Comparative Evaluation of scFv- and Peptide-Based CAR-T Cells for T-ALL

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by the uncontrolled proliferation of immature T cell precursors. Although current treatment protocols have substantially improved clinical outcomes, patients with relapsed or refractory disease or persistent measurable residual disease continue to have poor prognosis. The development of more selective therapeutic strategies is therefore needed. Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable clinical efficacy in several B-cell malignancies, but its application to T cell neoplasms remains challenging because of the shared expression of target antigens between malignant and normal T cells. CCR9 represents a potentially attractive target for T-ALL because it is expressed in a substantial proportion of leukemic cells while showing a limited expression in normal circulating T cells. This thesis aimed to generate and compare two CCR9-targeting CAR constructs: a conventional scFv-based CAR and a novel peptide-based CAR. CAR-T cells were generated from healthy-donor lymphocytes and characterized for CAR expression and immunophenotype. Their target-specific cytotoxic activity and functional persistence were subsequently evaluated in vitro using co-culture assays with CCR9-positive and CCR9-negative T-ALL cell lines. Non-transduced T cells and anti-CD19 CAR-T cells were included as negative controls. Both CCR9-targeting CAR-T cell products displayed cytotoxic activity against CCR9-positive tumor cells compared with non-transduced T cells and anti-CD19 CAR-T cells, while their activity against CCR9-negative cells was limited, supporting the target specificity of the response. Repeated antigen-challenge experiments were also performed to compare the persistence of their antitumor activity over time. Overall, these findings support CCR9 as a promising target for T-ALL and provide preliminary evidence that a peptide-based recognition domain may represent a potential alternative to conventional scFv-based CAR designs. Further studies will be required to confirm the efficacy, safety and translational potential of this strategy in more representative preclinical models.