MODIFIED NATURAL KILLER CELLS AND NATURAL KILLER CELL LINES TARGETTING TUMOUR CELLS

Abstract

NK cells and NK cell lines are modified to increase their selectivity for cancer cells by providing an ability to bind tumour associated MUC-1 antigen. Production of such modified NK cells and NK cell lines is via genetic modification to produce NK-CARs that are optionally further modified to have increased cytotoxicity against cancer cells.

Claims

1-17. (canceled)

18. A natural killer (NK) cell or NK cell line modified to express a chimeric antigen receptor (CAR) that binds a tumour-associated Mucin-1 (MUC-1) glycoform.

19. The NK cell or NK cell line of claim 18, wherein the CAR binds the tumour-associated MUC-1 glycoform with increased affinity relative to wildtype MUC-1 glycoforms.

20. The NK cell or NK cell line of claim 19, wherein the increased affinity is at least 10%.

21. The NK cell or NK cell line of claim 19, wherein the increased affinity is at least 50%.

22. The NK cell or NK cell line of claim 18, wherein the CAR comprises a short-chain variable fragment (scFv) derived from 5E5, SM3 or HMFG2.

23. The NK cell or NK cell line of claim 18, wherein the tumour-associated MUC-1 glycoform comprises a preponderance of shorter glycans relative to wildtype glycoforms, and wherein the shorter glycans are selected from the group consisting of Tn, sialyl Tn (STn), T, sialyl T (ST) glycans, and combinations thereof.

24. The NK cell or cell line of claim 18, wherein the NK cell or cell line has reduced propensity to form tumours relative to wildtype NK cells or wildtype NK cell lines.

25. The NK cell or NK cell line of claim 24, wherein the NK cell or cell line is rendered incapable of division.

26. The NK cell or cell line of claim 18, wherein the NK cell or cell line is modified to reduce expression of cancer-related MUC-1 glycoforms.

27. The NK cell or NK cell line of claim 18, wherein the NK cell or cell line is further modified to express a mutant TRAIL ligand with at least 10% higher affinity for one or more TRAIL death receptors relative to wildtype TRAIL.

28. The NK cell or NK cell line of claim 27, wherein the mutant TRAIL ligand comprises a D269H/E195R mutation.

29. The NK cell or NK cell line of claim 18, wherein the NK cell or cell line is further modified to reduce function of one or more checkpoint inhibitory receptors.

30. The NK cell or NK cell line of claim 29, wherein the one or more checkpoint inhibitory receptors are selected from the group consisting of CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7), SIGLEC9, TIGIT, TIM-3, and combinations thereof.

31. The NK cell or NK cell line of claim 18, wherein the NK cell line is a derivative of a KHYG-1 cell line.

32. A method of treating a cancer in a patient, the method comprising administering to the patient a NK cell or NK cell line modified to express a CAR that binds a tumour-associated MUC-1 glycoform with at least 10% increased affinity relative to wildtype MUC-1 glycoforms.

33. The method of claim 32, wherein the cancer is a solid cancer.

34. The method of claim 33, wherein the solid cancer is selected from the group consisting of breast cancer, ovarian cancer and colorectal cancer.

35. The method of claim 32, wherein the cancer is a blood cancer.

36. The method of claim 35, wherein the blood cancer is selected from the group consisting of 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, asymptomatic myeloma, smoldering multiple myeloma (SMM), active myeloma and light chain myeloma.

37. A method of treating multiple myeloma in a human patient, the method comprising administering to the human patient a NK cell or NK cell line modified to express a CAR that binds a tumour-associated MUC-1 glycoform with at least 10% increased affinity relative to wildtype MUC-1 glycoforms.

Description

EXAMPLES

[0102] The present invention is now described in more and specific details in relation to the production of NK cell line KHYG-1 derivatives, modified to exhibit more cytotoxic activity through an ability to bind aberrantly glycosylated MUC-1.

