First-in-human novel bispecific epitope anti-CD5 nanobody CAR-T cells for refractory or relapsed T-cell malignancies.

7015 Background: Refractory/relapse (r/r) aggressive T-cell lymphomas have poor prognosis with 5-year PFS of 20~30%, meanwhile CAR-T therapy brings new hope to this progressive disease. We previously reported allogeneic humanized CD5 CAR-T (huCD5 CAR) therapy achieved 100% ORR in r/r acute T lymphoblastic leukemia/lymphoma (T-ALL) patients. However, huCD5 CAR-T exhibit insufficient expansion activity, necessitating the optimization of CAR-T structure. Here, we developed a llama-derived nanobody-based bispecific epitope anti-CD5 CAR (NbCD5 CAR) construct, and evaluated its efficacy and safety in r/r T-ALL and T-cell non-Hodgkin’s Lymphoma (T-NHL). Methods: llama-derived nanobody library was utilized to screen for CD5-specific nanobodies. The efficacy was then examined in NSG mice inoculated with Jurkat cell line. Tumor burden was assessed by flow cytometry and bioluminescent imaging. Finally, the efficacy and safety of the NbCAR vector were evaluated in the trials of r/r T-ALL (NCT07070323) and r/r T-NHL (NCT07022964). Results: We selected the NbCD5 CAR-T cells with superior cytotoxicity against T-cell lines compared to huCD5 CAR-T constructs. Then we determined its efficacy in NSG mice inoculated with Jurkat cell line. On day 7 post-tumor inoculation, mice received either Mock-T, huCD5 CAR-T or NbCD5 CAR-T cells. We observed that the NbCD5 CAR completely controlled tumor burden in mice and showed superior efficacy compared to huCD5 CAR. Then we proceed to the clinical trials containing 5 cases of T-ALL and 9 cases of T-NHL. In the T-ALL cohort, 3 patients have history of allo SCT, 4 patients received prior CD7 CAR-T therapy. All patients have infused 1.0×10 6 /kg NbCD5 CAR-T cells. The adverse events (AEs) included grade 1 CRS (5/5), grade 2 ICANS (1/5), and grade 1~2 GVHD (2/5). All patients achieved CR at day 30 evaluation. In the median follow-up time of 80 days, 2 patients bridged to allo SCT, 1 patient relapsed at day 45 and died shortly thereafter. In the T-NHL cohort, 9 patients aged 30 to 64 have failed of at least 2 lines of chemotherapy, and 1 patient had history of auto SCT. The patients have received 1.0×10 6 /kg NbCD5 CAR-T cells. All patients had grade 1-2 CRS, no ICANS was reported. Notably, EBV reactivation (5/9) was observed, including Grade 3-5 EBV reactivation (3/9). The ORR was 66.7% at day 30, 2 patients failed to respond for CAR-T expansion failure or weak CD5 expression. The CAR-T expansion was reached its peak at day 12 (range: 11-17), with the clearance of CD5 + T cells in blood. In the median follow-up time of 94 days, 5 patients bridged to allo SCT. Conclusions: We optimized the nanobody NbCD5 CAR construct and showed superior cytotoxicity in vitro and in vivo . We also got preliminary safety and efficacy profiles in r/r T-ALL and T-NHL. This clinical trial is still ongoing, and long-term efficacy and clinical complication of CD5 CAR-T cell therapy still require further investigation. Clinical trial information: NCT07070323 , NCT07022964 .