WO2021209625A1 - High potency natural killer cells - Google Patents

Abstract

Modified NK cells and NK cell lines with a more cytotoxic phenotype and a reduced susceptibility to being evaded by cancer cells, methods of making the modified cells and cell lines, compositions comprising the modified cells and cell lines, as well as uses of said cells, cell lines and compositions in the treatment of cancer.

Description

HIGH POTENCY NATURAL KILLER CELLS

Introduction

The present invention relates to the modification of natural killer (NK) cells and NK cell lines to produce derivatives thereof with a more cytotoxic phenotype. Furthermore, the present invention relates to methods of producing modified NK cells and NK cell lines, compositions containing the cells and cell lines and uses of said compositions in the treatment of cancer.

Background to the Invention

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.

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

NK cell cytotoxicity refers to the ability of NK 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 NK cell.

NK cells are known to kill cancer cells by expressing TRAIL on their surface. TRAIL ligand is able to bind TRAIL receptors on cancer cells and induce apoptosis of said cancer cells. One speculative approach describes overexpressing TRAIL on NK cells, in order to take advantage of this anti-cancer mechanism (EP1621550). Furthermore, IL-12 has been reported to upregulate TRAIL expression on NK cells (Smyth et al. 2001).

Nevertheless, cancer cells have developed evasive and protective mechanisms for dealing with NK cells expressing TRAIL. Decoy TRAIL receptors are often expressed on cancer cell membranes, and binding of TRAIL to these decoy receptors is unable to induce apoptosis.

Chimeric antigen receptors (CARs) have recently been developed to target cytotoxic T cells to particular cell types and tissues. Most CARs possess an antigen recognition domain derived from an antibody, and a transmembrane and intracellular portion derived from an immune signaling protein that is involved in T cell signal transduction, thus allowing activation of a T cell’s cytotoxic function upon binding to a target antigen expressed on a target cell population.

CARs are recombinant antigen receptors that introduce a certain antigen specificity to an immune effector cell, e.g. a T cell or an NK cell. The CAR comprises a defined polypeptide sequence expressed from an exogenous polynucleotide that has been introduced into the immune effector cell. CARs comprise a leader sequence, a targeting domain, a transmembrane domain, and one or more intracellular signaling domains. The targeting domain is typically derived from an antibody molecule and comprises one or more complementarity determining regions (CDRs) from the antibody molecule that confer antigen specificity on the CAR. The targeting domain of the CAR may therefore be a single chain variable fragment (scFv) of an antibody. An scFv comprises the variable chain portion of an immunoglobulin light chain and an immunoglobulin heavy chain molecule separated by a flexible linker polypeptide. The flexible polypeptide linker allows the heavy and light chains to associate with one another and reconstitute an immunoglobulin antigen binding domain.

Adoptive cellular immunotherapy for use in cancer treatment commonly involves administration of natural and modified T cells to a patient. T cells can be modified in various ways, e.g. genetically, so as to express receptors and/or ligands that bind specifically to certain target cancer cells. Transfection of T cells with high-affinity T cell receptors (TCRs) and chimeric antigen receptors (CARs), specific for cancer cell antigens, can give rise to highly reactive cancer-specific T cell responses. A major limitation of this immunotherapeutic approach is that T cells must either be obtained from the patient for autologous ex vivo expansion or MHC-matched T cells must be used to avoid immunological eradication immediately following transfer of the cells to the patient or, in some cases, the onset of graft-vs-host disease (GVHD). Additionally, successfully transferred T cells often survive for prolonged periods of time in the circulation, making it difficult to control persistent side-effects resulting from treatment.

In haplotype transplantation, the graft-versus-leukemia effect is believed to be mediated by NK cells when there is a KIR inhibitory receptor-ligand mismatch, which can lead to improved survival in the treatment of AML (Ruggeri, Capanni et al. 2002; Ruggeri, Mancusi et al. 2005). Furthermore, rapid NK recovery is associated with better outcome and a stronger graft-vs-leukemia (GVL) effect in patients undergoing haplotype T-depleted hematopoietic cell transplantation (HCT) in AML (Savani, Mielke et al. 2007). Other trials have used haploidentical NK cells expanded ex vivo to treat AML in adults (Miller, Soignier et al. 2005) and children (Rubnitz, Inaba et al. 2010).

A considerable amount of research into NK cell cytotoxicity has been performed using mouse models. One example is the finding that perforin and granzyme B mRNA are constitutively transcribed in mouse NK cells, but minimal levels of protein are detected until stimulation or activation of the NK cells (Fehniger et al, 2007). Although this work and other work using mouse NK cells is of interest, it cannot be relied upon as conclusive evidence for NK cell cytotoxicity in humans. In contrast to the above example, human NK cells express high levels of perforin and granzyme B protein prior to stimulation (Leong et al, 2011). The result being that when either mouse or human NK cells are freshly isolated in culture, the mouse NK cells have weak cytolytic activity, whereas the human NK cells exhibit strong cytolytic capabilities.

Mouse and human NK cells also vary greatly in their expression markers, signalling cascades and tissue distribution. For example, CD56 is used as a marker for human NK cells, whereas mouse NK cells do not express this marker at all. Furthermore, a well-established mechanism for regulating NK cell cytotoxicity is via ligand binding NK activation and inhibitory receptors. Two of the most prominent human NK activation receptors are known to be NKp30 and NKp44, neither of which are expressed on mouse NK cells. With regards to NK inhibitory receptors, whilst human NK cells express KIRs that recognise MHC class I and dampen cytotoxic activity, mouse NK cells do not express KIRs at all but, instead, express Ly49s (Trowsdale et al, 2001). All in all, despite mouse NK cells achieving the same function as human NK cells in their natural physiological environment, the mechanisms that fulfil this role vary significantly between species.

In any case, cancer cells have a unique ability to evade immune responses, for example via expressing ligands for inhibitory receptors and by expressing TRAIL decoy receptors. Cancer cells can also show resistance to therapies based on targeted CARs by reducing expression of the target.

Thus, there exists a need for alternative and preferably improved human NK cells and human NK cell lines, e.g. with a more cytotoxic profile and a reduced susceptibility to evasion by cancer cells.

An object of the invention is to provide NK cells and NK cell lines with a more targeted and cytotoxic phenotype. A further object is to provide methods for producing modified NK cells and NK cell lines, compositions containing the cells or cell lines and uses of said compositions in the treatment of cancers. More particular embodiments aim to provide treatments for identified cancers, e.g. blood cancers, such as lymphomas. Specific embodiments aim at combining multiple modifications of NK cells and NK cell lines to further enhance the cytotoxicity of the modified cells and/or reduce the extent to which cancers can evade CAR NK-based therapies.

Summary of the Invention

The invention provides modified NK cells and NK cell lines with a more cytotoxic phenotype and a reduced susceptibility to being evaded by cancer cells, methods of making the modified cells and cell lines, compositions comprising the modified cells and cell lines, as well as uses of said cells, cell lines and compositions for treating cancer.

The invention provides NK cells and NK cell lines modified to express chimeric antigen receptors (CARs) and TRAIL ligands. Further modifications may also be present, providing the NK cells and NK cells with a more cytotoxic phenotype.

The invention further provides methods of treating cancer, e.g. blood cancer, using the modified NK cells and NK cell lines. Diseases particularly treatable according to the invention include cancers, including blood cancers, specifically lymphomas. Tumours and cancers in humans in particular can be treated. References to tumours herein include references to neoplasms.

Details of the Invention

Accordingly, the present invention provides an NK cell that has been modified so as to improve its targeting of cancer cells and increase its cytotoxicity against the targeted cancer cells.

References to NK cells herein include autologous NK cells, umbilical cord-derived NK cells, allogeneic NK cells, iPSC-derived NK cells, and NK cell lines such as KHYG-1 and NK-92. Preferably, the NK cells are human NK cells.

Increased or enhanced cytotoxicity resulting from modification of an NK cell is defined by comparison to the cytotoxicity of a wildtype NK cell not having such modification. A wildtype cell is defined as a cell of the same type as that comprising the modification but not having the modification itself.

