Optimised il-15 constructs

Abstract

Immune effector cells, e.g. NK cells and T cells, are modified to increase their cytotoxicity; the cells and compositions thereof have a use in the treatment of cancer. Production of modified immune effector cells and cell lines is via genetic modification to knock down expression of the CISH gene, CD38 gene and / or TGFBR2 gene, and to knock in expression of a gene encoding IL-15.

Description

[0001] OPTIMISED IL-15 CONSTRUCTS

[0002] Introduction

[0003] The present invention relates to the modification of immune effector cells e.g. natural killer (NK) cells or T cells, and cell lines thereof to produce cells with a more cytotoxic phenotype. Furthermore, the present invention relates to methods of producing modified immune effector cells and cell lines, compositions containing the cells and cell lines and uses of said cells, lines and compositions in the treatment of cancer.

[0004] Background to the Invention

[0005] Typically, immune cells require a target cell to present antigen via major histocompatibility complex (MHC) before triggering an immune response resulting in the death of the target cell. This allows cancer cells not presenting MHC class I to evade the majority of immune responses.

[0006] NK cells, however, are able to recognize cancer cells in the absence of MHC class I expression. Hence, they perform a critical role in the body’s defense against cancer.

[0007] On the other hand, in certain circumstances, cancer cells demonstrate an ability to dampen the cytotoxic activity of immune effector cells, through expression of ligands that bind inhibitory receptors on the immune effector cell membrane. Resistance to cancer therapy can involve a balance between these and other factors.

[0008] Cytotoxicity, in this context, refers to the ability of immune effector cells, e.g. NK cells or T cells, to induce cancer cell death, e.g. by releasing cytolytic compounds or by binding receptors on cancer cell membranes and inducing apoptosis of said cancer cells. Cytotoxicity is affected not only by signals that induce release of cytolytic compounds but also by signals that inhibit their release. An increase in cytotoxicity will therefore lead to more efficient killing of cancer cells, with less chance of the cancer cell dampening the cytotoxic activity of the immune effector cells, as mentioned above.

[0009] Cytokine-inducible SH2-containing protein (CIS) expression is induced by certain growth cytokines and is a key negative regulator of interleukin-15 (IL-15) signaling in immune effector cells e.g. natural killer (NK) cells. CIS is encoded by the CISH gene. CIS expression is associated with limited cell expansion and decreased cytotoxic activity against multiple cancer cell lines when maintained at low cytokine concentrations. As such, knocking out CIS expression by deletion of the CISH gene in NK cells has recently been reported as beneficial for NK cell cytotoxicity against cancer cells (Zhu et al. 2020 “Metabolic Reprograming via Deletion of CISH in Human iPSC-Derived NK Cells Promotes In Vivo Persistence and Enhances Antitumor Activity” Cell: Vol. 27(2): pp. 224-237).

[0010] TGF beta receptor (TGFBR) signaling in immune effector cells is known to be immunosuppressive and affect the function of immune effector cells. TGF beta can alter the expression of effector molecules and of activating and chemokine receptors, influence metabolism, and induce the NK cell conversion toward the less cytotoxic ILC-1 phenotype (Regis et al. 2020 “NK Cell Function Regulation by TGF-|3-lnduced Epigenetic Mechanisms” Front Immunol. 11 : 311).

[0011] Immune effector cells e.g. NK cells or T cells express the IL-15 receptor and are thus responsive to IL-15 in the cancer microenvironment, typically expressed by other cells of the immune system in both a soluble form and in a membrane-bound form.

[0012] Whilst the theoretical interplay between CISH expression and IL-15 signaling is known, it is not known whether any interplay can be exploited in order to provide new and improved cancer therapies.

[0013] There exists a need for alternative and preferably improved cell-based therapies with improved cytotoxicity and persistence, particularly against cancer cells.

[0014] An object of the present invention is to provide immune effector cells and cell lines that target cancer cells with a more cytotoxic and / or persistent phenotype. A further object is to provide methods for producing modified immune effector cells and cell lines, compositions containing the cells or cell lines and uses of such in therapy, specifically the treatment of cancers. More particular embodiments aim to provide treatments for identified cancers. Specific embodiments aim at combining two or more modifications of immune effector cells and cell lines to enhance the cytotoxicity and persistence of the modified cells.

[0015] Summary of the Invention There are provided herein modified immune effector cells e.g. NK cells or T cells with a more cytotoxic phenotype, and methods of making the cells and cell lines. Also provided are compositions of immune effector cells e.g. modified NK cells or T cells and cell lines thereof, and uses of said cells / compositions for treating cancer. Cytotoxicity in this context, as above, refers to the killing of cancer cells.

[0016] The invention provides immune effector cells e.g. NK cells or T cells and cell lines thereof that exhibit (e.g. have been modified to have) reduced expression of CISH and express (e.g. have been modified to express) IL-15 (e.g., a soluble, secreted form of IL-15, referred to herein as “sclL-15”).

[0017] Additionally, the invention also provides immune effector cells e.g. NK cells or T cells and cell lines thereof that exhibit (e.g. have been modified to have) reduced expression of CD38 and express (e.g. have been modified to express) IL-15.

[0018] The invention also provides immune effector cells e.g. NK cells or T cells and cell lines thereof that exhibit (e.g. have been modified to have) reduced expression of TGFBR2 and express (e.g. have been modified to express) IL-15.

[0019] According to the invention, there are further provided methods of treating cancer, e.g. solid tumors and blood cancers, using the immune effector cells e.g. modified NK or T cells and cell lines thereof, both generally and specifically, wherein the modified NK cells and lines are engineered to have reduced expression of CISH, CD38 and / or TGFBR2 and to express IL-15 and, optionally, to express one or more chimeric antigen receptors.

[0020] Diseases particularly treatable according to the invention include cancers, specifically solid cancers or blood cancers, and more particularly myeloma, ovarian or breast cancers. Tumors and cancers in humans in particular can be treated. References to tumors herein include references to neoplasms.

[0021] Details of the Invention

[0022] Accordingly, the present invention provides an immune effector cell, e.g. a natural killer (NK) cell or T cell, that has been modified to have reduced expression of CISH and to express IL-15, wherein the IL-15 expression is driven by an attenuated / weak promoter. Preferably, a soluble, secreted form of IL-15 (sclL-15) is expressed. Preferably, the IL-15 transgene is positioned downstream of the attenuated / weak promoter and upstream of at least one other transgene. Preferably, the transgenes are expressed at the CISH locus. More preferably, the transgenes are expressed within exon 4 of the CISH gene. Preferably, the at least one other transgene encodes a CAR, e.g., a CD38 CAR.

[0023] The present invention also provides an immune effector cell, e.g. a natural killer (N K) cell or T cell, that has been modified using a polycistronic (e.g., bicistronic) vector to express IL-15 and at least one other transgene, wherein the IL-15 transgene is positioned upstream of the at least one other transgene. Preferably, a soluble, secreted form of IL-15 (sclL-15) is expressed. Preferably, the IL-15 transgene is positioned downstream of an attenuated / weak promoter. Preferably, the transgenes are expressed at the CISH locus. More preferably, the transgenes are expressed within exon 4 of the CISH gene. Preferably, the at least one other transgene encodes a CAR, e.g., a CD38 CAR.

[0024] The present invention provides an immune effector cell, e.g. a natural killer (NK) cell or T cell, that has been modified to express IL-15 at the CISH locus, wherein the IL- 15 expression is driven by an attenuated / weak promoter. Preferably, a soluble, secreted form of IL-15 (sclL-15) is expressed. Preferably, the IL-15 transgene is positioned downstream of the attenuated / weak promoter and upstream of at least one other transgene. More preferably, the transgenes are expressed within exon 4 of the CISH gene. Preferably, the at least one other transgene encodes a CAR, e.g., a CD38 CAR.

[0025] For the avoidance of doubt, an immune effector cell according to the invention may be an immune effector cell line.

[0026] Preferably, the immune effector cell of the invention has further been modified to have reduced expression of CD38. The present invention may therefore provide an immune effector cell, e.g. a natural killer (NK) cell or T cell, that has been modified to have reduced expression of CD38, as well as reduced expression of CISH and expression of IL-15, preferably wherein the IL-15 expression is driven by an attenuated / weak promoter. Preferably, a soluble, secreted form of IL-15 (sclL-15) is expressed. The immune effector cell of the invention may also be modified to have reduced expression of TGFBR2. The present invention may therefore provide an immune effector cell, e.g. a natural killer (NK) cell or T cell, that has been further modified to have reduced expression of TGFBR2, as well as reduced expression of CISH and expression of IL-15, preferably wherein the IL-15 expression is driven by an attenuated / weak promoter. Preferably, a soluble, secreted form of IL-15 (sclL-15) is expressed.

[0027] The immune effector cell is preferably human. The immune effector cell is preferably an NK cell or a T cell. More preferably, the immune effector cell is an NK cell or NK cell line. Together, the NK cells and NK cell lines of the invention will also be referred to as the NK cells (unless the context requires otherwise).

[0028] The immune effector cell may be derived from umbilical cord blood or peripheral blood. More preferably, the immune effector cell is derived from umbilical cord blood, e.g. a cord blood-derived NK cell (CB-NK cell).

[0029] CB-NK cells possess an immature phenotype, when compared to other known NK cells, e.g., peripheral blood NK cells, presenting in reduced cytotoxicity, with higher expression of classical inhibitory receptors, such as NKG2A. For this reason, along with the difficulties experienced in successfully isolating these cells from umbilical cord blood, CB-NK cells have hitherto been regarded as an unfavorable option for NK cells to be used in cell therapies for cancer.

[0030] It is preferred that the immune effector cell is modified to have reduced expression of CISH and CD38, as well as to express a soluble, secreted form of IL-15.

[0031] Preferably, CISH expression is reduced by at least 50%, at least 75%, at least 90%, at least 95%, more preferably at least 99%, compared to the same immune effector cell or immune effector cell line without the modification (wildtype immune effector cell).

[0032] It is particularly preferred that immune effector cells according to the invention have CISH expression knocked out. Preferably, CD38 expression is reduced by at least 50%, at least 75%, at least 90%, at least 95%, more preferably at least 99%, compared to the same immune effector cell or immune effector cell line without the modification (wildtype immune effector cell).

[0033] It is particularly preferred that immune effector cells according to the invention have CD38 expression knocked out.

[0034] Preferably, TGFBR2 expression is reduced by at least 50%, at least 75%, at least 90%, at least 95%, more preferably at least 99%, compared to the same immune effector cell or immune effector cell line without the modification (wildtype immune effector cell).

[0035] It is particularly preferred that immune effector cells according to the invention have TGFBR2 expression knocked out.

[0036] As such, it is preferred that CISH and / or CD38 expression is genetically knocked out and IL-15 is genetically knocked in. Preferably, the IL-15 gene is knocked into the locus of the CISH gene, such that CISH expression is simultaneously knocked out.

[0037] Advantageously, the inventors have found that, when the sclL-15 gene is inserted into the CISH gene locus, thereby knocking out CISH expression, NK cells according to the invention have a significantly improved rate of expansion. Surprisingly, it has also been found that levels of IL-15 expression are significantly higher in NK cells expressing the IL-15 at the CISH gene locus, compared to NK cells expressing the IL-15 gene extra-genomically, i.e., outside of the CISH gene loci.

[0038] In some cases, however, a new observation in the present invention, believed unreported previously, is that immune effector cells such as NK cells can become over-stimulated by the high levels of IL-15 expression at the CISH gene locus and lead to undesirable effects e.g. overactivation of immune effector cells and potential toxicity. This occurs due to the down regulation of CISH expression making the immune effector cells highly sensitive to IL-15 signaling; IL-15 then can become toxic to the modified NK cells. In addition to the significantly improved rate for expansion and increased levels of IL- 15 expression, advantageously, by inserting IL-15 into the CISH gene locus, it was possible to modulate the level of transcription of the IL-15 gene using different upstream promoters with varying strengths while simultaneously knocking out CISH expression; this would not have been possible if the IL-15 gene was knocked in at a random locus.

[0039] The inventors have found that an immune effector cell e.g. NK cell that has been modified to have reduced expression of CISH, further comprising a nucleotide encoding IL-15 operably linked to an attenuated / weak promoter advantageously produced non-toxic levels of IL-15, yet the cell is capable of maintaining high levels of cytotoxicity against cancer cells i.e. expressing useful levels of IL-15 but avoiding toxic levels of IL-15 while maintaining cytotoxicity and expressing useful levels, where present, of other transgenes, e.g., for a CAR.

[0040] In an additional, parallel experiment, the inventors found that insertion of the IL-15 gene driven by the elongation factor- 1 alpha (EF-1a) promoter into the CISH locus resulted in more than 1 ,100 pg / ml of IL-15 expression, whereas the use of an attenuated / weak promoter e.g. MND promoter lowers the IL-15 expression by at least 50% and in specific examples approximately 10-fold, Preferably, the IL-15 gene is inserted into exon 4 of the CISH gene.

[0041] The weak or attenuated promoter referred to herein results in lower transgene and / or reporter expression when compared to the EF-1a promoter. The weak or attenuated promoter may result in at least 25% less induction of transgene, e.g. IL-15 or CAR, expression, in comparison to the EF-1a promoter. Preferably, the weak or attenuated promoter results in at least 50% less induction of transgene, e.g. IL-15 or CAR, expression in comparison to the EF-1a promoter. More preferably, the weak or attenuated promoter results in at least 75% less induction of transgene e.g. IL-15 or CAR expression in comparison to EF-1a promoter. Even more preferably, the weak or attenuated promoter results in at least 90% less induction of transgene e.g. IL-15 or CAR expression in comparison to EF-1a promoter.

[0042] The promoter may be selected from MND, UBC, PGK, EF-1a, CMV, CAGG, SV40 and TRE. Preferably, the attenuated / weak promoter may be selected from MND, UBC and PGK. Even more preferably, the promoter is MND. Strength of different promoters can be assessed by measuring expression of a target gene downstream of the promoter e.g. quantifying GFP reporter expression in immune effector cells using flow cytometry.

[0043] The promoter is preferably not positioned within a long terminal repeat (LTR).

