Senza5 TM CART5: An Autologous CD5-Deleted Anti-CD5 CART Product with Enhanced Anti-T-Cell Lymphoma Activity
Authors: Snook Adam E. , Patel Ruchi P. , Ghilardi Guido , Nimmagadda Aditya , Yang Steven , Qian Dean , Porazzi Patrizia , Mazanet Rosemary , Schuster Stephen J , Barta Stefan K. , Vu Trang , Ruella Marco , Siciliano Nicholas A. 
Published: December 2023

Introduction: Treatment with adoptive T cell therapies expressing Chimeric Antigen Receptors (CART) produces unprecedented clinical responses in relapsed or refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL), non-Hodgkin lymphomas (NHL), and multiple myeloma. Despite the transformative potential of these therapies for cancer, most patients treated with CART therapy experience incomplete responses or eventual relapse. Moreover, CART therapy is not yet effective for solid cancers or other hematological malignancies such as T-cell neoplasms. Thus, there is a dire need to enhance CART efficacy and extend this approach to more cancer types. We previously presented preliminary preclinical results of CD5-directed CAR-T cells (CART5) that had CD5 knocked out (KO) [ Blood (2020) 136 (Supplement 1): 51-52]. CD5 KO not only prevented CART fratricide but also dramatically enhanced CART function in multiple in vivo models. Enhanced CART function reflected disinhibition of early T-cell activation by abrogating CD5 signaling. Here, we combine the functional advantage of CD5 KO CART5 with a clinical-grade “rapid” 5-day manufacturing process, characterize the resulting drug product, and evaluate its in vivo antitumor efficacy in a human xenograft model of T-cell lymphoma/leukemia.

Methods and Results: Using normal healthy donor T cells, we developed a proprietary, “rapid” 5-day GMP-validated manufacturing process for CD5 KO CART5, completing the process in 5 days (CART5 rapid) instead of the conventional 14+ days (CART5 conv). At the end of manufacturing, the two products possessed similar ratios of CD8 + and CD4 + T cells, CD5 editing, and CAR5 expression. The CART5 rapid product did not exhibit any genetic or functional abnormalities characteristic of tumorigenic risk, demonstrated by low off-target gene editing in low-risk genes, no in vitro soft agar colony formation, and normal karyotypes. Importantly, the CART5 rapid manufacturing process did not skew TCRβ profiles, indicating that the CART5 rapid product retains a diverse T-cell repertoire. CD5 KO CART5 rapid cells possessed high specificity for T-cell leukemia/lymphoma, reflected by the absence of recognition of ex vivo normal human cells (cardiomyocytes, hepatocytes, neurons, monocytes, etc). Interestingly, CD5 KO CART5 rapid cells possessed a more central memory-like phenotype than CD5 KO CART5 conv cells which has been correlated with improved clinical efficacy. To compare their antitumor efficacy, we performed an in vivo experiment with CD5 + Jurkat cells utilizing a suboptimal dose of CART5 cells. Immunodeficient NSG mice were engrafted with 10 6 Jurkat cells on day -7 and treated intravenously on day 0 with a suboptimal dose (0.5x10 6 CAR + cells) of CD5 KO CART5 rapid or CD5 KO CART5 conv cells. Controls received an equivalent total number of Mock KO UTD conv. Mice treated with CD5 KO CART5 rapid cells demonstrated superior antitumor responses compared to CD5 KO CART5 conv. Improved tumor control correlated with dramatically increased overall survival of mice treated with CD5 KO CART5 rapid (median overall survival Mock KO UTD = 21 days; CD5 KO CART5 conv = 44 days vs. CD5 KO CART5 rapid = not reached, p=0.0019, Mantel-Cox; Figure 1).

Conclusion: This novel CD5 KO anti-CD5 CART product manufactured in a 5-day GMP process shows high specificity and significantly enhanced antitumor efficacy resulting from the synergistic effect of improved early T-cell activation due to CD5 deletion and preservation of T-cell memory phenotypes conferred by a novel, 5-day manufacturing processes. Autologous Senza5 TM CART5 for the treatment of CD5 + nodal T-cell lymphomas is expected to enter the clinic in a Phase I study later this year.