Preferably, the NK cell of the invention is modified to express TRAIL ligand (in addition to any TRAIL ligand naturally expressed by the NK cell) and to express a chimeric antigen receptor (CAR).

A CAR comprises a leader sequence, a targeting domain, a transmembrane domain, and one or more intracellular domains. The leader sequence is cleaved in the endoplasmic reticulum and thus does not form part of the mature CAR as expressed on the cell surface. The targeting domain of the CAR is often derived from a single chain variable fragment (scFv) of an antibody. As is well known in the art, a scFv comprises the variable heavy chain (VH) of an antibody linked to the variable light chain (VL) of an antibody. The transmembrane (TM) domain of the CAR functions to anchor the CAR to the cell membrane. The intracellular domain of the CAR optionally works to increase potency through immune cell signal transduction. As such, a common intracellular domain used in the construction of CARs is that from CD3zeta of the T cell receptor. Furthermore, it is known to include more than one intracellular domain in the CAR, in order to provide a highly potent CAR. Co-stimulatory domains (e.g. 4-1 BB and CD28) are excellent choices for this purpose. Suitably, the CAR binds an antigen expressed on one or more cancer cells, e.g. CD38, CD319/SLAMF-7, TNFRSF17/BCMA, SYND1/CD138, CD229, CD47,

Her2/Neu, epidermal growth factor receptor (EGFR), CD123/IL3-RA, CD19, CD20, CD22, Mesothelin, EpCAM, MUC1 , MUC16, Tn antigen, NEU5GC, NeuGcGM3, GD2, CLL-1 , or HERV-K. When referring to the CAR herein, reference to the targeted antigen indicates CAR function. Thus, a “CD19 CAR” is a CAR that binds to CD19 expressed on the surface of a target cell, usually a cancer cell.

Preferably, the CAR binds CD19.

The NK cell of the invention may express wildtype TRAIL ligand, in which case it preferably overexpresses wildtype TRAIL ligand. Preferably, expression of the wildtype TRAIL ligand is increased at least 1.5-fold, more preferably at least 2-fold, more preferably at least 5-fold, more preferably at least 10-fold, compared to expression of wildtype TRAIL on the wildtype NK cell.

The NK cell of the invention may express mutant TRAIL ligand, also referred to as variant TRAIL ligand or just TRAIL variant; in these cases, it is preferred that the NK cell expresses a TRAIL mutant / variant with increased affinity for TRAIL death receptors, e.g. DR4 and/or DR5, compared with the affinity of wildtype TRAIL ligand for TRAIL death receptors. The mutants / variants also preferably have lower affinity (or in effect no affinity) for ‘decoy’ receptors, compared with the binding of wildtype 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.

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%, more preferably at least 25%, more preferably at least 50%, more preferably at least 100%, more preferably at least 500%, compared with that exhibited by wildtype TRAIL.

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.

In certain embodiments, the TRAIL variant comprises at least one substitution selected from the group consisting of G131 R, G131 K, R149I, R149M, R149N, R149K, S159R, G160E, Y189A, Y189Q, R191 K, Q193H, Q193K, Q193S, Q193R,

E195R, N199V, N199R, N199H, T200H, K201 R, K201 H, D203A, K204E, K204D,

K204Y, K212R, Y213W, T214R, S215D, S215E, S215H, S215K, S215D, D218H,

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

D269H, and H270D.

In certain embodiments, the TRAIL variant comprises at least two substitutions selected from the group consisting of G131 R, G131 K, R149I, R149M, R149N, R149K, S159R, G160E, Y189A, Y189Q, R191 K, Q193H, Q193K, Q193S, Q193R,

E195R, N199V, N199R, N199H, T200H, K201 R, K201 H, D203A, K204E, K204D,

K204Y, K212R, Y213W, T214R, S215D, S215E, S215H, S215K, S215D, D218H,

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

D269H, and H270D.

In certain embodiments, the TRAIL variant comprises at least three substitutions selected from the group consisting of G131 R, G131 K, R149I, R149M, R149N, R149K, S159R, G160E, Y189A, Y189Q, R191 K, Q193H, Q193K, Q193S, Q193R,

E195R, N199V, N199R, N199H, T200H, K201 R, K201 H, D203A, K204E, K204D,

K204Y, K212R, Y213W, T214R, S215D, S215E, S215H, S215K, S215D, D218H,

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

D269H, and H270D.

In certain embodiments, amino acid substitution of the TRAIL variant is selected from the group consisting of G131 R, G131 K, R149I, R149M, R149N, R149K, S159R, G160E, Y189A, Y189Q, R191 K, Q193H, Q193K, Q193S, Q193R, E195R, N199V, N199R, N199H, T200H, K201 R, K201 H, D203A, K204E, K204D, K204Y, K212R, Y213W, T214R, S215D, S215E, S215H, S215K, S215D, D218H, D218A, Y240A, K251 D, K251 E, K251Q, T261 L, H264R, I266L, D267Q, D269A, D269H, H270D, T214R / E195R, T214R / D269H, Y189A / Q193S / N199V / K201 R / Y213W / S215D, Y213W / S215D, N199R / K201 H, N199H / K201 R, G131 R / N199R / K201 H, G131 R / N199R / K201 H / R149I / S159R / S215D, G131 R / R149I / S159R / S215D, G131 R / N199R / K201 H / R149I / S159R / S215D, G131 R / D218H, Y189Q / R191 K / Q193R / H264R / I266L / D267Q, T261 L / G160E, T261 L / H270D, T261 L / G160E / H270D, and T261 L / G160E / H270D / T200H (use of 7” indicates multiple amino acid substitutions).

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 / K201 R / Y213W / S215D, Y213W / S215D, D269A and Y240A.

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 G131 R, G131 K, R149I, R149M, R149N, R149K, S159R, Q193H, W193K, N199R, N199R / K201 H, N199H / K201 R, G131 R / N199R / K201 H, G131 R / N199R / K201 H, G131 R / N199R / K201 H / R149I / S159R / S215D, G131 R / R149I / S159R / S215D, G131 R / 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.

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 T261 L, H270D, T200H, T261 L / G160E, T261 L / H270D, T261 L / G160E / H270D, T261 L / G160E / H270D / T200H, D203A and D218A.

In an optional embodiment, treatment of a cancer using modified NK cells expressing TRAIL and/or TRAIL variant is 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 NK cells. It is preferable, however, that the agent is administered prior to administering the modified NK cells. In a preferred embodiment the agent upregulates expression of DR5 and/or DR4 on cancer cells. The agent may optionally be a chemotherapeutic medication, e.g. Bortezomib, and administered in a low dose capable of upregulating TRAIL receptor expression on the cancer. The invention is not limited to any particular agents capable of upregulating TRAIL receptor expression, but examples of agents include SMAC mimetics, Bortezomib, Gefitinib, Piperlongumine, Doxorubicin, Alpha-tocopheryl succinate and HDAC inhibitors.

It is preferred that the NK cell has been genetically modified to express

(i) a CD19 chimeric antigen receptor (CAR); and

(ii) (a) a TRAIL ligand, in addition to any TRAIL ligand naturally expressed by the NK cell or NK cell line, or

(b) a mutant TRAIL ligand, as described elsewhere herein, i.e. wherein the mutant TRAIL ligand has an increased affinity for TRAIL receptors, e.g. DR4 and/or DR5, and/or a reduced affinity for decoy TRAIL receptors, e.g. DcR1 and/or DcR2, compared with the affinity of wildtype TRAIL ligand for the receptors.

According to a preferred embodiment of the invention, the TRAIL ligand / mutant TRAIL ligand is linked to one or more NK cell co-stimulatory domains, e.g. 4-1 BB, CD28, 2B4, DAP-10, DAP-12, CD278 (ICOS) and/or 0X40. Binding of the ligand to its receptor on a target cell thus promotes apoptotic signals within the target cell, as well as stimulating cytotoxic signals in the NK cell.

It is also preferred that the intracellular domain of the CAR comprises one or more co-stimulatory domains, e.g. CD3zeta, 4-1 BB, CD28, 2B4, DAP-10, DAP-12, CD278 (ICOS) and/or 0X40.

