costs: Linear. Unless stated otherwise, sequence identity values provided herein refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively. Reference to identical sequences without specification of a percentage value implies 100% identical sequences (i.e. the same sequence).

[0041] Particular embodiments make use of the sequences as disclosed herein (i.e. 100% identical). General Biochemistry: Peptides, Amino Acid Sequences

[0042] The term polypeptide in the context of the present specification relates to a molecule consisting of 50 or more amino acids that form a linear chain wherein the amino acids are connected by peptide bonds. The amino acid sequence of a polypeptide may represent the amino acid sequence of a whole (as found physiologically) protein or fragments thereof. The term "polypeptides" and "protein" are used interchangeably herein and include proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences. Amino acid residue sequences are given from amino to carboxyl terminus. Capital letters for sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, 3rded. p. 21). Lower case letters for amino acid sequence positions refer to the corresponding D- or (2R)-amino acids. Sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (lie, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V).

[0043] General Molecular Biology: Nucleic Acid Sequences, Expression

[0044] The term gene refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. A polynucleotide sequence can be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.

[0045] The term transgene in the context of the present specification relates to a gene or genetic material that has been transferred from one organism to another. In the present context, the term may also refer to transfer of the natural or physiologically intact variant of a genetic sequence into tissue of a patient where it is missing. It may further refer to transfer of a natural encoded sequence the expression of which is driven by a promoter absent or silenced in the targeted tissue.

[0046] The term recombinant in the context of the present specification relates to a nucleic acid, which is the product of one or several steps of cloning, restriction and / or ligation and which is different from the naturally occurring nucleic acid. A recombinant virus particle comprises a recombinant nucleic acid.

[0047] The terms gene expression or expression, or alternatively the term gene product, may refer to either of, or both of, the processes - and products thereof - of generation of nucleic acids (RNA) or the generation of a peptide or polypeptide, also referred to transcription and translation, respectively, or any of the intermediate processes that regulate the processing of genetic information to yield polypeptide products. The term gene expression may also be applied to the transcription and processing of a RNA gene product, for example a regulatory RNA or a structural (e.g. ribosomal) RNA. If an expressed polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Expression may be assayed both on the level of transcription and translation, in other words mRNA and / or protein product.

[0048] The term Nucleotides in the context of the present specification relates to nucleic acid or nucleic acid analogue building blocks, oligomers of which are capable of forming selective hybrids with RNA or DNA oligomers on the basis of base pairing. The term nucleotides in this context includes the classic ribonucleotide building blocks adenosine, guanosine, uridine (and ribosylthymine), cytidine, the classic deoxyribonucleotides deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. It further includes analogues of nucleic acids such as phosphothioates, 2’0-methylphosphothioates, peptide nucleic acids (PNA; N-(2-aminoethyl)-glycine units linked by peptide linkage, with the nucleobase attached to the alpha-carbon of the glycine) or locked nucleic acids (LNA; 2’0, 4’C methylene bridged RNA building blocks). Wherever reference is made herein to a hybridizing sequence, such hybridizing sequence may be composed of any of the above nucleotides, or mixtures thereof. The terms capable of forming a hybrid or hybridizing sequence in the context of the present specification relate to sequences that under the conditions existing within the cytosol of a mammalian cell, are able to bind selectively to their target sequence. Such hybridizing sequences may be contiguously reverse-complimentary to the target sequence, or may comprise gaps, mismatches or additional non-matching nucleotides. The minimal length for a sequence to be capable of forming a hybrid depends on its composition, with C or G nucleotides contributing more to the energy of binding than A or T / ll nucleotides, and on the backbone chemistry.

[0049] The term nucleic acid expression vector in the context of the present specification relates to a plasmid, a viral genome or an RNA, which is used to transfect (in case of a plasmid or an RNA) or transduce (in case of a viral genome) a target cell with a certain gene of interest, or -in the case of an RNA construct being transfected- to translate the corresponding protein of interest from a transfected mRNA. For vectors operating on the level of transcription and subsequent translation, the gene of interest is under control of a promoter sequence and the promoter sequence is operational inside the target cell, thus, the gene of interest is transcribed either constitutively or in response to a stimulus or dependent on the cell’s status. In certain embodiments, the viral genome is packaged into a capsid to become a viral vector, which is able to transduce the target cell. (Cancer) Immunotherapy

[0050] In the context of the present specification, the term adoptive cell therapy (ACT), also used as adoptive therapy or adoptive T-cell therapy represents a recent immunotherapeutic approach within the realm of cancer treatment, which uses the potent anti-tumour capabilities of autologous (a patient's own) or allogeneic (another individual’s) immune cells by engineering or activating the cells for enhanced tumour recognition and destruction. The process of adoptive cell therapy begins with the extraction of specific immune cells, e.g. NK cells, from a patient's bloodstream; alternatively, the cells may be derived from a healthy donor cell bank. These cells are then modified ex vivo through genetic engineering techniques or activated ex vivo through various stimulation methods, to enhance their tumour-specific recognition and cytotoxicity. The genetic modification may involve the introduction of chimeric antigen receptors (CARs), which are synthetic receptors designed to target tumour-associated antigens, thus endowing the engineered immune cells with precise tumour recognition capabilities. The engineered or activated NK cells are then expanded to generate a substantial population, often referred to as a cell product. Once the cell product is prepared, it is infused into the patient. Adoptive therapy encompasses modalities such as Chimeric Antigen Receptor (CAR) Cell Therapy, wherein cells are genetically engineered to express CARs specific to tumour antigens; Natural Killer (NK) Cell Therapy.

[0051] The term cancer as used in the context of the present specification relates to malignant neoplastic disease; the terms “cancer” and “malignant neoplastic disease” are used synonymously herein. They specifically include carcinoma (epithelial derived cancer), sarcoma (connective tissue derived cancer), lymphoma and leukemia, germ-cell derived tumours and blastomas. Particular alternatives of any of the aspects and embodiments disclosed herein are directed at the use of the compounds and compositions of the invention in treatment of solid tumours. Other alternatives of any of the aspects and embodiments disclosed herein are directed at the use of the combinations of the invention in treatment of liquid cancers such as myelogenous or granulocytic leukemia, particularly AML, multiple myeloma, lymphatic, lymphocytic, or lymphoblastic leukemia and lymphoma, myeloproliferative neoplasms or myelodysplastic syndromes.

[0052] As used herein, the term treating or treatment of any disease or disorder (e.g. cancer) refers in one embodiment to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and / or prevention of disease are generally known in the art, unless specifically described hereinbelow.

[0053] Detailed Description of the Invention

[0054] Natural killer (NK) cell

[0055] A first aspect of the invention relates to an isolated natural killer cell characterized by

[0056] a. presence of a gene encoding an anti-CD19 chimeric antigen receptor (CAR) under control of a promoter of a KLRC1 gene; and

[0057] b. engineered, reduced or absent expression (= expression below a baseline level or knock out) of a KLRC1 gene. The baseline level is determined prior to knockout, meaning the baseline level is the expression level of a wildtype NK cell. The expression after KLRC1 gene knockout is below 50% of the baseline level. Some expression may remain due to the heterozygosity, meaning that only one allele of the KLRC1 gene may be knocked out. In certain embodiments, the expression after KLRC1 gene knockout is below 60% of the baseline level. In certain embodiments, the expression after KLRC1 gene knockout is below 70% of the baseline level. In certain embodiments, the expression after KLRC1 gene knockout is below 80% of the baseline level.

[0058] An alternative of the first aspect relates to an isolated natural killer cell, wherein a (endogenous) KLRC1 gene is replaced by a sequence encoding an anti-CD19 chimeric antigen receptor (CAR). The nucleic acid sequence encoding the CAR is inserted into the locus of the KLRC1 gene, thereby disrupting the KLRC1 gene.

[0059] In certain embodiments, the natural killer cell additionally is characterized by a lack of expression of a FAS gene. In certain embodiments, FAS is knocked out using CRISPR / Cas. In certain embodiments, a genome of the natural killer cell comprises a nucleic acid sequence having at least >75% identity with a sequence of SEQ ID NO 14. In certain embodiments, a genome of the natural killer cell comprises a nucleic acid sequence having at least >80% identity with a sequence of SEQ ID NO 14. In certain embodiments, a genome of the natural killer cell comprises a nucleic acid sequence having at least >85% identity with a sequence of SEQ ID NO 14. In certain embodiments, a genome of the natural killer cell comprises a nucleic acid sequence having at least >90% identity with a sequence of SEQ ID NO 14. In certain embodiments, a genome of the natural killer cell comprises a nucleic acid sequence having at least >92% identity with a sequence of SEQ ID NO 14. In certain embodiments, a genome of the natural killer cell comprises a nucleic acid sequence having at least >94% identity with a sequence of SEQ ID NO 14. In certain embodiments, a genome of the natural killer cell comprises a nucleic acid sequence having at least >95% identity with a sequence of SEQ ID NO 14. In certain embodiments, a genome of the natural killer cell comprises a nucleic acid sequence having at least >97% identity with a sequence of SEQ ID NO 14. In certain embodiments, a genome of the natural killer cell comprises a nucleic acid sequence having at least >98% identity with a sequence of SEQ ID NO 14. In certain embodiments, a genome of the natural killer cell comprises a nucleic acid sequence having at least >99% identity with a sequence of SEQ ID NO 14. In certain embodiments, a genome of the natural killer cell comprises a nucleic acid sequence having 100% identity with a sequence of SEQ ID NO 14.

[0060] Nucleic acid molecule

[0061] A second aspect of the invention relates to an isolated nucleic acid molecule (template) comprising

[0062] a. a coding sequence tract sequence encoding an anti-CD19 chimeric antigen receptor (CAR), wherein the encoded CAR comprises:

[0063] i. an extracellular domain comprising (or essentially consisting of) a single chain variable region moiety (scFv) against CD19;

[0064] ii. a transmembrane domain comprising (or essentially consisting of) a transmembrane domain of CD28; and

[0065] iii. an intracellular domain comprising (or essentially consisting of) an N-terminal part of an intracellular domain of CD28 and a C-terminal part of an intracellular domain of TCR-£;

[0066] b. two flanking regions situated immediately in 5’ and 3’ of the coding sequence tract, respectively, allowing for insertion (by homologous recombination) of the nucleic acid molecule into a genetic locus of a KLRC1 gene. Insertion by homologous recombination is a genetic engineering technique where a DNA sequence is introduced into a specific location within a genome by using a segment of DNA that shares homologous sequences with the target site. This method exploits the natural process of homologous recombination, allowing the introduced DNA to precisely integrate at the desired site, replacing or inserting next to the target sequences. In certain embodiments, the two flanking regions allowing for insertion comprise a first nucleic acid sequence (5’ of the gene encoding CAR) having at least >75% identity with a sequence of SEQ ID NO 04 and a second nucleic acid sequence (3’ of the gene encoding CAR) having at least >75% identity with a sequence of SEQ ID NO 05.

