NK CELLS FOR USE WITH ANITBODIES IN CANCER THERAPY

Abstract

Natural Killer (NK) cells and NK cell lines are modified to increase cytotoxicity, wherein the cells and compositions thereof have a use in the treatment of cancer. Modified NK cells and NK cell lines are produced via genetic modification of CD38.sup.low NK cells to transiently express the Fc receptor CD16 (F158V) from mRNA introduced into the NK cell, rather than from a chromosomal coding sequence. The cytotoxicity of the modified NK cells against CD38-expressing cancer cells is increased by administration of these modified cells in combination with a CD38-binding antibody. Separately, cytotoxicity against breast cancer is exhibited by the modified NK cells in combination with

Herceptin.

Claims

1. A natural killer (NK) cell transiently expressing an Fc receptor from an extra-chromosomal nucleic acid.

2. The NK cell of claim 1, wherein the extra-chromosomal nucleic acid is mRNA.

3. The NK cell of claim 1, wherein the Fc receptor is CD16.

4. The NK cell of claim 3, wherein the CD16 receptor comprises the amino acid substitution mutation F158V.

5. The NK cell of claim 1, wherein the NK cell is CD38.sup.low.

6. The NK cell of claim 1, wherein the NK cell is of the KHYG-1 cell line.

7. A method of treating cancer in a human patient, comprising administering to the patient an NK cell in combination with an antibody, wherein the NK cell transiently expresses an Fc receptor from an extra-chromosomal nucleic acid.

8. The method of claim 7, wherein the cancer is selected from the group consisting of acute myeloid leukemia, multiple myeloma and breast cancer.

9. The method of claim 7, wherein the antibody is selected from the group consisting of Daratumumab, Trastuzumab, Alemtuzumab, Brentuximab, Blinatumomab, Pankomab, Avelumab, Durvalumab and Atezolizumab.

10. The method of claim 7, wherein the extra-chromosomal nucleic acid is mRNA.

11. The method of claim 7, wherein the Fc receptor is CD16.

12. The method of claim 11, wherein the CD16 receptor comprises the amino acid substitution mutation F158V.

13. The method of claim 7, wherein the NK cell is CD38.sup.low.

14. The method of claim 7, wherein the NK cell is of the KHYG-1 cell line.

15. A pharmaceutical kit comprising (a) the NK cell of claim 1; (b) an antibody; and (c) instructions for administration of the NK cell and the antibody to a patient.

16. The pharmaceutical kit of claim 15, wherein the antibody binds CD38.

17. The pharmaceutical kit of claim 15, wherein the antibody binds HER2.

18. The pharmaceutical kit of claim 15, wherein the antibody is selected from the group consisting of Daratumumab, Trastuzumab, Alemtuzumab, Brentuximab, Blinatumomab, Pankomab, Avelumab, Durvalumab and Atezolizumab.

19. A method of treating a CD38-expressing cancer in a human patient, comprising administering to the patient a CD38.sup.low NK cell expressing an Fc receptor in combination with a CD38-binding antibody.

20. The method of claim 19, wherein the CD38-binding antibody is Daratumumab.

Description

EXAMPLES

[0063] The disclosure is now illustrated in specific embodiments with reference to the accompanying drawings in which:

[0064] FIGS. 1A-1C show KHYG1 NK cells lack KIR inhibitory receptors on their surface.

[0065] FIGS. 2A-2D show KHYG1 NK cells exhibit a low abundance of CD38 receptors on their surface (i.e. they are CD38.sup.low). Sample size n=4.

[0066] FIGS. 3A-3E show that KHYG1 cells electroporated with CD16 mRNA maintain viability and express CD16 receptors on their surface over a period of 120 hours.

[0067] FIGS. 4A-4F show the level of CD38 expression on multiple myeloma and NK cell lines

[0068] FIGS. 5A-5B show that CD16.sup.+ KHYG1 NK cells enhance the therapeutic activity of Daratumumab against myeloma cell lines more than mock electroporated CD16.sup.negative KHYG1 NK cells.

[0069] FIGS. 6A-6B show Daratumumab per se is not toxic to CD38.sup.low CD16+ KHYG1 NK cells, i.e. Daratumumab treatment causes minimal collateral damage to these cells.

[0070] FIGS. 7A-7B show that CD16.sup.+ KHYG1 NK cells in combination with Daratumumab are more potent in killing CD38.sup.high myeloma cell lines at multiple effector:target ratios compared to CD16.sup.+ KHYG1 NK cells alone.

