(P-BT-8) Development Of In Vitro Potency Assay To Assess The Effect Of T-Cells To Kill Cancer Cells

Background/Case Studies: With the rising numbers of phase I/II clinical trials using CAR-T cell therapies, there is an urgent need to reach a global consensus on the definition of “potency.” The emphasis is to manufacture functional therapeutics eliciting a consistent clinical response. We have developed a novel potency assay to assess the function of cellular therapeutics to kill cancer cells. This in vitro assay quantifies the potency of autologous and allogeneic products by overcoming the current drawbacks associated with the bioluminescence, radiolabelled, and enzymatic assays. This assay could also be modeled to screen the potency of several allogeneic products engineered to kill cancer cells isolated from a patient.

Study

Design/Methods: T-cells were isolated from three buffy coats derived from healthy donors, co-cultured with cancer cells, and the effect of T-cells killing the cancer cells was evaluated. RA-1 (Ramos) cells, B-lymphocytes isolated from Burkitts Lymphoma patient, were used as a model cancer cell line. The potency of T-cells was measured at different time points (24,48 and 72 hours), and the increase in the number of dead cancer cells was quantified. Flow cytometry was used to identify subsets of T-cells such as cytotoxic T-cells (Tc) and helper T-cells (Th) involved in killing the RA-1 cells. RA-1 cells treated with doxorubicin was used as a control. The release of TNF-α and IFN-ɤ by T-cells was also quantified as an adjunct measure of the T-cell killing assay.

Results/Findings: Our results demonstrated that activated T-cells killed cancer cells, and the degree of anti-tumor response varied between CD4+ and CD8+ T-cells. The kinetics of cancer cell death depended on the ratio of T-cells to cancer cells (Figure A). The assay was sensitive to delineating the differential response between T-cells derived from three buffy coats. The flow cytometry analysis selectively quantified the population of dead cancer cells mixed with the T-cells. The expression of TNF-α and IFN-ɤ is not sufficient to serve as an indirect measure of potency.

Conclusions: The function of T-cells to kill cancer cells was quantified using our potency assay. This assay can be modified to simulate other tumor models, such as lung cancer, ovarian cancer, etc. This potency assay can also be modified to test the function of acellular therapeutics like extracellular vesicles and exosomes.

Importance of research: With the rising numbers of phase I/II clinical trials using CAR-T cell therapies, there is an urgent need to reach a global consensus on the definition of “potency.” We have developed a novel potency assay to assess the function of cellular therapeutics to kill cancer cells. This in vitro assay quantifies the potency of autologous and allogeneic products by overcoming the current drawbacks associated with the bioluminescence, radiolabeled, and enzymatic assays.