[0067] In certain embodiments, the two flanking regions allowing for insertion comprise a first nucleic acid sequence (5’ of the gene encoding CAR) having at least >80% identity with a sequence of SEQ ID NO 04 and a second nucleic acid sequence (3’ of the gene encoding CAR) having at least >80% identity with a sequence of SEQ ID NO 05.

[0068] In certain embodiments, the two flanking regions allowing for insertion comprise a first nucleic acid sequence (5’ of the gene encoding CAR) having at least >85% identity with a sequence of SEQ ID NO 04 and a second nucleic acid sequence (3’ of the gene encoding CAR) having at least >85% identity with a sequence of SEQ ID NO 05.

[0069] In certain embodiments, the two flanking regions allowing for insertion comprise a first nucleic acid sequence (5’ of the gene encoding CAR) having at least >90% (particularly >92%, >94%, >95%, >97%, >98%, or >99%) identity with a sequence of SEQ ID NO 04 and a second nucleic acid sequence (3’ of the gene encoding CAR) having at least >90% (particularly >92%, >94%, >95%, >97%, >98%, or >99%) identity with a sequence of SEQ ID NO 05.

[0070] In certain embodiments, the two flanking regions allowing for insertion comprise a first nucleic acid sequence (5’ of the gene encoding CAR) having 100% identity with a sequence of SEQ ID NO 04 and a second nucleic acid sequence (3’ of the gene encoding CAR) having 100% identity with a sequence of SEQ ID NO 05.

[0071] In certain embodiments, the two flanking regions comprising a certain sequence or a sequence tract are characterized in that the two flanking regions have (retain) a biological activity of (at least) > 30% of an activity of two flanking regions of SEQ ID NO 04 and SEQ ID NO 05. In certain embodiments, the two flanking regions comprising a certain sequence or a sequence tract are characterized in that the two flanking regions have (retain) a biological activity of (at least) > 50% of an activity of two flanking regions of SEQ ID NO 04 and SEQ ID NO 05. In certain embodiments, the two flanking regions comprising a certain sequence or a sequence tract are characterized in that the two flanking regions have (retain) a biological activity of (at least) > 80% of an activity of two flanking regions of SEQ ID NO 04 and SEQ ID NO 05. In certain embodiments, the two flanking regions comprising a certain sequence or a sequence tract are characterized in that the two flanking regions have (retain) a biological activity of (at least) > 90% of an activity of two flanking regions of SEQ ID NO 04 and SEQ ID NO 05. Biological activity in this context is to allow for insertion of the nucleic acid molecule in the genetic locus of a KLRC1 gene with a certain efficacy compared to the efficacy of a nucleic acid sequence of SEQ ID NO 04 and SEQ ID NO 05.

[0072] The assay for insertion efficacy comprises the following steps: Insertion efficacy is determined by flow cytometric analysis, in which the anti-CD19 CAR is stained with recombinant, fluorescently labelled CD19 protein. In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least >75% identity with a sequence of SEQ ID NO 14. In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least >80% identity with a sequence of SEQ ID NO 14. In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least >85% identity with a sequence of SEQ ID NO 14. In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least >90% identity with a sequence of SEQ ID NO 14. In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least >92% identity with a sequence of SEQ ID NO 14. In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least >94% identity with a sequence of SEQ ID NO 14. In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least >95% identity with a sequence of SEQ ID NO 14. In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least >97% identity with a sequence of SEQ ID NO 14. In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least >98% identity with a sequence of SEQ ID NO 14. In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least >99% identity with a sequence of SEQ ID NO 14. In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence having 100% identity with a sequence of SEQ ID NO 14.

[0073] Sequence identity of determined via the BLASTN algorithm with the default parameters as described above.

[0074] An alternative of the second aspect relates to a combination of isolated nucleic acid molecules comprising or consisting of

[0075] a. a first isolated nucleic acid molecule according to the second aspect, and b. a second isolated nucleic acid molecule being capable of abolishing an expression of a FAS gene inside a cell, particularly wherein the second isolated nucleic acid molecule is of SEQ ID NO: 03.

[0076] Chimeric antigen receptor (CAR)

[0077] The chimeric antigen receptor of the NK cell or of the nucleic acid molecule is described here in further detail.

[0078] In certain embodiments, the CAR is monomeric. In certain embodiments, further associated proteins are not needed for the CAR to fulfil its function.

[0079] In certain embodiments, the encoded CAR comprises an extracellular domain comprising (or essentially consisting of) a single chain variable region moiety (scFv) against CD19. In certain embodiments, the encoded CAR comprises a transmembrane domain comprising (or essentially consisting of) a transmembrane domain of CD28.

[0080] In certain embodiments, the encoded CAR comprises an intracellular domain comprising (or essentially consisting of) an N-terminal part of an intracellular domain of CD28 and a C-terminal part of an intracellular domain of TCR-£.

[0081] In certain embodiments, the CAR is characterized in that the extracellular domain comprises (or essentially consists of) an amino acid sequence having at least >85% identity with a sequence of SEQ ID NO 08. In certain embodiments, the CAR is characterized in that the extracellular domain comprises (or essentially consists of) an amino acid sequence having at least >90% (particularly >92% or >94%) identity with a sequence of SEQ ID NO 08. In certain embodiments, the CAR is characterized in that the extracellular domain comprises (or essentially consists of) an amino acid sequence having at least >95% (particularly >97% or >98% or >99%) identity with a sequence of SEQ ID NO 08. In certain embodiments, the CAR is characterized in that the extracellular domain comprises (or essentially consists of) an amino acid sequence having 100% identity with a sequence of SEQ ID NO 08.

[0082] In certain embodiments, the CAR is characterized in that the transmembrane domain comprises (or essentially consists of) an amino acid sequence having at least >85% identity with a sequence of SEQ ID NO 10. In certain embodiments, the CAR is characterized in that the transmembrane domain comprises (or essentially consists of) an amino acid sequence having at least >90% (particularly >92% or >94%) identity with a sequence of SEQ ID NO 10. In certain embodiments, the CAR is characterized in that the transmembrane domain comprises (or essentially consists of) an amino acid sequence having at least >95% (particularly >97% or >98% or >99%) identity with a sequence of SEQ ID NO 10. In certain embodiments, the CAR is characterized in that the transmembrane domain comprises (or essentially consists of) an amino acid sequence having 100% identity with a sequence of SEQ ID NO 10.

[0083] In certain embodiments, the CAR is characterized in that the intracellular domain comprises (or essentially consists of) an amino acid sequence having at least >85% identity with a sequence of SEQ ID NO 12. In certain embodiments, the CAR is characterized in that the intracellular domain comprises (or essentially consists of) an amino acid sequence having at least >90% (particularly >92% or >94%) identity with a sequence of SEQ ID NO 12. In certain embodiments, the CAR is characterized in that the intracellular domain comprises (or essentially consists of) an amino acid sequence having at least >95% (particularly >97% or >98% or >99%) identity with a sequence of SEQ ID NO 12. In certain embodiments, the CAR is characterized in that the intracellular domain comprises (or essentially consists of) an amino acid sequence having 100% identity with a sequence of SEQ ID NO 12. In certain embodiments, the CAR is characterized in that the extracellular domain is encoded by a nucleic acid sequence having at least >75% identity with a sequence of SEQ ID NO 09. In certain embodiments, the CAR is characterized in that the extracellular domain is encoded by a nucleic acid sequence having at least >80% identity with a sequence of SEQ ID NO 09. In certain embodiments, the CAR is characterized in that the extracellular domain is encoded by a nucleic acid sequence having at least >85% identity with a sequence of SEQ ID NO 09. In certain embodiments, the CAR is characterized in that the extracellular domain is encoded by a nucleic acid sequence having at least >90% (particularly >92%, >94%, >95%, >97%, >98%, or >99%) identity with a sequence of SEQ ID NO 09. In certain embodiments, the CAR is characterized in that the extracellular domain is encoded by a nucleic acid sequence having 100% identity with a sequence of SEQ ID NO 09.

[0084] In certain embodiments, the CAR is characterized in that the transmembrane domain is encoded by a nucleic acid sequence having at least >75% identity with a sequence of SEQ ID NO 11. In certain embodiments, the CAR is characterized in that the transmembrane domain is encoded by a nucleic acid sequence having at least >80% identity with a sequence of SEQ ID NO 11. In certain embodiments, the CAR is characterized in that the transmembrane domain is encoded by a nucleic acid sequence having at least >85% identity with a sequence of SEQ ID NO 11. In certain embodiments, the CAR is characterized in that the transmembrane domain is encoded by a nucleic acid sequence having at least >90% (particularly >92%, >94%, >95%, >97%, >98%, or >99%) identity with a sequence of SEQ ID NO 11. In certain embodiments, the CAR is characterized in that the transmembrane domain is encoded by a nucleic acid sequence having 100% identity with a sequence of SEQ ID NO 11.

[0085] In certain embodiments, the CAR is characterized in that the intracellular domain is encoded by a nucleic acid sequence having at least >75% identity with a sequence of SEQ ID NO 13. In certain embodiments, the CAR is characterized in that the intracellular domain is encoded by a nucleic acid sequence having at least >80% identity with a sequence of SEQ ID NO 13. In certain embodiments, the CAR is characterized in that the intracellular domain is encoded by a nucleic acid sequence having at least >85% identity with a sequence of SEQ ID NO 13. In certain embodiments, the CAR is characterized in that the intracellular domain is encoded by a nucleic acid sequence having at least >90% (particularly >92%, >94%, >95%, >97%, >98%, or >99%) identity with a sequence of SEQ ID NO 13. In certain embodiments, the CAR is characterized in that the intracellular domain is encoded by a nucleic acid sequence having 100% identity with a sequence of SEQ ID NO 13.

[0086] In certain embodiments, the CAR comprises (or essentially consists of) an amino acid sequence having at least >85% identity with a sequence of SEQ ID NO 06. In certain embodiments, the CAR comprises (or essentially consists of) an amino acid sequence having at least >90% identity with a sequence of SEQ ID NO 06. In certain embodiments, the CAR comprises (or essentially consists of) an amino acid sequence having at least >92% identity with a sequence of SEQ ID NO 06. In certain embodiments, the CAR comprises (or essentially consists of) an amino acid sequence having at least >94% identity with a sequence of SEQ ID NO 06. In certain embodiments, the CAR comprises (or essentially consists of) an amino acid sequence having at least >95% identity with a sequence of SEQ ID NO 06. In certain embodiments, the CAR comprises (or essentially consists of) an amino acid sequence having at least >97% identity with a sequence of SEQ ID NO 06. In certain embodiments, the CAR comprises (or essentially consists of) an amino acid sequence having at least >98% identity with a sequence of SEQ ID NO 06. In certain embodiments, the CAR comprises (or essentially consists of) an amino acid sequence having at least >99% identity with a sequence of SEQ ID NO 06. In certain embodiments, the CAR comprises (or essentially consists of) an amino acid sequence having 100% identity with a sequence of SEQ ID NO 06.

