CN-121978342-A - Cell-based assays for determining in vitro tumor killing activity of immune cells expressing chimeric antigens

Abstract

The present disclosure relates to cell-based assays for determining in vitro tumor killing activity of immune cells expressing chimeric antigens. The present disclosure provides an in vitro method for determining the efficacy (e.g., cytotoxicity) of immune cells expressing a Chimeric Antigen Receptor (CAR) molecule. In the test sample, the immune cells expressing the CAR are incubated with target cells that express the antigen that interacts with the CAR. In the control sample, the immune cells expressing the CAR are incubated with the target cells and an inhibitory molecule that prevents interaction between the CAR and the target cells. The amount of target cell death in both the test sample and the control sample was determined and compared.

Inventors

• J.K. Williams

Assignees

• 詹森生物科技公司

Dates

Publication Date

20260505

Application Date

20210607

Priority Date

20200608

Claims (20)

1. 1. An in vitro method for determining the efficacy of an immune cell expressing a Chimeric Antigen Receptor (CAR) molecule, the method comprising: a) Contacting the CAR-expressing immune cells with target cells in a test sample, wherein the target cells express an antigen that interacts with the CAR, B) Contacting the CAR-expressing immune cells with the target cells in a first control sample, wherein (i) the contacting is performed in the presence of an inhibitory molecule, or (ii) the CAR-expressing immune cells and/or the target cells have been pre-incubated with the inhibitory molecule prior to the contacting, wherein the inhibitory molecule inhibits interactions between the CAR and the target cells, C) Determining the amount of target cell death in the test sample, D) Determining the amount of target cell death in the first control sample, and E) Determining the efficacy of the CAR-expressing immune cells based on comparing the amount of target cell death determined in steps (c) and (d), Wherein the contact time, the amount of CAR-expressing immune cells, and the amount of target cells are substantially the same in the test sample and the first control sample.
2. 2. The method of claim 1, wherein the contacting steps (a) and (b) are performed simultaneously.
3. 3. The method of claim 1, wherein the determining steps (c) and (d) are performed simultaneously.
4. 4. The method of claim 1, wherein in step (b) (i), the CAR-expressing immune cells and/or the target cells have been pre-incubated with the inhibitory molecule prior to the contacting step.
5. 5. The method of claim 1, wherein the method further comprises comparing the amount of target cell death determined in step (c) to the amount of target cell death determined in a second control sample, wherein the target cells are incubated in the absence of the CAR-expressing immune cells.
6. 6. The method of claim 1, wherein the method further comprises comparing the amount of target cell death determined in step (c) to the amount of target cell death determined in a third control sample, wherein the target cell is incubated in the absence of the CAR-expressing immune cells but in the presence of a detergent that causes the target cell to die.
7. 7. The method of claim 6, wherein the detergent is Triton X-100.
8. 8. The method of claim 1, wherein the target cells produce a detectable reporter signal upon death of the target cells, and step (c) comprises assaying the reporter signal in the test sample, step (d) comprises assaying the reporter signal in the first control sample, and step (e) comprises comparing the reporter signals assayed in steps (c) and (d).
9. 9. The method of claim 8, wherein the reporting signal is luminescent.
10. 10. The method of claim 8, wherein the reporter signal is fluorescence.
11. 11. The method of claim 8, wherein the target cell expresses a reporter protein that produces a signal when the target cell undergoes cell death.
12. 12. The method of claim 11, wherein the reporter protein is β -galactosidase, luciferase, green Fluorescent Protein (GFP), or a variant or derivative thereof.
13. 13. The method of claim 1, wherein the inhibitory molecule specifically binds the antigen on the target cell that interacts with the CAR.
14. 14. The method of claim 1, wherein the inhibitory molecule specifically binds the CAR.
15. 15. The method of claim 14, wherein the inhibitory molecule specifically binds to a region within the CAR that specifically binds to the antigen expressed on the target cell.
16. 16. The method of claim 13, wherein the inhibitory molecule is an antibody or antibody fragment.
17. 17. The method of claim 16, wherein the antibody is an anti-idiotype antibody.
18. 18. The method of claim 16, wherein the antibody fragment is a Fab, fab ', F (ab') 2 , fv, or Fd fragment, single chain antibody (scFv), linear antibody, single domain antibody, heavy chain variable region (VH) domain, or light chain variable region (VL) domain.
19. 19. The method of claim 16, wherein the antibody or antibody fragment specifically binds to an antigen within the scFv domain of the CAR.
20. 20. The method of claim 19, wherein the antibody or antibody fragment specifically binds to a Complementarity Determining Region (CDR) within the scFv domain of the CAR.

