IMPEDANCE ASSAY

Abstract

The present invention provides a method of determining the cytotoxic effect of an effector molecule or cell on a target cell presenting a heterologous peptide comprising: (a) incubating said effector molecule or cell with said target cell, wherein said target cell is immobilised on a substrate comprising at least one pair of electrodes; and (b) determining the impedance during and/or after step a) wherein a decrease in impedance is indicative of target cell death. The invention further provides the use of impedance to determine the cytotoxic effect of an effector cell and/or molecule on a target cell presenting a heterologous peptide and a kit for use in the method of the invention, comprising (a) a target cell presenting a heterologous peptide or a target cell and a heterologous peptide or a nucleic acid comprising a nucleotide sequence encoding said heterologous peptide; (b) a capture molecule for immobilising said target cell to a substrate; (c) instructions for use of (a) and (b) in a method of the invention; and optionally (d) a substrate comprising at least one pair of electrodes; and/or (e) an effector molecule and/or cell which specifically binds to the heterologous peptide presented by the target cell, or an effector cell and a nucleic acid comprising a nucleotide sequence encoding a receptor which specifically binds to the heterologous peptide presented by the target cell.

Claims

1. A method of determining the cytotoxic effect of an effector molecule or cell on a target cell presenting a heterologous peptide comprising: (a) incubating said effector molecule or cell with said target cell, wherein said target cell is immobilised on a substrate comprising at least one pair of electrodes; and (b) determining the impedance during and/or after step a), wherein a decrease in impedance is indicative of a cytotoxic effect.

2. The method of claim 1 wherein said target cell is an antigen presenting cell.

3. The method of claim 1 or 2 wherein said target cell is non-adherent.

4. The method of any one of claims 1 to 3 wherein said target cell is immobilised to said substrate using a capture molecule which specifically binds to said target cell.

5. The method of claim 4 wherein said capture molecule is an antibody which specifically binds to said target cell.

6. The method of claim 5 wherein said antibody is an anti-CD40 antibody.

7. The method of claim 3 wherein said target cell is a T2 cell.

8. The method of any one of claims 1 to 7, wherein said effector molecule is an antibody or a soluble TCR.

9. The method of any one of claims 1 to 7 wherein said effector cell is an immune cell, such as a T cell or NK cell.

10. The method according to claim 9 wherein said immune cell comprises one or more receptors which specifically bind to said heterologous peptide presented by said target cell.

11. The method according to claim 10 wherein said effector cell comprises at least one TCR and/or at least one CAR which specifically bind to said heterologous peptide presented by said target cell.

12. The method according to any one of claims 1 to 11 wherein said heterologous peptide is introduced into said target cell by pulsing or is expressed in said target cell from an introduced nucleic acid comprising a nucleotide sequence which encodes the peptide.

13. The method of anyone of claims 1 to 12 which comprises an additional step before step (a) of pulsing the target cells with the heterologous peptide.

14. The method of claim 13 wherein the additional pulsing step is carried out before immobilisation of the target cells to the substrate.

15. The method of any one of claims 1 to 14 wherein said heterologous peptide is a tumour associated antigen, such as WT1 or an epitope sequence therefrom.

16. The method of claim 15 wherein said heterologous peptide comprises or consists of SEQ ID NO. 1.

17. The method of any one of claims 1 to 16 wherein the amount of heterologous peptide presented by the target cell corresponds to that presented by a diseased or non-diseased cell from a subject.

18. The method of any one of claims 1 to 17 wherein said method comprises a step of measuring of determining impedance before step (a).

19. The method of claim 18 wherein said method comprises a step of comparing the impedance measurement obtained before step (a) and the impedance measurement obtained in step (b) to determine any change in impedance.

20. Use of impedance to determine the cytotoxic effect of an effector cell and/or molecule on a target cell presenting a heterologous peptide, wherein a decrease in impedance is indicative of a cytotoxic effect.

21. The use of claim 20, wherein said target cell is immobilised to a substrate comprising at least one pair of electrodes.

22. A method for increasing the cell index of non-adherent peptide pulsed target cells on a substrate comprising at least one pair of electrodes, comprising pulsing said cells with peptide prior to attachment to said substrate.