The NK cells of the invention may also be modified to have increased resistance to TRAIL-induced cell death. The cells may be less vulnerable to TRAIL-induced cell death or fratricide as a result.

The NK cells may be modified to have reduced function of one or more TRAIL receptors. This is optionally achieved using gene knockout or knockdown (e.g. using siRNA) or restricting the expression of the TRAIL receptor within the cell endoplasmic reticulum. Preferably, DR4 and/or DR5 function is reduced on the NK cells of the invention. It is particularly preferred that the DR4 and/or DR5 genes are knocked out. If multiple copies of the genes are present, it is preferred that all are knocked out.

The NK cells may be modified in a way that both reduces TRAIL-induced death of the cells and provides the cells with a more cytotoxic phenotype. Preferably the same modification can achieve both of these advantages. It is preferred that the NK cells are modified to express a TRAIL receptor linked to a co-stimulatory domain. The cells may express a TRAIL receptor linked to one or more co-stimulatory domains. Preferably, the TRAIL receptor is selected from DR4 and DR5. Preferably, the co stimulatory domain is selected from one or more of 4-1 BB, CD28, 2B4, DAP-10, DAP-12, CD278 (ICOS) and 0X40. More preferably, the co-stimulatory domain is 4- 1 BB linked to CD3zeta.

The resistance of the modified NK cells against TRAIL-induced cell death is preferably increased by at least 5%, more preferably at least 10%, more preferably at least 25%, most preferably at least 50%, relative to wildtype NK cells. Resistance to cell death can be measured in a number of ways known to the skilled person, e.g. by performing a cell viability assay. Preferably, increased resistance to cell death is measured using a flow cytometric propidium iodide cell viability assay. In an example, an NK cell population modified according to the invention to exhibit at least 10% increased resistance to TRAIL-induced cell death would be identified through an assay where soluble TRAIL is incubated with (1) the modified cells and (2) the wildtype cells, and then after staining each cell population with propidium iodide, the modified cell population is found to have a cell viability at least 10% higher than the wildtype population.

It is further preferred that the NK cells have been modified to have reduced expression of the CISH gene. Preferably, the modification is a stable knockout of the CISH gene, e.g. via CRISPR gene editing.

Preferably, CISH function and/or 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 NK cell or NK cell line without the modification. A significant advantage of this modification is in the mechanism by which the NK cells respond to IL-15 signaling. IL-15 is a positive regulator of NK cell proliferation and activation. Reduced CISH function in NK cells makes the cells hypersensitive to IL-15, leading to a further enhanced and prolonged increase in NK cell cytotoxicity. The result is a modified NK cell with potent cytotoxicity.

Optionally, the NK cells are further modified to have reduced or absent checkpoint inhibitory receptor function. Preferably, these receptors are specific checkpoint inhibitory receptors. Preferably still, these checkpoint inhibitory receptors are one or more or all of CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7), SIGLEC9, and/or TIGIT. Reduced or absent checkpoint inhibitory receptor function may be achieved, for example, by knocking down or knocking out one or more checkpoint inhibitory receptor genes.

The NK cells are optionally further modified to express an Fc receptor, in addition to any Fc receptor naturally expressed by the NK cells. Preferably, the Fc receptor is CD16. More preferably, the Fc receptor is a high-affinity variant of CD16, e.g. CD16 having a valine at amino acid position 158.

In an embodiment, the cells are modified by genetic modification. Optionally, this modification occurs before the cell has differentiated into an NK cell. For example, pluripotent stem cells (e.g. iPSCs) can be genetically modified to express CARs and TRAIL ligands before being differentiated into NK cells.

In another embodiment, the invention provides a composition comprising: a CRISPR / Cas9 guide sequence, comprising a guide, a tracr mate and a tracr sequence, adapted to target a locus in an NK cell, and a recombination template comprising a nucleotide sequence encoding a CAR and/or a TRAIL ligand, adapted to insert the nucleotide sequence at the locus.

In use, the composition modifies an NK cell, e.g. in vitro or ex vivo, to generate a genetically modified NK cell of the invention as defined elsewhere herein.

As per the objects of the invention, the modified NK cell, modified NK cell line, or composition thereof is for use in treating cancer in a patient, especially blood cancers and solid cancers. In preferred embodiments, the modified NK cell, NK cell line or composition is for use in treating blood cancers including acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, including T-cell lymphomas and B-cell lymphomas, multiple myeloma, asymptomatic myeloma, smoldering multiple myeloma (SMM), active myeloma or light chain myeloma. Preferably, the cancer is a human cancer.

Preferably, the cancer is lymphoma. More preferably, the lymphoma is B-cell lymphoma. Most preferably, the cancer is diffuse large B-cell lymphoma.

Various routes of administering the modified NK cells to a patient in need thereof will be known to the skilled person. Administration of the modified NK cells can be systemic or localized, e.g. via the intraperitoneally or intratumourally (suitable especially for solid tumours).

Advantageously, an NK cell, preferably treated to reduce its tumourigenicity, for example by rendering it mortal and/or incapable of dividing, can be obtained from a blood cancer cell line and used in methods of the invention to treat cancer.

To render a cancer-derived cell more acceptable for therapeutic use, it is generally treated or pre-treated in some way to reduce or remove its propensity to form tumours in the patient. Specific modified NK cell lines used in examples are safe because they have been rendered incapable of division; they are irradiated and retain their killing ability but die within about 3-4 days. Specific cells and cell lines are hence incapable of proliferation, e.g. as a result of irradiation. Treatments of potential NK cells for use in the methods herein include irradiation to prevent them from dividing and forming a tumour in vivo and genetic modification to reduce tumourigenicity, e.g. to insert a sequence encoding a suicide gene that can be activated to prevent the cells from dividing and forming a tumour in vivo. Suicide genes can be turned on by exogenous, e.g. circulating, agents that then cause cell death in those cells expressing the gene. A further alternative is the use of monoclonal antibodies targeting specific NK cells of the therapy. CD52, for example, is expressed on KHYG-1 cells and binding of monoclonal antibodies to this marker can result in antibody-dependent cell-mediated cytotoxicity (ADCC) and KHYG-1 cell death.

As discussed in an article published by Suck et al, 2006, cancer-derived NK cells and cell lines are easily irradiated using irradiators such as the Gammacell 3000 Elan. A source of Cesium-137 is used to control the dosing of radiation and a dose-response curve between, for example, 1 Gy and 50 Gy can be used to determine the optimal dose for eliminating the proliferative capacity of the cells, whilst maintaining the benefits of increased cytotoxicity. This is achieved by assaying the cells for cytotoxicity after each dose of radiation has been administered.

There are significant benefits of using an irradiated NK cell line for adoptive cellular immunotherapy over the well-established autologous or MHC-matched T cell approach. Firstly, the use of a NK cell line with a highly proliferative nature means expansion of modified NK cell lines can be achieved more easily and on a commercial level. Irradiation of the modified NK cell line can then be carried out prior to administration of the cells to the patient. These irradiated cells, which retain their useful cytotoxicity, have a limited life span and, unlike modified T cells, will not circulate for long periods of time causing persistent side-effects.

Additionally, the use of allogeneic modified NK cells and NK cell lines means that MHC class I expressing cells in the patient are unable to inhibit NK cytotoxic responses in the same way as they can to autologous NK cytotoxic responses. The use of allogeneic NK cells and cell lines for cancer cell killing benefits from the previously mentioned GVL effect and, unlike for T cells, allogeneic NK cells and cell lines do not stimulate the onset of GVHD, making them a much preferred option for the treatment of cancer via adoptive cellular immunotherapy.