[0071] FIGS. 8A-8B show that CD16.sup.+ KHYG1 NK cells in combination with Daratumumab are more potent in killing CD38.sup.low myeloma cell lines at multiple effector:target ratios as compared to CD16.sup.+ KHYG1 NK cells alone.

[0072] FIGS. 9A-9E show that CD16.sup.+ KHYG1 NK in combination with Daratumumab eliminate patient derived primary myeloma cells (n=5) more effectively than mock electroporated CD16.sup.negative KHYG1 in combination with Daratumumab. FIGS. 9A-9E show the results of a 14-hour ADCC assay with mock nucleofected or CD16 mRNA nucleofected KHYG1 against primary multiple myeloma (MM) cells in combination with Daratumumab. To obtain the results shown in this figure primary MM cells from 5 patients were independently tested and the data then pooled.

[0073] FIGS. 9F-9G show the discernible, but non-significant increase in Daratumumab induced NK cell fratricide on CD38.sup.low CD16 expressing KHYG1 in the absence of any target cells. Greater than 80% of the genetically modified cells are viable for experimental purposes.

[0074] FIGS. 10A-10D show that CD16.sup.+ KHYG1 NK in combination with Daratumumab exhibit less shedding of the CD16 receptor upon interaction with cells of the H929 (multiple myeloma) cell line.

[0075] FIGS. 11 shows the results of a 14-hour ADCC assay with CD16 mRNA nucleofected KHYG1 against H929 cells with or without Daratumumab.

[0076] FIGS. 12A-12D show cytokine release during a 14-hour NK cell-MM cell co-culture measured by ELISA for (a) interferon gamma (IFN), and (b) TNF-. Mock nucleofected or CD16 m-RNA nucleofected KHYG1 cells were co-cultured with MM cell lines, in the presence of Daratumumab. As control, mock nucleofected or CD16 m-RNA nucleofected KHYG1 cells were cultured alone in absence of any target tumor cells in presence or absence or Daratumumab to determine if the genetic modification alone induced cytokine production in the absence of any relevant target tumor cells. Standard curves for the ELISA assays were carried out and it was confirmed that the quantities measured for the experiments presented fell within the linear range of the ELISA.

[0077] FIGS. 13A-13D show expression levels of HER2 in 3 breast cancer cell lines.

[0078] FIGS. 14A-14C show the cytotoxicity of mock KHYG1 cells vs CD16 mRNA electroporated KHYG1 cells against 3 breast cancer cell lines in an ADCC assay.

[0079] FIGS. 15A-15C show the ability of CD16 mRNA electroporated KHYG1 cells to maintain cytotoxicity against 3 breast cancer cell lines in the presence of immunosuppressive factors.

Example 1

Measuring the Level of CD38 Expression on Multiple Myeloma and NK Cell Lines

[0080] CD38 expression was determined for a panel of cell lines: RPMI-8226, H929, MM.1S, U266 and JJN3 by employing the staining protocol for CD38 expression as set out below. The stained cells were then analysed by flow cytometry (FACS). The results of these experiments are shown in FIGS. 4A-4F (which are representative of 4 separate experiments) and reveal that multiple myeloma cell lines have a broad-spectrum cell surface expression of CD38. The expression of CD38 on KHYG1 cells was low in comparison with at least cell lines MM.1S, RPMI 8226 and H929.

Example 2

KYHG1 as a Candidate NK Cell for CD16 Expression

[0081] CD38 and KIR expression was determined for expanded primary NK cells, and cell lines NK92 and KHYG1 by employing the staining protocols for CD38 and KIR expression as set out below. The stained cells were then analysed by flow cytometry (FACS). The results of these experiments are shown in FIGS. 1A-1C and FIGS. 2A-2D. The expression of both CD38 and the KIR inhibitory receptors is much lower in KHYG1 in comparison to the mean fluorescence intensities and expression levels seen for NK92 and expanded primary NK cells.

Example 3

Assessing the Viability and Kinetics of CD16 m-RNA Electroporated KHYG1 NK Cells

[0082] mRNA transcripts coding for high affinity (HA) CD16 protein were synthesized using in vitro transcription (IVT), and KHYG1 cells were subsequently electroporated with the CD16 mRNA according to the protocol for Electroporation of CD16 mRNA into KHYG1 NK cells and time course experiments that are set out below. The results of this experiment are shown in FIGS. 3A-3E and show that CD38.sup.low KHYG1 NK cells can transiently overexpress CD16 receptor over a period of 120 hours.

