3D ADCC NK FACS assay

Abstract

Herein is reported a cell analytical technology based on a three-dimensional spheroid/aggregate co-culture assay, wherein the spheroid or aggregate is formed of tumor and natural killer cells. This method is useful for the in vitro functional analysis of antibodies in single and high-throughput format.

Claims

1. A method for the in vitro detection of effector function of an antibody, the method comprising the following steps: a) mixing natural killer cells and tumor cells, wherein the tumor cells are labeled with a first fluorescent dye capable of indicating the viability of the tumor cells, b) adding about 10.sup.4 mixed natural killer cells and tumor cells per 200 l cell suspension to each of the wells of a multi-well plate, c) centrifuging the multi-well plate to induce the formation of a three-dimensional spheroid or a three-dimensional aggregate, d) adding the antibody at a concentration of 0.1 g/ml to 15 g/ml to the wells of the multi-well plate, e) incubating the multi-well plate for about 20 hours to about 72 hours, f) labeling dead cells in the wells with a second fluorescent dye, g) resuspending the spheroid or aggregate into individual cells and analyzing the percentage of viable cells in the wells of the multi-well plate by fluorescence activated cell sorting, and h) detecting the effector function of the antibody, wherein the detection of the effector function is percentage of viable cells and the effector function is antibody-dependent cellular cytotoxicity.

2. The method according to claim 1, wherein the natural killer cells are human natural killer cells.

3. The method according to claim 1, wherein the ratio of natural killer cells to tumor cells is from 10:1 to 1:10, from 1:2 to 1:4, or from 3:1 to 4:1.

4. The method according to claim 1, wherein the incubating is for about 20 hours to about 28 hours.

5. The method according to claim 1, wherein the centrifuging is for 10 minutes at 100 to 1,000 rpm.

6. The method according to claim 1, wherein the tumor cell is a lymphoma cell.

7. The method according to claim 6, wherein the lymphoma cell is selected from the group consisting of a Raji-cell, a SU-DHL4 cell, and a Z138 cell.

8. The method according to claim 1, wherein the antibody is added at a concentration of from 8 g/ml to 12 g/ml.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A-C FACS analysis of viable and dead Raji-cells and natural killer (NK) cells in the absence of an antibody. Quadrants: lower left: viable NK cells, upper left: dead NK cells, lower right: viable Raji-cells, upper right: dead Raji-cells. FIG. 1A: Raji-cells only, FIG. 1B: Raji-cells and NK cells at a ratio of 1:1, FIG. 1C: Raji-cells and NK-cells 1:10.

(2) FIG. 2A-C FACS analysis of viable and dead Raji-cells and natural killer cells in the presence of an added anti-CD20 antibody (10 g/ml). Quadrants: lower left: viable NK cells, upper left: dead NK cells, lower right: viable Raji-cells, upper right: dead Raji-cells. FIG. 2A: Raji-cells only, FIG. 2B: Raji-cells and NK cells at a ratio of 1:1, FIG. 2C: Raji-cells and NK cells at a ratio of 1:10.

(3) FIG. 3 Optimization of lymphoma/spheroid-aggregate co-culture by variation of lymphoma cell, NK cell and antibody application schedule. Lymphoma cells: Raji-cells.

(4) FIG. 4A-C Percentages of viable lymphoma cells in the presence of NK cells as a function of the anti-CD20 antibody concentration. FIG. 4A: Raji-cells, FIG. 4B: SU-DHL4 cells, FIG. 4C: Z138 cells. NK cell to lymphoma cell ratio (E:T ratio) of 3:1.

(5) FIG. 5A-C Percentages of viable lymphoma cells in the presence of the anti-CD20 antibody concentration as a function of the effector (NK):target (lymphoma) ratio. FIG. 5A: Raji-cells, FIG. 5B: SU-DHL4 cells, FIG. 5C: Z138 cells. Anti-CD20 antibody concentration: 10 g/ml.

(6) FIG. 6 Schematic exemplary method.

(7) FIG. 7 Microscopic images of Raji-cells only and Raji cell co-cultivated with purified NK cells.

EXAMPLE 1

(8) Material and Methods

(9) Cell Lines:

(10) Raji-cells, SU-DHL4 cells and Z138 cell lines were obtained from ATCC (Manassas, Va., USA), from DSMZ (Braunschweig, Germany) and from Prof. M. Dyer (University of Leicester, UK), respectively. Raji-cells and SU-DHL4 cells were cultivated in RPMI 1640 medium (PAN Biotech, Cat. no. P04-18500) and Z138 in DMEM medium (PAN Biotech, Cat. no. P04-02500) supplemented with 10% FCS (Gibco, Cat. no. 10500-064) and Pen/Strep (Roche, Cat. no. 11 074 440 001) at 37 C. in a humidified incubator. Exponential growing cells with cell viability of 90% or more were used for the NK cell co-cultivation experiments.