[0087] Sequence identity of determined via the BLASTP algorithm with the default parameters as described above.

[0088] In certain embodiments, the CAR comprising a certain sequence or a sequence tract is characterized in that the CAR has (retains) a biological activity of (at least) > 30% of an activity of a CAR of SEQ ID NO 06. In certain embodiments, the CAR comprising a certain sequence or a sequence tract is characterized in that the CAR has (retains) a biological activity of (at least) > 50% of an activity of a CAR of SEQ ID NO 06. In certain embodiments, the CAR comprising a certain sequence or a sequence tract is characterized in that the CAR has (retains) a biological activity of (at least) > 80% of an activity of a CAR of SEQ ID NO 06. In certain embodiments, the CAR comprising a certain sequence or a sequence tract is characterized in that the CAR has (retains) a biological activity of (at least) > 90% of an activity of a CAR of SEQ ID NO 06.

[0089] The assay for this biological activity comprises the following steps: Biological activity is determined by a cytotoxicity assay against HLA-I proficient CD19+ target cells. In the assay, fluorescently labelled target cells are cocultured with CAR cells and the frequency of dead target cells is determined with a viability stain and read out by flow cytometry. The measurement for the biological activity of CAR cells is the frequency of dead target cells. Medical use

[0090] A third aspect of the invention relates to an isolated natural killer cell of the first aspect or an isolated nucleic acid molecule of the second aspect for use in medicine. A fourth aspect of the invention relates to an isolated natural killer cell of the first aspect or an isolated nucleic acid molecule or a combination of the second aspect for use in treatment of cancer.

[0091] In certain embodiments, the cancer is selected from lymphoma and leukemia.

[0092] A fifth aspect of the invention relates to an isolated natural killer cell of the first aspect or an isolated nucleic acid molecule or a combination of the second aspect for use in treatment of an autoimmune disease.

[0093] In certain embodiments, the autoimmune disease is selected from the group of systemic lupus erythematosus, myositis, systemic sclerosis and ulcerative colitis.

[0094] CD19 directed CAR cell therapy, including CAR NK cells (Wang et al. Cell. 2025 Aug 7;188(16):4225-4238) represents a promising and potentially transformative approach for autoimmune diseases such as systemic lupus erythematosus, myositis, systemic sclerosis and ulcerative colitis, offering deeper and more durable B-cell depletion than conventional therapies and showing early evidence of sustained immune reset, safety and remission (Mackensen et al. Nat Med. 2022 Oct;28(10):2124-2132, Muller et al. N Engl J Med. 2024 Feb 22;390(8):687-700, Wang et al. Cell. 2024 Sep 5;187(18):4890-4904, Tur et al. Ann Rheum Dis. 2025 Jul 11:80003-4967(25)04174-3., Muller et al. N Engl J Med. 2025 Sep 25;393(12):1239-1241).

[0095] Building on this therapeutic concept, the engineered CD19 CAR NK cells described herein are particularly well suited for these autoimmune indications. NK cells combine potent cytotoxicity with a favorable safety profile, as they do not induce graft-versus-host disease and exhibit limited cytokine release compared to CAR T cells. The targeted disruption of KLRC 7(encoding NKG2A) and the optional use of an NKG2A-promoter-driven CAR design further enhance their activity by releasing NK cells from inhibitory checkpoint control via the NKG2A / HI-A-E axis. This modification may promote stronger and more sustained depletion of autoreactive B cells.

[0096] Moreover, co-editing of the death receptor FAS improves CAR NK cell persistence, potentially supporting durable immune modulation and minimizing relapse of autoreactivity after initial remission. Together, these features position NKG2A- and FAS-edited CD19 CAR NK cells as a highly rational and innovative therapeutic approach for refractory or relapsing autoimmune diseases, combining effective B-cell targeting with enhanced cytotoxicity, persistence, and safety. Production method

[0097] A further aspect of the invention relates to a method for preparation of a natural killer cell as specified in the first aspect, the method comprising the steps:

[0098] a. providing a plurality of peripheral blood mononuclear cells (PBMCs); b. isolating an NKG2A+ natural killer (NK) cell and keeping the NK cell under cell culture conditions;

[0099] c. contacting the NKG2A+ NK cell with a vector comprising the isolated nucleic acid molecule as specified in the second aspect;

[0100] d. optionally, further knocking out FAS.

[0101] In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is selected from an adeno-associated viral (AAV) vector, an adenoviral vector, a retroviral vector, and a lentiviral vector. In certain embodiments, the vector is an AAV vector. In certain embodiments, the delivery of the vector is non-viral. The template can be provided in the form of double-stranded DNA and delivered by electroporation together with a KLRC1-targeting Cas9 RNP.

[0102] In certain embodiments, FAS is knocked out using CRISPR / Cas.

[0103] NK cell features

[0104] The NK cell of the present invention is characterized by expression of NKG2A and CD56 (NKG2A+ and CD56+) and lack of expression of CD3 (CD3-). This expression profile may be assessed via flow cytometry.

[0105] The term cell culture conditions in the context of the present specification of the NK cell culture relates to a co-culture with K562-mblL-21 feeder cells with a supplement of IL-2 (further details under “Isolation and expansion of NK cells”).

[0106] Pharmaceutical Compositions and Administration / Dosage Forms

[0107] According to one aspect of the cell according to the invention, the cell according to the invention is provided as a pharmaceutical composition, pharmaceutical administration form, or pharmaceutical dosage form.

[0108] Certain embodiments of the invention relate to a dosage form for parenteral administration, such as intravenous injection forms. Optionally, a pharmaceutically acceptable carrier and / or excipient may be present.

[0109] The dosage regimen for the cell of the present invention will vary depending upon known factors, such as the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the effect desired. In certain embodiments, the cell of the invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.

[0110] The pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical operations such as sterilization and / or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc. They may be produced by standard processes, for instance by conventional mixing, granulating, dissolving or lyophilizing processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see for example L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892).

[0111] Method of Manufacture and Method of Treatment according to the invention

[0112] The invention further encompasses, as an additional aspect, the use of a cell as identified herein, for use in a method of manufacture of a medicament for the treatment of cancer. Similarly, the invention encompasses methods of treatment of cancer, comprising administering to a patient in need thereof a therapeutically effective amount of the cell of the invention as specified in detail herein.

[0113] The invention further encompasses the following items:

[0114] Items:

[0115] 1. An isolated nucleic acid molecule comprising

[0116] a. a sequence encoding an anti-CD19 chimeric antigen receptor (CAR), wherein the CAR comprises:

[0117] i. an extracellular domain comprising a single chain variable region moiety (scFv) against CD19;

[0118] ii. a transmembrane domain comprising a transmembrane domain of CD28; and

[0119] iii. an intracellular domain comprising an intracellular domain of CD28 and TCR- ;

[0120] b. two regions allowing for insertion of the nucleic acid molecule into a genetic locus of a KLRC1 gene.

[0121] 2. The isolated nucleic acid molecule according to item 1, wherein the CAR is characterized in that

[0122] i. the extracellular domain comprises an amino acid sequence having at least >85%, particularly >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 08. The isolated nucleic acid molecule according to any one of the preceding items, wherein the CAR is characterized in that

[0123] ii. the transmembrane domain comprises an amino acid sequence having at least >85%, particularly >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 10.

[0124] The isolated nucleic acid molecule according to any one of the preceding items, wherein the CAR is characterized in that

[0125] iii. the intracellular domain comprises an amino acid sequence having at least >85%, particularly >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 12.

[0126] The isolated nucleic acid molecule according to any one of the preceding items, wherein the CAR is characterized in that

[0127] i. the extracellular domain is encoded by a nucleic acid sequence having at least >75%, particularly >80%, >85%, >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 09.

[0128] The isolated nucleic acid molecule according to any one of the preceding items, wherein the CAR is characterized in that

[0129] ii. the transmembrane domain is encoded by a nucleic acid sequence having at least >75%, particularly >80%, >85%, >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 11.

[0130] The isolated nucleic acid molecule according to any one of the preceding items, wherein the CAR is characterized in that

[0131] iii. the intracellular domain is encoded by a nucleic acid sequence having at least >75%, particularly >80%, >85%, >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 13.

[0132] The isolated nucleic acid molecule according to any one of the preceding items, wherein the CAR comprises an amino acid sequence having at least >85%, particularly >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 06.

[0133] The isolated nucleic acid molecule according to any one of the preceding items, wherein the two regions allowing for insertion comprise a first nucleic acid sequence having at least >75%, particularly >80%, >85%, >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 04 and a second nucleic acid sequence having at least >75%, particularly >80%, >85%, >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 05.

[0134] 10. The isolated nucleic acid molecule according to any one of the preceding items, wherein the nucleic acid molecule comprises a nucleic acid sequence having at least >75%, particularly >80%, >85%, >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 14.

[0135] 11. An isolated natural killer cell characterized by

[0136] a. presence of a gene encoding an anti-CD19 chimeric antigen receptor (CAR) under control of a promoter of a KLRC1 gene; and

[0137] b. reduced or absent expression of a KLRC1 gene.

[0138] 12. An isolated natural killer cell, wherein a KLRC1 gene is replaced by a sequence encoding an anti-CD19 chimeric antigen receptor (CAR).

[0139] 13. The isolated natural killer cell according to item 11 or 12, wherein the natural killer cell additionally is characterized by a lack of expression of a FAS gene.

[0140] 14. The isolated natural killer cell according to any one of items 11 to 13, wherein the CAR comprises:

[0141] iv. an extracellular domain comprising a single chain variable region moiety (scFv) against CD19;

[0142] v. a transmembrane domain comprising a transmembrane domain of CD28; and

[0143] vi. an intracellular domain comprising an intracellular domain of CD28 and TCR- ;

[0144] 15. The isolated natural killer cell according to any one of items 11 to 14, wherein the CAR is characterized in that

[0145] i. the extracellular domain comprises an amino acid sequence having at least >85%, particularly >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 08.

[0146] 16. The isolated natural killer cell according to any one of items 11 to 15, wherein the CAR is characterized in that

[0147] ii. the transmembrane domain comprises an amino acid sequence having at least >85%, particularly >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 10.