[0044] Optionally, the IL-15 gene is knocked into the locus of the CD38 or TGFBR2 gene.

[0045] For the avoidance of doubt, references herein to “modified” immune effector cells include the progeny of those cells, at least to the extent that the progeny carry the modification.

[0046] Genetic knockout of CISH, CD38 and / or TGFBR2, as well as genetic knock-in of IL- 15, is preferred, and further preferably by CRISPR gene editing.

[0047] Immune effector cells of the invention are for use in therapy; preferably, for use in treating cancer in a patient. The cancer is suitably a solid tumor or a blood cancer. Even more preferably, the cancer is myeloma, ovarian cancer or breast cancer.

[0048] In a particularly preferred embodiment, the invention provides a human NK cell, genetically modified to have CISH expression knocked out and to express IL-15 driven by an attenuated / weak promoter, for use in cancer therapy.

[0049] There are many ways in which IL-15 can be administered to or with immune effector cells, e.g., in soluble form or complexed with the IL-15 receptor alpha chain. Additionally, immune effector cells may be modified to express IL-15 as a membranebound cytokine.

[0050] However, modifying an immune effector cell to express IL-15 under the control of an attenuated / weak promoter is less common. Moreover, until now, this has never been performed in an immune effector cell setting in which the CISH is knocked out.

[0051] With that said, the present inventors have worked to identify and optimize immune effector cell modifications that have promising value in cancer therapeutics. Surprisingly, it has been found that when IL-15 driven by an attenuated / weak promoter is expressed in a CISH KO NK cell, the cell becomes potently cytotoxic against solid tumors or blood cancers. Advantageously, these two modifications have been shown to work synergistically with each other to provide an NK cell that is significantly more cytotoxic towards solid tumor cells than either a CISH KO NK cell or an NK cell with a knock-in of IL-15.

[0052] In a particularly preferred embodiment, the invention provides a human NK cell, genetically modified to have CD38 expression knocked out and to express IL-15 driven by an attenuated / weak promoter, for use in solid tumor therapy.

[0053] There are many ways in which IL-15 can be administered to or with immune effector cells, e.g., in soluble form or complexed with the IL-15 receptor alpha chain. Additionally, immune effector cells may be modified to express IL-15 as a membranebound cytokine.

[0054] However, as above, modifying an immune effector cell to express IL-15 under the control of an attenuated / weak promoter is less common is less common. Moreover, until now, this has never been performed in a NK cell setting in which the CD38 gene is knocked out.

[0055] With that said, the present inventors have worked to identify and optimize NK cell modifications that have promising value in cancer therapeutics. Surprisingly, it has been found that when IL-15 driven by an attenuated / weak promoter is expressed in a CD38 KO NK cell, the cell becomes potently cytotoxic against solid tumors and blood cancers. Advantageously, these two modifications have been shown to work effectively with each other to provide an NK cell that is significantly more cytotoxic towards solid tumor cells than either a CD38 KO NK cell or a NK cell with a knock-in of IL-15.

[0056] In a particularly preferred embodiment, the invention provides a human NK cell, genetically modified to have both CISH expression and CD38 expression knocked out and to express IL-15 driven by an attenuated / weak promoter, for use in cancer therapy. Preferably, a soluble, secreted form of IL-15 (sclL-15) is expressed.

[0057] There are many ways in which IL-15 can be administered to or with immune effector cells, e.g., in soluble form or complexed with the IL-15 receptor alpha chain. Additionally, immune effector cells may be modified to express IL-15 as a membranebound cytokine.

[0058] However, as above, modifying an immune effector cell to express of IL-15 under the control of an attenuated / weak promoter is less common. Moreover, until now, this has never been performed in an immune effector cell setting, in which both the CISH gene and the CD38 gene is knocked out.

[0059] With that said, the present inventors have worked to identify and optimize NK cell modifications that have promising value in cancer therapeutics. Surprisingly, it has been found that when IL-15 driven by an attenuated / weak promoter is expressed in a CISH / CD38 dual knock out (DKO) NK cell, the cell becomes potently cytotoxic against cancer cells. Advantageously, these three modifications have been shown to work effectively with each other to provide a NK cell that is significantly more cytotoxic towards cancer cells but with reduced toxicity than either a CISH KO NK cell, a CD38 KO NK cell or an NK cell with a knock-in of IL-15.

[0060] A specific embodiment of the invention, described in more detail below provides a human NK cell or NK cell line, wherein the cell is modified to have: a) CISH expression knocked out; and b) a gene encoding IL-15 knocked in, wherein expression of the IL-15 is driven by a weak promoter, and wherein the weak promoter results in at least 50% less induction of IL-15 expression in comparison to an EF-1a promoter.

[0061] Preferably, a soluble, secreted form of IL-15 (sclL-15) is expressed.

[0062] Another specific embodiment of the invention, described in more detail below provides a human NK cell or NK cell line, wherein the cell is modified to have: a) CISH and CD38 expression knocked out; and b) genes encoding IL-15 and CD38 CAR knocked into exon 4 of the CISH locus; wherein expression of the IL-15 and CD38 CAR are driven by an attenuated / weak promoter, e.g. MND; and wherein the IL-15 and CD38 CAR are expressed from a bicistronic vector, with the IL-15 coding sequence upstream of that for the CD38 CAR. Preferably, a soluble, secreted form of IL-15 (sclL-15) is expressed.

[0063] In a particularly preferred embodiment, the invention provides a human NK cell, genetically modified to have both CISH expression and TGFBR2 expression knocked out and to express IL-15 driven by an attenuated / weak promoter, for use in cancer therapy.

[0064] There are many ways in which IL-15 can be administered to or with immune effector cells, e.g., in soluble form or complexed with the IL-15 receptor alpha chain. Additionally, immune effector cells may be modified to express IL- 15 as a membranebound cytokine.

[0065] However, as above, modifying an immune effector cell to express IL-15 under the control of an attenuated / weak promoter is less common. Moreover, until now, this has never been performed in an immune effector cell setting, in which both the CISH gene and the TGFBR2 gene is knocked out.

[0066] Again, CB-NK cells possess an immature phenotype, when compared to other known NK cells, e.g., peripheral blood NK cells, presenting in reduced cytotoxicity, with higher expression of classical inhibitory receptors, such as NKG2A. For this reason, along with the difficulties experienced in successfully isolating these cells from umbilical cord blood, CB-NK cells hitherto been regarded as an unfavorable option for NK cells to be used in cell therapies for cancer.

[0067] With that said, the present inventors have worked to identify and optimize NK cell modifications that have promising value in cancer therapeutics. Surprisingly, it has been found that when IL-15 driven by an attenuated / weak promoter is expressed in a CISH / TGFBR2 DKO NK cell, the cell becomes potently cytotoxic against solid tumors. Advantageously, these two modifications have been shown to work effectively with each other to provide an NK cell that is significantly more cytotoxic towards solid tumor cells than either a TGFBR2 KO NK cell or a NK cell with a knock- in of IL-15.

[0068] The invention also provides a method of making an immune effector cell e.g. modified NK cell or T cell, or cell line thereof as described above or below, the method comprising the following steps: a) isolating an immune effector cell, for example from human umbilical cord blood or peripheral blood; b) genetically modifying the cell to have CISH expression knocked out; and c) expressing a gene encoding IL-15 driven by an attenuated / weak promoter.

[0069] Preferably, a soluble, secreted form of IL-15 (sclL-15) is expressed.

[0070] The method may further comprise the step of genetically modifying the cell to have CD38 and / or TGFBR2 expression knocked out. Preferably, the method may also comprise the step of expressing the gene encoding IL-15 at the locus of either the CISH gene, CD38 gene or the TGFBR2 gene. More preferably, the gene encoding IL-15 is expressed at the CISH locus. Even more preferably, the gene encoding IL-15 is expressed at exon 4 of the CISH locus.

[0071] Compositions of the modified immune effector cells of the invention are also provided, as are methods of using the modified immune effector cells or compositions thereof in therapy, e.g., cancer therapy. Pharmaceutical compositions are preferred.

[0072] In other embodiments of the invention, immune effector cells are provided that also express or overexpress a TRAIL ligand. Preferably, the TRAIL ligand is a mutant (variant) TRAIL ligand. The resulting immune effector cells exhibit increased binding to TRAIL receptors and, as a result, increased cytotoxicity against cancers, especially solid cancers, in particular ovarian, breast and colorectal cancers, and blood cancers, in particular leukemias. The immune effector cells with this combined activity may also be effective in reducing cancer metastases.

[0073] The TRAIL mutants / variants preferably have lower affinity (or in effect no affinity) for ‘decoy’ receptors, compared with the binding of wild type TRAIL to decoy receptors. Such decoy receptors represent a class of TRAIL receptors that bind TRAIL ligand but do not have the capacity to initiate cell death and, in some cases, act to antagonize the death signaling pathway. Mutant / variant TRAIL ligands may be prepared according to WO 2009 / 077857.

[0074] The mutants / variants may separately have increased affinity for TRAIL receptors, e.g. DR4 and DR5. Wildtype TRAIL is typically known to have a KD of >2 nM for DR4, >5 nM for DR5 and >20 nM for the decoy receptor DcR1 (WO 2009 / 077857; measured by surface plasmon resonance), or around 50 to 100 nM for DR4, 1 to 10 nM for DR5 and 175 to 225 nM for DcR1 (Truneh, A. et al. 2000; measured by isothermal titration calorimetry and ELISA). Therefore, an increased affinity for DR4 is suitably defined as a KD of <2 nM or <50 nM, respectively, whereas an increased affinity for DR5 is suitably defined as a KD of <5 nM or <1 nM, respectively. A reduced affinity for decoy receptor DcR1 is suitably defined as a KD of >50 nM or >225 nM, respectively. In any case, an increase or decrease in affinity exhibited by the TRAIL variant / mutant is relative to a baseline affinity exhibited by wildtype TRAIL. The affinity is preferably increased at least 10%, at least 25%, at least 50%, at least 100%, more preferably at least 1000%, compared with that exhibited by wildtype TRAIL.

[0075] The TRAIL variant preferably has an increased affinity for DR5 as compared with its affinity for DR4, DcR1 and DcR2. Preferably, the affinity is at least 1.5-fold, 2-fold, 5- fold, 10-fold, 100-fold, or even 1 ,000-fold or greater for DR5 than for one or more of DR4, DcR1 and DcR2. More preferably, the affinity is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 100-fold, or even 1 ,000-fold or greater for DR5 than for at least two, and preferably all, of DR4, DcR1 and DcR2.

[0076] The TRAIL variant preferably has an increased affinity for one or both of DR4 and DR5 as compared with its affinity for wildtype TRAIL. Preferably, the affinity is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 100-fold, or even 1 ,000-fold or greater for DR4 and / or DR5 than for wildtype TRAIL.

[0077] Further specific embodiments comprise an immune effector cell expressing a mutant TRAIL ligand that has reduced or no affinity for TRAIL decoy receptors. Further specific embodiments comprise an immune effector cell expressing a mutant TRAIL ligand that has reduced or no affinity for TRAIL decoy receptors and increased affinity for DR4 and / or DR5.

[0078] Binding affinity may be measured according to any suitable method known in the art. Preferably, binding affinity is measured using surface plasmon resonance, isothermal titration calorimetry or ELISA. In certain embodiments, the TRAIL variant comprises at least one amino acid substitution at a position selected from the group consisting of 131 , 149, 159, 160, 189, 191 , 193, 195, 199, 200, 201 , 203, 204, 212, 213, 214, 215, 218, 240, 251 , 261 , 264, 266, 267, 269, and 270.

[0079] In certain embodiments, the TRAIL variant comprises at least one substitution selected from the group consisting of G131R, G131K, R149I, R149M, R149N, R149K, S159R, G160E, Y189A, Y189Q, R191K, Q193H, Q193K, Q193S, Q193R,

[0080] E195R, N199V, N199R, N199H, T200H, K201R, K201H, D203A, K204E, K204D,

[0081] K204Y, K212R, Y213W, T214R, S215D, S215E, S215H, S215K, S215D, D218H,

[0082] D218A, Y240A, K251 D, K251 E, K251Q, T261 L, H264R, I266L, D267Q, D269A,

[0083] D269H, and H270D.

[0084] In certain embodiments, the TRAIL variant comprises at least two substitutions selected from the group consisting of G131R, G131K, R149I, R149M, R149N, R149K, S159R, G160E, Y189A, Y189Q, R191K, Q193H, Q193K, Q193S, Q193R,

[0085] E195R, N199V, N199R, N199H, T200H, K201R, K201H, D203A, K204E, K204D,

[0086] K204Y, K212R, Y213W, T214R, S215D, S215E, S215H, S215K, S215D, D218H,

[0087] D218A, Y240A, K251 D, K251 E, K251Q, T261 L, H264R, I266L, D267Q, D269A,

[0088] D269H, and H270D.

[0089] In certain embodiments, the TRAIL variant comprises at least three substitutions selected from the group consisting of G131R, G131K, R149I, R149M, R149N, R149K, S159R, G160E, Y189A, Y189Q, R191K, Q193H, Q193K, Q193S, Q193R,

[0090] E195R, N199V, N199R, N199H, T200H, K201R, K201H, D203A, K204E, K204D,

[0091] K204Y, K212R, Y213W, T214R, S215D, S215E, S215H, S215K, S215D, D218H,

[0092] D218A, Y240A, K251 D, K251 E, K251Q, T261 L, H264R, I266L, D267Q, D269A,

[0093] D269H, and H270D.