Example 4

Demonstration that CD16 Expressing KHYG1 Induces Cell Death in Daratumumab Treated Multiple Myeloma Cells

[0083] CD16 expressing KHYG1 cells were analyzed for surface expression of CD16, and further co-cultured with multiple myeloma cell lines RMPI 8226, H929, JJN3 and U266 either alone or in combination with Daramutumab in an ADCC assay. NK cell induced cytotoxicity was measured by FACS-based methods as described below. The results are shown in FIGS. 5A-5B. The boxed panel to the right of the FACS frequency plots indicates gating for dead cells as determined by propidium iodide staining.

[0084] HA CD16 nucleofected KHYG1 in combination with Daramutumab was significantly more cytotoxic towards NK resistant multiple myeloma cell lines JJN3 and H929; data represents mean of 4 independent experiments) at E:T (Effector:Target ratio) of 0.5:1, 1:1, and 2:1, as compared to HA-CD16 KHYG1 alone. Furthermore, the combination was also significantly cytotoxic against NK sensitive cell line RPMI 8226, albeit at a lower NK:MM E:T ratio E:T 0.25:1, 1:1.

Example 5

Demonstration that Daratumumab Induces Minimal Collateral Damage on CD16.SUP.+ KHYG.1 NK Cells.

[0085] CD16 expressing KHYG1 cells were co-cultured with multiple myeloma cell lines RPMI 8226, H929, JJN3 and U266 or primary CD38.sup.+ multiple myeloma cells either alone or in combination with Daramutumab in an ADCC assay. NK cell induced cytotoxicity was measured by FACS-based methods as described below. The results for multiple myeloma cell lines RMPI 8226, H929, JJN3 and U266 are shown in FIGS. 6A-6B, which demonstrate that the presence of Daramutumab in the assay had no significant effect on the viability of the CD16 expressing KHYG1 NK cells. The results for the primary CD38.sup.+ multiple myeloma cells are shown in FIGS. 10A-10D which also demonstrate that the presence of Daramutumab in the assay had no significant effect on the viability of the CD16 expressing KHYG1 NK cells.

Example 6

The Combination of Daratumumab and CD16.SUP.+ .KHYG1 Eliminates Cells of Multiple Myeloma Cell Lines

[0086] CD16 expressing KHYG1 cells were co-cultured with multiple myeloma cell lines RMPI 8226, H929, JJN3 and U266 or primary CD38.sup.+ multiple myeloma cells either alone or in combination with Daramutumab in an ADCC assay at E:T (Effector:Target) ratios of 0:1, 0.25:1, 0.5:1, 1:1 and 2:1. FIGS. 7A-7B show the results for CD38.sup.high multiple myeloma cell lines RPMI-8266 and H929. FIGS. 8A-8B show the results for CD38.sup.low multiple myeloma cell lines JJN3 and U266. FIGS. 8A-8B show the results for primary CD38.sup.+ multiple myeloma cells. All of these experiments demonstrate that CD16 expressing KHYG1 cells are cytotoxic to the multiple myeloma cell lines and primary multiple myeloma cells tested.

Example 7

[0087] Demonstration of CD16 receptor shedding upon interaction with multiple myeloma cells CD16 expressing KHYG1 cells were co-cultured for 24 hours with multiple myeloma cell line RMPI 8226, H929, JJN3 and U266 or primary CD38.sup.+ multiple myeloma cells either alone or in combination with Daramutumab in an ADCC assay. FIGS. 10A-10D show the results of a 24-hour ADCC assay with CD16 mRNA nucleofected KHYG1 against H929 cells with or without Daratumumab. FIGS. 10A-10D show that CD16.sup.+ KHYG1 NK in combination with Daratumumab exhibits very limited shedding of the CD16 receptor upon interaction (i.e. activation) with cells of the H929 (multiple myeloma) cell line.

Example 8

Demonstration that CD16 Expressing KHYG1 Induces Cell Death in Daratumumab Treated Primary Multiple Myeloma Cells

[0088] CD16 expressing KHYG1 cells were analyzed for surface expression of CD16, and further co-cultured with primary multiple myeloma cells from 5 different multiple myeloma patients in combination with Daramutumab in an ADCC assay. As control, primary myeloma cells were cultured with mock nucleofected KHYG1 in the presence of Daratumumab at different E:T (Effector:Target) ratios.