(11) Purification of NK Cells:

(12) Whole blood was withdrawn from normal healthy donors into vaccutainer tubes (Becton Dickinson, Cat. no. 368484). PBMC were obtained by Ficoll preparation (PAN Biotech Cat. no. P04-60125). To leave the NK cells untouched, the NK cells were purified using a NK cell, negative selection kit (Miltenyi, Cat. no. 130-092-657). In short, the Ficoll isolated PBMCs were resuspended in MACS-buffer (PBS/0.5% BSA/2 mM EDTA) at 110.sup.7 cells/40 l. 10 l of an NK-Cell-Biotin-Antibody cocktail was added to the cells and incubated for 10 min. at 4 C., followed by the addition of 30 l MACS buffer. Thereafter, 20 l of the NK-Cell-Microbead cocktail was added to the cells and incubated for 15 min. at 4 C. 2 ml of MACS-buffer was added and the cells were centrifuged for 10 min. at 300 g. The pellet was resuspended in 500 l MACS-buffer and loaded onto the separation column which was equilibrated with 500 l MACS-buffer before. The column was washed subsequently three times with 500 l MACS-buffer and the cell number was determined in the total eluate using the CASY Cell Counter (Scharfe System).

(13) The purity of the NK cell preparation was determined by staining of an aliquot of the MACS eluate. In short about 210.sup.5 cells were resuspended in 100 l RPMI 1640/10% FCS and stained with 10 l each of anti-CD56-PE and anti-CD3-FITC antibodies (Becton Dickinson, Cat. no. 555516 and 555339, respectively) for 15 min at 4 C. Thereafter, 2 ml of RPMI 1640/10% FCS were added to the cells which were centrifuged for 5 min. at 400 g. The pellet was resuspended in 0.5 ml RPMI 1640/10% FCS and the percentage of the CD56 positive but CD3 negative cell fraction within the lymphocyte scatter gate was analyzed using a FACS Scan or FACS Canto II instrument (Becton Dickinson).

(14) Purification of Monocytes:

(15) Whole blood was withdrawn from normal healthy donors into vaccutainer tubes (Becton Dickinson, Cat. no. 368484). PBMC were obtained by Ficoll preparation (PAN Biotech Cat. no. P04-60125). To leave the monocytes untouched, the monocytes were purified using a negative selection, monocyte enrichment kit (Stem Cell Technologies, Cat No.: 19059).

(16) CMFDA Staining of Lymphoma Cells:

(17) The CMFDA lyophilizate (Invitrogen Cat. no. C7025) was resuspended in DMSO to obtain a 10 mM stock solution. 110.sup.6 lymphoma cells were incubated for 30 min. at 37 C. in 1 ml complete medium supplemented with 1 M CMFDA. Thereafter, cells were pelleted, washed once in complete medium and resuspended finally in complete medium at 110.sup.6 cells/ml.

EXAMPLE 2

(18) Generation of 3D Spheroids/Aggregates from Lymphoma Cell Lines

(19) The lymphoma cell number was determined using a CASY instrument (Scharfe-Systems, Reutlingen) and the cell suspension was diluted in ice cold medium to 2.510.sup.4 cells/ml (for 5,000 cells per spheroid/aggregate) and 510.sup.4 cells/ml (for 10,000 cells per spheroid/aggregate). A volume of 200 l of the cell suspension was added to each well of a 96-well plate with round (Corning Inc., New York, USA) or conical (Nunc, Roskilde, The Netherlands) bottom. To prevent cell attachment the plates were pre-coated with 50 l 0.5% polyHEMA (Polysciences, Eppelheim, Germany) in 95% ethanol (v/v) and air dried at 37 C. for three days. The spheroid formation was initiated by centrifugation of the plates at 1,000 g for 10 min. using an Eppendorf 5810 centrifuge (Eppendorf AG, Hamburg, Germany) with swinging buckets. The plates were incubated under standard cell culture conditions at 37 C. and 7% CO.sub.2 in humidified incubators.