[0103] The invention is now illustrated in specific embodiments with reference to the accompanying drawings in which:

[0104] FIG. 1 shows the gene sequence organisation of a MUC-1 chimeric antigen receptor (CAR);

[0105] FIG. 2 shows the vector map of Lenti-EF1a-MUC1-CAR used to express the CAR in KHYG-1 cells;

[0106] FIGS. 3a 3b and 3c show the killing of different breast cancer cells using MUC-1 CAR-NK cells; and

[0107] FIG. 4 shows the killing of MDA-MB-453 breast cancer cells using MUC-1 CAR-NK cells at an effector:target ratio of 5:1.

[0108] DNA, RNA and amino acid sequences are referred to below, in which: [0109] SEQ ID NO: 1 is the MUC-1 CAR DNA sequence; [0110] SEQ ID NO: 2 is the MUC-1 CAR peptide sequence; and [0111] SEQ ID NO: 3 is the 24-mer peptide staining sequence used for MUC-1 binding.

TABLE-US-00001 SEQIDNO:1 ATGTGGCAACTGCTGCTGCCTACAGCTCTGCTGCTTCTGGTGTCCGCCGA TATCGTGGTCACACAAGAGAGCGCCCTGACCACCTCTCCTGGCGAAACAG TGACCCTGACCTGCAGATCTTCTACAGGCGCCGTGACCACAAGCAACTAC GCCAACTGGGTGCAAGAGAAGCCCGATCACCTGTTCACAGGCCTGATCGG CGGCACAAACAATAGAGCACCTGGCGTGCCAGCCAGATTCAGCGGATCTC TGATCGGAGACAAGGCCGCACTGACAATCACAGGCGCCCAGACAGAGGAC GAGGCCATCTACTTTTGCGCCCTGTGGTACAGCAACCACTGGGTTTTCGG CGGAGGCACCAAGCTGACAGTGCTGGGATCTGAAGGTGGCGGAGGATCTG GCGGAGGTGGAAGCGGAGGCGGAGGTTCTGAAGTTCAGCTGCAACAATCT GGCGGCGGACTGGTTCAACCTGGCGGCTCTATGAAGCTGAGCTGTGTGGC CAGCGGCTTCACCTTCAGCAACTACTGGATGAACTGGGTCCGACAGAGCC CCGAGAAAGGCCTGGAATGGGTTGCCGAGATCAGACTGAAGTCCAACAAT TACGCCACACACTACGCCGAGAGCGTGAAGGGCAGATTCACCATCAGCCG GGACGACAGCAAGAGCAGCGTGTACCTCCAGATGAACAACCTGAGAGCCG AGGACACCGGCATCTACTACTGCACCTTCGGCAACAGCTTCGCCTATTGG GGCCAGGGAACCACCGTGACCGTGTCCAGCACCTTCACCTGTTTTGTCGT GGGCAGCGACCTGAAGGATGCCCACCTGACATGGGAAGTCGCCGGCAAAG TTCCTACCGGTGGCGTGGAAGAAGGCCTGCTGGAAAGACACAGCAACGGC AGCCAGAGCCAGCACAGCAGACTGACACTGCCTAGAAGCCTGTGGAATGC CGGCACCAGCGTGACCTGCACACTGAATCATCCTAGCCTGCCTCCACAGA GACTGATGGCCCTGAGAGAACCTGCTGCTCAGGCCCCTGTGAAGCTGTCC CTGAATCTGCTCGCCAGCAGCGATCCTCCTGAAGCCGCCAATGTGAACCA CAAGCCTAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCG ACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTGCTCGGCGGA CCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAG CAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATC CCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTCGAGGTGCACAACGCC AAGACAAAGCCCAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTC CGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGT GCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCAGC AAGGCCAAGGGCCAGCCTAGAGAACCCCAGGTGTACACACTGCCTCCAAG CAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGG GCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAATGGACAGCCC GAGAACAACTACAAGACAACCCCTCCTGTGCTGGACTCCGACGGCTCATT CTTCCTGTACAGCAAACTGACCGTGGACAAGTCCAGATGGCAGCAGGGCA ACGTGTTCTCCTGCAGCGTGATGCACGAGGCCCTGCACTTTTGGGTGCTC GTGGTTGTTGGCGGAGTGCTGGCCTGTTACAGCCTGCTGGTTACCGTGGC CTTCATCATCTTTTGGGTCCGAAGCAAGCGGAGCCGGCTGCTGCACAGCG ACTACATGAACATGACCCCTAGACGGCCCGGACCTACCAGAAAGCACTAC CAGCCTTACGCTCCTCCTAGAGACTTCGCCGCCTACAGAAGCAGACGGGA CCAAAGACTGCCTCCTGACGCTCACAAACCTCCAGGCGGCGGAAGCTTCA GGACCCCTATCCAAGAAGAACAGGCTGACGCCCACAGCACCCTGGCCAAG ATCCGCGTGAAGTTCTCCAGATCCGCCGACGCTCCTGCCTATCAGCAGGG ACAGAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACG ACGTGCTGGATAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGAAAGCCC CAGCGGAGAAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAGAAAGA CAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAA GAGGCAAGGGACACGATGGACTGTACCAGGGACTGAGCACCGCCACCAAG GATACCTATGACGCCCTGCACATGCAGGCTCTGCCTCCA SEQIDNO:2 MWQLLLPTALLLLVSADIVVTQESALTTSPGETVTLTCRSSTGAVTTSNY ANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTED EAIYFCALWYSNHWVFGGGTKLTVLGSEGGGGSGGGGSGGGGSEVQLQQS GGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNN YATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYW GQGTTVTVSSTFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNG SQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLS LNLLASSDPPEAANVNHKPSNTKVDI(KVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHFWV LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKH YQPYAPPRDFAAYRSRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLA KIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPP SEQIDNO:3 TAPPAHGVTSAPDTRPAPGSTAPP