[0089] CD16 nucleofected KHYG1 in combination with Daramutumab was significantly more cytotoxic towards the primary multiple myeloma cells (FIGS. 9A-9E; data represents mean of 5 independent experiments at E:T ratios of 0.5:1, 1:1, 2.5:1 and 5:1) as compared to mock nucleofected KHYG1 and Daratumumab.

Example 9

[0090] Demonstration of cytokine release during a 14-hour ADCC measured by ELISA for interferon gamma (IFN-) (FIGS. 12A-12B); and TNF- (FIGS. 12C-12D) in CD16 expressing KHYG1 in combination with Daratumumab when co-cultured with multiple myeloma cell lines H929 and JJN3. Standard curves for the ELISA assays were carried out and it was confirmed that the quantities measured for the experiments presented fell within the linear range of the ELISA.

Example 10

[0091] Breast cancer cell lines HCC-1954, MDA-MB-453 and ZR-75-1 were shown (via FACS) to express HER2 at differing levels (see FIGS. 13A, 13B, 13C and 13D). HCC-1954 was shown to express HER2 to a further extent than either of the other two cell lines.

[0092] In FIGS. 14A-14C, it is shown that at different E:T ratios KHYG1 cells transfected to transiently express CD16 mRNA in combination with Herceptin are more effective at killing the 3 cancer cell lines than mock KHYG1 cells with Herceptin. This specifically shows that ADCC is enhanced when the KHYG1 cells are transfected with CD16 mRNA.

[0093] Finally, it is shown in FIGS. 15A-15C that the addition of immunosuppressive factors lactate (50 mM), PGE.sub.2 (100 ng/mL) and TGF- (5 ng/mL) individually or in combination was not effective at reducing the enhanced ADCC demonstrated against the 3 breast cancer cell lines by the KHYG1 cells transfected with CD16 mRNA. This provides evidence that expression of CD16 in NK cells, according to the disclosure, is effective at mitigating immunosuppression.

Materials and Methods

Electroporation of CD16 mRNA into KHYG1 NK cells

Electroporation

[0094] One Sample Contains

[0095] 100 l (OC-100 cuvette Maxcyte GT)

[0096] Cell number: 210.sup.6 cells

[0097] Maxcyte Buffer: 100 l (for each sample)

[0098] CD16 mRNA 12.5 ug/100 l sample [0099] 1. Passage cells at 1:1 (10 ml cells+10 ml media) on the day before electroporation in T75 flask, cells must be in logarithmic growth phase. [0100] 2. Pre-warm the Maxcyte Buffer to room temperature. [0101] 3. Prepare a fresh 10 ml aliquot of culture medium (CM) containing 2 ml FBS, 8 ml RPMI 1640, and supplements 1 l IL-2 (RPMI1640+20% FBS+100 IU/ml IL-2) at 37 C. in a 15 ml tube (no antibiotics). [0102] 4. Take 10 ml cell culture in 15 ml tubes and count the cells to determine the cell density. [0103] 5. Spin cells at 1200 rpm/5 min and discard the supernatant. [0104] 6. Wash cells once with 5 ml Maxcyte Buffer. [0105] 7. Resuspend 210.sup.6 in 100 l Maxcyte Buffer. [0106] 8. Transfer the sample into an OC-100 cuvette. Add 12.5 ug mRNA 12.5 ug/100 l sample to the CD16+ KHYG1 cuvette. The MOCK KHYG1 cuvette will not contain any mRNA. Make sure that the sample covers the bottom of the cuvette, avoid air bubbles while pipetting. [0107] 9. Close cuvette with the cap. [0108] 10. Select a program for natural killer cells on the Maxcyte GT. Insert the MOCK KHYG1 cuvette into the cuvette holder press the start program. Repeat this for CD16+ KHYG1 cuvette. [0109] 11. Remove the cuvette and transfer the cells as a bubble to a 6 well plate. Use two separate wells, one for CD16+ KHYG1 and another for MOCK KHYG1 [0110] 12. Incubate cells in a humidified 37 C. for 20 minutes. After 20 minutes add 3 ml of CM to each well. [0111] 13. Incubate cells in a humidified 37 C. incubator for another 24 hours. [0112] 14. Measure CD16 expression on the MOCK KHYG1 and CD16+ KHYG1 [0113] 15. Set up cytotoxicity assay as described below with MM cell lines.

Time Course Experiments

[0114] 16. Incubate cells suspension for up to 120 hours at 37 C. & 5% CO.sub.2 post electroporation. Add 1-4 ml fresh media (RPMI1640+10% FBS+100 IU/ml IL-2) as per cell growth requirements.

