Comparison of the functional and immunophenotypical profi le of anti-CD19 CAR T-cells derived from healthy donors and patients with B-cell acute lymphoblastic leukemia during prolonged restimulation in vitro

Introduction. An important characteristic of therapy with modified T-cells expressing the chimeric antigen receptor (CAR) is the duration of CAR T-cell persistence in the patient’s body. Prolonged re-stimulation of CAR T-cell product in vitro with further analysis of its subpopulation composition and cytotoxic activity is one of the approaches to model cell behavior in patients.

Aim: to study the immunophenotype, exhaustion and functional characteristics of anti-CD19 CAR T-cell products derived from healthy donors and patients with B-cell acute lymphoblastic leukemia (B-ALL) at prolonged re-stimulation conditions in vitro.

Materials and methods. Anti-CD19 CAR T-cell products were generated from 5 healthy donors and 3 patients with B-ALL. The immunophenotype of the obtained cell products was studied under conditions of repeated antigenic re-stimulation in vitro using the target cell line NALM6 carrying the CD19 antigen over 7-10 days. At each experimental timepoint, the following parameters were assessed: proliferative and cytotoxic activity, expression of exhaustion markers, and changes in the subpopulation composition of CAR T-memory cells (immunophenotype).

Results. Anti-CD19 CAR T-cells had significant cytotoxic activity regardless of the lymphocyte source (healthy donors/patients). During prolonged antigenic re-stimulation of all products, a decrease in the proportion of naive (TN) and effector (TE) CAR T-cells, and an increase in the proportion of central (TCM) and effector memory (TEM) cells was observed. CAR T-cells showed increased expression of exhaustion markers (PD1, TIM3) irrespective of the origin of the cell and E:T ratio.

Conclusion. Analysis of cytotoxic activity and immunophenotypic composition of CAR T-cell products under conditions of prolonged re-stimulation revealed a trend toward decreased cytotoxicity, as well as differences in proliferation dynamics and population composition between cell products obtained from B-ALL patients and healthy donors.

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