[0094] In certain embodiments, amino acid substitution of the TRAIL variant is selected from the group consisting of G131R, G131K, R149I, R149M, R149N, R149K, S159R, G160E, Y189A, Y189Q, R191K, Q193H, Q193K, Q193S, Q193R, E195R, N199V,

[0095] N199R, N199H, T200H, K201 R, K201 H, D203A, K204E, K204D, K204Y, K212R,

[0096] Y213W, T214R, S215D, S215E, S215H, S215K, S215D, D218H, D218A, Y240A,

[0097] K251 D, K251 E, K251Q, T261 L, H264R, I266L, D267Q, D269A, D269H, H270D,

[0098] T214R / E195R, T214R / D269H, Y189A / Q193S / N199V / K201 R / Y213W / S215D, Y213W / S215D, N199R / K201H, N199H / K201R, G131R / N199R / K201H, G131 R / N199R / K201H / R149I / S159R / S215D, G131R / R149I / S159R / S215D, G131 R / N199R / K201H / R149I / S159R / S215D, G131R / D218H, Y189Q / R191K / Q193R / H264R / I266L / D267Q, T261L / G160E, T261L / H270D, T261 L / G160E / H270D, and T261L / G160E / H270D / T200H (use indicates multiple amino acid substitutions).

[0099] In certain embodiments, amino acid substitution of the TRAIL variant is selected based on the variant having an increased affinity for DR5; a substitution of this kind may be selected from the group consisting of D269H, E195R, T214R, D269H / E195R, T214R / E195R, T214R / D269H, N199V, Y189A / Q193S / N199V / K201R / Y213W / S215D, Y213W / S215D, D269A and Y240A.

[0100] In certain embodiments, amino acid substitution of the TRAIL variant is selected based on the variant having an increased affinity for DR4; a substitution of this kind may be selected from the group consisting of G131R, G131K, R149I, R149M, R149N, R149K, S159R, Q193H, W193K, N199R, N199R / K201H, N199H / K201R, G131R / N199R / K201 H, G131R / N199R / K201 H, G131R / N199R / K201 H / R149I / S159R / S215D, G131R / R149I / S159R / S215D, G131R / D218H, K201 R, K201 H, K204E, K204D, K204L, K204Y, K212R, S215E, S215H, S215K, S215D, D218H, K251 D, K251 E, K251 Q and Y189Q / R191 K / Q193R / H264R / I266L / D267Q.

[0101] In certain embodiments, amino acid substitution of the TRAIL variant is selected based on the variant having a decreased affinity for TRAIL decoy receptors; a substitution of this kind may be selected from the group consisting of T261L, H270D, T200H, T261 L / G160E, T261 L / H270D, T261L / G160E / H270D, T261L / G160E / H270D / T200H, D203A and D218A.

[0102] Treatment of a cancer using modified immune effector cells expressing TRAIL or a TRAIL variant is optionally enhanced by administering to a patient an agent capable of upregulating expression of TRAIL death receptors on cancer cells. This agent may be administered prior to, in combination with or subsequently to administration of the modified immune effector cells. It is preferable, however, that the agent is administered prior to administering the modified immune effector cells. The agent upregulates expression of DR5 on cancer cells. The agent may optionally be a chemotherapeutic medication, e.g. a proteasome inhibitor, one of which is Bortezomib, and administered in a low dose capable of upregulating DR5 expression on the cancer. Other examples of DR5-inducing agents include Gefitinib, Piperlongumine, Doxorubicin, Alpha-tocopheryl succinate and HDAC inhibitors.

[0103] In certain embodiments of the invention, immune effector cells are provided that are further modified so as to have reduced or absent function of a checkpoint inhibitory receptor. Immune effector cells may be produced that have one or more checkpoint inhibitory receptor genes knocked down / out. Preferably, these receptors are specific checkpoint inhibitory receptors. Preferably still, these checkpoint inhibitory receptors are one or more or all of TIGIT, CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7), SIGLEC9 and / or TIM-3. In other embodiments, immune effector cells are provided in which one or more inhibitory receptor signaling pathways are knocked out or exhibit reduced function - the result again being reduced or absent inhibitory receptor function. For example, signaling pathways mediated by SHP-1 , SHP-2 and / or SHIP are knocked out by genetic modification of the cells.

[0104] In cases where the immune effector cell is an NK cell, it is preferred to reduce function of checkpoint inhibitory receptors over other inhibitory receptors, due to the expression of the former following NK cell activation. The normal or ‘classical’ inhibitory receptors, such as the majority of the KIR family, NKG2A and UR-2, bind MHC class I and are therefore primarily involved in reducing the problem of selftargeting. Preferably, therefore, checkpoint inhibitory receptors are knocked out. Reduced or absent function of these receptors according to the invention prevents cancer cells from suppressing immune effector function (which might otherwise occur if the receptors were fully functional). Thus, a key advantage of these embodiments of the invention lies in immune effector cells that are less susceptible to suppression of their cytotoxic activities by cancer cells; as a result, they are useful in cancer treatment.

[0105] As used herein, references to inhibitory receptors generally refer to a receptor expressed on the plasma membrane of an immune effector cell, e.g. a NK cell, whereupon binding its complementary ligand resulting intracellular signals are responsible for reducing the cytotoxicity of said immune effector cell. These inhibitory receptors are expressed during both ‘resting’ and ‘activated’ states of the immune effector cell and are often associated with providing the immune system with a ‘self- tolerance’ mechanism that inhibits cytotoxic responses against cells and tissues of the body. An example is the inhibitory receptor family ‘KIR’ which are expressed on NK cells and recognize MHC class I expressed on healthy cells of the body.

[0106] Also as used herein, checkpoint inhibitory receptors are usually regarded as a subset of the inhibitory receptors above. Unlike other inhibitory receptors, however, checkpoint inhibitory receptors are expressed at higher levels during prolonged activation and cytotoxicity of an immune effector cell, e.g. a NK cell or T cell. This phenomenon is useful for dampening chronic cytotoxicity at, for example, sites of inflammation. Examples include the checkpoint inhibitory receptors PD-1 , CTLA-4 and CD96, all of which are expressed on NK or T cells.

[0107] It is preferred in the invention not to reduce the function of inhibitory receptors that bind MHC class I.

[0108] The modified cells of the invention may also express a chimeric antigen receptor (CAR). The membrane-bound CAR typically comprises a targeting sequence (commonly an antibody-derived single-chain fragment (scFv)) and usually a hinge (to overcome steric hindrance issues), a spacer, a membrane-spanning element and a signalling endodomain.

[0109] The bicistronic or polycistronic sequence expressing the genes encoding IL-15 and CAR may be used for targeted insertion into an immune effector cell. Preferably, the genes for targeted insertion may be inserted into a bicistronic or polycistronic vector. Even more preferably, the vector expressing the gene encoding IL-15 is bicistronic. It is also preferred that the gene encoding the CAR is inserted with the IL-15 gene into a bicistronic vector. The bicistronic or polycistronic sequence may also contain one or more intervening skipping sequences, e.g. a furin-glycine-serine-glycine-T2A linker.

[0110] The vector used for targeted gene insertion may be selected from adeno-associated virus (AAV), lentivirus, retrovirus, adenovirus or herpes virus. Preferably, the vector used for targeted gene insertion is AAV.

[0111] Preferably, this bicistronic vector is used to modify the immune effector cells of the invention to express the exogenous genes at the CISH gene locus, simultaneously disrupting and knocking out expression of CISH. Even more preferably, the In a preferred embodiment, the CAR immune effector cell binds the CD38 antigen. Preferably, when the CAR binds CD38, the immune effector cell is further modified to have a reduced expression of CD38. Even more preferably, when the CAR binds CD38, the immune effector cell is further modified to have CD38 knocked out.

[0112] In a preferred embodiment, the CAR immune effector cell binds the HER2 antigen.

[0113] In a preferred embodiment, the CAR immune effector cell binds the VEGF antigen.

[0114] In a preferred embodiment, the CAR immune effector cell binds the EpCAM antigen.

[0115] In a preferred embodiment, the CAR immune effector cell binds the EGFR antigen.

[0116] The CAR may be bispecific and bind two of the above-mentioned antigens. In another embodiment, a second CAR may be expressed and targets an antigen different to the first CAR.

[0117] Examples of sequences that are known to bind CD38 can be used as the targeting sequence, such sequences including daratumumab, isatuximab and those disclosed in WO 2018 / 104562, and are suitably incorporated in a CAR of the invention. It is preferred that the CD38 CAR has reduced affinity for CD38, compared to the affinity of daratumumab for CD38. Preferably, this reduced affinity is by at least 10%, at least 25%, more preferably at least 45%, compared to the affinity of daratumumab. Preferably, also, this reduced affinity is no more than 90%, no more than 75%, more preferably no more than 55%, compared to the affinity of daratumumab. Advantageously, an anti-CD38 CAR with a lower affinity than daratumumab reduces fratricide risk i.e. self-killing of NK cells which also express CD38 during expansion.

[0118] It is preferred that the CD38 CAR of the present invention has a binding domain that comprises a heavy chain variable region comprising SEQ ID NO: 11. Optionally, the heavy chain variable region comprises SEQ ID NO: 12. It is preferred that the CD38 CAR binding domain comprises a light chain variable region comprising SEQ ID NO: 19 or SEQ ID NO: 20.

[0119] The preferred CD38 CAR of the examples has a binding domain that comprises a heavy chain variable region comprising SEQ ID NO: 11 and a light chain variable region comprising SEQ ID NO: 19. Immune effector cells of the invention are for use in therapy, especially in treating cancer in a patient. The cancer is suitably a solid tumor or a blood cancer. Preferably, the cancer is ovarian cancer, breast cancer or myeloma.

[0120] NK cell resistant cancers in general are well-known in the art (Pardoll, D.M. Immunity (2015) 42:605-606). Sensitivity of cancer cells to NK cell-mediated killing is determined by a number of factors. There exists a balance of positive and negative signals, largely delivered through membrane receptors on NK cells interacting with ligands on cancer cells. It is often the balance of expression of the ligands for these receptors that determines whether a cancer is sensitive or resistant to killing by NK cells (Yokoyama, W.M. Immunol Res (2005) 32:317-325).

[0121] Cancer sensitivity to immune effector cell-mediated cytotoxicity is generally understood as falling into one of the following categories: highly resistant, resistant, sensitive and highly sensitive. In the laboratory, cancer cells can be screened for their sensitivity to immune effector cell-mediated cytotoxicity through the use of cytotoxicity assays. Each category is then understood as corresponding to the percentage of cancer cells that are killed during exposure to NK cells at a specific effector : target (E:T) ratio and for a specific amount of time.

[0122] In examples of the invention, a cancer is said to be highly resistant to NK cell- mediated killing if < 25% of the cancer cells are killed after incubation with NK cells for up to 15 hours at an E:T ratio of up to 5:1.

[0123] In examples of the invention, a cancer is said to be resistant to NK cell-mediated killing if < 50% of the cancer cells are killed after incubation with NK cells for up to 15 hours at an E:T ratio of up to 5:1.

[0124] In examples of the invention, a cancer is said to be sensitive to NK cell-mediated killing if > 50% of the cancer cells are killed after incubation with NK cells for up to 15 hours at an E:T ratio of up to 5:1.

[0125] In examples of the invention, a cancer is said to be highly sensitive to NK cell- mediated killing if > 75% of the cancer cells are killed after incubation with NK cells for up to 15 hours at an E:T ratio of up to 5:1. Optional features of the invention include providing further modifications to the immune effector cells and immune effector cell lines described above, wherein, for example, a Fc receptor (which can be CD16, CD32 or CD64, including subtypes and derivatives) is expressed on the surface of the cell. In use, these cells can show increased recognition of antibody-coated cancer cells and improved activation of the cytotoxic response.

[0126] Modified immune effector cells and compositions thereof described herein, above and below, are suitable for treatment of cancer, in particular cancer in humans, e.g. for treatment of solid cancers. The immune effector cells and derivatives thereof are preferably human immune effector cells. For human therapy, human NK and T cells are preferably used. The invention also provides methods of treating cancer in humans comprising administering an effective amount of the immune effector cells or compositions comprising the same.

[0127] Various routes of administration will be known to the skilled person to deliver active agents and combinations thereof to a patient in need. Administration of the modified immune effector cells can be systemic or localized, such as for example via the intraperitoneal route. In other embodiments, active agent is administered more directly. Thus, administration can be directly intratumorally, suitable especially for solid tumors.

[0128] Examples

[0129] The invention is now illustrated in specific embodiments with reference to the accompanying drawings in which:

[0130] Fig. 1 shows that CISH KO and sclL-15 knock-in in NK cells synergistically enhances the cytotoxic profile against human breast cancer cells;

[0131] Fig. 2 shows that CISH KO and sclL-15 knock-in in NK cells synergistically enhances the cytotoxic profile against human ovarian cancer cells;

[0132] Fig. 3 shows that NK cells with CISH and TGFBRII double KO (DKO) and sclL-15 knock-in are highly effective in cancer cell killing;

[0133] Fig. 4 shows that sclL-15 genomic insertion at the CISH locus increases NK cell expansion; and

[0134] Fig. 5 shows that sclL-15 genomic insertion at the CISH locus increases IL-15 expression in NK cells, compared to those cells expressing the sclL-15 gene extra- genomically. Fig. 6 shows the transfer “cis” plasmid construct “85A” designed for targeted knock-in into the double-strand break (DSB) in CISH.

[0135] Fig. 7 shows the transfer “cis” plasmid construct “86A” designed for targeted knock-in into the double-strand break (DSB) in CISH.

[0136] Fig. 8 shows that successful expansion of CB-NK cells transfected with the 85A or 86A transfer “cis” plasmid construct was achieved by Day 18.

[0137] Fig. 9 shows that CD38 expression was successfully knocked out at Day 7 in CD56 / CD16+ NK cells, while anti-CD38 CARs were successfully knocked in using the 85A or 86A transfer “cis" plasmid construct.

[0138] Fig. 10 shows that stronger expression level of anti-CD38 CAR was detected in NK cells transfected with the 86A transfer “cis” plasmid construct at Day 7.

[0139] Fig. 11 shows that the strong expression level of anti-CD38 CAR was detected in NK cells transfected with the 86A transfer “cis” plasmid construct was maintained at Day 18.

[0140] Fig. 12 shows that lower levels of soluble IL-15 were produced by NK cells transfected with the 86A transfer “cis” plasmid construct.

[0141] Fig. 13 shows that CB136 donor derived NK cells transfected with the 85A transfer “cis” plasmid construct are highly effective in cancer cell killing.