[0148] 17. The isolated natural killer cell according to any one of items 11 to 16, wherein the CAR is characterized in that

[0149] iii. the intracellular domain comprises an amino acid sequence having at least >85%, particularly >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 12. The isolated natural killer cell according to any one of items 11 to 17, wherein the CAR is characterized in that

[0150] i. the extracellular domain is encoded by a nucleic acid sequence having at least >75%, particularly >80%, >85%, >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 09.

[0151] The isolated natural killer cell according to any one of items 11 to 18, wherein the CAR is characterized in that

[0152] ii. the transmembrane domain is encoded by a nucleic acid sequence having at least >75%, particularly >80%, >85%, >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 11.

[0153] The isolated natural killer cell according to any one of items 11 to 19, wherein the CAR is characterized in that

[0154] iii. the intracellular domain is encoded by a nucleic acid sequence having at least >75%, particularly >80%, >85%, >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 13.

[0155] The isolated natural killer cell according to any one of items 11 to 20, wherein the CAR comprises an amino acid sequence having at least >85%, particularly >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 06.

[0156] The isolated natural killer cell according to any one of items 11 to 21 , wherein a genome of the natural killer cell comprises a nucleic acid sequence having at least >75%, particularly >80%, >85%, >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 14.

[0157] The isolated nucleic acid molecule according to any one of items 1 to 10 or the isolated natural killer cell according to any one of items 11 to 22 for use in medicine. The isolated nucleic acid molecule according to any one of items 1 to 10 or the isolated natural killer cell according to any one of items 11 to 22 for use in treatment of cancer.

[0158] The isolated nucleic acid molecule or the isolated natural killer cell for use according to item 24, wherein the cancer is selected from lymphoma and leukemia.

[0159] A method for preparation of a natural killer cell as specified in any one of items 11 to 22, the method comprising the steps:

[0160] a. providing a peripheral blood mononuclear cell (PBMC); b. isolating an NKG2A+ natural killer (NK) cell and keeping the NK cell under cell culture conditions;

[0161] c. contacting the NKG2A+ NK cell with a vector comprising the isolated nucleic acid molecule according to any one of items 1 to 10;

[0162] d. optionally, further knocking out FAS.

[0163] 27. The method according to item 26, wherein the vector is a viral vector, particularly the vector is selected from an adeno-associated viral (AAV) vector, an adenoviral vector, a retroviral vector, and a lentiviral vector, more particularly the vector is an AAV vector.

[0164] 28. The method according to any one of items 26 to 27, wherein FAS is knocked out using CRISPR / Cas.

[0165] Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein. Thus, any of the alternative embodiments for a cell type may be combined with any of the alternative embodiments of an additional knockout and these combinations may be combined with any CAR mentioned herein.

[0166] The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

[0167]

[0168] Fig. 1 shows generation of NKG2A edited CAR NK cells. A, Expression of NKG2A and CAR after electroporation with non-targeting control RNP (NKG2A+), NKG2A RNP (NKG2AKO), NKG2A RNP + CAR AAV as HDR repair template (CARAAV), non-targeting control RNP + retroviral CAR transduction (CARRVNKG2A+) or NKG2A RNP + retroviral CAR transduction (CARRVNKG2AKO). Cells were initially sorted as NKG2A+NK cells (CD3'CD56+NKG2A+), except for bulk NK cells (CD3-CD56+). Cells were electroporated and transduced after 4 days of expansion and FACS analysis was performed 7 days after electroporation and transduction. B, Frequency of CAR expressing NK cells (left) and MFI of CAR (middle + right). C, Frequency of NKG2A+NK cells (left) and MFI of NKG2A (middle + right). D, expansion of NK cells after electroporation / transduction using K562-mblL-21 feeder cells (1x / week) and 200 lll / ml IL-2 (2x / week). E, frequency of successful and failed cell expansions. Failed expansion was defined as < 1-fold expansion from the start of expansion to day 20 of expansion. B-E: n=7, each replicate presents an individual donor. One-way ANOVA except D, 2way ANOVA. MFI: mean fluorescence intensity. RNP: ribonucleoprotein.

[0169] Fig. 2 shows editing of NKG2A increases cytotoxicity of NK cells and CAR NK cells.

[0170] A, specific lysis of LCL 721.45 (wt) or LCL 721.45 HLA-EKO. Cells were cocultured for 4-5 hours at an effector: target ratio of 5:1. n=6-10, each replicate presents an individual donor. B, specific lysis of LCL 721.45 by non-CAR NK cells (left) or CAR NK cells (right). Cells were co-cultured for 4-5 hours at an effector: target ratio of 5:1. n=6-10 (individual donors). C, IFN-y secretion after 4-5 hours co-culture with LCL 721.45. Background IFN-y secretion of effector cells without target cells is subtracted. n=2-3 (individual donors). D, specific lysis of Raji cells by non-CAR NK cells (left) or CAR NK cells (right). Cells were cocultured for 4-5 hours at an effector: target ratio of 5: 1. n=5-8 (individual donors). E, experimental scheme (left). 0.1x106effector cells were seeded on day 0, and 0.1x106target cells were added every 2-3 days for 14 days. IL-2 was added 2x / week until day 11 / 12. Target cells on day 14 were labelled with PKH67 and specific killing of PKH67+cells was measured after 4-5 hours of co-culture. Specific lysis of LCL 721.45 by non-CAR NK cells (middle) or CAR NK cells (right). n=6-7 (individual donors). F, G, numbers of effector and target cells after the assay described in (E). Shown are cell numbers of non-CAR (F) or CAR NK (effector) (G) cells (left) and total target cells (right) after 14 days. n= 6-7 (individual donors). Dashed line shows number of initially seeded effector cells. One-way ANOVA. wt: wildtype.

[0171] Fig. 3 shows reduced expansion of NKG2A edited NK cells can partly be restored by FasKO. A, expression of FAS on non-CAR NK cells (left) or CAR NK cells (right) after culture in the absence of feeder or target cells (E only) or after co-culture with LCL 721.45 target cells (added every 2 days over the course of one week) (E+T). n=6-7 (individual donors). B, Top row: Cells electroporated with nontargeting control RNP (NKG2A+), NKG2A RNP (NKG2AKO) or NKG2A RNP + FAS RNP (NKG2AKOFasKO). Bottom row: Cells retrovirally transduced with CAR and electroporated with non-targeting control RNP (CARRVNKG2A+), NKG2A RNP (CARRVNKG2AKO) or NKG2A RNP + FAS RNP (CARRVNKG2AKOFasKO); or site-specific CAR insertion in KLRC1 locus by electroporation with NKG2A RNP + AAV6 (CAR^ ) or NKG2A RNP + FAS RNP + AAV6 (CAR^ FasKO). MFI of FAS is shown on the right. N= 6-7 (individual donors). C, fold-expansion of non-CAR NK cells (top) or CAR NK cells (bottom) with or without FasKO. n=7 (individual donors). D, frequency of live non-CAR (left) or CAR NK cells (right) after 7 days of culture (without feeder cells). E, cell number of non-CAR (left) or CAR NK cells (right) after 7 days of culture (without feeder cells). Dashed line shows number of initially seeded effector cells. n=7 (individual donors). F, G, 0.1x106effector cells were seeded on day 0, and 0.1x106target cells were added every 2-3 days for 14 days. IL-2 was added 2x / week until day 11 / 12. Shown are cell numbers of non-CAR (F) or CAR NK (effector) (G) cells (left) and total target cells (right) after 14 days. n= 6-7 (individual donors). One-way ANOVA except B, 2way ANOVA. MFI: mean fluorescence intensity. RNP: ribonucleoprotein. E: effector, T: target.

[0172] Fig. 4 shows NKG2A edited CAR NK cells kill CAR NK cell resistant CD19negtarget cells. A, specific lysis of LCL 721.45 CD19KOcells by non-CAR NK cells (left) or CAR NK cells (right). Cells were co-cultured for 4-5 hours at an effector to target ratio of 5:1. n=6-9 (individual donors). B, schematic of long-term killing assay.

[0173] 0.1x106non-CAR or CAR NK cells were seeded at day 0 and 0.1x106LCL 721.45 cells were added every 2-3 days for 14 days. IL-2 was added 2x / week until day 12. Target cells added on day 7 were labelled with PKH26 and on day 14 with PKH67. C, relative MFI of CD19 on total target cells (CD56-) on day 14 after co-culture with non-CAR (top) or CAR (bottom) NK cells. Relative MFI is calculated by dividing through MFI of CD19 on target cells cultured alone. n=6- 7 (individual donors). D, Representative expression of PKH26 (target cells added day 7) and PKH67 (target cells added day 14) on total target cells (CD56-) after co-culture with non-CAR or CAR NK cells for 14 days. E, Quantification of remaining PKH26+target cells (added on day 7) of total target cells on day 14 after co-culture with non-CAR cells (left) or CAR NK cells (right). Target cells alone (LCL only) were not included in the statistical analysis. n=6-7 (individual donors). F, Quantification of remaining PKH67+target cells (added on day 14 and co-cultured for 4-5 hours before readout) of total target cells after co-culture with non-CAR cells (left) or CAR NK cells (right). Target cells alone (LCL only) were not included in the statistical analysis. n=6-7 (individual donors). One-way ANOVA. MFI: mean fluorescence intensity.

[0174] Fig. 5 shows non-viral generation of CAR cells with site-specific knock-in in KLRC1 locus. A, Schematic of HDR template construct with anti-CD19 CAR cassette and knock-in into the KLRC1 locus. CAR expression is driven from the endogenous KLRC1 promoter. B, Conformation of site-specific knock-in by PCR. The forward primer binds to a genomic region within NKG2A, which is upstream and outside of the HDR repair template. The reverse primer binds to a region within the CAR sequence. Lane 1: NKG2AKO. Lane 2: CARdsDNA. Lane 3: unedited NK cells. Lane 4: CARRVNKG2A+. Lane 5: 1kb ladder. Expected band size: 847 bp. C, Transgene expression after non-viral, site-specific knock- in of CAR or GFP into the KLRC1 locus. NKG2A+NK cells were electroporated with NKG2A RNP + dsDNA HDR template carrying either CAR or GFP. n=5 (individual donors). D, CAR+NK cells after flow cytometric cell sorting of CAR cells generated by electroporation with NKG2A RNP + dsDNA HDR template (CARdsDNA) or retroviral transduction (CARRV). n=3 (individual donors). One-way ANOVA.

