Novel bispecific epitope anti-CD5 nanobody CAR-T cells for refractory or relapsed T-cell malignancies

Abstract

Abstract Introduction: Due to the lack of effective therapeutic approaches, refractory/relapse (r/r) aggressive T-cell lymphomas have poor prognosis with 5-year progress free survival (PFS) of 20~30%, necessitating the novel cellular therapies. As allogeneic CD5 CAR (IASO Biotech) T-cell therapy can induce 100% response rate (1 month) in 19 r/r acute T lymphoblastic leukemia (T-ALL) patients (Pan J, et al. Nat Med 2024), targeting CD5 on r/r T-cell lymphoma with CAR T-cell therapy might be feasible. We developed a novel bispecific epitope anti-CD5 nanobody CAR construct, comparing the cytotoxicity to previous CD5 CARs. Furthermore, we evaluated efficacy and safety of autologous or allogeneic CD5 CAR vector in r/r peripheral T-cell lymphomas (PTCL) and T lymphoblastic lymphomas (T-LBL) with the novel bispecific epitope anti-CD5 nanobody CAR-T Cells (NCT07022964). Methods: To enhance the efficacy of CD5 CAR, we employed a llama-derived nanobody library to screen for CD5-specific nanobodies, constructing dual-epitope CAR-T cells targeting the CD5 antigen. After comparing the expression efficiency, expansion capacity, and phenotypes of the dual-epitope construct to 2 previously published CD5 CAR-T cells (H65 and CD5 CARs [IASO Biotech]), we evaluated efficacy and safety of autologous or allogeneic this novel CD5 CAR vector in r/r peripheral T-cell lymphomas patients (NCT07022964). This trial has design to contain 2 cohorts: patients in cohort A received standard lymphodepletion and autologous CAR-T cells, and patients in cohort B received enhanced lymphodepletion and new donor CAR-T cells as previously published (Pan J, et al. Nat Med, 2024). Results: Using phage display technology, we initially identified 1344 sequences potentially binding CD5, subsequent positive selection against CD5 and negative selection narrowed this down to 2 candidates. The dual-epitope CAR-T cells (H65 and CD5 CARs [IASO Biotech]) showed superior cytotoxicity against CD5+ Jurkat and SUP-T1 cell lines compared to previous CAR-T constructs. Therefore, we utilized a subcutaneous xenograft model in NSG mice to further validate the efficacy of the CAR-T cells in vivo. Female NSG mice (6-8 weeks old) were subcutaneously inoculated with 5×10⁶ Jurkat-GFP-luc tumor cells per mouse. Tumor burden was assessed 6 days post-inoculation to stratify mice into experimental groups. Beginning on day 7 post-tumor inoculation, mice received either Mock-T cells or CAR-T cells. We observed that the dual-epitope anti-CD5 CAR-T cells completely controlled tumor burden in mice and prolonged their survival. To date, 4 patients were enrolled in this trial with a median age of 48 (range: 6~61) years, including 2 case of PTCL-NOS, 1 case of CTCL, and 1 case of T-LBL. Baseline evaluation shows that 4 patients have extramedullary involvement, and 2 patients had bone marrow involvement. The median lines of previous therapy were 2.5 (range 2-6), and 1 case of T-LBL had history of allo-HCT. All patients received 1.0×106/kg CAR-T cells. No DLTs were observed post-infusion. All patients had grade 1 cytokine release syndrome. No ICANS was documented. Severe adverse events (AEs) included cytopenia (4, Grade 3 or 4), which resolved by G-CSF within 30 days. Other AEs included 2 cases of EBV re-activation (grade 2 or 3). At 1-month evaluation, 3 patients achieved complete remission (Deauville score of 1 point) by PET/CT, with negative minimal residual disease (MRD) in bone marrow assessments. 1 patient showed significant regression of extramedullary lesions and transient reduction of lymphoma cells in peripheral blood. 3 CR patients had CAR-T expansion peak of 138.42 (range: 58.6-714.56) cells/μL at day 14 (range: 11-16). With the expansion of CAR-T cells, the patient's peripheral blood CD5+ T cells turned persistently negative at a median time of 10 days. In the median follow-up time of 35 (range: 30-97) days, 3 patients bridged to allo-HCT, all of them remain remission, however 1 patient died of infection at day 14 post-HCT. Conclusion: We optimized the novel bispecific epitope anti-CD5 nanobody CAR construct, and shows superior cytotoxic activity comparing with CD5 CAR (IASO Biotech) in vitro. We also got preliminary safety and efficacy profiles in r/r T-cell lymphoma. This clinical trial is still ongoing, and long-term efficacy and clinical complication of CD5 CAR-T cell therapy still require further investigation.