[0142] Fig. 14 shows that CB137 donor derived NK cells transfected with the 85A transfer “cis” plasmid construct achieved 100% cancer cell killing.

[0143] Fig. 15 shows that CB136 donor derived NK cells transfected with the 86A transfer “cis” plasmid construct are highly effective in cancer cell killing.

[0144] Fig. 16 shows that CB137 donor derived NK cells transfected with the 86A transfer “cis” plasmid construct achieved 100% cancer cell killing.

[0145] Fig. 17 shows that CISH was simultaneously knocked out via targeted insertion of the sclL-15 and anti-CD38 CAR construct.

[0146] DNA, RNA and amino acid sequences are referred to below, in which:

[0147] SEQ ID NO: 1 refers to the DNA sequence encoding the full-length codon- optimized sclL-15 peptide; and

[0148] SEQ ID NO: 2 refers to the amino acid sequence of the full-length sclL-15 peptide.

[0149] SEQ ID NO: 3 refers to the sequence of the DNA sequence of the transfer “cis” plasmid construct “85A”.

[0150] SEQ ID NO: 4 refers to the sequence of the DNA sequence of the transfer “cis” plasmid construct “86A”. SEQ ID NO: 5 refers to a single CDR sequence from exemplary CD38 CAR variable heavy chain (SEQ ID NO: 11).

[0151] SEQ ID NO: 6 refers to a single CDR sequence from exemplary CD38 CAR variable heavy chain (SEQ ID NO: 11).

[0152] SEQ ID NO: 7 refers to a single CDR sequence from exemplary CD38 CAR variable heavy chain (SEQ ID NO: 11).

[0153] SEQ ID NO: 8 refers to a single CDR sequence from exemplary CD38 CAR variable heavy chain (SEQ ID NO: 12).

[0154] SEQ ID NO: 9 refers to a single CDR sequence from exemplary CD38 CAR variable heavy chain (SEQ ID NO: 12).

[0155] SEQ ID NO: 10 refers to a single CDR sequence from exemplary CD38 CAR variable heavy chain (SEQ ID NO: 12).

[0156] SEQ ID NO: 11 refers to a heavy chain variable region sequence of an exemplary CD38 CAR.

[0157] SEQ ID NO: 12 refers to a heavy chain variable region sequence of an exemplary CD38 CAR.

[0158] SEQ ID NO: 13 refers to a single CDR sequence from exemplary CD38 CAR variable light chain (SEQ ID NO: 19).

[0159] SEQ ID NO: 14 refers to a single CDR sequence from exemplary CD38 CAR variable light chain (SEQ ID NO: 19).

[0160] SEQ ID NO: 15 refers to a single CDR sequence from exemplary CD38 CAR variable light chain (SEQ ID NO: 19).

[0161] SEQ ID NO: 16 refers to a single CDR sequence from exemplary CD38 CAR variable light chain (SEQ ID NO: 20).

[0162] SEQ ID NO: 17 refers to a single CDR sequence from an exemplary CD38 CAR variable light chain (SEQ ID NO: 20).

[0163] SEQ ID NO: 18 refers to a single CDR sequence from exemplary CD38 CAR variable light chain (SEQ ID NO: 20).

[0164] SEQ ID NO: 19 refers to a light chain variable region sequence of an exemplary CD38 CAR.

[0165] SEQ ID NO: 20 refers to a light chain variable region sequence of an exemplary CD38 CAR.

[0166] SEQ ID NOs: 21-35 refer to the 5’ - 3’ end DNA sequences of PCR primers used for Gibson assembly to assemble the variable heavy and light chain DNA fragments into the CAR construct.

[0167] SEQ ID NO: 36 refers to the animo acid sequence of CD3zeta domain. SEQ ID NO: 37 refers to the amino acid sequence of 41 BB co-stimulatory domain.

[0168] SEQ ID NO: 38 refers to the amino acid sequence of CD28 co-stimulatory domain.

[0169] Example 1 - Design Protocol for Knockout of CISH in NK Cells

[0170] NK cells can be prepared as follows, having CIS function removed. gRNA constructs can be designed and prepared to target the CISH gene in NK cells. CRISPR / Cas9 genome editing can then used to knock out CISH expression.

[0171] A total of 3 gRNA candidates may be selected for the CISH gene and their cleavage efficacies in primary expanded NK cells should be determined. The cells can then be electroporated with the gRNA:Cas9 ribonucleoprotein (RNP) complex using Maxcyte® GT and subsequently knockout of CISH can be analysed using flow cytometry. The cleavage activity of the gRNA can also be determined using an in vitro mismatch detection assay. T7E1 endonuclease recognises and cleaves non- perfectly matched DNA, allowing the parental CISH gene to be compared to the mutated gene following CRISPR / Cas9 transfection and non-homologous end joining (NHEJ).

[0172] The gRNA with highest KO efficiency can be selected for further experiments to knockout CISH in NK cells. Knockout of CISH can be determined by flow cytometrybased assays.

[0173] Example 2 - Knockout of CISH in NK Cells

[0174] Materials

[0175] 1. Enriched cord blood-derived NK cells.

[0176] 2. NK-MACS medium, Premium grade IL-15, anti-CD3 VioBlue, Anti-CD56VB515, Inside Stain Kit, MACSquant 16 (Miltenyi Biosciences).

[0177] 3. Human Serum Albumin (Sigma).

[0178] 4. Gene knockout Kit V2 for CISH and Cas9 recombinant protein (Synthego).

[0179] 5. Cloudz™ Cell Activation Kits (R&D systems).

[0180] 6. Anti-CISH antibody (D4D9) (Cell Signaling Technology). 7. Electroporation buffer (EB buffer), OC-100X2 processing assembly (PA), MaxCyte ATx electroporation system (Maxcyte).

[0181] 8. Solu-Cortef (Hydrocortisone) (Pfizer).

[0182] 9. Ficoll (Cytiva).

[0183] Protocol

[0184] Cord Blood Mononuclear Cells (CBMCs) can be isolated by the Ficoll method and enriched for 15 days in NK-MACS medium with 10% human serum albumin, CD2 / NKp46 microsphere (Cloudz™), anti-CD3, anti-CD16, IL-15 and hydrocortisone.

[0185] NK cell enrichment of over 90% can be confirmed by FACS (MACSquant 16) analysis by staining with anti-CD3 and anti-CD56 antibodies. CB-NK cells should be washed twice with Maxcyte electroporation buffer (EB buffer) 300xg for 10mins and 5 million cells can be taken in 75pl of EB buffer. sgRNA for CISH can be dissolved in nuclease-free TE buffer to get a stock of 200pM. 1200pmol of sgRNA can be mixed with 160 pmol Cas9 at a 7.5: 1 ratio. The volume can be adjusted with EB buffer to 25pl and incubated at room temperature for 10mins for formation of RNP complex.

[0186] 25|jl RNP complex can be mixed with 75pl CB-NK cells and transferred to the OC- 100X2 processing assembly. The cells can then be electroporated using the NK5 protocol and left 15mins to rest at room temperature.

[0187] Electroporated cells can then be transferred to the Grex 6 well plate in NK-MACS medium with 10% AB and 20ng / mL IL-15 and incubated at 37°C in a 5% CO2 incubator for 13 days. The cells should be analysed for CISH knockout every 3-4 days. Intracellular expression of CISH can be analysed using Inside Stain Kit and an anti-CISH antibody.

[0188] Example 3 - Design Protocol for Knockout of TGFBRII in NK Cells

[0189] NK cells can be prepared as follows, having TGFBRII function removed. gRNA constructs can be designed and prepared to target the TGFBRII gene in NK cells. CRISPR / Cas9 genome editing may then be used to knock out TGFBRII expression. A total of 3 gRNA candidates may be selected for the TGFBRII gene and their cleavage efficacies in primary expanded NK cells determined. The cells can be electroporated with the gRNA:Cas9 ribonucleoprotein (RNP) complex using Maxcyte® GT and subsequently knockout of TGFBRII can be analysed by flow cytometry. The cleavage activity of the gRNA can also be determined using an in vitro mismatch detection assay. T7E1 endonuclease recognises and cleaves non- perfectly matched DNA, allowing the parental TGFBRII gene to be compared to the mutated gene following CRISPR / Cas9 transfection and non-homologous end joining (NHEJ).

[0190] The gRNA with highest KO efficiency can be selected for further experiments to knockout TGFBRII in NK cells. Knockout of TGFBRII can then be determined by flow cytometry-based assays.

[0191] Example 4 - Knockout of TGFBRII in NK Cells

[0192] Materials

[0193] 1. Enriched cord blood-derived NK cells.

[0194] 2. NK-MACS medium, Premium grade IL-15, anti-CD3 VioBlue, Anti-CD56VB515, Inside Stain Kit, MACSquant 16 (Miltenyi Biosciences).

[0195] 3. Human Serum Albumin (Sigma).

[0196] 4. Gene knockout Kit for TGFBRII and Cas9 recombinant protein.

[0197] 5. Cloudz™ Cell Activation Kits (R&D systems).

[0198] 6. pSMAD2 antibody.

[0199] 7. Electroporation buffer (EB buffer), OC-100X2 processing assembly (PA), MaxCyte ATx electroporation system (Maxcyte).

[0200] 8. Solu-Cortef (Hydrocortisone) (Pfizer).

[0201] 9. Ficoll (Cytiva).

[0202] Protocol

[0203] Cord Blood Mononuclear Cells (CBMCs) can be isolated by the Ficoll method and enriched for 15 days in NK-MACS medium with 10% human serum albumin, CD2 / NKp46 microsphere (Cloudz™), anti-CD3, anti-CD16, IL-15 and hydrocortisone.

[0204] NK cell enrichment of over 90% can be confirmed by FACS (MACSquant 16) analysis by staining with anti-CD3 and anti-CD56 antibodies. CB-NK cells should be washed twice with Maxcyte electroporation buffer (EB buffer) 300xg for 10mins and 5 million cells can be taken in 75pl of EB buffer. sgRNA for TGFBRII can be dissolved in nuclease-free TE buffer to get a stock of 200pM. 1200pmol of sgRNA can be mixed with 160 pmol Cas9 at a 7.5: 1 ratio. The volume can be adjusted with EB buffer to 25pl and incubated at room temperature for 10mins for formation of RNP complex.

[0205] 25pl RNP complex can be mixed with 75pl CB-NK cells and transferred to the OC- 100X2 processing assembly. The cells can then be electroporated using the NK5 protocol and left 15mins to rest at room temperature.

[0206] Electroporated cells can subsequently be transferred to the Grex 6 well plate in NK- MACS medium with 10% AB and 20ng / mL IL-15 and incubated at 37°C in a 5% CO2 incubator for 13 days. The cells should be analysed for TGFBRII knockout every 3-4 days. Successful TGFBRII knockout can be determined by pSMAD2 expression using an anti-pSMAD2 antibody following stimulation with 50ng / ml of TGFB1 for 15 minutes.

[0207] Example 5 - Design Protocol for Knockout of CD38 in NK Cells

[0208] NK cells can be prepared as follows, having CD38 function removed. gRNA constructs can be designed and prepared to target the CD38 gene in NK cells. CRISPR / Cas9 genome editing may then be used to knock out CD38 expression.

[0209] A total of 3 gRNA candidates may be selected for the CD38 gene and their cleavage efficacies in primary expanded NK cells determined. The cells can be electroporated with the gRNA:Cas9 ribonucleoprotein (RNP) complex using Maxcyte® GT and subsequently knockout of CD38 can be analysed by flow cytometry. The cleavage activity of the gRNA can also be determined using an in vitro mismatch detection assay. T7E1 endonuclease recognises and cleaves non-perfectly matched DNA, allowing the parental CD38 gene to be compared to the mutated gene following CRISPR / Cas9 transfection and non-homologous end joining (NHEJ). The gRNA with highest KO efficiency can be selected for further experiments to knockout CD38 in NK cells. Knockout of CD38 can then be determined by flow cytometry-based assays.

[0210] Example 6 - Knockout of CD38 in NK Cells

[0211] Materials

[0212] 1. Enriched cord blood-derived NK cells.

[0213] 2. NK-MACS medium, Premium grade IL-15, anti-CD3 VioBlue, Anti-CD56VB515, Inside Stain Kit, MACSquant 16 (Miltenyi Biosciences).

[0214] 3. Human Serum Albumin (Sigma).

[0215] 4. Gene knockout Kit for CD38 and Cas9 recombinant protein.

[0216] 5. Cloudz™ Cell Activation Kits (R&D systems).

[0217] 6. anti-CD38 antibody.

[0218] 7. Electroporation buffer (EB buffer), OC-100X2 processing assembly (PA), MaxCyte ATx electroporation system (Maxcyte).

[0219] 8. Solu-Cortef (Hydrocortisone) (Pfizer).

[0220] 9. Ficoll (Cytiva).

[0221] Protocol

[0222] Cord Blood Mononuclear Cells (CBMCs) can be isolated by the Ficoll method and enriched for 15 days in NK-MACS medium with 10% human serum albumin, CD2 / NKp46 microsphere (Cloudz™), anti-CD3, anti-CD16, IL-15 and hydrocortisone.

[0223] NK cell enrichment of over 90% can be confirmed by FACS (MACSquant 16) analysis by staining with anti-CD3 and anti-CD56 antibodies. CB-NK cells should be washed twice with Maxcyte electroporation buffer (EB buffer) 300xg for 10mins and 5 million cells can be taken in 75pl of EB buffer. sgRNA for CD38 can be dissolved in nuclease-free TE buffer to get a stock of 200pM. 1200pmol of sgRNA can be mixed with 160 pmol Cas9 at a 7.5: 1 ratio. The volume can be adjusted with EB buffer to 25pl and incubated at room temperature for 10mins for formation of RNP complex. 25yl RNP complex can be mixed with 75pl CB-NK cells and transferred to the OC- 100X2 processing assembly. The cells can then be electroporated using the NK5 protocol and left 15mins to rest at room temperature.

[0224] Electroporated cells can subsequently be transferred to the Grex 6 well plate in NK- MACS medium with 10% AB and 20ng / mL IL-15 and incubated at 37°C in a 5% CO2 incubator for 13 days. The cells should be analysed for CD38 knockout every 3-4 days. Expression of CD38 can be measured using flow cytometry and anti-CD38 antibody.