[0112] The expected molecular weight of the MUC1 chimeric antigen receptor is 813 amino acids=89.6 kDa, within which the sequence of single chain variable fragment and activating domains of the MUC1 CAR are as follows:

TABLE-US-00002 Sequence position Sequence Type Base Pair length 1 Signal peptide 48 bp 2 VL (Variable region light chain) 336 bp 3 (G4S)3 Linker sequence 45 bp 4 VH (Variable region heavy chain) 351 bp 5 IgD Hinge 309 bp 6 IgG1 Fc+ Hinge 699 bp 7 CD28 204 bp 8 OX40 111 bp 9 CD3zeta 336 bp

[0113] For related examples of knockout/knockdown of inhibitory receptor function and knock in of mutant TRAIL we refer to WO 2017/017184 the contents of which are incorporated herein by reference.

Example 1Lentiviral Plasmids Encoding MUC-1-CARs

[0114] DNA nucleotides encoding a single chain variable fragment (scFv) derived from an HMFG2 clone that recognises tumour-associated antigen MUC-1 (SEQ ID NO: 1), were cloned into pCDCAR1-GFP vectors. This scFv sequence was followed by an additional immunoglobulin-based hinge region to overcome the stearic hindrance of MUC-1. The hinge regions were followed by the co-stimulatory activating domains of CD28, OX40 and CD3zeta, in order to provide activating signals for NK cell cytotoxicity, thereby triggering cytolysis of MUC-1 positive tumour cells. Following the chimeric antigen receptor (CAR) protein (SEQ ID NO: 2), the selection marker Enhanced Green Fluorescent Protein (EGFP) was positioned downstream of the T2A sequence, separating the CAR and the EGFP. The sequence was cloned between EcoRI and XbaI restriction sites of the pCDCAR1-GFP vector (see FIGS. 1 and 2). 150 l vials of competent E. coli bacteria were thawed on ice. In the meantime, unlabelled tubes were chilled on ice. Once the last crystal had melted, 50 l of the cells were promptly transferred to each chilled tube, along with 1 l of the appropriate diluted plasmid solution (5 ng/l). The tubes were then left on ice for 30 minutes. The cells were heat-shocked by submerging the tubes in a water bath set at 42 C. for 30 seconds and then allowing them to cool back on the ice for 5 minutes. 950 l of room temperature LB broth was added to each tube and then the tubes were flicked gently to make a uniform mixture. The tubes were placed on a shaker (250 rpm) in the incubator (37 C.) for 1 hour. Next, 50 l of each plasmid solution was added into its corresponding agar plate containing ampicillin. The plates were stored in the incubator upside down overnight to allow bacteria containing the plasmid to grow out and form single colonies (14-16 hrs). A colony was selected from one of the plates. Using an autoclaved pipette tip, a colony was taken up and smeared against the inner surface of the corresponding culture tube. The tubes were then left on a shaker (125 rpm) in the incubator (37 C.) overnight for a maximum of 16 hours. The plasmid DNA was isolated according to the manufacturer's instructions. The plasmid was verified by restriction digestion with restriction enzymes. The bacterial culture (2.5 ml) was added to 247.5 ml LB broth containing Ampicillin. The 250 ml bacterial solution was then split into 50 ml tubes and stored at 20 C.