Examples

The present invention is now described in more and specific details in relation to the production of NK cells, modified to exhibit increased cytotoxic activity and hence improved ability to cause cancer cell death in humans, wherein specific embodiments are illustrated with reference to the accompanying drawings in which:

Fig. 1 shows a FACS plot of the baseline expression of TRAIL on KHYG-1 cells; Fig. 2 shows a FACS plot of the expression of TRAIL and TRAIL variant after transfection of KHYG-1 cells;

Fig. 3 shows a FACS plot of the expression of CD107a after transfection of KHYG-1 cells;

Fig. 4 shows the effects of transfecting KHYG-1 cells with TRAIL and TRAIL variant on cell viability;

Fig. 5 shows a FACS plot of the baseline expression of DR4, DR5, DcR1 and DcR2 on both KHYG-1 cells and NK-92 cells;

Fig.s 6, 7 and 8 show the effects of expressing TRAIL or TRAIL variant in KHYG-1 cells on apoptosis of three target cell populations: K562, RPMI8226 and MM1.S, respectively;

Fig. 9 shows mitigation of NK cell fratricide by knocking down DR5 expression; Fig. 10 shows expression of both CD19 and DR5 on B-cell lymphoma RIVA cells;

Fig. 11 shows TRAIL expression and CD19 CAR expression in NK cells before and after electroporation;

Fig. 12 shows five independent experiments at five different E:T ratios to demonstrate the enhanced cytotoxic activity of the modified NK cells of the invention against B-cell lymphoma cells;

Fig. 13 shows the p values between each of the samples present in Fig. 12; and

Fig. 14 illustrates the superior killing of NK cells according to the invention compared to NK cells expressing the CD19 CAR only.

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

SEQ ID NO: 1 is an example gRNA for DR5;

SEQ ID NO: 2 is an example gRNA for DR4; and SEQ ID NO: 3 is a second example gRNA for DR4.

Example 1 - Knock-in of CD19 CARs in primary NK cells

The anti-CD19^, anti-CD19-BB^, and anti-CD19-truncated (control) plasmids used have been described previously (Imai et al. 2004. Leukemia. 18(4):676-84). The cDNA encoding the intracellular domains of human DAP10 and 4-1 BB ligand (4- 1 BBL), and interleukin-15 (IL-15) with long signal peptide were sub-cloned by polymerase chain reaction (PCR) with a human spleen cDNA library used as a template. An anti-CD19-DAP10 plasmid was constructed by replacing the sequence encoding Oϋ3z with that encoding DAP10, using the splicing by overlapping extension by PCR (SOE-PCR) method. The cDNA encoding the signal peptide of CD8a, the mature peptide of IL-15 and the transmembrane domain of CD8a were assembled by SOE-PCR to encode a “membrane-bound” form of IL-15. The resulting expression cassettes were sub-cloned into EcoRI and Xhol sites of murine stem-cell virus-internal ribosome entry site-green fluorescent protein (MSCV-IRES-GFP).

The RD114-pseudotyped retrovirus was generated as described previously (Imai et al. 2004. Leukemia. 18(4):676-84). A calcium phosphate DNA precipitation was used to transfect 293T cells with anti-CD19^, anti-CD19-DAP10, anti-CD19-BB^, or anti- CD19-truncated; pEQ-PAM3(-E); and pRDF. Conditioned medium containing retrovirus was harvested at 48 hours and 72 hours after transfection, immediately frozen in dry ice, and stored at -80°C until use.

K562 cells were transduced with the construct encoding the “membrane-bound” form of IL-15. Cells were cloned by limiting dilution, and a single-cell clone with high expression of GFP and surface IL-15 (K562-mb15) was expanded. This clone was subsequently transduced with human 4-1 BBL (K562-mb15-41 BBL). K562 cells expressing wildtype IL-15 (K562-wt15) or 4-1 BBL (K562-41 BBL) were produced by a similar procedure. Peripheral blood mononuclear cells (1.5x10⁶) were incubated in a 24-well tissue-culture plate with or without 10⁶ K562-derivative stimulator cells in the presence of 10 lU/mL human IL-2 in RPMI 1640 and 10% FCS.

Mononuclear cells stimulated with K562-mb15-41 BBL were transduced with retroviruses, as described previously (Imai et al. 2004. Leukemia. 18(4):676-84). Briefly, 14mL polypropylene centrifuge tubes were coated with human fibronectin (100 pg/mL) or RetroNectin (50 pg/mL). Pre-stimulated cells (2x10⁵) were re suspended in the tubes in 2-3 mL virus-conditioned medium with Polybrene (4 pg/mL) and centrifuged at 2400g for 2 hours (centrifugation was omitted when RetroNectin was used). The multiplicity of infection (4-6) was identical in each experiment comparing the activity of different CARs. After centrifugation, cells were left undisturbed for 24 hours in a humidified incubator at 37°C, 5% CO2. The transduction procedure was repeated on 2 successive days. After a second transduction, the cells were re-stimulated with K562-mb15-41 BBL in the presence of 10 lU/mL IL-2. Cells were maintained in RPMI 1640, 10% FCS and 10 lU/mL IL-2. Transduced NK cells were stained with goat anti-mouse (Fab)2 polyclonal antibody conjugated with biotin followed by streptavidin conjugated to peridinin chlorophyll protein. For Western blotting, cells were lysed in RIPA buffer (PBS, 1% Triton-X100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) containing 3 pg/mL pepstatin, 3 pg/mL leupeptin, 1mM phenylmethylsulfonyl fluoride (PMSF), 2 mM ethylenediaminetetraacetic acid (EDTA) and 5 pg/mL aprotinin. Centrifuged lysate supernatants were boiled with an equal volume of loading buffer, with or without 0.1 M dithiothreitol (DTT), and then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a precast 10-20% gradient acrylamide gel. The proteins were transferred to a polyvinylidene fluoride (PVDF) membrane which was incubated with primary mouse anti-human Oϋ3z monoclonal antibody (clone 8D3). Membranes were then washed, incubated with a goat anti-mouse IgG horseradish peroxidase-conjugated second antibody and developed using an enhanced chemiluminescence system.

The following antibodies were used for immunophenotypic characterization of expanded and transduced cells: anti-CD3 conjugated to fluorescein isothiocyanate (FITC), to PerCP or to energy-coupled dye (ECD); anti-CD10 conjugated to phycoerythrin (PE); anti-CD19 PE; anti-CD22 PE; anti-CD56 FITC, PE or allophycocyanin (APC); anti-CD16 CyChrome; and anti-CD25 PE. Surface expression of KIR and NK activation molecules was determined with specific antibodies conjugated to FITC or PE, as described previously (Leung et al. 2004. Journal of Immunology. 172:644-650). Antibody staining was detected with a FACScan or an LSR II flow cytometer.

Example 2 - Knock-in of TRAIL / TRAIL variant in NK cells

KHYG-1 cells were transfected with both TRAIL and TRAIL variant, in order to assess their viability and ability to kill cancer cells following transfection.

The TRAIL variant used is that described in WO 2009/077857. It is encoded by the wildtype TRAIL gene containing the D269H/E195R mutation. This mutation significantly increases the affinity of the TRAIL variant for DR5, whilst reducing the affinity for both decoy receptors (DcR1 and DcR2).

Baseline TRAIL Expression Baseline TRAIL (CD253) expression in KHYG-1 cells was assayed using flow cytometry.

Mouse anti-human CD253-APC (Biolegend catalog number: 308210) and isotype control (Biolegend catalog number: 400122) were used to stain cell samples and were analyzed on a BD FACS Canto II flow cytometer.

KHYG-1 cells were cultured in RPMI 1640 medium containing 10% FBS, 2mM L- glutamine, penicillin (100 U/mL)/streptomycin (100 mg/mL) and IL-2 (10ng/mL). 0.5-

I .0 x 10⁶ cells/test were collected by centrifugation (1500rpm x 5 minutes) and the supernatant was aspirated. The cells (single cell suspension) were washed with 4 mL ice cold FACS Buffer (PBS, 0.5-1% BSA, 0.1% NaN3 sodium azide). The cells were re-suspended in 100 pL ice cold FACS Buffer, add 5uL antibody was added to each tube and incubated for 30 minutes on ice. The cells were washed 3 times by centrifugation at 1500 rpm for 5 minutes. The cells were then re-suspended in 500 pL ice cold FACS Buffer and temporarily kept in the dark on ice.

The cells were subsequently analyzed on the flow cytometer (BD FACS Canto II) and the generated data were processed using FlowJo 7.6.2 software.