[0225] Example 7 - CISH KO NK cells with IL-15 knocked in (KI) demonstrated synergy in enhancing NK cell cytotoxicity against cancer cells

[0226] NK cell expansion

[0227] Fresh cord blood was processed using Lymphoprep followed by ACK lysis and CD3 depletion. Derivative CD3 negative CBMCs were seeded in NK MACS medium at 500,000 / ml with 250IU / ml human IL-2, and EBV-LCL feeders at CBMC: feeder ratio 1 :2.5 and cultured in 37C incubator with 5% CO2. IL-2 was spiked in the medium at day 3 of culture. CISH gene was knocked out using LNP on day 5, and the sclL-15 gene (SEQ ID NO: 1) was knocked-in by electroporation on day 7 using Tc Buster Transposon nanoplasmid and Transposase enzyme. Cells were then restimulated with EBV-LCL feeders at 4:1 EBV:CBMC ratio at day 9, along with 200nM methotrexate for selection and 250IU / ml IL-2. Cells were then expanded in NK MACS for total of 19 days after which cells were harvested and used for tumor killing assays.

[0228] CISH knockout by CRISPR / CasQ

[0229] CBMC from day 5 of culture were washed with CTS-Optmizer and resuspended at 10 million cells / ml CTS-Optmizer. CISH gene knockout was done by incubating CBMC cells with 91mer CISH guide encapsulated in a LNP at a concentration of 500,000 cells / ml with 1.25ug / ml CISH LNP and 2.5ug / ml ApoE3 in CTS-OpTmizer containing 250IU / ml human IL-2 and 5ng / ml human IL-15. sclL-15 knock-in using Tc Buster Transposase

[0230] CBMC from day 7 of culture were washed with Opti-MEM medium. 20 million cells were resuspended in 95ul cold Opti-MEM, and 2.5ug of sclL-15 transposon Plasmid and 2.5ug of TcBuster transposase were added to the cells. Total 10Oul reaction were added to OC100x2 cuvette and electroporated using MaxCyte NK-6 program. The cells were then recovered in NK MACS medium with 250IU / ml IL-2 and 5ng / ml IL-15.

[0231] NK cell killing assay using 3D spheroid model

[0232] Cord blood-derived NK cells (CB-NKs) were isolated for use in wildtype form or for use in the presence of a CISH gene knockout and / or sclL-15 gene knock-in.

[0233] The cytotoxicity of wildtype (WT) or CISH KO NK cells alone, or with the addition of sclL-15 knock-in, against ZR-75 GFP breast cancer cells or SKOV-3 GFP ovarian cancer cells was evaluated in the following experimental setup. Human breast cancer or ovarian cancer target cells were plated in a 96 well eSight plate and allowed to adhere for 3 days before the addition of fresh NK cells at 10:1 or 3:1 effector : target (E:T) ratio for 120 hours. The change in tumour growth was estimated by GFP expression using the Incucyte Spheroid module. The experiments were conducted using 1) Cancer target cells alone (ZR-75 GFP or SKOV-3 GFP), 2) WT NK cells alone, 3) WT NK cells + sclL-15 KI, 4) CISH KO NK cells alone and 5) CISH KO NK cells + sclL-15 KI.

[0234] As can be seen from Figure 1 , CISH KO NK cells or NK cells with sclL-15 KI alone exhibited limited cytotoxicity in the ZR-75 human breast cancer model, with a steady increase in cancer cell GFP signal detected in both groups. At both 10:1 and 3:1 E:T ratios, the CISH KO + sclL-15 KI NK cells demonstrate potent cytotoxicity against breast cancer, when compared to CISH KO NK cells or sclL-15 KI NK cells alone.

[0235] As seen from Figure 2, a similar effect was observed in the SKOV-3 human ovarian breast cancer model. No difference in cytotoxic effect was detected between WT NK cells, NK cells with sclL-15 KI, or CISH KO NK cells alone. On the other hand, the CISH KO + sclL-15 KI NK cells demonstrated potent cytotoxicity.

[0236] These data demonstrate that a genetic knockout of CISH works synergistically with a genetic knock-in of sclL-15, resulting in an enhanced cytotoxic phenotype against cancer cells. Example 8 - CISH and TGFBRII Double KO with IL-15 KI NK cells are highly effective in mediating cancer cell killing

[0237] NK cell expansion

[0238] Fresh cord blood was processed using Lymphoprep followed by ACK lysis and CD3 depletion. Derivative CD3 negative CBMCs were seeded in NK MACS medium at 500,000 / ml with 250IU / ml human IL-2, and EBV-LCL feeders at CBMC: feeder ratio 1 :2.5, and cultured in 37C incubator with 5% CO2. IL-2 was spiked in the medium at day 3 of culture. CISH gene was knocked out using LNP on day 5, TGFpRII gene was knocked out using LNP on day 6, and the sclL-15 gene was knocked-in by electroporation on day 7 using Tc Buster Transposon nanoplasmid and Transposase enzyme. Cells were then restimulated with EBV-LCL feeders at 4:1 EBV:CBMC ratio at day 9, along with 200nM methotrexate for selection and 250IU / ml IL-2. Cells were then expanded in NK MACS for total of 19 days which cells were harvested and used for tumor killing assays

[0239] CISH and TGFBRII knockout by CRISPR / Cas9

[0240] CBMC from day 5 of culture were washed with CTS-Optmizer and resuspended at 10 million cells / ml CTS-Optmizer. CISH gene knockout was done by incubating CBMC cells with 91mer CISH guide encapsulated in an LNP at a concentration of 500,000 cells / ml with 1.25ug / ml CISH LNP and 2.5ug / ml ApoE3 in CTS-OpTmizer containing 250IU / ml human IL-2 and 5ng / ml human IL-15.

[0241] On day 6, TGFpRII gene knockout was achieved by incubating CBMC cells with TGFpRII LNP and ApoE3 at concentration of 500,000 cells / ml with 1.25ug / ml TGFBRII LNP and 2.5ug / ml ApoE3 in CTS-OpTmizer containing 250IU / ml human IL- 2 and 5ng / ml human IL-15. sclL-15 knock-in using Tc Buster Transposase system

[0242] CBMC from day 7 of culture were washed with Opti-MEM medium. 20 million cells were resuspended in 95ul cold Opti-MEM, and 2.5ug of sclL-15 transposon transposon Plasmid and 2.5ug of TcBuster transposase were added to the cells. Total 100ul reaction were added to OC100x2 cuvette and electroporated using MaxCyte NK-6 program. The cells were then recovered in NK MACs medium with 250IU / ml IL-2 and 5ng / ml IL-15. NK cell killing assay using 3D spheroid model

[0243] Cord blood-derived NK cells (CB-NK cells) were isolated for use in wildtype form or for use in the presence of a CISH and / or TGFBRII knockout with scll_-15 knock-in.

[0244] The cytotoxicity of wildtype (WT), CISH KO NK cells, TFGBRII KO NK cells, and CISH + TGFBRII double KO (DKO) NK cells with the addition of sclL-15 knock-in against SKOV-3 GFP ovarian cancer target cells was evaluated in the following experimental setup. Human ovarian cancer target cells were plated in a 96 well eSight plate and allowed to adhere for 3 days before the addition of fresh NK cells at 10:1 or 3:1 effector : target (E:T) ratios for 120 hours in the presence of TGFB1. The change in tumour growth was estimated by GFP expression using the Incucyte Spheroid module. The experiments were conducted using 1) Cancer target cells alone (SKOV-3 GFP), 2) WT NK cells with sclL-15 KI, 3) CISH KO NK cells + sclL-15 KI, 4) TGFBRII KO NK cells with sclL-15 KI, and 5) CISH + TGFBRII DKO NK cells with sclL-15 KI.

[0245] As can be seen from Figure 3, NK cells with the CISH + TGFBRII DKO and the scIL- 15 KI were highly effective in mediating SKOV-3 cancer cell killing, in comparison to WT NK with sclL-15 KI, as well as both CISH or TGFBRII KO NK cells with sclL-15 KI at both 10:1 and 3:1 ratios.

[0246] These data demonstrate CISH + TGFBRII DKO CB-NK cells with sclL-15 knocked in result in an enhanced cytotoxic phenotype against cancer cells.

[0247] Example 9 - Insertion of IL-15 gene into CISH gene locus increases NK cell expansion

[0248] NK cell expansion

[0249] Fresh cord blood was processed using Lymphoprep followed by ACK lysis and CD3 depletion. Derivative CD3 negative CBMCs were seeded in NK MACs medium at 500,000 / ml with 250IU / ml human IL-2, and EBV-LCL feeders at CBMC: feeder ratio 1 :2.5, and cultured in 37C incubator with 5% CO2. IL-2 was spiked in the medium at day 3 of culture. CISH gene knockout and sclL-15 knock-in using GenWand DNA was done in one step on day 5. The peptide sequence of sclL-15 corresponded to amino acids 49-162 of the full-length sequence (SEQ ID NO: 2), i.e., having the signal peptide removed. Cells were then restimulated with EBV-LCL feeders at 4:1 was done in one step on day 5. Cells were then restimulated with EBV-LCL feeders at 4:1 EBV:CBMC ratio at day 9, along with 200nM methotrexate for selection and 250IU / ml IL-2. Cells were then expanded in NK MACs for total of 18 days.

[0250] CISH knockout and IL-15 knockin using GenWand

[0251] 10 million CBMC from day 5 of culture were washed with Opti-MEM and resuspended in 95ul Opti-MEM medium. 21.3ug 91mer CISH gRNA, 20ug Cas9, and 10ug sclL-15 GenWand DNA were added to the cells, Total 10Oul reaction were added to 00100x2 cuvette and electroporated using MaxCyte NK-6 program. The cells were then recovered in NK MACS medium with 250IU / ml IL-2 and 5ng / ml IL-15.

[0252] NK cell IL-15 expression assay

[0253] Cord blood-derived NK cells were isolated for use in wildtype form or for use in the presence of a CISH gene knockout with or without sclL-15 knock-in.

[0254] The expression of sclL-15 in wildtype (WT) NK cells, CISH KO NK cells alone, or CISH KO NK cells with the addition of sclL-15 knock-in was evaluated in the following experimental setup. The supernatant was collected on Day 18 of NK cell expansion from the donor cord blood samples S-CB87 and S-CB88. The sclL-15 expression was measured using the ELISA MAX DELUXE Human IL-15 set and the concentration was calculated based on the standard curve provided by the kit. The experiments were conducted using 1) WT control NK cells, 2) CISH KO NK cells, 3) WT NK cells with extra-genomic sclL-15, 4) CISH KO NK cells with sclL-15 genomic insertion into the CISH locus.

[0255] As can be seen from Figure 5, NK cells with CISH KO alone failed to increase IL-15 levels when compared to the WT NK cells. In addition, WT NK cells with extra- genomic sclL-15 only mildly increased levels of IL-15 when compared with the WT NK cell. The CISH KO NK cells with sclL-15 insertion into the CISH locus greatly increased IL-15 levels, with both examples expressing more than 200 pg / ml of IL-15.

[0256] Interestingly, although CISH is a negative regulator of IL-15, NK cells with CISH KO alone did not show increased IL-15 levels compared to WT NK cells. These data also show that extra-genomic expression of sclL-15 only has mild effects on IL-15 levels, while the genetic insertion of sclL-15 into the CISH locus results in significantly enhanced IL-15 levels. Taken together, these data demonstrate that CISH KO alone is not sufficient to increase IL-15 levels significantly and the importance of having sclL-15 genomically inserted into the CISH locus.

[0257] Example 11 - Generation of a construct with an atenuated / weak promoter and anti-CD38 CAR transgene in the first position for targeted knock-in of IL-15 and anti-CD38 CAR transgenes into the CISH locus.

[0258] Generation of 85A “cis” transfer plasmid (SEQ ID NO: 3)

[0259] As indicated in FIG. 6, the 85A “cis” transfer plasmid was generated and was designed for achieving targeted sclL-15 and anti-CD38 CAR knock-in. Simultaneously, knock out of CISH was achieved as direct targeted insertion of an anti-CD38 CAR and a sclL-15 transgene into the double strand break (DSB) site in the endogenous CISH gene via homologous recombination. The DSB in CISH was accomplished using CRISPR / Cas9 gene editing by electroporating a ribonucleoprotein (RNP) consisting of a sgRNA targeting exon 4 of CISH and a GMP v1.2 Cas9 protein (GenScript), followed by addition of an rAAV6 vector containing the 85A construct to serve as a homology repair template.

[0260] The 85A template is 4288bp from 5’ AAV ITR to 3’ AAV ITR. The construct contains an MND core promoter, a kozac motif, sclL-15 transgene, a furin-glycine-serine- glycihi1 11"iker, - isgene plus CD3 and CD28 domains and a bGH poly(A) signal. The gene encoding the anti-CD38 CAR (driven by the weak MND promoter) is in the first position and the gene encoding the slL-15 protein (wild type sequence) is in the second position, separated by the furin-glycine-serine- glycii iker.

[0261] Example 12 - Generation of a construct with an atenuated / weak promoter and IL-15 transgene in the first position for targeted knock-in of IL-15 and anti- CD38 CAR transgene into the CISH locus.

[0262] Generation of 86A “cis” transfer plasmid (SEQ ID NO: 4)

[0263] Alternatively, as indicated in FIG. 7, the 86A “cis” transfer plasmid was generated. Similarly to the 85A “cis” transfer plasmid, the 86A “cis” transfer plasmid was designed for achieving targeted sclL-15 and anti-CD38 CAR knock-in, while simultaneously knocking out CISH expression by directing targeted insertion of an anti-CD38 CAR and a sclL-15 transgene into the DSB site in the endogenous CISH gene via homologous recombination. The DSB in CISH was accomplished using CRISPR / Cas9 gene editing by electroporating an RNP consisting of a sgRNA targeting exon 4 of CISH and a GMP-grade Cas9 protein, followed by addition of an rAAV6 vector containing the 86A construct to serve as a homology repair template.

