Abstract
B-Chronic lymphocytic leukemia (B-CLL) is characterized by a progressive accumulation of mature B-lymphocytes expressing CD19, CD20dim and aberrantly expressing the CD5 T-cell marker. Moreover, they over-express the B-cell activation marker CD23. Chimeric Antigen Receptors (CAR) are engineered molecules able to redirect T-cell killing/effector activity towards a selected target in a non MHC-restricted manner. First trials targeting B-CLL were based on both monoclonal antibodies and anti-CD19/anti-CD20 CAR-transduced T cells. However, this approach causes the elimination of normal B-lymphocytes and B-precursors with consequent impairment of humoral responses. Selective CD23 expression on B-CLL cells renders it an optimal target to design a specific CAR. A new CD23-targeting CAR to redirect T cells against CD23+ B-CLL has been generated. After transduction, modified T cells were tested for cytotoxicity against different CD23+-targets, using a classic chromium release assay and for specific cytokine release by multiplex flow cytomix assay. The anti-CD23 CAR was stably expressed by healthy donor-derived primary T cells after transduction (average expression,20%;range,10%–60%;n=10) and conferred them a strong cytotoxicity against CD23+ tumor cell lines: Epstein Barr Virus transformed lymphoblastoid cell line (EBV-LCL) (average lysis, 50%; range 15%–70%, at 40:1 Effector:Target (E:T) ratio; n=5); Bjab and Jeko cell lines transduced with human CD23 antigen (average lysis, 60%; range, 20%–75%, at 40:1 E:T ratio; n=3). On the contrary, anti-CD23 transduced T-cells displayed no relevant killing versus normal B cells (average lysis, 8%; range, 1%–15% at 40:1 E:T ratio; n=3), differently from anti-CD19 CAR redirected T-cells, which killed tumor and normal B cells in an indistinct manner. T cells from B-CLL patients were also efficiently transduced with the anti-CD23 CAR (average expression, 80%; range, 70%–90%; n=3) and redirected specifically toward autologous blasts (average lysis, 29%; range, 21%–35% at 20:1 E:T ratio; n=3), without being inhibited by soluble CD23-enriched autologous plasma. Moreover, we demonstrated that expression of the anti-CD23 CAR caused a significant increase in cytokine release from transduced in vitro activated T cells after 48h stimulation with irradiated EBV-LCL at 1:1 ratio, both in healthy donors (n=3) and B-CLL patients (n=2). Anti-CD23 CAR expressing T cells from healthy donors secreted 5.5-fold more INF-gamma (3079 pg/ml vs 561pg/mL, p=0.05) and 11-fold more TNF-alpha (187.17 pg/ml vs 16.53 pg/mL, p=0.05), 147-fold more IL-5 (147 pg/ml vs 0 pg/mL, p=0.05) and 13-fold more IL-8 (590 pg/ml vs 43.24pg/mL, p=0.05), compared to non transduced T cells (n=3). In line with these findings, T cells expressing anti-CD23 CAR from B-CLL donors secreted 8.8-fold more INF-gamma (2988 pg/ml vs 337pg/mL, p=0.05) and 17-fold more TNF-gamma (187.17 pg/ml vs 17.34 pg/mL, p=0.05); 25.8-fold more IL-5 (3483.14 pg/ml vs 134.785 pg/mL, p=0.05), 173-fold more IL-8 (2154 pg/ml vs 12.415 pg/mL, p=0.05), compared to non transduced T cells. Altogether these results suggest that for the potentiality to get selective and potent killing of tumor cells, while sparing normal B cells, and for the capability to induce the selective release of immunostimulatory cytokines, CD23-targeting through a specific CAR holds great promises for adoptive immunotherapy of B-CLL.
CD3xCD19 DART molecule treatment induces non-apoptotic killing and is efficient against high-risk chemotherapy and venetoclax-resistant chronic lymphocytic leukemia cells