As can be seen in Figure 1 , FACS analysis showed weak baseline expression of TRAIL on the KHYG-1 cell surface.

TRAIL / TRAIL variant Knock-in by Electroporation

Wildtype TRAIL mRNA and TRAIL variant (D269H/195R) mRNA was synthesized by TriLink BioTechnologies, aliquoted and stored as -80°C. Mouse anti-human CD253- APC (Biolegend catalog number: 308210) and isotype control (Biolegend catalog number: 400122), and Mouse anti-human CD107a-PE (eBioscience catalog number: 12-1079-42) and isotype control (eBioscience catalog number: 12-4714) antibodies were used to stain cell samples and were analyzed on a BD FACS Canto II flow cytometer. DNA dye SYTOX-Green (Life Technologies catalog number: S7020; 5 mM Solution in DMSO) was used. To achieve transfection efficiencies of up to 90% with high cell viability in KHYG-1 cells with the Nucleofector™ Device (Nucleofector

II, Lonza), a Nucleofector™ Kit T from Lonza was used. Antibiotics-free RPMI 1640 containing 10% FBS, L-glutamine (2mM) and IL-2 (10ng/mL) was used for post- Nucleofection culture. KHYG-1 and NK-92 cells were passaged one or two days before Nucleofection, as the cells must be in the logarithmic growth phase. The Nucleofector solution was pre warmed to room temperature (100 pi per sample), along with an aliquot of culture medium containing serum and supplements at 37°C in a 50 ml_ tube. 6-well plates were prepared by filling with 1.5 ml_ culture medium containing serum and supplements and pre-incubated in a humidified 37°C / 5% CO2 incubator. An aliquot of cell culture was prepared and the cells counted to determine the cell density. The required number of cells was centrifuged at 1500rpm for 5 min, before discarding the supernatant completely. The cell pellet was re-suspended in room temperature Nucleofector Solution to a final concentration of 2x10⁶ cells/100mI (maximum time in suspension = 20 minutes). 100 mI cell suspension was mixed with 10 pg mRNA (volume of RNA < 10 mI_). The sample was transferred into an Amaxa-certified cuvette (making sure the sample covered the bottom of the cuvette and avoiding air bubbles). The appropriate Nucleofector program was selected (i.e. U-001 for KHYG- 1 cells). The cuvettes were then inserted into the cuvette holder. 500 pi pre-warmed culture medium was added to the cuvette and the sample transferred into a prepared 6-well plate immediately after the program had finished, in order to avoid damage to the cells. The cells were incubated in a humidified 37°C / 5% CO2 incubator. Flow cytometric analysis and cytotoxicity assays were performed 12-16 hours after electroporation. Flow cytometry staining was carried out as above.

As can be seen in Figures 2 and 3, expression of TRAIL / TRAIL variant and CD107a (NK activation marker) increased post-transfection, confirming the successful knock- in of the TRAIL genes into KHYG-1 cells.

Figure 4 provides evidence of KHYG-1 cell viability before and after transfection via electroporation. It can be seen that no statistically significant differences in cell viability are observed following transfection of the cells with TRAIL / TRAIL variant, confirming that the expression of wildtype or variant TRAIL is not toxic to the cells. This observation contradicts corresponding findings in NK-92 cells, which suggest the TRAIL variant gene knock-in is toxic to the cells (data not shown). Nevertheless, this is likely explained by the relatively high expression levels of TRAIL receptors DR4 and DR5 on the NK-92 cell surface (see Figure 5).

Effects of TRAIL / TRAIL variant on KHYG-1 Cell Cytotoxicity Mouse anti-human CD2-APC antibody (BD Pharmingen catalog number: 560642) was used. Annexin V-FITC antibody (ImmunoTools catalog number: 31490013) was used. DNA dye SYTOX-Green (Life Technologies catalog number: S7020) was used. A 24-well cell culture plate (SARSTEDT AG catalog number: 83.3922) was used. Myelogenous leukemia cell line K562, multiple myeloma cell line RPMI8226 and MM1.S were used as target cells. K562, RPMI8226, MM1.S were cultured in RPMI 1640 medium containing 10% FBS, 2mM L-glutamine and penicillin (100 U/mL)/streptomycin (100 mg/mL).

As explained above, KHYG-1 cells were transfected with TRAIL / TRAIL variant.

The target cells were washed and pelleted via centrifugation at 1500rpm for 5 minutes. Transfected KHYG-1 cells were diluted to 0.5x10⁶/mL. The target cell density was then adjusted in pre-warmed RPMI 1640 medium, in order to produce effectortarget (E:T) ratios of 1 :1.

0.5 mL KHYG-1 cells and 0.5 mL target cells were then mixed in a 24-well culture plate and placed in a humidified 37°C / 5% CO2 incubator for 12 hours. Flow cytometric analysis was then used to assay KHYG-1 cell cytotoxicity; co-cultured cells (at different time points) were washed and then stained with CD2-APC antibody (5 pL/test), Annexin V-FITC (5 pL/test) and SYTOX-Green (5 pL/test) using Annexin V binding buffer.

Data were further analyzed using FlowJo 7.6.2 software. CD2-positive and CD2- negative gates were set, which represent KHYG-1 cell and target cell populations, respectively. The Annexin V-FITC and SYTOX-Green positive cells in the CD2- negative population were then analyzed for TRAIL-induced apoptosis.

Figures 6, 7 and 8 show the effects of both KHYG-1 cells expressing TRAIL or TRAIL variant on apoptosis for the three target cell lines: K562, RPMI8226 and MM1.S, respectively. It is apparent for all target cell populations that TRAIL expression on KHYG-1 cells increased the level of apoptosis, when compared to normal KHYG-1 cells (not transfected with TRAIL). Moreover, TRAIL variant expression on KHYG-1 cells further increased apoptosis in all target cell lines, when compared to KHYG-1 cells transfected with wildtype TRAIL. Example 3 - Knock-in of CD19 CARs and TRAIL variants in primary NK cells

Anti-CD19-CD28(TM)-CD3C, anti-CD19-41 BB(TM)-003z, and anti-CD19-truncated (control) plasmids were used. The cDNA encoding the CD19 scFv, with transmembrane domains of human Oϋ3z, CD28 or 4-1 BB ligand (4-1 BBL), and with intracellular domains of Oϋ3z were used as a template mRNA. The gene cassette containing the combination of CD19 CAR and TRAIL variant was synthesized as mRNA. CD19 CAR and high affinity TRAIL DR5 variant was delivered to the NK cells as two separate in vitro synthesized mRNAs at the same time.

The TRAIL variant used is that described in WO 2009/077857. It is encoded by the wildtype TRAIL gene containing the D269H/E195R mutation. This mutation significantly increases the affinity of the TRAIL variant for DR5, whilst reducing the affinity for both decoy receptors (DcR1 and DcR2).

Electroporated NK cells were stained with goat anti-mouse (Fab)2 polyclonal antibody conjugated with biotin followed by streptavidin conjugated to PE or FITC flurophore. For Western blotting, cells were lysed in RIPA buffer (PBS, 1% Triton- X100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) containing 3 pg/mL pepstatin, 3 pg/mL leupeptin, 1mM phenylmethylsulfonyl fluoride (PMSF), 2 mM ethylenediaminetetraacetic acid (EDTA) and 5 pg/mL aprotinin. Centrifuged lysate supernatants were boiled with an equal volume of loading buffer, with or without 0.1 M dithiothreitol (DTT), and then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a precast 10-20% gradient acrylamide gel. The proteins were transferred to a polyvinylidene fluoride (PVDF) membrane which was incubated with primary mouse anti-human ΰϋ3z monoclonal antibody (clone 8D3). Membranes were then washed, incubated with a goat anti-mouse IgG horseradish peroxidase-conjugated second antibody and developed using an enhanced chemiluminescence system.