[0115] The labelled 50 ml tubes containing the bacterial cell pellets were removed from the 20 C. freezer and allowed to thaw on the bench for a few minutes. The first cell pellet was vortexed and re-suspended in 12 ml of re-suspension buffer containing RNase A. 12 ml of Lysis Buffer was added to the suspensions. The suspensions were inverted for mixing and then incubated at room temperature for 5 minutes. 12 ml of neutralization buffer was added to each suspension, followed by inverting the tubes until the sample turned from blue to colourless. The crude lysates were then incubated on ice for 5 minutes. A column filter was inserted into NucleoBond Xtra columns, before applying 15 ml of Equilibration buffer onto the rim of each column filter to ensure moistening of the entire column and its circumference. The column was emptied by gravity flow into a basin under a column rack. The lysates were poured into their designated column filters. The column filters were washed with 5 ml Filter Wash Buffer, making sure to apply the buffer to the funnel shaped rim of the filter as mentioned above. The column was then emptied by gravity flow. The column filter was removed and discarded. The NucleoBond Xtra Column was washed with 35 ml of Wash Buffer, before allowing the column to empty by gravity flow. For washes of the column, the buffer was gently pipetted directly into the centre as opposed to on the rim. This wash step was then repeated. A 15 ml tube was positioned underneath each of the columns to collect elute. The plasmid DNA was eluted from the columns with 5 ml of Elution Buffer. 3.5 ml of room-temperature isopropanol was pipetted into each 15 ml tube, in order to precipitate the eluted plasmid DNA. The tubes were then vortexed thoroughly. The tubes were centrifuged at 4500 rpm for 40 minutes at 4 C., before carefully aspirating the supernatants. 2 ml of endotoxin-free room-temperature 70% ethanol was added to each pellet. The tubes were then centrifuged at 4500g for 10 minutes at room temperature. The ethanol from each tube was removed using a pipette and left to dry under a hood at room temperature until the pellets turned from being slightly opaque to a more translucent glassy appearance. The DNA concentration was then measured using a Nanodrop spectrophotometer, and the DNA pellets were dissolved in 400 l of H2O-EF.

Example 2Nucleofection of MUC-1 CAR Plasmids into KHYG-1 Cells

[0116] KHYG-1 cells were passaged at 1:1 (5 ml cells+5 ml culture media) the day before nucleofection in T25 flasks, while the cells were in the logarithmic growth phase. The Lonza Nucleofection kit T (Cat: VCA-1002) was used; one nucleofection sample contained 100 l nucleofection solution (standard cuvette) and 210.sup.6 cells. The nucleofection solution contained 18 l Supplement and 82 l Nulceofector solution (per sample) prepared fresh and incubated at 37 C. for 10 minutes.

[0117] Solution T was warmed to room temperature. A fresh 12 ml aliquot of culture medium (CM) was prepared containing 2.4 ml FBS, 9.6 ml RPMI 1640 and supplements 6 l IL-2 (RPMI1640+20% FBS+500 IU/ml IL-2) at 37 C. in a 15 ml tube (no antibiotics). 4 ml of CM was aliquoted into T25 flasks, and plates were pre-incubated in a humidified 37 C. incubator for 20 minutes. 10 ml cell culture in 15 ml tubes was taken and the cells were counted to determine the cell density. A 210.sup.6 sample was dispensed into 15 ml tubes and centrifuged at 1000 RPM for 5 min (acc. 9/decc.7).

