Evaluating Efficacy and Safety of Rapid-Manufactured CD5KO CART5 Cells in Preclinical Models of T Cell Malignancies

Introduction: Chimeric antigen receptor T (CAR T) cell therapy has revolutionized the treatment of hematological malignancies, with FDA approvals for B cell lymphoblastic leukemia, B cell non-Hodgkin lymphoma, and multiple myeloma. However, CAR T therapy for T cell non-Hodgkin lymphomas (T-NHL) remains underdeveloped, with limited clinical data and poor prognoses for patients. This underscores an urgent need for effective and durable therapies. We have previously reported the development of an anti-CD5 CAR T cell product, knocked out (KO) for CD5 (Blood (2022) 140 (Supplement 1): 1604-1605; Blood (2023) 142 (Supplement 1): 3462), which is now being tested in a phase I clinical trial for relapsed and refractory CD5+ nodal T cell lymphomas (NCT06420089). Critical challenges in developing CAR T cells for T-NHL include CAR T fratricide and toxicity to normal T cells. In this study, we conduct an in-depth investigation into the safety and efficacy of a rapid-manufactured CD5KO CART5 product in preclinical models. Methods and Results: To enhance our GMP-grade product, we streamlined our manufacturing process, reducing the duration from >10 days (conventional) to 5 days (rapid). We previously demonstrated that CD5KO CART5 exhibited enhanced efficacy compared to CD5 wild-type (WT) CART5 in a small-scale, conventional manufacturing setting (Blood(2020) 136 (Supplement 1): 51-52). To confirm the superior efficacy of CD5KO in the rapid manufacturing setting, we also compared CD5KO and CD5 WT cells produced at a small scale with rapid-manufacturing (small-rapid). As expected, CD5 deletion in small-rapid cells resulted in enhanced efficacy and T cell expansion in NSG mice. We then conducted a comparative analysis of CD5KO CART5 produced using both small-scale, conventional-manufacturing (small-conv) and large-scale, rapid-manufacturing (large-rapid) protocols. Using the CD5+ Jurkat T cell leukemia model, we treated NOD-SCID IL2Rgnull (NSG) mice with suboptimal doses of large-rapid and small-conv CD5KO CART5. Mice treated with the large-rapid product displayed significantly stronger anti-tumor responses compared to those treated with the small-conv product, likely due to the heightened activation state of the large-rapid cells at the time of injection. This increased tumor suppression correlated with improved overall survival rates (median overall survival: small-conv = 52 days vs. large-rapid = undefined; p=0.0134, Mantel-Cox) and greater expansion of large-rapid cells in the peripheral blood. Ten days post-CAR T injections, a subset of animals was euthanized for comprehensive necropsy. Mice treated with the large-rapid product exhibited no detectable treatment-related lesions and no significant differences in overall weight or organ weight compared to mice treated with untransduced (UTD) cells. Additionally, bloodwork analyses showed no differences in clinical chemistry profiles or complete blood counts between the groups. Previously, we developed a dual-population technology to mitigate potential toxicity in patients receiving CD5KO CART5 treatment (Blood (2022) 140 (Supplement 1): 1604-1605). We optimized our manufacturing to achieve >95% CD5 knockout efficiency with ~50% CAR transduction, resulting in two main cell populations: CD5KO CART5 cells and CD5KO normal UTD cells. While CD5KO CART5 cells target CD5+ T-NHL cells, CD5KO normal UTD cells are intended to mitigate normal T cell toxicity. We have already demonstrated CD5KO UTD reactivity to CMV and EBV, but have since conducted T cell receptor (TCR) sequencing indicating little to no loss of viral-reactive TCRs during the 5-day manufacturing process. Conclusion: This study presents the development of a novel dual-population GMP-grade CD5KO CART5 product for T cell lymphomas, characterized by robust anti-tumor activity and a strategic approach to mitigate toxicity. This autologous CD5KO CART5 product is being tested in a phase I clinical trials for patients with relapsed and refractory CD5+ nodal T cell lymphomas, marking a significant step towards addressing the unmet needs in T-NHL treatment.