Description

Cell-based assays for determining in vitro tumor killing activity of immune cells expressing chimeric antigens The application is applied for 2021, 6, 7 and 202180049139.3 (PCT/IB 2021/054996), the invention is a divisional application of the patent entitled "cell-based assay for determining in vitro tumor killing activity of immune cells expressing chimeric antigens". Cross Reference to Related Applications The present application claims the benefit of U.S. provisional application Ser. No. 63/036,249 filed on 6-8-2020 and U.S. provisional application Ser. No. 63/125,173 filed on 12-14-2020. The entire contents of the above-mentioned application are incorporated herein by reference in their entirety. Technical Field The present invention provides improved assays for determining the efficacy (e.g., cytotoxicity) of immune cells expressing chimeric antigen receptors. These improved assays allow avoiding the use of mock transfected immune cells as assay control, but rather the use of inhibitory molecules preventing the chimeric antigen receptor of an immune cell from interacting with its target cell as assay control. Background Current methods for determining specific in vitro cytotoxicity of T cells expressing chimeric antigen receptors (CAR-T cells) involve the use of autologous non-transduced expanded T cells (mock cells) as a baseline control. These controls were used to calculate the specific cytotoxicity of transduced CAR-T cells. However, generating non-transduced expanded autologous or allogeneic control T cells (mock cells) is expensive and time consuming, particularly because these cells are typically produced by the patient's own T cells. Furthermore, the production of these mock cells is prone to failure to achieve the required yield, which may delay treatment or prevent proper administration of CAR-T cells during immunotherapy. The method of determining in vitro cytotoxicity of CAR-T cells involves using autologous non-transduced expanded T cells (mock cells) as a baseline control. The baseline control was used to calculate the percent increase in CAR-T cell specific cytotoxicity (CAR-T kill%). If no autologous mimetic cells are available, a qualified batch of allergen mimetic cells is used instead. However, the use of these qualified batches results in efficacy relative to allogeneic mock cells, and thus may not reflect the true efficacy of CAR-T cells. Alternatively, the baseline control was omitted and total cytotoxic activity was used. However, the total cytotoxic activity does not indicate whether any enhancement of the cytotoxic activity of the immune cell/target cell interaction has occurred due to CAR-T cells. The total cytotoxic activity does not distinguish between the contribution of the drug product and the spontaneous death of the target cells themselves. Alternative assays (such as cytokine ELISA) have been used in place of functional assays as alternatives to measuring activity, but these methods do not allow direct measurement of cytotoxicity. Thus, there is a need for improved assay controls in order to simplify the production and testing of CAR-T cells, while maintaining the accuracy of CAR-T cell potency, and while reducing the high cost and complexity associated with using mock cells and/or additional alternative assays when mock cells are not available. The subject matter described throughout this disclosure meets this need by providing a novel assay that does not require the use of mock cells as a control. Disclosure of Invention In one aspect, an in vitro method for determining the efficacy of an immune cell expressing a Chimeric Antigen Receptor (CAR) molecule is provided, the method comprising: a) Contacting, in a test sample, an immune cell expressing a CAR with a target cell, wherein the target cell expresses an antigen that interacts with the CAR, B) Contacting the CAR-expressing immune cells with the target cells in a first control sample, wherein (i) the contacting is performed in the presence of an inhibitory molecule, or (ii) prior to the contacting, the CAR-expressing immune cells and/or the target cells have been pre-incubated with an inhibitory molecule, wherein the inhibitory molecule inhibits interaction between the CAR and the target cells, C) Determining the amount of target cell death in the test sample, D) Determining the amount of target cell death in a first control sample, and E) Determining the efficacy of the CAR-expressing immune cells based on comparing the amount of target cell death determined in steps (c) and (d), Wherein the contact time, the amount of CAR-expressing immune cells, and the amount of target cells are substantially the same in the test sample and the first control sample. In some embodiments, contacting steps (a) and (b) are performed simultaneously. In some embodiments, determining steps (c) and (d) are performed simultaneously. In some embodiments, in step (b) (i), the CAR-expressing immune cells and/or target cells have been pre-