The following antibodies were used for immunophenotypic characterization of expanded and transduced cells: anti-CD3 conjugated to fluorescein isothiocyanate (FITC), to PerCP or to energy-coupled dye (ECD); anti-CD10 conjugated to phycoerythrin (PE); anti-CD19 PE; anti-CD22 PE; anti-CD56 FITC, PE or allophycocyanin (APC); anti-CD16 CyChrome; and anti-CD25 PE. Surface expression of KIR and NK activation molecules was determined with specific antibodies conjugated to FITC or PE, as described previously (Leung et al. 2004. Journal of Immunology. 172:644-650). Antibody staining was detected with a FACS Canto II flow cytometer.

Baseline TRAIL (CD253) expression in naive or expanded NK cells was assayed using flow cytometry.

Mouse anti-human CD253-APC (Biolegend catalog number: 308210) and isotype control (Biolegend catalog number: 400122) were used to stain cell samples and were analyzed on a BD FACS Canto II flow cytometer.

Primary NK cells were cultured in Miltenyi’s NK cell expansion medium containing 10% human AB serum, penicillin (100 U/mL)/streptomycin (100 mg/mL) and IL-2 (500U/ml). 0.5-1.0 x 10⁶ cells/test were collected by centrifugation (1500rpm x 5 minutes) and the supernatant was aspirated. The cells (single cell suspension) were washed with 4 mL ice cold FACS Buffer (PBS, 0.5-1% BSA, 0.1% NaN3 sodium azide). The cells were re-suspended in 100 pL ice cold FACS Buffer and 5pL antibody was added to each tube and incubated for 30 minutes on ice. The cells were washed 3 times by centrifugation at 1500 rpm for 5 minutes. The cells were then re-suspended in 500 pL ice cold FACS Buffer and temporarily kept in the dark on ice.

The cells were subsequently analyzed by flow cytometer (BD FACS Canto II) and the generated data were processed using FlowJo 7.6.2 software.

FACS analysis showed weak baseline expression of TRAIL on the NK cell surface.

TRAIL variant (D269H/195R) mRNA was synthesized by TriLink BioTechnologies, aliquoted and stored as -80°C. Mouse anti-human CD253-APC (Biolegend catalog number: 308210) and isotype control (Biolegend catalog number: 400122), antibodies were used to stain cell samples and were analyzed on a BD FACS Canto II flow cytometer. Propidium Iodide was used for cell viability. In order to achieve transfection efficiencies of up to 90% with high NK cell viability, an electroporation based technique was implemented using Maxcyte GT. Cells were processed in Maxcyte buffer prior to electroporation and cells were electroporated with 5-10pg/ml of each individual mRNA (i.e. 5pg/ml of CD19 CAR and 5pg/ml TRAIL variant) Naive or expanded NK cells were passaged one or two days before electroporation, as the cells must be in the logarithmic growth phase. 6-well plates were prepared by filling with 1.5 ml_ culture medium containing serum and supplements and pre incubated in a humidified 37°C / 5% CO2 incubator. An aliquot of cell culture was prepared and the cells counted to determine the cell density. The required number of cells was centrifuged at 1500rpm for 5 min, before discarding the supernatant completely. The cell pellet was re-suspended in room temperature Maxcyte buffer to a final concentration of 2x10⁶ cells/1 OOmI (maximum time in suspension = 20 minutes). 100 pi cell suspension was mixed with 5 pg mRNA. The samples were transferred into Maxcyte-certified cuvettes OC-100x2 (making sure the samples covered the bottom of the cuvettes and avoiding air bubbles). The appropriate electroporation program was selected (i.e. NK-4). The cuvettes were then inserted into the cuvette holder. Immediately after the program had finished, in order to avoid damage to the cells, the cells were transferred to 6-well plates and incubated for 20 mins at 37°C. Flow cytometric analysis and cytotoxicity assays were performed 20-24 hours after electroporation. Flow cytometry staining was carried out as above.

Expression of CD19 CAR and TRAIL variant was shown to increase post transfection, confirming the successful knock-in of the CD19 CAR and TRAIL variant genes into primary NK cells.

There was evidence of NK cell viability before and after transfection via electroporation. It can be seen that no statistically significant differences in cell viability are observed following transfection of the cells with TRAIL variant, confirming that the expression of variant TRAIL is not toxic to the cells.

The effects of the TRAIL variant on NK cell cytotoxicity were also measured. Mouse anti-human CD2-APC antibody (BD Pharmingen catalog number: 560642) was used. Annexin V-FITC antibody (ImmunoTools catalog number: 31490013) was used. A 96-well cell culture plate (SARSTEDT AG catalog number: 83.3922) was used. B-cell lymphoma cell lines OCI-LY10, RIVA, and SU-DHL6 were cultured in RPMI 1640 medium containing 10% FBS, 2mM L-glutamine and penicillin (100 U/mL)/streptomycin (100 mg/mL).

The target cells were washed and pelleted via centrifugation at 1500rpm for 5 minutes. Transfected NK cells were diluted to achieve a concentration of 2x10⁶/mL cells. The target cell density was then adjusted in pre-warmed RPMI 1640 medium, in order to produce effectortarget (E:T) ratios of 5:1 , 2.5, and 1.25:1.

0.1 ml_ electroporated NK cells and 0.1 mL target cells were then mixed in a 96-well culture plate and placed in a humidified 37°C / 5% CO2 incubator for 16 hours. Flow cytometric analysis was then used to assay NK cell cytotoxicity; co-cultured cells (at different time points) were washed and then stained with CD2-APC antibody (5 pL/test), and cell viability was assessed using propidium iodide.

Data were further analyzed using FlowJo 7.6.2 software. CD2-positive and CD2- negative gates were set, which represented NK cell and target cell populations, respectively. The PI cells in the CD2-negative population were then analyzed for TRAIL-induced apoptosis.

The effects of NK cells expressing TRAIL variant on apoptosis were assessed for the three target cell lines: OCI-LY10, RIVA, and SU-DHL6, respectively. It is apparent for all target cell populations that TRAIL variant expression on NK cells increased the level of apoptosis, when compared to normal NK cells (not transfected with TRAIL variant).

Cells of the invention, expressing both the CD19 CAR and the TRAIL variant, offer a significant advantage in cancer therapy, due to their ability to specifically target cancer cells with high affinity and then kill those cells via the death receptor DR5. When challenged by the cells of the invention, cancer cells are prevented from developing defensive strategies to circumvent death via a single pathway. For example, cancers cannot effectively circumvent TRAIL-induced cell death by upregulating TRAIL decoy receptors, as cells of the invention are modified so that they remain cytotoxic in those circumstances.

Example 4 - Knockout of NK Cell TRAIL Receptors DR4 and DR5

NK cells are prepared as follows, having death receptor 5 (DR5) and/or death receptor 4 (DR4) function removed. gRNA constructs targeting TRAIL-R2 (DR5) and TRAIL-R1 (DR4) are designed (e.g. SEQ ID NO:1 : CCCAUCUUGAACAUACCAG (DR5),

SEQ ID NO:2: AACCGGUGCACAGAGGGUGU (DR4) and SEQ ID NO:3: AUUUACAAGCUGUACAUGGG (DR4)) and prepared to target endogenous genes encoding DR5 and DR4 gene(s) in NK cells. CRISPR/Cas9 genome editing is then used to knock out the DR5 and/or DR4 target genes.

A total of 3 gRNA candidates are selected for the DR5 gene and their cleavage efficacies in RPMI8226 cells determined. A total of 3 gRNA candidates are selected for the DR4 gene and their cleavage efficacies in HL60 cells determined. RPMI8226 cells and HL60 are electroporated with the gRNA:Cas9 ribonucleoprotein (RNP) complex using Maxcyte® GT and subsequently knockout of DR5 and/or is analyzed by flowcytometry. The cleavage activity of the gRNA is also determined using an in vitro mismatch detection assay. T7E1 endonuclease recognizes and cleaves non- perfectly matched DNA, allowing the parental DR5 gene / DR4 gene to be compared to the mutated gene following CRISPR/Cas9 transfection and non-homologous end joining (NHEJ).

The gRNA with highest KO efficiency is selected to further experiments to knockout DR5 / DR4 in primary NK cells, NK cell lines or CD34+ progenitors (for subsequent differentiation and expansion to NK cells). Knockout of DR4 / DR5 is determined by flowcytometry based assays.