[0264] The 86A template is 4288bp from 5’ AAV ITR to 3’ AAV ITR. The construct contains an MND core promoter, a kozac motif, sclL-15 transgene, a furin-glycine-serine- glycihv I 'v h iker, am; GT sgene plus CD3 and CD28 domains and a bGH poly(A) signal. The sclL-15 gene (driven by the weak MND promoter) is in the first position and the gene encoding the anti-CD38 CAR is in the second position, separated by the furin-glycine-serine-glyc1- > ' linker.

[0265] Example 13 - Generation of CISH / CD38 DKO NK cells with IL-15 and anti- CD38 CAR knocked in.

[0266] Cord blood-derived NK cells were isolated for use in wildtype form or for use in the presence of CISH / CD38 DKO with sclL-15 and anti-CD38 CAR knock-in.

[0267] Frozen cord blood was thawed and processed using Ficoll DGCF 1 / 0, followed by CD3 depletion on Day 0. Derivative CD3 negative CBMCs were seeded in stromal cell growth medium (SCGM) with 10% Human AB serum at 500,000 / ml with 250IU / ml human IL-2, and K562-mblL-21-4-1 BBL feeder cells at 1 :1 cord blood mononuclear cell (CBMC): feeder ratio and cultured in 37°C incubator with 5% CO2. CBMC from day 3 of culture were washed with CTS-Optmizer and resuspended in CTS-Optmizer at 10 million cells / ml. CD38 gene knockout was done by incubating CBMC cells with CD38 guide encapsulated in a LNP at a concentration of 500,000 cells / ml with 0.625ug / ml CD38 LNP and 2.5ug / ml ApoE3 in CTS-OpTmizer containing 250IU / ml human IL-2 and 5ng / ml human IL-15. CISH gene knockout, sclL-15 and anti-CD38 CAR knock-in via electroporation using 85A or 86A “cis” transfer plasmid was performed as described in Examples 11 and 12, respectively. This was performed in one step on day 5 at 25,000, 50,000, and 100,000 multiplicity of infection (MOI) concentrations. IL-2 (250IU / ml) and IL-15 (5ng / ml) was added during post-editing recovery at both day 3 and 5. The cells were then restimulated with K562-m b IL-21-4- 1 BBL feeders at 1 :1 feeder:CBMC ratio at day 7. The cells were subsequently expanded until day 18. Example 14 - CD38 / CISH DKO NK cells with IL-15 and anti-CD38 CAR knocked in demonstrated enhanced expansion.

[0268] To examine the growth kinetics of the NK cells generated according to Examples 11- 13, the number of NK cells was measured using the following set up at day 0, day 7, day 11 , day 14 and day 18 time points.

[0269] The growth kinetics were evaluated for the following groups: 1) negative unmodified WT; 2) DKO NK cells transfected with 85A plasmid at an MOI of 25,000; 3) DKO NK cells transfected with 85A plasmid at an MOI of 50,000; 4) DKO NK cells transfected with 85A plasmid at an MOI of 100,000; 5) DKO NK cells transfected with 86A plasmid at an MOI of 25,000; 6) DKO NK cells transfected with 86A plasmid at an MOI of 50,000; 7) DKO NK cells transfected with 86A plasmid at an MOI of 100,000; and 8) positive control DKO NK cells transfected with Signagen anti-CD38 CAR plasmid at an MOI of 25,000. Both unmodified WT NK cells and DKO Signagen anti- CD38 CAR NK cells do not have sclL-15 knocked in.

[0270] As seen in FIG. 8, anti-CD38 CAR expressing DKO NK cell groups with sclL-15 knocked in were shown to have higher numbers of NK cells than both the unmodified WT group and the positive control DKO Signagen anti-CD38 CAR NK cell group by day 18.

[0271] Advantageously, methotrexate which is toxic to cells, was no longer required; neither the 85A nor the 86A construct include the selection marker DHFR. As such, the cells were exposed to less toxicity and the process of obtaining >2 billion cells decreased from 28 days to 14 days. Additionally, both 85A and 86A NK cell groups transfected at an MOI of 25,000 were shown to have the highest number of NK cells at day 18 in both donor samples CB136 and CB137, indicating a lower MOI may be advantageous for cell viability.

[0272] Example 15 - Anti-CD38 CAR expression detected in DKO NK cells with IL-15 knocked in.

[0273] To examine the CD38 KO and anti-CD38 CAR knock in efficiency of the NK cells generated according to Examples 11-13, the percentage of NK cells expressing endogenous CD38 and anti-CD38 CAR was measured on day 7 using flow cytometry.

[0274] The CD38 and anti-CD38 CAR expression of NK cells was evaluated in the following setup: 1) negative unmodified WT; 2) DKO NK cells transfected with 85A plasmid at an MOI of 25,000; 3) DKO NK cells transfected with 85A plasmid at an MOI of 50,000; 4) DKO NK cells transfected with 85A plasmid at an MOI of 100,000; 5) DKO NK cells transfected with 86A plasmid at an MOI of 25,000; 6) DKO NK cells transfected with 86A plasmid at an MOI of 50,000; 7) DKO NK cells transfected with 86A plasmid at an MOI of 100,000; and 8) positive control DKO NK cells transfected with Signagen anti-CD38 CAR plasmid at an MOI of 25,000. Neither unmodified WT NK cells nor DKO Signagen anti-CD38 CAR NK cells had sclL-15 knocked in.

[0275] As shown in FIG. 9, little to no endogenous CD38 expression was detected in all NK cell groups other than the unmodified WT group, as expected, indicating the endogenous expression of CD38 was successfully knocked out.

[0276] Furthermore, anti-CD38 CAR expression was detected by day 7 in all groups except for the unmodified WT group.

[0277] Similar levels of anti-CD38 CAR expression were detected across different MOI concentrations in groups transfected with either the 85A or 86A constructs, respectively.

[0278] Surprisingly, despite having the anti-CD38 CAR transgene in the second position, a higher percentage of NK cells expressing anti-CD38 CAR was detected in groups transfected with the 86A construct.

[0279] Example 16 - Significantly higher surface expression of anti-CD38 CAR was observed when the IL-15 transgene was in the first position and the anti-CD38 CAR transgene was in the second position.

[0280] To examine the level of anti-CD38 CAR expression on NK cells generated according to Examples 11-13, the mean fluorescence intensity (MFI) of NK cells expressing the anti-CD38 CAR was measured on day 7 using flow cytometry. J

[0281] Example 17 transgenes.

[0282] 1 86A plasmid at an MOI of 50,000; 7) DKO NK cells transfected with 86A plasmid at an MOI of 100,000; and 8) positive control DKO NK cells transfected with Signagen anti-CD38 CAR plasmid at an MOI of 25,000. Neither unmodified WT NK cells nor DKO Signagen anti-CD38 CAR NK cells had sclL-15 knocked in.

[0283] As shown in FIG. 11 , NK cells transfected with the 86A “cis” transfer plasmid at all three MOI concentrations in both CB136 and CD137 samples maintained a high level of anti-CD38 CAR expression at day 18, measuring at approximately 40,000-50,000 MFI. As such, it was shown that stable integration of genomic anti-CD38 CAR and sclL-15 transgenes was achieved and maintained following an extended period of time.

[0284] Example 18 - Lower levels of IL-15 were detected in NK cells transfected with a construct using the MND promoter.

[0285] The expression of IL-15 in NK cells generated according to Examples 11-13 was evaluated in the following experimental setup.

[0286] On day 18, the NK cells were washed twice and 5 x 106of cells were reseeded per well in a GREX24-well plate supplemented with IL-2. The supernatant was collected after at 24 and 48 hours. The IL-15 expression was measured at 24 and 48-hour time points using the ELISA MAX DELUXE Human IL-15 set and the concentration was calculated based on the standard curve provided by the kit.

[0287] The experiments were conducted using 1) negative unmodified WT; 2) DKO NK cells transfected with 85A plasmid at an MOI of 25,000; 3) DKO NK cells transfected with 85A plasmid at an MOI of 50,000; 4) DKO NK cells transfected with 85A plasmid at an MOI of 100,000; 5) DKO NK cells transfected with 86A plasmid at an MOI of 25,000; 6) DKO NK cells transfected with 86A plasmid at an MOI of 50,000; 7) DKO NK cells transfected with 86A plasmid at an MOI of 100,000; and 8) positive control DKO NK cells transfected with Signagen anti-CD38 CAR plasmid at an MOI of 25,000. Neither unmodified WT NK cells nor DKO Signagen anti-CD38 CAR NK cells had sclL-15 knocked in. As shown in FIG. 12, all NK cell groups transfected with the sclL-15 gene expressed increased levels of IL-15 in comparison to the unmodified WT group and Signagen group.

[0288] Overall, lower levels of IL-15 were detected in NK cells transfected with the 86A “cis” transfer plasmid, wherein the sclL-15 transgene was placed in the first position and driven by the MND promoter.

[0289] Surprisingly, taken together with the results outlined in FIG. 11 , NK cells transfected with the 86A “cis” transfer plasmid advantageously had higher levels of anti-CD38 CAR expression, yet exhibited desirable, non-toxic levels of IL-15 expression.

[0290] Example 19 - Effective killing of cancer cells using NK cells transfected with constructs having the anti-CD38 CAR transgene in the first position.

[0291] NK cells were generated according to Examples 11 and 13 and DKO NK cells with 85A “cis” transfer construct inserted were used. The cytotoxicity of these DKO NK cells with sclL-15 and anti-CD38 CAR KI was evaluated against cancer cells in the following experimental setup.

[0292] The unmodified WT NK cell group; the positive control DKO NK cells with anti- CD38CAR knock-in only group; and the DKO NK cells with anti-CD38 CAR / sclL-15 knock-in group were separately co-cultured with human-derived H929 myeloma cancer target cells to examine NK cell cytotoxicity.

[0293] 2 x 104red fluorescent protein (RFP) expressing H929 cancer target cells were plated in a poly-L-ornithine coated 96 well plate for 30 minutes before the addition of day 18 NK cells at 1 :1 or 3:1 effector : target (E:T) ratios and co-cultured for 24 hours. The change in tumour growth was estimated by RFP expression using the Incucyte basic analyser module. Tumour growths were normalised to time point t=0 for each condition and displayed as percentage change in graphs.

[0294] The experiments were conducted using 1) negative unmodified WT; 2) DKO NK cells transfected with 85A plasmid at an MOI of 25,000; 3) DKO NK cells transfected with 85A plasmid at an MOI of 50,000; 4) DKO NK cells transfected with 85A plasmid at an MOI of 100,000; 5) positive control DKO NK cells transfected with Signagen anti- CD38 CAR plasmid at an MOI of 25,000 and 6) H929 tumour cells only. Neither unmodified WT NK cells nor DKO Signagen anti-CD38 CAR NK had scll_-15 knocked in.

[0295] As shown in FIG. 13 and 14, in both CB136 and CD137 samples, DKO NK cells with anti-CD38 CAR in addition of sclL-15 knock-in were highly effective in mediating H929 cancer cell killing at all three MOI concentrations in comparison to unmodified WT NK cells and positive control DKO NK cells with anti-CD38 CAR knocked in at both 1 :1 and 3:1 ratios.

[0296] These data demonstrate that NK cells transfected with 85A constructs having the anti-CD38 CAR transgene in the first position, and driven by a weak promoter, resulted in an enhanced cytotoxic phenotype against cancer cells.

[0297] Surprisingly, enhanced cytotoxic activity against cancer cells was still achieved at the lowest MOI, indicating a more cost-effective approach can be taken with less viral particles required, as well as achieving improved cell viability as shown in Example 14.

[0298] Example 20 - Efficient killing and complete ablation of cancer cells using NK cells transfected with constructs having the IL-15 transgene in the first position.

[0299] NK cells were generated according to Examples 12 and 13 and DKO NK cells with 86A “cis” transfer constructed inserted were used. The cytotoxicity of these DKO NK cells with sclL-15 and anti-CD38 CAR KI was evaluated against cancer cells in the following experimental setup.

[0300] The unmodified WT NK cell group; the positive control DKO NK cells with anti- CD38CAR knock-in only group; and the DKO NK cells with anti-CD38 CAR / sclL-15 knock-in group were separately co-cultured with human-derived H929 myeloma cancer target cells to examine NK cell cytotoxicity.

[0301] 2 x 104RFP expressing H929 cancer target cells were plated in a poly-L-ornithine coated 96 well plate for 30 minutes before the addition of day 18 NK cells at 1 :1 or 3:1 effector : target (E:T) ratios for 24 hours. The change in tumour growth was estimated by RFP expression using the Incucyte basic analyser module. Tumor growths were normalised to time point t=0 for each condition and displayed as percentage change in graphs.

[0302] The experiments were conducted using 1) negative unmodified WT; 2) DKO NK cells transfected with 86A plasmid at an MOI of 25,000; 3) DKO NK cells transfected with 86A plasmid at an MOI of 50,000; 4) DKO NK cells transfected with 86A plasmid at an MOI of 100,000; 5) positive control DKO NK cells transfected with Signagen anti- CD38 CAR plasmid at an MOI of 25,000 and 6) H929 tumour cells only. Neither unmodified WT NK cells nor DKO Signagen anti-CD38 CAR NK cells had sclL-15 knocked in.

[0303] As shown in FIG. 15 and 16, in both CB136 and CD137 samples, DKO NK cells with anti-CD38 CAR in addition of sclL-15 knock-in were highly effective in mediating cancer cell killing at all three MOI concentration, achieving 100% killing within 12-15 hours at 3:1 E:T ratio and near 100% killing within 24 hours at 1 :1 E:T ratio.

[0304] Surprisingly, near complete elimination of cancer cells was still achieved at the lowest MOI, indicating a more cost-effective approach can be taken with less viral particles required, while also achieving improved cell viability as shown in Example 14.

[0305] Advantageously, these data demonstrate that NK cells transfected with constructs having a weak promoter driving the sclL-15 transgene expression in the first position, and upstream of the anti-CD38 CAR transgene, resulted in complete ablation of cancer cells.