EXAMPLE 3

(20) Generation of 3D Spheroids/Aggregates from Solid Tumor Cell Lines

(21) Monolayer cells were detached with Accutase (PAA Laboratories GmbH, Innsbruck, Austria) to generate a single cell suspension. The cell number was determined using a CASY instrument (Schrfe-Systems, Reutlingen) and the cell suspension was diluted in ice cold medium to 2.510.sup.4 cells/ml (for 5,000 cells per spheroid/aggregate) and 510.sup.4 cells/ml (for 10,000 cells per spheroid/aggregate). The rBM was thawed on ice overnight and added at a final concentration of 2.5% (v/v) with ice cold pipette tips to the cell suspension. A volume of 200 l of the cell suspension was added to each well of a 96-well plate with round (Corning Inc., New York, USA) or conical (Nunc, Roskilde, The Netherlands) bottom. To prevent cell attachment the plates were pre-coated with 50 l 0.5% polyHEMA (Polysciences, Eppelheim, Germany) in 95% ethanol and air dried at 37 C. for three days. The spheroid formation was initiated by centrifugation of the plates at 1,000 g for 10 min. using an Eppendorf 5810 centrifuge (Eppendorf AG, Hamburg, Germany) with swinging buckets. The plates were incubated under standard cell culture conditions at 37 C. and 7% CO.sub.2 in humidified incubators.

EXAMPLE 4

(22) Spheroid/Aggregate Lymphoma/NK Co-Cultivation and Incubation with Antibody

(23) The sequence of cell co-cultivation and antibody addition can be varied. In an exemplary co-cultivation experiment, lymphoma cells (CMFDA labeled) and NK cells were mixed at ratios as indicated in 6 well plates. For example, an E:T (NK cell to lymphoma cell) ratio of 3:1 corresponds to a cell mixture of 3+1 (e. g. 75% NK cells and 25% lymphoma cells). 200 l of the cell suspension was added to a single well of a polyHEMA coated 96 well V-plate (Nunc, Cat. no. 249662). PolyHEMA coating: 50 l 0.5% polyHEMA in 95% ethanol per well; drying for 72 h at 37 C. (Polysciences, Cat. No. 18894). The plates were centrifuged for 10 min. at 1,000 g. The antibodies were added thereafter at concentrations as indicated above, and cell aggregates/spheroids were incubated at 37 C., 7% CO.sub.2 in a humidified incubator. Microscopic images of Raji-cells only and Raji cell co-cultivated with purified NK cells are shown in FIG. 7, as well as images of co-cultivated Raji and monocyte cells to illustrate 3D co-cultivation of tumor cells with other immune cells then NK cells.

EXAMPLE 5

(24) Viable Cell and Cell Death Analysis

(25) Spheroids/aggregates were generated using 10,000 cells and incubated with the antibody as outlined in Examples 2 and 3. The identification of viable lymphoma tumor cells was as follows: Individual aggregates from individual wells representing identical experimental conditions were pooled, dissociated by pipetting and centrifuged at 300 g for 10 min. Individual spheroids were pooled, washed once with phosphate buffered saline (PBS), resuspended in Accutase solution, and incubated at 37 C. Every five minutes, the spheroids/aggregates were resuspended by pipetting and dissociation was complete within 5 to 15 min. Cells were washed using complete medium, centrifuged and cell pellets were resuspended in complete medium and propidium iodide was added at a concentration of 1 g/ml (Sigma, Cat. no. P4170). Fluorescence analysis was performed by FACS analysis (Becton Dickinson, Canto II instrument).

(26) Viable lymphoma cells were identified as shown in FIG. 1b. The upper right quadrant of PI and CMFDA positive cells represent the dead lymphoma cells, and the lower right quadrant of PI negative but CMFDA positive cells represent the viable lymphoma tumor target cell fraction. In the lower left quadrant are viable NK cells, whereas dead NK cells are located in the upper left quadrant.

(27) In an alternative setting an apoptosis assay can be performed. Spheroids/aggregates were generated using 10,000 cells and incubated with the antibody as outlined in Examples 2 and 3. For apoptosis analysis, the spheroids/aggregates were transferred into a 96-well conical-bottom plate, washed once with phosphate buffered saline (PBS), resuspended in Accutase solution, and incubated at 37 C. Every five minutes, the spheroids/aggregates were resuspended by pipetting and dissociation was complete within 5 to 15 min. The single cell suspensions from eight spheroids/aggregates were pooled and cells were stained with annexin-V-fluos and propidium iodide in the presence of supplemented 2 mM CaCl.sub.2, (annexin-V-fluos staining kit, Roche Diagnostics GmbH, Mannheim, Germany). The fluorescence of 10,000 cells was acquired using a flow cytometer (FACS scan instrument, Becton Dickinson, San Jose, Calif., USA). Quadrant statistics was applied on the dot plots, with the number of viable cells located in the lower-left quadrant.

(28) To obtain the absolute number of dead and viable cells, the number of total cells from the spheroids/aggregates were counted using a Fuchs-Rosenthal cell counting chamber and multiplied with the percentage of viable or dead cells of the same spheroids/aggregates as determined from the annexin-V-fluos/PI staining.