23. A kit for use in a method of any one of claim 1 to 19 or 22, wherein said kit comprises: (a) a target cell presenting a heterologous peptide or a target cell and a heterologous peptide or a nucleic acid comprising a nucleotide sequence encoding said heterologous peptide; (b) a capture molecule for immobilising said target cell to a substrate; (c) instructions for use of (a) and (b) in a method of the invention; and optionally (d) a substrate comprising at least one pair of electrodes; and/or (e) an effector molecule and/or cell which specifically binds to the heterologous peptide presented by the target cell, or an effector cell and a nucleic acid comprising a nucleotide sequence encoding a receptor which specifically binds to the heterologous peptide presented by the target cell.

24. A method of assessing the affinity and/or avidity of an effector cell or molecule for a heterologous peptide presented by a target cell wherein said target cell is immobilised to a substrate comprising at least one pair of electrodes comprising a) incubating said effector cell or molecule with the immobilised target cell and b) measuring the impedance during and/or after step a) wherein a decrease in impedance is indicative of a cytotoxic effect of the effector cell or molecule and of the effector cell or molecule having an affinity and/or avidity for the heterologous peptide.

Description

[0161] The invention will now be described in terms of the figures and non-limiting examples as set out below:

[0162] FIG. 1 shows the mechanisms involved in cytotoxic T lymphocyte (CTL) and natural killer (NK) mediated target cell killing.

[0163] FIG. 2 shows diagrammatically how the presence of cells on a substrate can impede electron flow.

[0164] FIG. 3 shows the optimisation of antibody concentration for non-adherent cell immobilisation and of cell seeding density for an impedance assay using WT1 pulsed APC (T2 cell line). FIG. 3A shows the amounts of antibodies and cells used. FIG. 3B shows the change of cell index (CI) on E-plates coated with 6 g/ml of CD19 and CD40 and 1.5 g/ml of IgM (50000 T2 cells). CD40 shows the highest increase of CI. CD19 shows moderate attachment and IgM showed a poor increase of CI. FIG. 3C shows the cell attachment impedance plot for CD40 using 100000 cells/well. 1.5 g/ml coating had the lowest binding efficiency, 3 g/ml showed a higher CI and there was no difference in the maximum CI between 4.5 and 6 g/ml. FIG. 3D shows cell index changes when using gradients of concentration of anti CD19 antibodies. FIG. 3E shows the optimisation of cell seeding density when using 6 g/ml of anti-CD40 antibody.

[0165] FIG. 4 shows the optimisation results for seeding density and antibody concentration, demonstrating the advantageous effect of using an anti-CD40 antibody for immobilisation.

[0166] FIG. 5 shows the cell density required for maximum cell index, using either anti-CD40 or anti-CD19 antibodies. The bar chart shows the seeding density on the x axis, where the left hand bar at each cell density measurement shows the CD40 cell index and the right hand bar at each cell density measurement shows the CD19 cell index.

[0167] FIG. 6 shows the effect of peptide pulsing on cell index, where cells pulsed prior to attachment result in a higher cell index. FIG. 6A shows pulsing before attachment and FIG. 6B shows pulsing after attachment.

[0168] FIG. 7 shows the optimisation of effector:target ratios for T2 cells pulsed with SEQ ID NO. 1 and for WT1 effector cells added after 4 hours of target cell attachment. The top line shows a 0:1 ratio, the second line from the top shows a 1:1 ratio, the third line from the top shows a 2:1 ratio and the line at the bottom shows a 5:1 ratio.

[0169] FIG. 8 shows a normalised WT1 potency assay using impedance. When using an effector:target ratio of E1:T1, the killing stops at 8-12 hours. 50% cell death is observed for effector:target ratios of 2:1 and 5:1 and EC50 is reached within 4 hours for an effector:target ratio of 5:1.

[0170] FIG. 9 shows there is no difference in T2 attachment in cells loaded with either WT126 or WT235.

[0171] FIG. 10 shows the killing of JY target cells which have been pulsed with WT126 as opposed to JY cells pulsed with WT-235, by effector cells.

[0172] FIG. 11 shows a peptide titration experiment in T2 cells, were different amounts of peptide was pulsed into the cells. The experiment demonstrates that the amount of peptide presented can be tailored by pulsing with different peptide amounts.

[0173] FIG. 12 shows a bar chart demonstrating the killing of JY cells pulsed with WT126 as opposed to WT235. Bar chart shows the results for the JY cell line before (Pre) and after (Post) treatment with WT-1 transduced cells. The height of the bars represents the mean across all replicates per peptide and time point. Error bars denote confidence intervals of the standard error of the mean (SEM) at 95% probability level. A post-hoc t-test demonstrated significant differences between WT 126 and WT 235 after the induction of WT-1 transduced cells (JY_WT126_Post vs. JY_WT235_Post, P=0.023).