[0306] Example 21 - Simultaneous knock out of CISH expression achieved by targeted knock in of IL-15 / anti-CD38 CAR construct into the CISH gene locos.

[0307] The CISH expression of NK cells generated according to Examples 11-13 was evaluated in the following experimental setup.

[0308] 3 x 106NK cells generated according to Examples 11-13 were collected for gDNA extraction. The CISH editing locus was amplified and then detected with a WT and INDEL probe. The editing efficiency was calculated as: 100%*(1 - (INDEL copy number / WT copy number)). The input genomic DNA is 40ng. Fluorescence threshold of 1300 CISH-INDEL was applied foi and 5000 CISH-INDEL was applied for FCB137. Fluorescence threshold of 2000 was applied for CISH-WT.

[0309] The data demonstrated that effective CISH knockout was achieved by targeted insertion of the 85A or 86A “cis” transfer plasmid into exon 4 of the CISH locus (see Fig. 17).

[0310] The invention thus provides CISH, CD38 and / or TGFBRII KO immune effector cells with IL-15 and / or CAR transgenes driven by an attenuated / weak promoter knocked in, as well as uses of this cell in cancer therapy.

[0311] Sequence Listing

[0312] SEQ ID NO: 1 (DNA sequence encoding full-length sclL-15 peptide)

[0313] ATGCGAATCTCAAAGCCCCACCTGAGAAGCATCTCCATCCAGTGCTACCTGTGC

[0314] CTGCTGCTGAATAGCCACTTTCTGACAGAGGCCGGCATCCATGTGTTCATCCTG

[0315] GGATGTTTTAGCGCCGGACTGCCTAAGACCGAGGCCAACTGGGTGAACGTGAT

[0316] TAGTGATCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCTAC

[0317] ACTGTACACCGAATCTGATGTGCACCCTTCTTGTAAAGTGACAGCCATGAAGTG

[0318] CTTCCTGCTCGAGCTGCAAGTGATCAGCCTGGAAAGCGGCGACGCCTCTATCC

[0319] ACGACACCGTCGAAAACCTGATCATCCTGGCCAACAACAGCCTGTCTAGCAACG

[0320] GCAATGTGACTGAATCCGGATGCAAGGAGTGCGAGGAACTGGAAGAGAAAAAT

[0321] ATCAAGGAATTCCTTCAAAGCTTCGTGCATATCGTGCAGATGTTCATCAACACCA GC

[0322] SEQ ID NO: 2 (Amino acid sequence of full-length sclL-15 peptide)

[0323] MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKI

[0324] EDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAN NSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0325] SEQ ID NO: 3 (DNA sequence of transfer “cis” plasmid construct “85A”) tctggggcataggggggtctctggagatgggggaagcatgggtcctccactcactgctcatccactgccttctagacct cgtcctttgctggctgtggagcggactgggcagcggcccctgtgggccccgtccctggaactgcccaagccagtcatg cagcccttgcctgctggggccttcctcgaggaggtggcagagggtaccccagcccagacagagagtgagccaaag gtgctggacccagaggaggatctgctgtgcatagccaagaccttctcctaccttcgggaatctggtgagtctgagggg ggaggcaggccttttcctgagtagtctggtgggagaagcttagttctgcctttgagtctgatttgggaggcatagcccggc ccaggagagtctgatgggagaggcacagccctctctagggaagcgtgaagctggaggcactgccttgacttgcact ggattggtaccccagctaatttccactatgtcccttggccccctctgcacttgcctaggctggtattggggttccattacgg ccagcgaggcccgacaacacctgcagaagatgccagaaggcacgttcttagtacgtgacagcacgcaccccagct acctgttcacgctgtcagtgaaaaccactcgtggccccaccaatgtacgcattgagtatgccgactccagcttccgtctg gactccaactgcttgtccaggccacgcatcctggcctttccggatgtggtcagccttgtgcagcactatgtggcctcctgc actgctgatacAcgaagcgaacagagagacagcagaatatgggccaaacaggatatctgtggtaagcagttcctg ccccggctcagggccaagaacagttggaacagcagaatatgggccaaacaggatatctgtggtaagcagttcctgc cccggctcagggccaagaacagatggtccccagatgcggtcccgccctcagcagtttctagagaaccatcagatgtt tccagggtgccccaaggacctgaaatgaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgc gcttctgctccccgagctctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgtt ttgacctccatagaagacaccgactctagaggatccaccggtgccaccAT GGCCCTGCCTGT GACCGC CCTGCTGCTGCCCCTGGCTCTGCTGCTGCACGCCGCTCGGCCCGACATCCAGA TGACCCAGAGCCCTAGCAGCCTGTCAGCCAGCGTGGGCGATCGGGTGACAATC ACCTGTAGAGCCAGCCAAGGCATCAGCAACTACCTGGCCTGGTTCCAACAGAA GCCCGGcAAGGCCCCAAAGAGCCTGATCTACGCCGCCAGCTCCCTGCAATCTG GCGTGCCCAGCAGATTCAGCGGCAGCGGCAGCGGAACCGATTTCACCCTGACA ATCAGCAGCCTGCAGCCAGAGGACTTCGCCACATACTACTGCCAGCAATACAAC AGCTACCCCTACACCTTCGGCCAGGGAACCAAGCTGGAGATCAAGGGAGGCG GAGGTTCCGGCGGCGGCGGCAGCGGCGGCGGCGGAAGCCAGGTGCAGCTGG TCCAGAGCGGAGCCGAGGTGAAGAAGCCAGGCTCTAGCGTGAAGGTGAGCTG TAAAGCGTTCGGCGGCACATTTTCTTCTTATGCCATTAGCTGGGTGCGGCAGGC CCCCGGCCAGGGCCTGGAATGGATGGGCAGAATCATCAGATTCCTGGGCATCG CCAACTACGCCCAAAAGTTCCAAGGCAGAGTGACGCTGATCGCCGAcAAGTCCA CAAACACCGCCTACATGGAACTGAGCAGCCTGAGATCTGAAGATACCGCCGTG TACTATTGTGCCGGAGAGCCCGGCGAGCGGGACCCCGACGCTGTTGATATCTG GGGCCAGGGCACCATGGTGACAGTGTCTTCTGCTGCTGCTATCGAGGTGATGT ACCCCCCTCCTTACCTGGACAATGAGAAGAGCAATGGCACTATCATCCACGTGA AGGGCAAACACCTGTGCCCCAGCCCCCTGTTCCCTGGCCCTTCCAAGCCTTTC TGGGTGCTGGTTGTGGTGGGCGGCGTGCTCGCCTGCTACAGCCTGCTGGTGA

[0326] CCGTGGCCTTTATCATCTTCTGGGTGCGGAGCAAGCGTTCCAGACTGTTGCACA GCGATTACATGAACATGACCCCTAGAAGGCCTGGCCCTACCAGAAAGCACTAC

[0327] CAGCCTTATGCCCCTCCTAGAGATTTTGCCGCCTATAGATCCAGAGTGAAATTTT CTAGAAGCGCCGATGCCCCTGCCTACCAGCAGGGACAGAATCAACTGTATAAC GAGCTGAACCTGGGCAGACGGGAAGAGTATGACGTGCTGGACAAGAGAAGAG GACGGGATCCGGAGATGGGCGGGAAACCTCGGAGGAAGAACCCCCAGGAAGG GCTGTATAACGAGCTGCAGAAAGACAAGATGGCCGAGGCCTATTCCGAAATCG GAATGAAGGGCGAACGGCGGAGAGGaAAGGGCCACGACGGCCTCTACCAGGG

[0328] ACTGAGCACAGCCACAAAAGACACCTACGACGCCCTGCACATGCAGGCTCTGC CCCCAAGAAGAAGAAAGAGAGGATCCGGAGAAGGCAGAGGTTCTTTACTGACA

[0329] TGCGGCGACGTGGAAGAGAACCCCGGACCTatgcgaatctcaaagccccacctgagaagcat ctccatccagtgctacctgtgcctgctgctgaatagccactttctgacagaggccggcatccatgtgttcatcctgggatg ttttagcgccggactgcctaagaccgaggccaactgggtgaacgtgattagtgatctgaagaagatcgaggacctgat ccagagcatgcacatcgacgctacactgtacaccgaatctgatgtgcacccttcttgtaaagtgacagccatgaagtg ctcctgctcgagctgcaagtgatcagcctggaaagcggcgacgcctctatccacgacaccgtcgaaaacctgatcat cctggccaacaacagcctgtctagcaacggcaatgtgactgaatccggatgcaaggagtgcgaggaactggaaga gaaaaatatcaaggaattccttcaaagcttcgtgcatatcgtgcagatgttcatcaacaccagcTGAacaagtaacg cggccgcctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactc ccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggg gcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgggacagc ccAgatcctgctcccaccccggccctgcctatgcctaaggaggatgcgcctagtgacccagcactgcctgctcctcca ccagccactgctgtacacctaaaactggtgcagccctttgtacgcagaagcagtgcccgcagcctgcaacacctgtg ccgccttgtcatcaaccgtctggtggccgacgtggactgcctgccactgccccggcgcatggccgactacctccgaca gtaccccttccagctctgactgtacggggcaatctgcccaccctcacccagtcgcaccctggaggggacatcagccc cagctggacttgggcccccactgtccctcctccaggcatcctggtgcctgcatacctctggcagctggcccaggaaga gccagcaagagcaaggcatgggagaggggaggtgtcacacaacttggaggtaaatgcccccaggccgcatgtgg cttcattatactgagccatgtgtcagaggatggggagacaggcaggaccttgtctcacctgtgggctgggcccagacc tccactcgcttgcctgccctggccacctgaactgtatgggcactctcagccctggtttttcaatccccagggtcgggtagg acccctactggcagccagcctctgtttctgggaggatgacatgcagaggaactgagatcgacagtgactagtgaccc cttgtgaggggtaagccaggctaggggactgcacaattatacactattatttatttattctccttggggttggtgtcaggg gcgagccaacccc

[0330] SEQ ID NO: 4 (DNA sequence of transfer “cis” plasmid construct “86A”) tctggggcataggggggtctctggagatgggggaagcatgggtcctccactcactgctcatccactgccttctagacct cgtcctttgctggctgtggagcggactgggcagcggcccctgtgggccccgtccctggaactgcccaagccagtcatg cagcccttgcctgctggggccttcctcgaggaggtggcagagggtaccccagcccagacagagagtgagccaaag gtgctggacccagaggaggatctgctgtgcatagccaagaccttctcctaccttcgggaatctggtgagtctgagggg ggaggcaggccttttcctgagtagtctggtgggagaagcttagttctgcctttgagtctgatttgggaggcatagcccggc ccaggagagtctgatgggagaggcacagccctctctagggaagcgtgaagctggaggcactgccttgacttgcact ggattggtaccccagctaatttccactatgtcccttggccccctctgcacttgcctaggctggtattggggttccattacgg ccagcgaggcccgacaacacctgcagaagatgccagaaggcacgttcttagtacgtgacagcacgcaccccagct acctgttcacgctgtcagtgaaaaccactcgtggccccaccaatgtacgcattgagtatgccgactccagcttccgtctg gactccaactgcttgtccaggccacgcatcctggcctttccggatgtggtcagccttgtgcagcactatgtggcctcctgc actgctgatacAcgaagcgaacagagagacagcagaatatgggccaaacaggatatctgtggtaagcagttcctg ccccggctcagggccaagaacagttggaacagcagaatatgggccaaacaggatatctgtggtaagcagttcctgc cccggctcagggccaagaacagatggtccccagatgcggtcccgccctcagcagtttctagagaaccatcagatgtt tccagggtgccccaaggacctgaaatgaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgc gcttctgctccccgagctctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgtt ttgacctccatagaagacaccgactctagaggatccaccggtgccaccatgcgaatctcaaagccccacctgagaa gcatctccatccagtgctacctgtgcctgctgctgaatagccactttctgacagaggccggcatccatgtgttcatcctgg gatgttttagcgccggactgcctaagaccgaggccaactgggtgaacgtgattagtgatctgaagaagatcgaggac ctgatccagagcatgcacatcgacgctacactgtacaccgaatctgatgtgcacccttcttgtaaagtgacagccatga agtgcttcctgctcgagctgcaagtgatcagcctggaaagcggcgacgcctctatccacgacaccgtcgaaaacctg atcatcctggccaacaacagcctgtctagcaacggcaatgtgactgaatccggatgcaaggagtgcgaggaactgg aagagaaaaatatcaaggaattccttcaaagcttcgtgcatatcgtgcagatgttcatcaacaccagcAGAAGAA