[0175] Fig. 6 shows phenotyping of NKG2AKONK cells and NKG2A edited CAR NK cells. NK cells were expanded using K562-mblL-21 feeder cells + IL-2. At the timepoint of phenotyping, no new feeder cells were added for at least 7 days. Phenotyping was performed approximately after 28 days of expansion. Data are shown as frequency of live CD3'CD56+NK cells. A, Top: expression of the indicated markers on non-CAR NK cells. Bottom: Histogram of indicated markers from one representative donor. B, Top: expression of the indicated markers on CAR NK cells. Bottom: Histogram of indicated markers from one representative donor. n=4-6 (individual donors). Repeated measures one-way ANOVA.

[0176] Fig. 7 shows additional data for killing assays. A, gating strategy of killing assay. B-E,

[0177] HLA-E and CD19 expression on different target cells compared to wildtype (wt) 721.45 cells. F, specific lysis of LCL 721.45 cells after 4-5 hours of co-culture (E:T=5:1) with non-virally generated CARdsDNANK cells or CARRVNK cells. CAR NK cells were enriched by FACS sorting prior to the assay. n=3 (individual donors). G, specific lysis of LCL 721.45 after 4-5 hours of co-culture CAR^ NK cells or CARRVNK cells at the indicated E:T ratios. CAR NK cells were enriched by FACS sorting prior to the assay. n=2 (individual donors). 2way-ANOVA. H, specific lysis of freshly generated, allogenic LCL derived from CD19+cells of human fetal liver (HFL) tissue by non-CAR NK cells (left) or CAR NK cells (right). Cells were co-cultured for 4-5 hours at an E:T ratio of 5:1. n=9 (individual donors).

[0178]

[0179] Cell lines and cell culture

[0180] K562-mblL-21 feeder cells (kindly provided by Dean A. Lee, Nationwide Children’s Hospital, Columbus, United States) were cultured in complete Roswell Park Memorial Institute (RPMI) medium (Gibco, Cat. 7001612) supplemented with 10% fetal calf serum (FCS) (Biochrome, Cat. S0615-500ML), 1% penicillin / streptomycin (Thermo Fisher, Cat. 7001592), and 2mM L-glutamine (Thermo Fisher, Cat. 25030081) (R10) and kept at 0.3-1x106cells / ml. The lymphoblastoid cell line (LCL) 721.45 was cultured in R10 at 0.5-1x106cells / ml. To generate LCL 721.45 CD19KO, LCL 721.45 cells were electroporated with CD19-RNP in Opti-MEM Reduced Serum Medium (Life Technologies, Cat. 31985-047) using the 4D Nucleofector (Lonza, Switzerland) with the pulse code DN-100. LCL 721.45 CD19KOcells were purified by fluorescence-activated cell sorting (FACS) approximately one week after electroporation. CD19 expression was assessed before assays by flow cytometry and FACS purification was repeated, if necessary. Raji cells were cultured in R10 at 0.1-1x106cells / ml. LCL from allogeneic human fetal liver (HFL) tissue were generated by first isolating CD19+cells by magnetic activated cell sorting with CD19 beads (Miltenyi, Cat. 130-050-301) and then infecting CD19+cells with Epstein-Barr Virus (EBV) B95-8; produced as previously described [1] at a multiplicity of infection (MOI) of 0.05-0.15.

[0181] Isolation and expansion of NK cells

[0182] Whole blood was collected from healthy donors and peripheral blood mononuclear cells (PBMC) were isolated by gradient centrifugation with Ficoll-Paque (GE Healthcare, Cat. 17-5442-03) at 1000g, 20°C for 20 minutes using slow deceleration. NK cells were isolated from PBMC by FACS sorting as NKG2A+NK cells (CDS’ CD56+NKG2A+) or bulk NK cells (CD3‘ CD56+). Sorted NK cells were cultured in R10 + 200 lU / ml IL-2 (Peprotech, Cat. 200-02) with irradiated (130 Gy) K562-mblL-21 feeder cells (2:1 feeder: NK ratio). NK cells were counted 2x / week and adjusted to a density of 1x106cells / ml with R10 and IL-2 was renewed. Feeder cells were added every 7 days at a ratio of 1 : 1.

[0183] Retrovirus production

[0184] Human embryonic kidney (HEK293T) cells were cultured in Dulbecco’s Modified Eagle’s Medium (DM EM) (Gibco, 41966-052) supplemented with 10% FBS, 2mM L-glutamine and 1% penicillin / streptomycin (D10) and passaged 2-3 times per week. For retrovirus production, HEK293T cells were transfected with packaging plasmid pUMVC (Addgene, Cat. #8449), BaEVRIess envelope plasmid (kindly provided by Els Verhoeyen, University of Lyon, France) and anti-CD19 CAR vector plasmid MSGV FMC63.28z (kindly provided by James N. Kochenderfer, NCI, Bethesda, USA), using polyethyleneimine (PEI). Supernatant was collected 48 hours after transfection and either used directly or stored at -80°C until further use.

[0185] Cloning of CARdsDNAHDR template plasmid

[0186] The CAR insert consisting of 300 bp KLRC1 homology arms left (HAL) and right (HAR) (HAL-Myc tag-FMC62-28-WPRE3-SV40pA-HAR) was ordered as synthetic gene fragment (IDT, gBIocks) and cloned into EcoRI (New England Biolabs, Cat. R3101)-linearized pUC19 (Addgene, Cat. #50005) by Gibson Assembly (New England Biolabs, Cat. E5510S) and introduced into competent NEB5alpha cells (New England Biolabs, Cat. C2987) by heat shock. AAV6 HDR template

[0187] AAV6 plasmid was cloned (using the plasmid described above) and AAV6 was generated at the Viral Vector Facility Zurich (VVF) of the University of Zurich.

[0188] Generation of CAR NK cells and NKG2AKO

[0189] CAR cells were generated on day 4 of expansion. For the generation of retrovirally transduced CAR NK cells, NK cells were electroporated with ribonucleoprotein complexes (RNP) consisting of Cas9 protein (MacroLab, UC Berkeley), non-targeting control crRNA (IDT, Cat.

[0190] 1072544) or NKG2A crRNA (IDT), and trRNA (IDT, Cat. 1072533) using the 4D Nucleofector (Lonza, Switzerland) with the P3 primary cell kit (Lonza, Cat. V4XP-3032) and the pulse code DK-100. To generate RNP, first crRNA and trRNA were incubated for 30 minutes at 37°C to form gRNA, then Cas9 was added and incubated for 15 minutes at 37°C and formed the RNP. If applicable, RNP was frozen and stored at -20°C. After electroporation, NK cells were rested for 30 min - 2 hours. For retroviral transduction, non-treated tissue culture 24-well plates were coated with Retronectin® (Takara, Cat. T100B) according to the manufacturer’s instructions. Retroviral supernatant (1ml / 0.5x106NK cells) was added to the plate and centrifuged for 2 hours at 32°C and 1500g. The viral supernatant was removed completely and electroporated NK cells were added to the plate.

[0191] For site-specific knock-in with AAV6 HDR template, NK cells were electroporated with NKG2A RNP (pulse code DK-100) and rested for 5 minutes in pre-warmed R10. Afterwards, NK cells were transferred into a 96 well plate containing AAV6 HDR template at a MOI of 1x105, HDR enhancer V2 (IDT, Cat. 10007921) at 1 .M and 200 lU / ml IL-2. Medium was changed after 24 hours and replaced with R10 + 200 I U / ml IL-2.

[0192] For site-specific knock-in with dsDNA HDR template, NKG2A RNP and dsDNA template (2 pg / 0.5x106NK cells) were incubated for 2 minutes and then added to the cell suspension in P3 buffer and electroporated (pulse code DK-100). Cells were cultured in R10 + 200 IU / IL-2 and 1 iM HDR enhancer V2. Medium was changed after 24 hours and replaced with R10 + 200 lU / ml IL-2.

[0193] Killing assay

[0194] Effector cells were supplemented with fresh IL-2 one day before the assay. For the assay, new medium without IL-2 was used. Target cells were labelled with PKH26 (Sigma Aldrich, Cat. MINI26) or PKH67 (Sigma Aldrich, Cat. MINI67) according to the manufacturer’s instructions. Effector and target cells were co-cultured at an E:T ratio of 5:1 and incubated for 4-5 hours at 37°C / 5% CO2. Afterwards, plates were stored in the fridge or on ice in the dark until acquisition. TO-PPRO3 (Invitrogen, Cat. T3605) was diluted 1:500 in PBS and 10pl TO-PRO3 / 1 OOpI cell suspension were added to the cell suspension just before FACS analysis on a BD LSRFortessa. Dead target cells were gated as debris? singlets / PKH26+ / TO-PRO3+.

[0195] For long-term killing assays, 0.1x106LCL 721.45 cells (unlabelled) were co-cultured with 0.1x106effector cells in R10 + 200 lU / ml IL-2. Fresh unlabelled LCL 721.45 cells (0.1x106) were added every 2-3 days and every second time, IL-2 was added. On day 7, LCL 721.45 cells were labelled with PKH26 and on day 14 with PKH67. To read out the specific lysis of day 14 target cells, TO-PRO3 was added to the sample and assessed as described above. To read out the frequency of remaining target and effector cells, cells were stained with CD19-PE-Cy5 (Biolegend, Clone HIB19, Cat. 302210) and CD56-PE-Cy7 (BD, Clone NCAM16, Cat.

[0196] 335826). To quantify effector and target cells, the total cell number was determined on day 14, 4-5 hours after addition of the last target cells by cell counting with trypan blue and multiplied by the frequency of live CD56+CD19' (effector cells) or live CD56' (target cells).

[0197] IFN-y ELISA

[0198] IFN-y ELISA was performed with the supernatant of 4-5-hourco-culture of 0.1x106LCL 721.45 cells and 0.1x106effector cells using the human IFN-y ELISA Flex kit (Mabtech, Cat. 3420-1 H-6), following the manufacturer’s instructions. Values from effector cells cultured alone were subtracted from the values of the co-culture. Plates were acquired using an ELISA reader (Infinite M200 Pro, Tecan) and analyzed with the i-control 2.0 software (Tecan).

[0199] Flow cytometry

[0200] For phenotyping after expansion, cells were labelled with surface markers and incubated for 20 minutes at 4°C. After washing, cells were permeabilized and fixed with the BD Pharmingen Transcription Factor Buffer Set (Cat. 562574) and incubated for 50-60 minutes at 4°C. After 2x washing, intracellular or intranuclear markers were stained and incubated for 45 minutes. After washing, cells were acquired on the same day on a Cytek Aurora analyzer. Used antibodies: CD16 - BUV737 (BD, 3G8, Cat. 564434), CD56 - BUV563 (BD, NCAM16.2, Cat.