Example 5 - Knockdown of TRAIL Receptors DR4 and DR5 in NK Cells siRNA knockdown of DR4 and/or DR5 in NK-92 cells, KHYG-1 cells and primary NK cells was performed by electroporation. siRNA based delivery was performed using the Maxcyte GT system.

The cells were then incubated in a humidified 37°C / 5% CO2 incubator until DR4 and/or DR5 knockdown analysis was performed. Flow cytometry analysis was performed 72 hours after electroporation, and (optionally) just prior to electroporation of TRAIL variant (e.g. E195R/D269H) mRNA in order to measure DR4 and/or DR5 expression levels. This electroporation protocol was found to reliably result in DR4 and DR5 knockdown in KHYG-1 cells, NK-92 cells and primary NK cells.

Example 6 - NK Cell Fratricide Resistance

As illustrated in Figure 9, NK cell fratricide in the following cells was assessed: (1) primary NK cells, otherwise referred to as mock or wildtype NK cells, (2) primary NK cells expressing high affinity membrane-bound TRAIL ligand DR5^e195R;D269H, (3) primary NK cells with a DR5^KD via siRNA and (4) primary NK cells with a DR5^KD via siRNA and also expressing high affinity membrane-bound TRAIL ligand

DP5E195R;D269H

Primary NK cells receiving the DR5 knockdown were electroporated with the DR5 siRNA on day 9 of the expansion, whereas primary NK cells receiving the DR5 TRAIL variant were electroporated with the variant mRNA on day 12 of the expansion.

It was observed that after prolonged expansion of the primary NK cells in IL-2 containing growth media that DR5 expression became upregulated, leading to increased fratricide when the DR5 TRAIL variant was expressed.

The data clearly indicate that knocking down DR5 expression using siRNA protects primary expanded NK cells from fratricide, regardless of whether those NK cells express wildtype TRAIL or the high-affinity DR5 TRAIL variant.

Example 7 - Lymphoma Treatment Protocol

The following protocol was developed for use in treating patients with lymphoma. Nevertheless, it is apparent that the invention is suitable for treating patients with many different CD19-expressing cancers.

Following diagnosis of a patient with a CD19 positive cancer, in this case lymphoma, an aliquot of NK cells is thawed and cultured prior to administration to the patient in an effective dose.

The aliquoted cells are modified to express a CD19 CAR and a high-affinity TRAIL variant; and, also, to knock out expression of the TRAIL receptor that is targeted by the TRAIL variant (this last modification being to protect the effector NK cells from fratricide). These modifications may be performed prior to freezing or, alternatively, a transient transfection can be prepared after thawing using e.g. viral means, electroporation etc. For electroporation, the MaxCyte Flow Electroporation platform offers a suitable solution for achieving fast large-scale transfections in the clinic. ln any case, the modified NK cells are cultured and then administered intravenously to the patient.

Example 8 - Expression of CD19 and DR5 on Target Cells

Materials, Reagents and Instruments

Mouse anti-human DR5 (CD262)-BV421 (catalog number: 744768), mouse anti human CD19-FITC and Zombie NIR Viability Dye (catalog number: 423106) were obtained from BioLegend. The stained cell samples were measured using BD FACS Celesta. Data was analysed using FlowJo™ v10.7.

Protocol

The B-cell lymphoma cell line RIVA was grown in Cytiva HyClone™ Iscove's Modified Dulbecco's Medium (IMDM from FisherScientific, catalog number: 10444102). The medium was supplemented with 0.01% of Pen/Strep (from Gibco™, catalog number: 15140122) and 10% fetal bovine serum (from Cytiva, catalog number: 10309433) in 5% CO2 atmosphere at 37°C.

For consistency, cells were used at passage numbers lower than ten. Cells were stained at the recommended antibody concentrations from the manufacturer. CD19 and DR5 basal expression were analysed by flow cytometry.

Results

As can be seen from Figure 10, RIVA cells were shown to express both CD19 (lower graph) and the TRAIL death receptor DR5 (upper graph).

Example 9 - Synergistic Effect of Therapy Against B-Cell Lymphomas

Materials, Reagents and Instruments

MaxCyte Electroporation Buffer and MaxCyte OC-100X2 cuvettes were obtained from MaxCyte, Inc. Complete NK expansion media (ExCellerate, catalog number: CCM032) supplemented with cytokines were purchased from Bio-Techne. 24-well cell culture plates and flat bottom 96-well cell culture plates were from Sarstedt. Tag- IT Violet (catalog number: 425101), Zombie NIR (catalog number: 423106) and Zombie Violet (catalog number: 423113) were purchased from BioLegend.

Protocol The required number of NK cells was prepared by washing in 10 ml MaxCyte Electroporation Buffer and centrifuged at 300 x g for 7 minutes. Supernatant was discarded and NK cells were then resuspended in MaxCyte Buffer at the density required for electroporation (final volume 100 pi per electroporation). 100 ul of cells was then aliquoted into DNase/RNase free sterile microtubes, mixed with the mRNA (5 pg per 10⁶ cells) and then transferred to a OC-100X2 cuvette. Cells were electroporated using the ExPERT ATx device and operating the NK-4 program. After electroporation, cells were transferred in a 24-well plate as a drop and incubated at 37°C for 20 minutes. Complete NK expansion media supplemented with cytokines was added to the cells to give a density that matches the number of NK cells at the highest E:T ratio in 100 pi. Cells were then left to rest at 37°C for 4 hours before co culture.

RIVA target cells were counted and the required number was transferred into a 50ml tube. After centrifugation at 200 x g for 7 mins, the supernatant was discarded and the cells were washed once with 10 ml PBS and centrifuged again as before. Tag-IT Violet was prepared in a 1 :2000 dilution in PBS. Target cells were then resuspended in staining solution (1 ml per 10⁶ cells). After 10 minutes incubation at 37°C, complete target cell culture media was added to 10ml volume and the cells were centrifuged again. Finally, target cells were resuspended in complete media at the density required for plating (3 x 10⁵/ml). 100mI of the suspension was transferred per well in a flat bottom 96-well cell culture plate following experimental design. Cells were then maintained at 37°C.

NK cells were transferred after 4 hours resting from the 24-well plate into sterile microtubes. The required volume of NK cells was then transferred into each well of the 96-well plate following experimental design (E:T ratios were the following 0.5, 1 , 2.5, 5 and 10). Additional NK cell expansion media plus cytokines was added in order to bring the volume of each up to 200 mI. The co-culture was then incubated at 37°C for 16 hours. The remaining NK cells were used to check the expression of targets and added mRNA. Quality control was performed at 4 hours (start of the co-culture) and 20 hours (end of the co-culture) post-electroporation. CD19-CAR was stained using the CD19-FITC protein (FITC-Labeled Human CD19 (20-291) Protein from ACROBiosystems, catalog number: CD9-HF251-200UG) and TRAILv was stained using APC anti-human CD253 (TRAIL) antibody (catalog number: 308209) from BioLegend. Zombie Violet viability dye (catalog number: 423113) was used to stain dead cells.

The co-cultured cells were collected after 16 hour and then stained using Zombie NIR Viability Dye (0.1 mI in 100 mI PBS) for 15 mins. After, cells were washed again with PBS and then resuspended in FACS Buffer (PBS + 2% FBS). Cells were then measured using FACS Celesta (from BD) using the HTP plate system. TAG-it Vio- negative populations represented the primary NK cells. TAG-it Vio-positive cells were the target cells. Data were further analysed using FlowJo. E:T ratios were re calculated by using the counted number of NK cells normalized to the counted numbers of lymphoma cells. Correction factors were applied to the specific killing percentages.

Results

As can be seen from Figure 11 , TRAIL and CD19 CAR expression was measured after electroporation with (1) TRAILv mRNA, (2) CD19 CAR mRNA and (3) TRAILv mRNA and CD19 CAR mRNA. Successful protein expression was confirmed via this exemplary density blot with TRAIL expression on the y-axis (4hr and 20hr post electroporation) and CD19 CAR expression on the x-axis (4hr and 20hr post electroporation).