[0331] AGAGAGGATCCGGAGAAGGCAGAGGTTCTTTACTGACATGCGGCGACGTGGAA

[0332] GAGAACCCCGGACCTATGGCCCTGCCTGTGACCGCCCTGCTGCTGCCCCTGG

[0333] CTCTGCTGCTGCACGCCGCTCGGCCCGACATCCAGATGACCCAGAGCCCTAGC

[0334] AGCCTGTCAGCCAGCGTGGGCGATCGGGTGACAATCACCTGTAGAGCCAGCCA

[0335] AGGCATCAGCAACTACCTGGCCTGGTTCCAACAGAAGCCCGGcAAGGCCCCAA

[0336] AGAGCCTGATCTACGCCGCCAGCTCCCTGCAATCTGGCGTGCCCAGCAGATTC

[0337] AGCGGCAGCGGCAGCGGAACCGATTTCACCCTGACAATCAGCAGCCTGCAGCC

[0338] AGAGGACTTCGCCACATACTACTGCCAGCAATACAACAGCTACCCCTACACCTT

[0339] CGGCCAGGGAACCAAGCTGGAGATCAAGGGAGGCGGAGGTTCCGGCGGCGG

[0340] CGGCAGCGGCGGCGGCGGAAGCCAGGTGCAGCTGGTCCAGAGCGGAGCCGA

[0341] GGTGAAGAAGCCAGGCTCTAGCGTGAAGGTGAGCTGTAAAGCGTTCGGCGGCA

[0342] CATTTTCTTCTTATGCCATTAGCTGGGTGCGGCAGGCCCCCGGCCAGGGCCTG

[0343] GAATGGATGGGCAGAATCATCAGATTCCTGGGCATCGCCAACTACGCCCAAAA

[0344] GTTCCAAGGCAGAGTGACGCTGATCGCCGAcAAGTCCACAAACACCGCCTACAT

[0345] GGAACTGAGCAGCCTGAGATCTGAAGATACCGCCGTGTACTATTGTGCCGGAG

[0346] AGCCCGGCGAGCGGGACCCCGACGCTGTTGATATCTGGGGCCAGGGCACCAT

[0347] GGTGACAGTGTCTTCTGCTGCTGCTATCGAGGTGATGTACCCCCCTCCTTACCT

[0348] GGACAATGAGAAGAGCAATGGCA( ACGTGAAGGGCAAACACCTGT

[0349] GCCCCAGCCCCCTGTTCCCTGGCCCTTCCAAGCCTTTCTGGGTGCTGGTTGTG

[0350] GTGGGCGGCGTGCTCGCCTGCTACAGCCTGCTGGTGACCGTGGCCTTTATCAT

[0351] CTTCTGGGTGCGGAGCAAGCGTTCCAGACTGTTGCACAGCGATTACATGAACAT

[0352] GACCCCTAGAAGGCCTGGCCCTACCAGAAAGCACTACCAGCCTTATGCCCCTC

[0353] CTAGAGATTTTGCCGCCTATAGATCCAGAGTGAAATTTTCTAGAAGCGCCGATG

[0354] CCCCTGCCTACCAGCAGGGACAGAATCAACTGTATAACGAGCTGAACCTGGGC AGACGGGAAGAGTATGACGTGCTGGACAAGAGAAGAGGACGGGATCCGGAGA TGGGCGGGAAACCTCGGAGGAAGAACCCCCAGGAAGGGCTGTATAACGAGCT

[0355] GCAGAAAGACAAGATGGCCGAGGCCTATTCCGAAATCGGAATGAAGGGCGAAC GGCGGAGAGGaAAGGGCCACGACGGCCTCTACCAGGGACTGAGCACAGCCAC

[0356] AAAAGACACCTACGACGCCCTGCACATGCAGGCTCTGCCCCCAAGATGAtgtacaa gtgcggccgcctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgcctccttgaccctggaaggtgcca ctcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtg gggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgggaca gcccAgatcctgctcccaccccggccctgcctatgcctaaggaggatgcgcctagtgacccagcactgcctgctcctc caccagccactgctgtacacctaaaactggtgcagccctttgtacgcagaagcagtgcccgcagcctgcaacacctg tgccgccttgtcatcaaccgtctggtggccgacgtggactgcctgccactgccccggcgcatggccgactacctccga cagtaccccttccagctctgactgtacggggcaatctgcccaccctcacccagtcgcaccctggaggggacatcagc cccagctggacttgggcccccactgtccctcctccaggcatcctggtgcctgcatacctctggcagctggcccaggaa gagccagcaagagcaaggcatgggagaggggaggtgtcacacaacttggaggtaaatgcccccaggccgcatgt ggcttcattatactgagccatgtgtcagaggatggggagacaggcaggaccttgtctcacctgtgggctgggcccaga cctccactcgcttgcctgccctggccacctgaactgtatgggcactctcagccctggtttttcaatccccagggtcgggta ggacccctactggcagccagcctctgtttctgggaggatgacatgcagaggaactgagatcgacagtgactagtgac cccttgttgaggggtaagccaggctaggggactgcacaattatacactatttatttatttattctccttggggttggtgtcag gggcgagccaacccc

[0357] SEQ ID NO: 5 (Amino acid sequence of a single CDR sequence from exemplary CD38 CAR variable heavy chain SEQ ID NO: 11)

[0358] FGGTFSSYAI

[0359] SEQ ID NO: 6 (Amino acid sequence of a single CDR sequence from exemplary CD38 CAR variable heavy chain SEQ ID NO: 11)

[0360] IIRFLGIANY

[0361] SEQ ID NO: 7 (Amino acid sequence of a single CDR sequence from exemplary CD38 CAR variable heavy chain SEQ ID NO: 11)

[0362] CAGEPGERDPDAVDIW

[0363] SEQ ID NO: 8 (Amino acid sequence of a single CDR sequence from exemplary

[0364] CD38 CAR variable heavy chain SEQ ID NO: 12)

[0365] SGYSFSNYWI SEQ ID NO: 9 (Amino acid sequence of a single CDR sequence from exemplary

[0366] CD38 CAR variable heavy chain SEQ ID NO: 12)

[0367] IIYPHDSDARY

[0368] SEQ ID NO: 10 (Amino acid sequence of a single CDR sequence from exemplary

[0369] CD38 CAR variable heavy chain SEQ ID NO: 12)

[0370] CARHVGWGSRYWYFDL

[0371] SEQ ID NO: 11 (Amino acid sequence of a heavy chain variable region sequence of an exemplary CD38 CAR)

[0372] QVQLVQSGAEVKKPGSSVKVSCKAFGGTFSSYAISVWRQAPGQGLEWMGRIIRFL

[0373] GIANYAQKFQGRVTLIADKSTNTAYMELSSLRSEDTAVYYCAGEPGERDPDAVDIW

[0374] GQGTMVTVSS

[0375] SEQ ID NO: 12 (Amino acid sequence of a heavy chain variable region sequence of an exemplary CD38 CAR)

[0376] EVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWIGWVRQMPGKGLEWMGIIYPHD

[0377] SDARYSPSFQGQVTFSADKSISTAYLQWSSLKASDTAMYYCARHVGWGSRYWYF

[0378] DLWGRGTLVTVSS

[0379] SEQ ID NO: 13 (Amino acid sequence of a single CDR sequence from exemplary

[0380] CD38 CAR variable light chain SEQ ID NO: 19)

[0381] QSIGSSL

[0382] SEQ ID NO: 14 (Amino acid sequence of a single CDR sequence from exemplary

[0383] CD38 CAR variable light chain SEQ ID NO: 19)

[0384] LIKYASQSF

[0385] SEQ ID NO: 15 (Amino acid sequence of a single CDR sequence from exemplary

[0386] CD38 CAR variable light chain SEQ ID NO: 19)

[0387] HQSSSLPYTF

[0388] SEQ ID NO: 16 (Amino acid sequence of a single CDR sequence from exemplary

[0389] CD38 CAR variable light chain SEQ ID NO: 20) SEQ ID NO: 17 (Amino acid sequence of a single CDR sequence from exemplary CD38 CAR variable light chain SEQ ID NO: 20)

[0390] LIYAASSLQ

[0391] SEQ ID NO: 18 (Amino acid sequence of a single CDR sequence from exemplary CD38 CAR variable light chain SEQ ID NO: 20)

[0392] QQYNSYPYTF

[0393] SEQ ID NO: 19 (Amino acid sequence of a light chain variable region sequence of an exemplary CD38 CAR)

[0394] EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQKPDQSPKLLIKYASQSFSG

[0395] VPSRFSGSGSGTDFTLTINSLEAEDAATYYCHQSSSLPYTFGQGTKLEIK

[0396] SEQ ID NO: 20 (Amino acid sequence of a light chain variable region sequence of an exemplary CD38 CAR)

[0397] DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSLIYAASSLQSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYT FGQGTKLEIK

[0398] SEQ ID NOs: 21-35 (5’ - 3’ end DNA sequences of PCR primers used for Gibson assembly to assemble the variable heavy and light chain DNA fragments into the CAR construct)

[0399] SEQ ID NO: 21

[0400] CTGCTGCTGCATGCGGCGCGCCCGGACATCCAGATGACCCAGAGC

[0401] SEQ ID NO: 22

[0402] CTGCTGCTGCATGCGGCGCGCCCGGACATCCAGCTGACCCAGAGC

[0403] SEQ ID NO: 23

[0404] CTGCTGCTGCATGCGGCGCGCCCGGCCATCCAGCTGACCCAGAGC

[0405] SEQ ID NO: 24

[0406] CTGCTGCTGCATGCGGCGCGCCCGGTGATCTGGATGACCCAGAGC

[0407] SEQ ID NO: 25

[0408] CTGCTGCTGCATGCGGCGCGCCCGGAGATCGTGCTGACCCAGAGC -

[0409] SEQ ID NO: 26

[0410] CTGCTGCTGCATGCGGCGCGCCCGGAGATCGTGATGACCCAGAGC

[0411] SEQ ID NO: 27

[0412] CTGCTGCTGCATGCGGCGCGCCCGGACATCGTGATGACCCAGAGC

[0413] SEQ ID NO: 28

[0414] GCGGCGGAGGATCTGGGGGAGGCGGCTCTCAGGTGCAGCTGGTGCAGAGCG

[0415] SEQ ID NO: 29

[0416] GCGGCGGAGGATCTGGGGGAGGCGGCTCTGAAGTGCAGCTGGTGCAGTCTGG

[0417] SEQ ID NO: 30

[0418] CAGATCCTCCGCCGCCAGATCCGCCTCCGCCCTTGATCTCCACCTT GGTGCC

[0419] SEQ ID NO: 31

[0420] CAGATCCTCCGCCGCCAGATCCGCCTCCGCCCTTGATTTCCAGCTT GGTGCC

[0421] SEQ ID NO: 32

[0422] CAGATCCTCCGCCGCCAGATCCGCCTCCGCCCTTGATGTCCACCTT GGTGCC

[0423] SEQ ID NO: 33

[0424] CAGATCCTCCGCCGCCAGATCCGCCTCCGCCCTTGATTTCCAGCCGGGTGCC

[0425] SEQ ID NO: 34

[0426] GGGGCGGGTGTAGGCGGCCGCGGAGCTGGTGTTGTTGTGCTGGACACGGTGA

[0427] CCATTGTG

[0428] SEQ ID NO: 35

[0429] GGGGCGGGTGTAGGCGGCCGCGGAGCTGGTGTTGTTGTGCTAGACACGGTCA

[0430] CGAGGGTG

[0431] SEQ ID NO: 36 (Amino acid sequence of a CD3zeta domain of an exemplary CD38

[0432] CAR) MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSAD

[0433] APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNEL

[0434] QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0435] SEQ ID NO: 37 (Amino acid sequence of a 41 BB co-stimulatory domain of an exemplary CD38 CAR)

[0436] MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSA

[0437] GGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQ ELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDWCGPSPADL

[0438] SPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSWKRGRKKLLYIF KQPFMRPVQTTQEED GCSCRFPEEEEGGCEL

[0439] SEQ ID NO: 38 (Amino acid sequence of a CD28 co-stimulatory domain of an exemplary CD38 CAR)

[0440] MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHK GLDSAVEVCWYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFC

[0441] KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLL VTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

Claims

Claims1. A human immune effector cell that has been modified to have reduced expression of CISH and to express IL-15, wherein the IL-15 expression is driven by an attenuated / weak promoter.

2. A human immune effector cell according to according to claim 1 , wherein the human immune effector cell is an NK cell.

3. A human immune effector cell according to any preceding claim, wherein the attenuated / weak promoter results in at least 25% less induction of IL-15 expression in comparison to an EF-1a promoter.

4. A human immune effector cell according to claim 3, wherein the attenuated / weak promoter results in at least 50% less induction of IL-15 expression in comparison to an EF-1a promoter.

5. A human immune effector cell according to any preceding claim, wherein the attenuated / weak promoter is selected from MND, UBC and PGK.

6. A human immune effector cell according to any preceding claim, wherein the attenuated / weak promoter is MND.

7. A human immune effector cell according to any preceding claim, wherein the attenuated / weak promoter is MND.

8. A human immune effector cell according to any preceding claim, wherein CISH expression is reduced by at least 50% compared to the same human immune effector cell without the modification.

9. A human immune effector cell according to any preceding claim, wherein CISH expression is knocked out.

10. A human immune effector cell according to any preceding claim, wherein the IL-15 is expressed at the CISH locus, e.g., within exon 4, thereby knocking out expression of CISH.11 A human immune effector cell according to any preceding claim, further expressing a chimeric antigen receptor (CAR) that binds e.g., CD38, HER2, VEGF, EpCAM, EGFR, BCMA, PD-L1 , CD138, CD229, MUC-1 , CLL-1 , SLAMF7, CD96, CD19, and / or CD47.

12. A human immune effector cell according to according to any preceding claim, wherein the human immune effector cell is further modified to have reduced expression of CD38, e.g., wherein CD38 expression is knocked out.

13. A human immune effector cell according to any preceding claim, comprising a bicistronic or polycistronic sequence expressing the genes encoding IL-15 and a second transgene, e.g. for the CAR of claim 10.

14. A human immune effector cell according to claim 13, wherein in the bicistronic or polycistronic sequence, the gene encoding IL-15 is upstream of the gene encoding the second transgene, e.g. the CAR of claim 10.

15. A human NK cell or NK cell line according to any preceding claim, wherein the cell is modified to have: a) CISH expression knocked out; and b) a gene encoding IL-15 knocked in, wherein expression of the IL-15 is driven by a promoter that results in at least 50% less induction of IL-15 expression compared to when IL-15 expression is driven by an EF-1a promoter.

16. A human NK cell or NK cell line according to any preceding claim, wherein the cell is modified to have: a) CISH and CD38 expression knocked out; and b) genes encoding IL-15 and a CD38 CAR knocked in to the CISH locus; wherein the IL-15 and CD38 CAR are driven by an attenuated / weak MND promoter; and wherein the IL-15 and CD38 CAR are expressed from a bicistronic vector, with the IL-15 coding sequence upstream of that for the CD38 CAR.

17. A human NK cell or NK cell line according to claim 16, wherein the genes encoding IL-15 and CD38 CAR are knocked in to exon 4 of the CISH gene.

18. A human NK cell or NK cell line, comprising a bicistronic vector that expresses IL-15 and a CAR, wherein the IL-15 transgene is positioned upstream of the CAR transgene.

19. A human immune effector cell according to any preceding claim, wherein the IL-15 is a soluble, secreted form of IL-15 (sclL-15)20. A human immune effector cell according to any preceding claim, for use in treating cancer, e.g., a blood cancer or a solid cancer.

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