[0118] The supernatant completely discarded so that no residual medium covered the cell pellet. The cell pellet was re-suspended in 100 l room temperature Nucleofector Solution (see above) to a final concentration of 210.sup.6 cells/100 l. Storing the cell suspension longer than 5 min in Nucleofector Solution was avoided, as this is known to reduce cell viability and gene transfer efficiency. 0.2-2 g Lenti-EF1a-MUC1-CAR Plasmid DNA was added to each tube. The sample was then transferred into an Amaxa certified cuvette, making sure that the sample covered the bottom of the cuvette, in order to avoid air bubbles while pipetting. The cuvette was closed with a blue cap and Nucleofector program A-024 was selected. The cuvette was inserted into the cuvette holder, before pressing the x button to start the program (NB: program U-001 can also be used). To avoid damage to the cells, the samples were removed from the cuvette immediately after the program had finished (display showing OK). The pre-warmed culture medium was added to the cuvette and the sample transferred into one T25 flask. The X button was pressed to reset the Nucleofector. The cells were incubated in a humidified 37 C./5% CO.sub.2 incubator for 48 hours. 2-4 ml fresh media (RPMI1640+10% FBS+100 IU/ml IL-2) was added and the cells were incubated for another 24-96 hours to establish optimal protein expression (NB: the cell culture was monitored after every 24 hours using a microscope to check the status and condition of the cells). Flow cytometric analysis was performed between 72 and 240 hours post-nucleofection.

[0119] The MUC-1 CAR plasmid had EGFP as a selection marker. Therefore, KHYG-1 cells expressing MUC-1 CARs can be determined by estimating the number of GFP-positive KHYG-1 cells.

[0120] 0.5 ml cell suspension (110.sup.5 cells) was centrifuged at 1200 rpm for 5 mins. The supernatant was discarded, before adding 1 ml FACS buffer and centrifuging again at 1200 rpm for 5 mins. The cells were re-suspended in a final volume of 200 l FACS buffer.

[0121] The cells were then analysed by flow cytometry using a 488 nm laser and detection filter of 530/30 on a FAGS Canto Apresence of GFP-positive cells confirmed successful transfection.

Example 3Killing of Blood Cancer Cells by MUC-1 CAR-KHYG-1 Cells

[0122] NK cell cytotoxicity was measured using U266 and RPMI-8226 target multiple myeloma cells (both FAGS screened in advance, confirming MUC-1 expression on the targets; RPMI-8226 also expresses siglec ligand) at effector:target (E:T) ratios of 0.125:1, 0.25:1, 0.5:1, 1:1 and 2:1 in a 14 hour assay with 40,000 target tumour cells per 10001, plated in a 96 well flat bottom plate. Mock transfected KHYG-1 cells or MUC-1-CAR transfected KHYG-1 cells were centrifuged, and the supernatant was discarded. Cells were re-suspended in 1 ml fresh media (RPMI1640+10% FBS+100 IU/ml IL-2) and counted. KHYG-1 cells were then brought to a final concentration of 800,000 cells/100 l and combined with the tumour cells in a final volume of 200 l. The IL-2 concentration in the final reaction was 50 IU/ml IL-2. The plates were then incubated at 37 C. and 5% CO.sub.2 for 14 hours.

[0123] NK cell-induced target cell death was determined by flow cytometry. FACS tubes were pre-filled with 200 l FACS buffer using Eppendorf repeater units, before centrifuging at 2000 RPM for 3 minutes. The supernatant was discarded by inverting and then blotting on dry clean paper. Cells were re-suspended in the tubes using a vortex. 4 l of diluted CD2 BV421 antibody was added and incubated for 20 mins on ice and in the dark. 200 l FACS buffer was added using an Eppendorf repeater unit, before spinning at 2000 RPM for 3 minutes and then discarding the supernatant by inverting and then blotting on clean dry paper. The cells were then re-suspended in the tubes using a vortex. 200 l FACS buffer was added to each of 30 tubes with Eppendorf repeater units. 2 l of Propidium Iodide was added to each tube, before waiting 2-3 mins and analysing each tube using flow cytometry.