EXAMPLES

Example 1: Antibody Mediated T2 Cell Attachment

Materials and Methods

T2 Cell Culture

[0174] T2 cells were obtained from ATCC. Cells were cultured in RPMI 1640 supplemented with Glutamax (ThermoFisher) containing 20% foetal bovine serum (FBS) at a seeding density of 0.510.sup.6 cells/ml. Cells were cultured for a minimum of 4 days before experimentation.

Attachment Substrate

[0175] Attachment substrate was prepared by making a 6 g/ml solution of anti-CD40 and anti-CD19 (both from R&D) in PBS (Sigma). IgM attachment substrate was prepared by making a 1.5 g/ml solution of IgM (Cambridge Biosciences) in PBS. 50 l of each substrate solution was added to each well of a 96-well E-Plate 96 (ACEA Bioscience) and the lid replaced and secured with parafilm. The plate was placed into a 4 C. fridge for 12-18 hours.

Cell Seeding

[0176] Attachment substrate was aspirated and the wells were washed three time using PBS. Cultured T2 cells were seeded at 50,000 cells/well. The plate was placed on the xCELLigence RTCA MP (ACEA Bioscience) module and sweeps were performed every 15 minutes for 24 hours.

Results

CD19, CD40 and IgM Mediated T2 Cell Attachment

[0177] First, the individual antibodies were compared for their binding efficiency. FIG. 3B shows the change of cell index (CI) on E-Plates coated with 6 g/ml of CD19 and CD40, and 1.5 g/ml of IgM. Each well was seeded with 50,000 T2 cells. CD40 shows the highest increase of CI with a maximum of 1.150.22 at 1.8 hours following attachment. CD19 shows moderate attachment with a maximum of 0.360.05 after 3 hours of attachment. In contrast, IgM showed poor increase of CI with a maximum of 0.080.018. From these results, CD19 and CD40 were chosen for the remaining optimisation experiments.

Example 2: T2 Cell Attachment Optimisation

Materials and Methods

As Above

Results

Antibody Concentration

[0178] Anti-CD40 and anti-CD19 were coated in increasing concentrations from 1.5 g/ml, 3 g/ml, 4.5 g/ml and 6 g/ml on the E-Plate 96. FIG. 3C shows the cell attachment impedance plot for CD40 using 100,000 cells/well over 24 hours. The increase of cell index (CI) over the first 3 hours shows the binding of the T2 cells onto the surface of the E-Plate. The graph shows a coating concentration of 1.5 g/ml had the lowest binding efficiency with a maximum CI of 1.350.067. The CI of 3 g/ml coated-wells showed a higher CI of 1.70.14. There was no difference in the maximum CI between the 4.5 g/ml and 6 g/ml coated wells at 1.900.14 and 2.060.13 respectively.

[0179] FIG. 3D cell index changes when using gradients of concentration of anti-CD19 antibody. There was no significant difference between well coated with 3 g/ml or 6 g/ml where the CI was 1.150.09 and 1.040.20.

Cell Seeding Concentration

[0180] The effect of cell seeding was then determined. FIG. 3E shows the plot of various seeding densities using anti-CD40 (6 g/ml). Under these conditions, the maximum cell index increased proportionally to the increasing seeding density, this ranged from a maximum CI of 1.010.03 in wells seeded with 25,000 T2 cells to 2.80.20 in wells seeded with 200,000 cells.

Effect of Antibody Concentration and Seeding Density on Cell Index

[0181] A range of both anti-body coating concentration and cell seeding densities were examined together to demonstrate the relationship between the two factors on CI. E-plates were coated with anti-CD40 and anti-CD19 at 1.5 g/ml, 3 g/ml, 4.5 g/ml and 6 g/ml for 16 hours before being seeded with 25K, 50K, 75K, 100K, 150K or 200K cells/well. Surface plots for anti-CD19 and anti-CD40 are shown in FIG. 4 where the CI was taken after cell attachment at 2.51 hours. With both antibodies there was a linear relationship between coating concentration and seeding density.