Five independent experiments were performed using different donor-derived NK cells at varying E:T ratios. This is illustrated by the individual dots in each bar of Figure 12. Figure 12 thus shows the synergistic effect of using both a DR5 TRAIL variant and a CD19 CAR in NK cells to treat a B-cell lymphoma. This is evident from the significant increase in killing of RIVA cells when the NK cells contain both modifications compared to when the NK cells express just one of the modifications (or the additive combination of both modifications).

To further demonstrate this synergy and rule out a merely additive effect, Figures 13 and 14 are provided. Figure 13 shows the p-values of side-by-side comparisons of the different samples (two-sided t-test applied). Figure 14 shows the side-by-side comparison of NK cells expressing the CD19 CAR vs NK cells expressing the TRAIL variant and CD19 CAR. Dots indicate different biological replicates and lines link the corresponding matched datasets. Example 10 - Further Knockout of CISH in the NK Cells

The NK cells according to the invention (expressing CD19 CAR and TRAILv) are further modified as follows, having CIS function removed. gRNA constructs are designed and prepared to target the endogenous CISH gene in the NK cells. CRISPR/Cas9 genome editing is then used to knock out the target gene.

A total of 3 gRNA candidates are selected for the CISH gene and their cleavage efficacies in primary expanded NK cells determined. The cells are electroporated with the gRNA:Cas9 ribonucleoprotein (RNP) complex using Maxcyte® GT and subsequently knockout of CISH is analysed by flowcytometry. The cleavage activity of the gRNA is also determined using an in vitro mismatch detection assay. T7E1 endonuclease recognizes 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).

The gRNA with highest KO efficiency is selected for further experiments to knockout CISH in the CD19 CAR / TRAILv NK cells. Knockout of CISH is determined by flow cytometry based assays.

The NK cells are optionally also transfected with a gene encoding IL-15.

The resulting cells can then be used in lymphoma therapy, e.g. in accordance with Example 7.

The invention thus provides highly cytotoxic NK cells for use in targeted cancer therapy, specifically lymphoma therapy.

Claims

Claims

1. A natural killer (NK) cell or NK cell line that has been genetically modified to express

(i) a CD19 chimeric antigen receptor (CAR); and

(ii) (a) a TRAIL ligand in addition to any TRAIL ligand naturally expressed by the NK cell or NK cell line, or

(b) a mutant TRAIL ligand, wherein the mutant TRAIL ligand has an increased affinity for TRAIL receptors, e.g. DR4 and/or DR5, and/or a reduced affinity for decoy TRAIL receptors, e.g. DcR1 and/or DcR2, when compared to the affinity of wildtype TRAIL for the receptors.

2. An NK cell or NK cell line according to claim 1 , wherein the mutant TRAIL ligand comprises at least one amino acid substitution at a position selected from 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.

3. An NK cell or NK cell line according to either claim 1 or claim 2, wherein the mutant TRAIL ligand comprises an amino acid substitution selected from the group consisting of G131 R, G131 K, R149I, R149M, R149N, R149K, S159R, G160E, Y189A, Y189Q, R191 K, Q193H, Q193K, Q193S, Q193R, E195R, N199V, N199R, N199H, T200H, K201 R, K201 H, D203A, K204E, K204D, K204Y, K212R, Y213W, T214R, S215D, S215E, S215H, S215K, S215D, D218H, D218A, Y240A, K251 D, K251 E, K251Q, T261 L, H264R, I266L, D267Q, D269A, D269H, H270D, T214R / E195R, T214R / D269H, Y189A / Q193S / N199V / K201 R / Y213W / S215D, Y213W / S215D, N199R / K201 H, N199H / K201 R, G131 R / N199R / K201 H, G131 R / N199R / K201 H / R149I / S159R / S215D, G131 R / R149I / S159R / S215D, G131 R / N199R / K201 H / R149I / S159R / S215D, G131 R / D218H, Y189Q / R191 K/ Q193R / H264R / I266L / D267Q, T261 L / G160E, T261 L / H270D, T261 L / G160E / H270D, and T261 L / G160E / H270D / T200H.

4. An NK cell or NK cell line according to either claim 1 or claim 2, wherein the mutant TRAIL ligand comprises an amino acid substitution selected from the group consisting of D269H, E195R, T214R, D269H / E195R, T214R / E195R, T214R / D269H, N199V, Y189A / Q193S / N199V / K201 R / Y213W / S215D, Y213W / S215D, D269A, and Y240A.

5. An NK cell or NK cell line according to either claim 1 or claim 2, wherein the mutant TRAIL ligand comprises an amino acid substitution selected from the group consisting of G131 R, G131 K, R149I, R149M, R149N, R149K, S159R, Q193H, W193K, N199R, N199R / K201 H, N199H / K201 R, G131 R / N199R / K201 H, G131 R / N199R / K201 H, G131 R / N199R / K201 H / R149I / S159R / S215D, G131 R / R149I / S159R / S215D, G131 R / D218H, K201 R, K201 H, K204E, K204D, K204L, K204Y, K212R, S215E, S215H, S215K, S215D, D218H, K251 D, K251 E, K251Q, and Y189Q / R191 K/ Q193R / H264R / I266L / D267Q.

6. An NK cell or NK cell line according to either claim 1 or claim 2, wherein the mutant TRAIL ligand comprises an amino acid substitution selected from the group consisting of T261 L, H270D, T200H, T261 L / G160E, T261 L / H270D, T261 L / G160E / H270D, T261 L / G160E / H270D / T200H, D203A, and D218A.

7. An NK cell or NK cell line according to any preceding claim, wherein the mutant TRAIL ligand has an affinity for a TRAIL receptor, e.g. DR4 and/or DR5, that is at least 25% greater than that of wildtype TRAIL for the same TRAIL receptor.

8. An NK cell or NK cell line according to any preceding claim, wherein TRAIL ligand expression is increased by at least 1.5-fold, compared to the expression of TRAIL ligand on a wildtype NK cell.

9. An NK cell or NK cell line according to any preceding claim, wherein the NK cell or NK cell line further comprises a modification that reduces or abolishes expression of a checkpoint inhibitory receptor selected from CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7), SIGLEC9, and TIGIT.

10. An NK cell or NK cell line according to any preceding claim, further modified to have reduced expression of TRAIL receptors DR4 and/or DR5, e.g. via genetic knockdown or knockout.

11. An NK cell or NK cell line according to any preceding claim, further modified to express a TRAIL receptor, e.g. DR4 or DR5, that is linked to a co-stimulatory domain.

12. An NK cell or NK cell line according to any preceding claim, wherein the TRAIL ligand is linked to a co-stimulatory domain.

13. An NK cell or NK cell line according to claim 11 or claim 12, wherein the co stimulatory domain is one or more of 4-1 BB, CD28, 2B4, DAP-10, DAP-12, CD278 (ICOS) and 0X40.

14. An NK cell or NK cell line according to any preceding claim, wherein the NK cell or NK cell line further comprises a modification that reduces or abolishes expression of CISH.

15. An NK cell or NK cell line according to claim 14, wherein CISH expression is genetically knocked out.

16. An NK cell or NK cell line according to any preceding claim for use in treating cancer.

17. An NK cell or NK cell line for use according to claim 16, wherein the cancer is a lymphoma.

18. An NK cell or NK cell line for use according to claim 17, wherein the lymphoma is a B-cell lymphoma.

19. A natural killer (NK) cell or NK cell line for use in treating lymphoma in humans, wherein the NK cell or NK cell line has been genetically modified to express

(i) a CD19 chimeric antigen receptor (CAR); and

(ii) a mutant TRAIL ligand having an increased affinity for TRAIL receptors, e.g. DR4 and/or DR5, and/or a reduced affinity for decoy TRAIL receptors, e.g. DcR1 and/or DcR2, when compared to the affinity of wildtype TRAIL for the receptors.

20. An NK cell or NK cell line for use according to claim 19, wherein the lymphoma is a B-cell lymphoma.

21. An NK cell or NK cell line for use according to claim 19 or claim 20, wherein the mutant TRAIL ligand has increased affinity for DR5, when compared to the affinity of wildtype TRAIL for DR5.