[0201] 612928), TIM-3 - APC-Cy7 (Biolegend, F38-2E2, Cat. 345026), LAG-3 - BV650 (Biolegend, 11C3C65, Cat. 369316), NKG2A - PE-Cy5 (Biolegend, S19004C, Cat. 375112), pan-KIR -APC (CD158a,h: Beckman Coulter, EB6B, Cat. A22332; CD158b1 / b2,j: Beckman Coulter, GL183, Cat. A22333; CD158e1 / e2: R&D, Cat. FAB1225A), T-bet - PerCP-Cy5.5 (Invitrogen, eBio4B10, Cat. 45-5825-82), EOMES - FITC (Invitrogen, WD1928, Cat. 11-4877, 42), CD19 - BUV661 (BD, HIB19, Cat. 741604), ILT2 - PE-Cy7 (eBioscience, HP-F1, Cat. 25-5129-42), Ki-67 - BV711 (Biolegend, Ki-67, Cat. 350516), Perforin - BV421 (Biolegend, dG9, Cat.

[0202] 308122), Granzyme B - Alexa Fluor 700 (BD, GB11, Cat. 560213), FAS - BV785 (Biolegend, dx2, Cat. 305645), Zombie Aqua Fixable Viability Kit (Biolegend, Cat. 423102). To measure CAR expression, cells were labelled with FITC-labelled human CD19 (20-291) protein (Aero Biosystems, CD9-HF2H3). Example 2: Generation of CAR-NKG2AKONK cells

[0203] We generated CAR-NKG2AKONK cells from NK cells derived from peripheral blood mononuclear cells (PBMC) of healthy donors. It was previously shown that targeting a CAR construct to the T-cell receptor a constant (TRAC) locus results in CAR T cells with enhanced anti-tumor function. This appeared to be based on the more favorable endogenous gene regulation of the TRAC locus compared to the exogenous promoter used in retroviral expression cassettes, like reduced tonic signaling or a lower CAR MFI. To investigate if a similar benefit of endogenous gene regulation can be achieved in CAR NK cells, we targeted the CAR to the KLRC1 locus (Fig. 5A) and site-specific integration was confirmed by PCR (Fig. 5B).

[0204] We initially optimized the homology directed repair (HDR) template using dsDNA with either a CAR or GFP transgene, which resulted in 6.8% ± 4.4 CAR+NK cells (CARdsDNA) or 15.3% ± 6.1 GFP+NK cells (GFPdsDNA) (Fig. 5C). CAR frequency could be increased by fluorescence-activated cell sorting (FACS) to up to 90% CAR+NK cells (Fig. 5D). Next, we generated an adeno-associated virus serotype 6 (AAV6) HDR template, which increased CAR knock-in rates to the range of retroviral transduction (Fig. 1A-B), which made cell sorting expendable. While the frequency of CAR+NK cells was comparable between Cas9-mediated site-specific CAR knock-in in the KLRC1 locus using AAV6 HDR template (hereafter called CARAAV) and retrovirally transduced CAR NK cells (hereafter called CARRV), CAR™7NK cells had a significantly lower CAR mean fluorescence intensity (MFI) compared to CARRVNK cells (Fig.

[0205] 1B).

[0206] The chosen knock-in strategy for the CAR insertion in the KLRC1 locus leads to simultaneous CAR expression and NKG2A downregulation (Figure 1A). To distinguish the effects of sitespecific CAR insertion and NKG2A knock-out on the killing capacity of NK cells, we generated CARRVNK cells with or without NKG2AKO. Although CAR™vand CARRVNKG2AKONK cells both showed significantly reduced expression of NKG2A, CAR™7NK cells seem to maintain expression of NKG2A on a moderately higher level (Fig. 1C).

[0207] We showed previously that genomic ablation of NKG2A leads to reduced expansion of NK cells, posing a potential limitation for the use of NKG2A edited CAR NK cells for cellular immunotherapy. While we similarly observed this effect in the present study for cells harboring NKG2AKO, we observed a trend towards better expansion for CAR™7NK cells compared to CARRVNKG2AKONK cells (Fig. 1 D). Additionally, expansion of CARRVNKG2AKONK cells failed (<1-fold expansion after approx. 20 days of expansion) in 3 out of 7 donors (42.8%), while CAR™7NK cells could be expanded successfully for 7 out of 7 donors (100%) (Fig. 1E). This could be due to maintained expansion mediated by low but sustained expression of NKG2A in CAR™7NK cells relative to CARRVNKG2AKONK cells, as previously reported. We analyzed the phenotype of NKG2AKONK cells and NKG2A edited CAR NK cells by flow cytometry and compared them to their respective NKG2A-proficient controls. NKG2AKONK cells showed moderately decreased expression of CD16, KIR and interestingly, l_AG-3. Frequency of EOMES+T-bet+NK cells as well as expression of Ki-67, granzyme B, perforin, Tim-3 and ILT2 was unaltered by NKG2AKO(Fig. 6A). NKG2A edited CAR NK cells showed the same results, with decreased CD16, KIR and LAG-3 in CARRVNKG2AKOand CAR^ NK cells compared to CARRVNKG2A+and CARRVbulk NK cells (Fig. 6B), indicating a lack of enhanced terminal differentiation in NKG2A edited CAR NK cells.

[0208] To confirm that the NKG2A-HLA-E axis confers inhibition to NK cells, we generated LCL721.45 HLA-EKOcells by genomic knockout of HLA-E (Fig. 7B). Indeed, killing of HLA-EKOtarget cells was significantly increased for bulk NK cells and for CARRVbulk NK cells compared to unedited target cells (Fig. 2A, gating of killing assay shown in Fig. 7A). To assess the interruption of the NKG2A-HLA-E axis from the effector cell side, we tested NKG2A edited CAR NK cells in killing assays against HLA-E expressing target cells. NKG2AKOincreased the specific lysis against LCL 721.45 for non-CAR NK cells as well as for CAR NK cells (Fig. 2B). Killing was increased for CARRVNKG2AKOcells as well as CAR^ , confirming that site-specific CAR insertion in the KLRC1 locus generates functional CAR NK cells (Fig. 2B and Fig. 7G). Similarly, the non-virally generated CARdsDNANK cells were functional and showed increased killing compared to CARRVNKG2A+NK cells (Fig. 7F). As a different functional readout, we measured IFN-y secretion after co-culture with LCL 721.45 and found increased IFN-y secretion in CAR^ NK cells compared to CARRVbulk NK cells (Fig. 2C). We explored killing of additional CD19+HLA-E+target cells and tested the specific lysis of Raji cells (Fig. 2D, Fig. 7C) as well as newly generated LCL (Fig. 7D and 7H) and consistently found increased lysis by NKG2AKOcells compared to unedited NK cells as well as increased lysis by CARAAVand CARRVNKG2AKONK cells compared to CARRVNKG2A+and bulk CARRVNK cells.

[0209] To model conditions of chronic antigen stimulation, we exposed effector cells to fresh target cells 3 times a week for a total of 2 weeks. After two weeks of repeated challenge, we no longer observed increased killing by NKG2AKOcells compared to NKG2A+or bulk NK cells, and while all CAR NK cells were still able to lyse target cells, we could no longer detect an increased effect of NKG2A edited CAR NK cells (Fig. 2E). To better understand this result, we quantified the fractions of remaining target and effector cells. For non-CAR NK cells, we found no difference in the number of remaining effector or target cells between NKG2AKOcells and NKG2A+or bulk NK cells (Fig. 2F). For CAR NK cells, we found significantly less effector cells for CARRVNKG2AKOand CARAAVNK cells compared to CARRVNKG2A+or CARRVbulk NK cells (Fig. 2G), again pointing to a decreased expansion capacity of CAR NK cells with NKG2AKO. The significantly lower number of CARRVNKG2AKOand CAR^ NK cells compared to CARRVNKG2A+and bulk CARRVNK cells could be one explanation for why killing converged after 2 weeks of repeated challenge for the different CAR NK cell products.

[0210] Taken together, NKG2AKOincreases the cytotoxicity against HI_A-E+target cells and can be combined with the transduction of a CAR by retroviral delivery as well as non-viral or AAV6 enabled site-specific insertion by Cas9-mediated HDR to generate NKG2A edited CAR NK cells with enhanced cytotoxicity.

[0211] Our lab has recently shown that the reduced expansion capacity of NKG2AKONK cells stems from increased apoptosis rather than from reduced cell cycling. Indeed, we observed that while NKG2A+or bulk NK cells downregulate expression of the death receptor FAS after co-culture with target cells, NKG2AKOcells show sustained levels of FAS expression (Fig. 3A). CARRVNKG2AKOand CAR™7NK cells even showed a tendency towards upregulation of FAS after co-culture with target cells (Fig. 3A). To increase the expansion capacity of NKG2AKONK cells, we thus hypothesized that genomic knock-out of FAS might reduce apoptosis. We successfully generated NK and CAR NK cells with significantly reduced expression of FAS after Cas9-mediated knock-out (Fig. 3B). We found that FAS knock-out increases cell expansion of NKG2AKONK cells more than two-fold (average expansion after 20 days: NKG2AKO8.0-fold vs. NKG2AKOFasKO17.2-fold). While knockout of FAS also showed a trend towards better expansion of CARRVNKG2AKOFasKOcompared to CARRVNKG2AKONK cells (CARRVNKG2AKO7.3-fold vs. CARRVNKG2AKOFasKO15.56-fold), no difference was observed between CAR™vand CARAAVFasKO(Fig.3C).

[0212] We also found that in 7 day mono-cultures of engineered NK cells in the absence of feeder or target cells, the viability of NKG2AKONK cells is restored to the level of NKG2A+NK cells when FAS is knocked out. Similarly, viability of CARRVNKG2AKOand CARAAVNK cells is restored by FasKOto the level of CARRVNKG2A+NK cells (Fig. 3D). Also, the cell number of NKG2AKOFasKONK cells tends to be increased compared to NKG2AKONK cells, while intriguingly, knockout of FAS leads to significantly higher cell numbers of CARRVNKG2AKOand CAR™7NK cells relative to FAS-proficient NKG2A edited CAR NK cells (Fig. 3E).

[0213] Next, we looked at cell numbers and expansion after 2 weeks of repeated challenge with target cells. While co-cultures with NKG2AKOFasKONK cells showed a trend towards higher effector cell numbers compared to NKG2AKONK cells, they still failed to control target cells (Fig. 3F). For CAR NK cells, co-cultures with FasKOled to a trend towards higher effector cell numbers with CARRVNKG2AKONK cells (CAR NKG2AKO1.53-fold expansion vs. CAR NKG2AKOFasKO3.64-fold expansion) as well as with CAR™7NK cells (CAR™72.0-fold expansion vs. CAR™7FasKO3.47-fold expansion). All of the CAR-bearing NK cell products could control target cell numbers to a comparable degree (Fig. 3G). In summary, FasKOcan partly restore the NKG2AKO-induced reduced viability and expansion capacity of NK cells. To model antigen-negative relapse, which is a known cause for CAR T cell therapy failure, we generated LCL 721.45 CD19KOcells (Fig. 7E). As expected, for non-CAR NK cells, we observed similar results as seen for killing of wildtype LCL 721.45 (Fig. 2B), with an increase in killing by NKG2AKONK cells (Fig. 4A). Likewise, we observed increased killing by CARRVNKG2AKOand CAR^ NK cells compared to CARRVNKG2A+or CARRVbulk NK cells. The extent of target cell lysis was in the same range comparing NKG2A+and CARRVNKG2A+as well as between NKG2AKOand CARRVNKG2AKOor CARAAVNK cells, due to the absence of CAR-redirected killing of the CD19 negative target cells (Fig. 4A).