[0115] Note: The cell culture is examined every 24 hours under a microscope to check the status and condition of the cells.

Nucleofection of CD16 mRNA into KHYG1 NK Cells

Nucleofection

[0116] One Nucleofection sample contains:

[0117] 100 l (standard cuvette)

[0118] Cell number: 210.sup.6 cells

[0119] Nucleofector solution: 100 l (Supplement 18 l+freshly prepared Nucleofector solution

[0120] T, 82 l, (Incubate at 37 C. incubator for 10 minutes) (15 ml tubes))

[0121] Amaxa Nucleofector 11 system [0122] 1. Pre-warm the solution T to room temperature. [0123] 2. Prepare a fresh 10 ml aliquot of culture medium containing 2 ml FBS, 8 ml RPMI 1640, and supplements 1 l IL-2 (IL-2 10 ng/ml) at 37 C. in a 15 ml tube (no antibiotics!!!). [0124] 3. Prepare 12-well plates by filling with 2 ml of culture medium containing the above media, and pre-incubate plates in a humidified 37 C. incubator for 20 minutes. [0125] 4. Take 10 ml cell culture in 15 ml tubes and count the cells to determine the cell density. [0126] 5. Centrifuge the required number of cells 210.sup.6 at 1200 rpm for 5 min. [0127] 6. Discard supernatant completely so that no residual medium covers the cell pellet. [0128] 7. Resuspend the cell pellet in room temperature 100 l Nucleofector Solution (see above) to a final concentration of 210.sup.6 cells/100 l. [0129] 8. Avoid storing the cell suspension longer than 5 min in Nucleofector Solution, as this reduces cell viability and gene transfer efficiency. [0130] 9. Add 12.5 ug m-RNA one tube. [0131] 10. Transfer the sample into an Amaxa certified cuvette. [0132] 11. Make sure that the sample covers the bottom of the cuvette, avoid air bubbles while pipetting. [0133] 12. Close cuvette with the blue cap. [0134] 13. Select Nucleofector program (A-024). Insert the cuvette into the cuvette holder press the X button to start the program. [0135] 14. To avoid damage to the cells, remove the samples from the cuvette immediately after the program has finished (display showing OK). [0136] 15. Add the pre-warmed culture medium into the cuvette and transfer the sample into the prepared 12-well plated. [0137] 16. Press the X button to reset the Nucleofector. [0138] 17. Incubate cells in a humidified 37 C. incubator for 24 hours. [0139] 18. Perform flow cytometric analysis at 24 hour time point. [0140] 19. Set up cytotoxicity assay as described below with MM cell lines

Cell Cytotoxicity Assay (Part 1 with Maxcyte GT against MM Cell Lines

[0141] 1. Count MM cell lines RPM1-8226, H929, JJN3, and U266. [0142] 2. Incubate 400,000 MM cells in 1 ml RPMI1640 media (+10% FBS+1%P/S) with Daratumumab at a final concentration of 10 ug/ml. [0143] 3. Incubate the cells at room temperature for 30 minutes with Daratumumab. [0144] 4. Plate 100 l (40,000 cells) of Daratumumab treated MM cells in 96 well plate, either alone, or in-combination with NK cellsMOCK KHYG1 or CD16+ KHYG1 as below. [0145] 5. Add 100 l of MOCK KHYG1 or CD16+ KHYG1 NK cells containing 40,000 cells to the MM cells [0146] 6. Incubate the co-cultures at 37 C. incubator for 14 hours. Perform Cell staining Protocol for cytotoxicity

Cell Cytotoxicity Assay (Part 2 with Amaxa Nucleofector II against MM Cell Lines)

[0147] 1. Count MM cell lines RPMI-8226, H929, JJN3, and U266. [0148] 2. Incubate 400,000 MM cells in 1 ml RPMI1640 media (+10% FBS+1% P/S) with or without Daratumumab at a final concentration of 10 ug/ml. [0149] 3. Incubate the cells at room temperature for 30 minutes with Daratumumab. [0150] 4. Plate 100 l (40,000 cells) of untreated MM cells or Daratumumab treated MM cells in 96 well plate, either alone or in-combination with CD16+ KHYG1 at multiple E:T ratios. [0151] 5. Add 100 l of CD16+KHYG1 NK cells to the MM cells at the E:T ratio of 0.25:1, 0.5:1, 1:1 and 2:1. [0152] 6. Incubate the co-cultures at 37 C. incubator for 14 hours. [0153] 7. Perform Cell staining Protocol for cytotoxicity