Determining Maximum Seeding Concentration

[0182] As seen in FIG. 4 the maximum saturation concentration of CD40 is 4.5 g/ml. To obtain the highest CI the seeding densities was increased to 50 k, 100 k, 200 k, 400 k, 600, 800 k and 1000 k cells/well. FIG. 5 shows the CI comparing CD19 and CD40 at 2.5 hours after seeding the T2 cells. In all densities, except 50 k cells/well, wells coated with anti-CD40 had significantly higher CI. Both antibodies showed maximum CI saturation at 400K cells/well.

Example 3: T2 Cell Peptide Loading and Attachment

Materials and Methods

Peptide Preparation and Loading

[0183] T2 cells were loaded using the relevant WT126 peptide (126-134; RMFPNAPYL) or irrelevant control WT235 peptide (235-243; CMTWNQMNL) (both from Bachem). WT126 was prepared to a stock concentration of 20 mM in sterile PBS, WT235 was prepared to a stock concentration of 20 mM in dimethyl sulfoxide (DMSO, Sigma). Cells were either loaded before or after attachment. To load before attachment, T2's were resuspended at 610.sup.6 cells/ml and the peptides added to the final desired concentration. They were loaded for 2 hours at 37 C. and 5% CO.sub.2 in a humidified incubator. The cells were then attached to the E-plate as before using anti-CD40 at 4.5 g/ml.

[0184] To load the cells during attachment of the cells, the peptide was added into the wells during initial 2-3 hour attachment phase.

Results

[0185] Cells were pre-loaded with 0, 25, 50, 100 or 200 M of WT126 or WT235 peptide and allowed to adhere to the anti-CD40 coated xCELLigence plate. There was no difference in T2 attachment in either the WT126 or WT235 loaded cells or between the different loading concentrations (FIG. 9).

Example 4: Killing Assay

Materials and Methods

Effector Cell Culture

[0186] Effector cells were cultured in X-Vivo 15 (Lonza) and 5% v/v human serum (SeraLabs) supplemented with IL-2 (1.2 U/l, Peprotech). Proliferation was stimulated by addition of CD3/CD28 DynaBeads (Life Technologies). Effector cells were cultured for 4 days before initiation of the killing assay.

Results

Effect Cell Mediated Killing

[0187] T2 cells were pulsed using 100 M of WT126 peptide and allowed to attach as detailed in Examples 2 and 3. Following attachment the effector cells were added at different effector:target ratios: 0:1, 1:1, 2:1 and 5:1.

[0188] FIG. 7 shows the full cell index plot over 24 hours from T2 attachment, effector cell addition and subsequent decrease of cell index due to the cytotoxic effects. There is no killing seen in the 0:1 control, whereas the decreased CI in the other ratios indicate target cell death. The killing is proportional to the number of effector cells where the 1:1 ratio shows a decrease from 2.5 to 1.75 compared to the 2:1 and 5:1 ratios where CI is reduced to 0.87 and 0.75 respectively.

Example 5: Impedance Assay with JY Target Cells

Materials and Methods

Day 1

[0189] T2, JY and WT-1 transduced cells where thawed and kept in culture for four days (37 C. and 5% CO.sub.2). T2 and JY cells were cultured in RPMI with L-Glutamine and 20% Foetal Bovine Serum. WT-1 transduced cells were cultured in X-VIVO 10 supplemented with 5% human AB serum, IL-2 (120 IU/mL) and activated with CD3/CD28 beads.

Day 2

Cells Remain in Culture

Day 3

[0190] E-96 plate was coated with Human CD40/TNFRSF5 at 4 C.

Day 4

[0191] T2 and JY were counted and seeded at 610.sup.6 cells/mL in 24 well plates and pulsed with WT-126 (relevant peptide) or WT-235 (non-relevant peptide) or a vehicle (DMSO) at a 20 M concentration for two hours at 37 C. and 5% CO.sub.2. In addition, T2 were pulsed with different concentrations of WT-126 peptide (10 M, 1 M, 100 nM, 10 nM and 1 nM). After two hours incubation, the impedance plate was washed with PBS. T2 and JY cells were resuspended at 210.sup.6 cells/mL prior seeding 200 L/well of the impedance plate (400.000 cells/well). Cell index was monitored until a plateau is reachednormally 1.5 h-2 h. WT-1 transduced cells were de-beaded and resuspended at 410.sup.6 cell/mL. 200 L/well were transferred to the impedance plate (800.000 cells/well). Cell index was monitored for 24 h.

Results

[0192] As can be seen in FIG. 10, JY cells pulsed with WT-126 were killed by effector cells, to a significantly larger amount than JY cells pulsed with WT-235.