[0214] Next, we performed a repetitive stimulation assay, in which the occurrence of target cells resistant to killing by CAR NK cells could be modeled (Fig. 4B). Fresh target cells were added every 2-3 days for 2 weeks, with target cells labelled differently on day 7 (PKH26) and on day 14 (PKH67) to track killing (or resistance to killing) over time. Interestingly, target cells cocultured with anti-CD19 CAR NK cells showed a strong decrease in CD19 expression compared to expression at baseline (Fig. 4C). The remaining target cells were analyzed for the abundance of PKH26+(added on day 7) and PKH67+(added on day 14) target cell fractions (Fig. 4D). In co-cultures with non-CAR NK cells, we found the frequency of day 7 PKH26+target cells being in the same range as target cells cultured alone, thus showing resistance to killing by NK cells, including to NKG2AKONK cells (Fig. 4E). In contrast, for CAR NK cells with edited NKG2A, we observed near complete depletion of the PKH26+target cell fraction. Of note, a fraction of PKH26+target cells was preserved in co-cultures with CARRVNKG2A+and CARRVbulk NK cells, indicating a deficiency in killing of CD19negtarget cells by CAR NK cells without disruption of KLRC1 (Fig. 4E). Analyzing PKH67+target cells added at the very end (day 14) of the repetitive stimulation assay 4 hours before readout, we again found a lack of killing of target cells by chronically stimulated non-CAR NK cells (Fig. 4F). In co-cultures with NKG2A edited CAR NK cells, the vast majority of target cells were recently added PKH67+cells (80-100%), indicating that almost all of the previously added target cells had been cleared, compared to 10% PKH67+cells in the target only control (Fig. 4F). In contrast, for CARRVNKG2A+or CARRVbulk NK cells, the frequency of PKH67+target cells was significantly lower at 25-50%, suggesting that previously added, non-PKH67 labelled target cells were not killed as efficiently as by NKG2A edited CAR NK cells. This finding again points to target cell resistance and evasion of killing by CAR NK cells without disruption of KLRC1 (Fig. 4F). Together, NKG2A edited CAR NK cells outperform CAR NK cells against CD19negtarget cells, possibly due to their increased innate killing capacity. This feature might offer therapeutic advantages for NKG2A edited CAR NK cells in preventing CD19negrelapses in patients treated with CD19 directed therapies. Example 3: Discussion

[0215] CAR NK cells have great potential as an off-the-shelf product for cancer immunotherapy. However, effector functions of CAR NK cells can be attenuated by engagement with tumor cells and the immunosuppressive tumor microenvironment, driven by mechanisms such as upregulation of HI_A-E, which interacts with the inhibitory NK cell receptor NKG2A. In this study we show that NKG2A edited anti-CD19 CAR NK cells have increased cytotoxicity against HLA-E+target cells compared to conventional CAR NK cells. Although we observed decreased cell expansion of NKG2A edited CAR NK cells, this limitation could partially be compensated by knock-out of FAS, a death receptor that regulates extrinsic apoptosis. We also found that NKG2A edited CAR NK cells have the potential to protect from CAR-antigen negative relapse, which frequently occurs after CAR T cell therapy, because NKG2A edited CAR NK cell could kill target cells that were CD19negand resistant to killing by conventional CAR NK cells. Our study confirms the finding that genomic knock-out of KLRC1 disrupts the inhibitory NKG2A-HI.A-E axis in primary human NK cells and thus, enhances NK cell cytotoxicity against HLA-E expressing cell lines. We extended the approach to CAR NK cells, producing NKG2A edited CAR NK cells, which resulted in an additive enhancement of cytotoxicity by abrogating inhibition by NKG2A and CAR-mediated killing.

[0216] While previous studies described no decrease in NK cell viability or expansion after genomic knock-out of KLRC1 we found a similar decrease of NK cell expansion as we had noted earlier. Different to the K562-mblL-21 feeder cell and IL-2 based expansion protocol we used, Bexte et al. used a feeder- free and IL- 15 based protocol [2], We hypothesize that the decreased proliferation we observe is due to increased activation-induced cell death (AICD) after coculture with feeder or target cells. NKG2AKONK cells lack inhibitory signaling and could be more prone to AICD, so the different expansion protocols might explain these differences. Mac Donald et al. used an expansion protocol comparable to ours, based on K562-mb-IL-21 feeder cells and IL-2 [3], However, these authors reported expression of inhibitory KIR2D of close to 100% expression on KLRC1K° NK cells, so inhibitory signaling by KIR2D molecules might have compensated for lack of inhibitory signaling by NKG2A. Indeed, in our previous study, we showed that only NKG2A' KIR but not NKG2A' KIR+NK cells exhibit a deficit in expansion capacity.

[0217] To counteract apoptosis in NKG2AKONK cells, which we have found to be increased in our previous study, we hypothesized that knock-out of the death receptor FAS might lead to improvement of cell survival. Indeed, it was shown that FAS is upregulated on NK cells after expansion with K562-mblL-21 feeder cells. In addition to FAS upregulation after ex vivo expansion, FAS is expressed on NK cells in patients with gastric cancer, which is accompanied by increased NK cell apoptosis. Moreover, although a majority of cancers appear not to express FAS ligand (FasL), FasL is expressed by several cell types within the TME including endothelial cells, cancer-associated fibroblasts and myeloid-derived suppressor cells. While these studies focused on apoptosis of antitumor CD8+T cells induced by FasL expression by TME cells, the same mechanism might also apply for NK cells. Building on this concept, FAS knock-out could not only enhance ex vivo NK cell expansion but could also improve persistence of NK cells within the TME. In terms of enhancing ex vivo cell expansion, we observed an improvement of NKG2AKOFasKONK cells relative to NKG2AKONK cells; however, NK cell expansion was still compromised compared to NKG2A+or bulk NK cells. Future research could investigate additional genes regulating apoptosis that might be altered together with FAS knock-out.

[0218] One mechanism of relapse after anti-CD19 CAR T cell therapy is the loss or downregulation of CD19 expression on malignant B cells. The risk of antigen-negative relapse differs between the type of B cell malignancy and the co-stimulatory domain utilized in the CAR construct. Nevertheless, the resistance to CAR T cells after loss of CAR antigen is a relevant contributor to CAR T cell therapy failure. Early clinical data on CAR NK cell therapy generated from allogeneic primary human NK cells are still just beginning to become available. In the most recent study, a small cohort of patients treated with CAR NK cells were tested for tumor CD19 expression after relapse. From the available samples, only 1 out of 8 patients showed modest reduction of CD19 expression in tumor cells. Given the limited data and research available, it is difficult to draw more general conclusions on the significance of CAR-antigen negative relapse following CAR NK cell therapy. Nevertheless, our in vitro data suggest that loss of CD19 may indeed occur after prolonged co-culture of CD19 positive target cells with CAR NK cells, and these cells could not be lysed efficiently by conventional CAR NK cells. By contrast, NKG2A edited CAR NK cells efficiently and completely eliminated emerging CD19 negative target cells. Future clinical data will make it possible to estimate the need for a prevention of CAR-antigen negative relapse after CAR NK cell therapy.

[0219] Cited references:

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[0222] [3] Mac Donald A, Guipouy D, Lemieux W, Harvey M, Bordeleau LJ, Guay D, Romero H, Li Y, Dion R, Beland K, Haddad E. KLRC1 knockout overcomes HLA-E-mediated inhibition and improves NK cell antitumor activity against solid tumors. Front Immunol. 2023 Aug 21;14:1231916. doi: 10.3389 / fimmu.2023.1231916. PMID: 37675109; PMCID: PMC10478211.

[0223] [4] Wang et al. Cell. 2025 Aug 7;188(16):4225-4238.

[0224] [5] Mackensen et al. Nat Med. 2022 Oct;28(10):2124-2132.

[0225] [6] Muller et al. N Engl J Med. 2024 Feb 22;390(8):687-700.

[0226] [7] Wang et al. Cell. 2024 Sep 5;187(18):4890-4904.

[0227] [8] Tur et al. Ann Rheum Dis. 2025 Jul 11:80003-4967(25)04174-3.

[0228] [9] Muller et al. N Engl J Med. 2025 Sep 25;393(12):1239-1241.

[0229] All scientific publications and patent documents cited in the present specification are incorporated by reference herein.

[0230] SEQUENCES:

[0231] In the event of discrepancies between the sequences shown in the present specification and those of the enclosed sequence protocol according to WIPO standard ST.26, the sequences shown herein shall prevail.