Cell Cytotoxicity Assay (Part 3 with Maxcyte GT against Primary Patient Derived CD38.SUP.+ .Cells

[0154] 1. Isolate CD38.sup.+ MM cells from the patient Bone marrow. Check the expression of CD38 on the isolated cells. [0155] 2. Count the CD38.sup.+ MM cells. [0156] 3. Incubate 100,000 MM cells in 0.5 ml RPMI1640 media (+10% FBS+1% P/S) with Daratumumab at a final concentration of 10 ug/ml. [0157] 4. Incubate the cells at room temperature for 30 minutes with Daratumumab. [0158] 5. Plate 100 l (20,000) of Daratumumab treated MM cells in 96 well plate, either alone, or in-combination with NK cellsMOCK KHYG1 or CD16+ KHYG1 as below. [0159] 6. Add 100 l of MOCK KHYG1 or CD16+ KHYG1 NK cells at the E:T ratio of 0.25:1, 0.5:1, 1:1 and 2:1. [0160] 7. Incubate the co-cultures at 37 C. incubator for 14 hours. Perform Cell staining Protocol for cytotoxicity

Cell Staining Protocol for Cytotoxicity

[0161] 1. Prefill FACS tubes with 200 l FACS buffer (15 tubes) with Eppendorf repeater unit. [0162] 2. Add the cell co-cultures to the FACS tubes. [0163] 3. Spin at 2000 RPM/3 MIN [0164] 4. Discard supernatant by inverting on a try and then botting on a dry paper. [0165] 5. Resuspend cells in the tubes by vortexing. [0166] 6. Add 1 l of diluted CD2 BV421 antibody [0167] 7. Incubate for 25 mins on dark/ice. [0168] 8. Add 200 l FACS buffer (30 tubes) with Eppendorf repeater unit [0169] 9. Spin at 2000 RPM/3 MIN [0170] 10. Discard supernatant by inverting on a try and then botting on a dry paper. [0171] 11. Resuspend cells in the tubes by vortexing [0172] 12. Add 200l FACS buffer (30 tubes) to each tube with Eppendorf repeater unit [0173] 13. Measure on FACS CANTO II [0174] 14. Add 2 l of propidium iodide in each tube, wait 2-3 mins and measure each tube.

Staining Protocol for CD38

[0175] 1. Obtain 110.sup.6 cells NK cells, NK92, KHYG1 and primary expanded NK cells [0176] 2. Centrifuge at 2000 rpm for 3 mins. [0177] 3. Discard supernatant, add 5 ml FACS buffer and centrifuge at 200 rpm for 3 mins. [0178] 4. Resuspend the 110.sup.6 cells in 250 l in FACS buffer. [0179] 5. Aliquot 50 l of cells and antibody in each tubes as per below: [0180] Unstained [0181] CD38-Pe antibody [0182] 6. Mix well, vortex for 1-3 seconds, and incubate for 15 minutes in the dark in the refrigerator (2-8 C.) (NB this step should not be carried out on ice). [0183] 7. Wash cells with 0.5 ml of buffer and centrifuge at 2000 rpm for 3 minutes. Aspirate the supernatant completely. [0184] 8. Resuspend cell pellet in a suitable amount of buffer (200 l) for analysis by FACS.

Staining Protocol for KIR expression

[0185] 9. Obtain 110.sup.6 cells NK cells, NK92, KHYG1 and primary expanded NK cells [0186] 10. Centrifuge at 2000 rpm for 3 mins. [0187] 11. Discard supernatant, add 5 ml FACS buffer and centrifuge at 200 rpm for 3 mins. [0188] 12. Resuspend the 110.sup.6 cells in 250 l in FACS buffer. [0189] 13. Aliquot 50 l of cells and antibody in each tubes as per below: [0190] i) Unstained [0191] ii) KIR 2DL1 [0192] iii) KIR2DL2/3 [0193] iv) KIR3DL1 [0194] 14. Mix well, vortex for 1-3 seconds, and incubate for 15 minutes in the dark in the refrigerator (2-8 C.) (NB this step should not be carried out on ice). [0195] 15. Wash cells with 0.5 ml of buffer and centrifuge at 2000 rpm for 3 minutes. Aspirate the supernatant completely. [0196] 16. Resuspend cell pellet in a suitable amount of buffer (200 l) for analysis by FACS.

[0197] The disclosure thus provides NK cells and cell lines, and production thereof, for use in blood cancer therapy.