[0124] CD2 expression on KHYG1 cells was measured using a 405 nm laser and detector 450/50, while Propidium Iodide was measured by exciting the cells with a 488 nm laser and detection filter of 585/42 on a FACS Canto II.

[0125] Results confirmed the MUC-1 CAR KHYG-1 cells demonstrated increased cytotoxicity over KHYG-1 cells not expressing the CAR against both U266 and RPMI8226 targets.

Example 4Killing of Breast Cancer Cells by MUC-1 CAR-KHYG-1 Cells

[0126] Expression of MUC-1 on Breast Cancer Cells

[0127] MUC-1 expression on breast cancer cell lines HCC-1954, MDA-MB-1954 and ZR-75-1 was analysed using flowcytometry. Briefly, cells were stained with anti-MUC1 (Clone: HMFG2)AF647 (Cat: BD 566590) for 25 minutes on ice, and subsequently MUC-1 expression was measured on a FACS Canto II using 633/660/20. Expression analysis revealed that all the breast cancer cell lines had expression of MUC-1 on the cell surface, albeit at different levels. MUC-1 therefore represented a suitable candidate target antigen for the MUC-1 NK-CARs of the invention.

[0128] Transfection of NK Cells with MUC-1 CARs

[0129] mRNA for the MUC-1 CAR was synthesized using in vitro transcription (IVT) at Trilink Biotech. The EGFP sequence was excluded from the mRNA construct to reduce the mRNA size without compromising CAR activity.

[0130] KHYG-1 cells were electroporated with 12.5 g of MUC-1 CAR expressing mRNA. KHYG-1 cells were mock electroporated for use as a control. Both samples were incubated for 24 hours to allow for protein expression of the MUC-1 CAR construct. After 24 hours, cells were stained with a 24-mer peptide sequence (TAPPAHGVTSAPDTRPAPGSTAPP-OH) and conjugated to 5(6)Carboxy-fluorescein (JPT Peptide Solutions) for 30 minutes; this peptide sequence binds MUC-1 CAR specifically. After electroporation, 86% KHYG-1 cells were positive for MUC-1 CAR expression. Mock electroporated KHYG-1 cells showed <1% MUC-1 CAR+ cells 24 hours post electroporation. Thus, successful expression of the MUC-1 CAR was demonstrated in NK cells.

[0131] Cytotoxicity of MUC-1 NK-CARs Against Breast Cancer Cells

[0132] NK cell cytotoxicity assays were performed in 96 well flat bottom plates in 200 l final volume. After 14 hour co-culture, cells were harvested and stained with CD2-BV-421 antibody (BD Biosciences) for 30 minutes to distinguish the CD2-negative breast cancer cells from CD2-negative KHYG-1 cells. Cell death was analysed by addition of 1.5 l of 100 g/ml Propidium Iodide solution and incubating the cells on ice for 2 minutes before flowcytometry analysis.

[0133] Upon co-culture with MUC-1 NK-CARs with a panel of breast cancer cell lines, it was observed that the MUC-1 NK-CARs were more cytotoxic towards NK cell resistant breast cancer cell lines (HCC-1954 and MDA-MB-453) than mock electroporated KHYG-1. This observation was valid at multiple effector:target (E:T) ratios (see FIGS. 3a, 3b and 4).

[0134] There was no difference in cytotoxicity between the MUC-1 NK-CARs of the invention and the control cells (mock electroporated KHYG-1) against ZR-75-1 cells. This observation can be explained as the ZR-75-1 cells are already sensitive to NK cell mediated killing, as can be seen in FIG. 3c. Data is representative of a 14-hour cytotoxicity co-culture assay. NK cell induced tumour lysis was determined by flowcytometry using Propidium Iodide, after gating for the CD2-negative breast cancer cells.

[0135] Thus, it is demonstrated that the MUC-1 NK-CARs of the invention are particularly effective at killing a range of cancer types, especially in cases where the cancer is inherently resistant to NK cell-mediated cytotoxicity.

[0136] The invention thus provides NK cells and cell lines, and production thereof, for use in blood cancer therapy.