[0232] > crRNA CD19, SEQ ID NO 01

[0233] CGAGGAACCTCTAGTGGTGA

[0234] > crRNA NKG2A, SEQ ID NO 02

[0235] ACTGCAGAGATGGATAACCA

[0236] > crRNA Fas, SEQ ID NO 03

[0237] GTGACTGACATCAACTCCAA

[0238] > NKG2A HAL, SEQ ID NO 04 CTCCTGACCTCGTGATCGGCATGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCCGGC CTAAAAATCTTTTTTTAAAACAAATATTCATAAGAAACGTGTTTAGGCTTGAAGAAAATCAGAGAAAGAACTTT AGATTATTTAATGCAAAATGAGCTCCAATACTCGTTCTCCACCTCACCCTTTTAATTGCACTAGGGAATCCTGT ATATAAACCATTTATTAACTTCTTAACTACTGTTATTATAGAGTACAGTCCCTGACATCACACACTGCAGAGAT G > NKG2A HAR, SEQ ID NO 05 GATAACCAAGGAGTAATCTACTCAGACCTGAATCTGCCCCCAAACCCAAAGAGGCAGCAACGAAAACCTAAAGG CAATAAAAACTCCATTTTAGCAACTGAACAAGAGATCACTTACGCCGAGTTGAATCTACAAAAAGCTTCTCAGG ATTTTCAAGGGAATGACAAAACCTATCACTGCAAAGGTAAAGCATTTAAAAGATCCTCAATATAACAGTCTAGG ATGTGCAGCTTGGGGTACAGGAATGTGGGGAAAGAGAAGGGAGTGCTCATATATCTTCTATTTGCAAAGATCAG AATTCCA

[0239] > CAR full-length, Amino acid sequence, (hGM-CSF receptor signal sequence -Myc tag - linker - FMC63 light chain - linker - FMC63 heavy chain - CD28-CD3z - linker) , SEQ ID NO 06 MLLLVTSLLLCELPHPAFLLIPEQKLISEEDLGGSGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQ KPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSG SGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNS ALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEVMYPPPYLDN EKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRP GPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGTGAGSG

[0240] > CAR full-length, Nucleic acid sequence, ( hGM-CSF receptor signal sequence - Myc tag - linker - FMC63 light chain - linker - FMC63 heavy chain -CD28-CD3Z - linker - WPRE3 - SV40 polyA) , SEQ ID NO 07 CTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGAACAAAAACT CATCTCAGAAGAGGATCTGGGAGGTTCTGGAGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTC TGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAA CCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGG CAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCC AACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACAGGCTCCACCTCTGGATCC GGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGC GCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTC GCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCT CTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAAC TGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTC AAGGAACCTCAGTCACCGTCTCCTCAGCGGCCGCAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAG AAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAA GCCCTTTTGGGTGCTGGTGGTGGTTGGGGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTA TTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGG CCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAG CAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAG AGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCT CAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGA GCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCC TTCACATGCAGGCCCTGCCCCCTCGCGGAACCGGTGCTGGAAGTGGTTAAGATAATCAACCTCTGGATTACAAA ATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCC TTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCA CGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTG GAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTT TTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAA

[0241] > CAR extracellular domain, Amino acid sequence, (hGM-CSF receptor signal sequence - Myc tag - linker - FMC63 light chain - linker - FMC63 heavy chain - CD28 extracellular part) , SEQ ID NO 08 MLLLVTSLLLCELPHPAFLLIPEQKLISEEDLGGSGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQ KPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSG SGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNS ALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEVMYPPPYLDN EKSNGTIIHVKGKHLCPSPLFPGPSKP

[0242] > CAR extracellular domain, Nucleic acid sequence, (hGM-CSF receptor signal sequence - Myc tag - linker - FMC63 light chain - linker - FMC63 heavy chain - CD28 extracellular part) SEQ ID NO 09 ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGAACAAAA ACTCATCTCAGAAGAGGATCTGggaggttctggaGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCT CTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAG AAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAG TGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTT GCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACAGGCTCCACCTCTGGA TCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGT GGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGA TTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCA GCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCA AACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGG GTCAAGGAACCTCAGTCACCGTCTCCTCAGCGGCCGCAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAAT GAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTC TAAGCCCTTTTGGGTGCTGGTGGTGGTTGGGGGAGTCCTGGCT > CAR transmembrane domain, Amino acid sequence, (CD28 transmembrane) , SEQ ID NO 10

[0243] F WVLWVGGVL ACYS L LVTVAF 1 I F WV

[0244] > CAR transmembrane domain, Nucleic acid sequence, (CD28 transmembrane) , SEQ ID NO 11

[0245] TGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG

[0246] > CAR intracellular domain, Amino acid sequence, (CD28 intracellular (underlined) -CD3z - linker) , SEQ ID NO 12

[0247]

[0248] VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPRGTGAGSG

[0249] > CAR intracellular domain, Nucleic acid sequence, (CD28 intracellular (underlined) -CD3z - linker - WPRE3 - SV40 polyA) , SEQ ID NO 13 AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAA GCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAG ACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGAT GTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCT GTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGG GCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG GCCCTGCCCCCTCGCGGAACCGGTGCTGGAAGTGGTTAAGATAATCAACCTCTGGATTACAAAATTTGTGAAAG ATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATG CTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTC ATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGAACTTGTTTA TTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCAT TCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAA

[0250] > CAR + flanking region, Nucleic acid sequence, (NKG2A HAL - hGM-CSF receptor signal sequence - Myc tag - linker - FMC63 light chain - linker - FMC63 heavy chain - CD28-CD3Z - linker- WPRE3 - SV40 polyA - NKG2A HAR) , SEQ ID NO 14 CTCCTGACCTCGTGATCGGCATGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCCGGC CTAAAAATCTTTTTTTAAAACAAATATTCATAAGAAACGTGTTTAGGCTTGAAGAAAATCAGAGAAAGAACTTT AGATTATTTAATGCAAAATGAGCTCCAATACTCGTTCTCCACCTCACCCTTTTAATTGCACTAGGGAATCCTGT ATATAAACCATTTATTAACTTCTTAACTACTGTTATTATAGAGTACAGTCCCTGACATCACACACTGCAGAGAT GCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGAACAAAAAC TCATCTCAGAAGAGGATCTGGGAGGTTCTGGAGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCT CTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAA ACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTG GCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGC CAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACAGGCTCCACCTCTGGATC CGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGG CGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATT CGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGC TCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAA CTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGT CAAGGAACCTCAGTCACCGTCTCCTCAGCGGCCGCAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGA GAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTA AGCCCTTTTGGGTGCTGGTGGTGGTTGGGGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATT ATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGG GCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCA GCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGA GAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCC TCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCG AGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCC CTTCACATGCAGGCCCTGCCCCCTCGCGGAACCGGTGCTGGAAGTGGTTAAGATAATCAACCTCTGGATTACAA AATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGC CTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCC ACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGT GGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTT TTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAAGATAACCAAGGAGTAATCTACTC AGACCTGAATCTGCCCCCAAACCCAAAGAGGCAGCAACGAAAACCTAAAGGCAATAAAAACTCCATTTTAGCAA CTGAACAAGAGATCACTTACGCCGAGTTGAATCTACAAAAAGCTTCTCAGGATTTTCAAGGGAATGACAAAACC TATCACTGCAAAGGTAAAGCATTTAAAAGATCCTCAATATAACAGTCTAGGATGTGCAGCTTGGGGTACAGGAA TGTGGGGAAAGAGAAGGGAGTGCTCATATATCTTCTATTTGCAAAGATCAGAATTCCA

Claims

Claims1. An isolated natural killer cell characterized bya. presence of a gene encoding an anti-CD19 chimeric antigen receptor (CAR) under control of a promoter of a KLRC1 gene; andb. reduced by >50% or absent expression of a KLRC1 genewherein the natural killer cell additionally is characterized by a lack of expression of a FAS gene.

2. The isolated natural killer cell according to claim 1, wherein a KLRC1 gene is replaced by a sequence encoding an anti-CD19 chimeric antigen receptor (CAR).

3. The isolated natural killer cell according to any one of the preceding claims, wherein the CAR comprises:i. an extracellular domain comprising a single chain variable region moiety (scFv) against CD19;ii. a transmembrane domain comprising a transmembrane domain of CD28; andiii. an intracellular domain comprising an intracellular domain of CD28 and TCR- 4. The isolated natural killer cell according to any one of the preceding claims, wherein the CAR is characterized in thati. the extracellular domain comprises an amino acid sequence having at least >85%, particularly >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 08; and ii. the transmembrane domain comprises an amino acid sequence having at least >85%, particularly >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 10; and iii. the intracellular domain comprises an amino acid sequence having at least >85%, particularly >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 12.

5. The isolated natural killer cell according to any one of the preceding claims, wherein the CAR comprises an amino acid sequence having at least >85%, particularly >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 06.

6. The isolated natural killer cell according to any one of the preceding claims, wherein a genome of the natural killer cell comprises a nucleic acid sequence having at least>75%, particularly >80%, >85%, >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 14.

7. An isolated nucleic acid molecule comprisinga. a sequence encoding an anti-CD19 chimeric antigen receptor (CAR), wherein the CAR comprises:i. an extracellular domain comprising a single chain variable region moiety (scFv) against CD19;ii. a transmembrane domain comprising a transmembrane domain of CD28; andiii. an intracellular domain comprising an intracellular domain of CD28 and TCR- ;b. two regions allowing for insertion of the nucleic acid molecule into a genetic locus of a KLRC1 gene.

8. The isolated nucleic acid molecule according to claim 7, wherein the CAR is characterized in thati. the extracellular domain comprises an amino acid sequence having at least >85%, particularly >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 08; and ii. the transmembrane domain comprises an amino acid sequence having at least >85%, particularly >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 10; and iii. the intracellular domain comprises an amino acid sequence having at least >85%, particularly >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 12.

9. The isolated nucleic acid molecule according to any one of the preceding claims 7 to 8, wherein the two regions allowing for insertion comprise a first nucleic acid sequence having at least >75%, particularly >80%, >85%, >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 04 and a second nucleic acid sequence having at least >75%, particularly >80%, >85%, >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 05.

10. The isolated nucleic acid molecule according to any one of the preceding claims 7 to 9, wherein the nucleic acid molecule comprises a nucleic acid sequence having at least >75%, particularly >80%, >85%, >90%, >92%, >94%, >95%, >97%, >98%, >99% or 100% identity with a sequence of SEQ ID NO 14.

11. A combination of isolated nucleic acid molecules comprising or consisting ofa. a first isolated nucleic acid molecule according to any one of the preceding claims 7 to 10, andb. a second isolated nucleic acid molecule being capable of abolishing an expression of a FAS gene inside a cell, particularly wherein the second isolated nucleic acid molecule is of SEQ ID NO: 03.

12. The isolated natural killer cell according to any one of claims 1 to 6 or the isolated nucleic acid molecule according to any one of claims 7 to 10 or the combination according to claim 11 for use in medicine.

13. The isolated natural killer cell according to any one of claims 1 to 6 or the isolated nucleic acid molecule according to any one of claims 7 to 10 or the combination according to claim 11for use in treatment of cancer.

14. The isolated nucleic acid molecule or the combination or the isolated natural killer cell for use according to claim 13, wherein the cancer is selected from lymphoma and leukemia.

15. The isolated natural killer cell according to any one of claims 1 to 6 or the isolated nucleic acid molecule according to any one of claims 7 to 10 or the combination according to claim 11 for use in treatment of an autoimmune disease.

16. The isolated nucleic acid molecule or the combination or the isolated natural killer cell for use according to claim 15, wherein the autoimmune disease is selected from the group of systemic lupus erythematosus, myositis, systemic sclerosis and ulcerative colitis.

17. A method for preparation of a natural killer cell as specified in any one of claims 1 to 6, the method comprising the steps:a. providing a peripheral blood mononuclear cell (PBMC);b. isolating an NKG2A+ natural killer (NK) cell and keeping the NK cell under cell culture conditions;c. contacting the NKG2A+ NK cell with a vector comprising the isolated nucleic acid molecule according to any one of claims 7 to 10;d. further knocking out FAS.

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