Clinical Development of Senza5TM CART5: a Novel Dual Population CD5 CRISPR-Cas9 Knocked out Anti-CD5 Chimeric Antigen Receptor T Cell Product for Relapsed and Refractory CD5+ Nodal T-Cell Lymphomas

Abstract

Introduction: Chimeric antigen receptor T-cell (CART) immunotherapy is FDA-approved for B-cell lymphoblastic leukemia, B-cell non-Hodgkin lymphomas, and multiple myeloma. However, CART immunotherapy for T-cell non-Hodgkin lymphomas (T-NHL) is lagging, with only limited clinical results. T-NHL have a poor prognosis and there is an unmet need for effective and long-lasting treatments. We previously presented preliminary preclinical results of anti-CD5 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. Critical challenges to developing CART cells for T-NHL are CART fratricide and normal T-cell toxicity. In this study, we addressed these challenges by improving manufacturing and including a dual-population technology to mitigate toxicity. We present the development of Senza5TM CART5 an autologous good-manufacturing practice (GMP)-grade product (Fig.1) that will be tested in a phase I clinical trial for relapsed and refractory CD5+ nodal T-cell lymphomas. Methods and Results: We first identified a single guide RNA (sgRNA) that deletes CD5 in T cells with >90% KO efficiency by flow cytometry and ICE analysis. We then assessed the specificity of the KO by performing iGUIDE sequencing. We confirmed high on-target CD5 cleavage with minimal off-targets (CALCP, C20orf85, INPP4B, XPO7, and SLC10A7). These genes have little to no expression in naïve CD4+ and CD8+ T cells by RNA-seq (DICE). Targeted functional assays are ongoing. For the CAR construct, we selected a lead CAR5 among six 4-1BB-constructs based on its superior anti-tumor function in NOD-SCID IL2Rgnull (NSG) mice engrafted with a Jurkat T-lymphoblastic leukemia cell line. Furthermore, to confirm the specificity of the CAR5 for CD5, we screened CAR5 T cells for interactions with HEK293 cells expressing 5,828 human plasma membrane proteins or cell-surface tethered secreted proteins and 398 human heterodimers (Retrogenix assay). The lead CAR5 did not bind to any targets besides CD5. For an optimized GMP-grade product, we modified our manufacturing protocol, shortening it from 10 to 5 days. Moreover, KO efficiency and safety were optimized to reduce off-target activity without loss of efficiency. We also optimized the activation strategy to decrease cell loss. Functional studies were performed to demonstrate the comparability of these methods. With these improvements, we were able to achieve >90% CD5 KO with >30% CAR transduction, thereby allowing the generation of two main populations: one of CD5KO CART5 cells and the other of CD5KO normal untransduced T cells. CD5KO CART5 are expected to target CD5+ T-NHL cells but could also lead to normal T-cell toxicity. This toxicity will be mitigated by the CD5KO normal T cells that will have a survival advantage after infusion (Fig.1) and have been demonstrated to maintain reactivity against opportunistic pathogens such as CMV and EBV. To evaluate Senza5TMCART5 safety, we performed cytokine-independent CART proliferation and long- and short-term xenograft models. Senza5TM CART5 cells did not grow aberrantly without growth/survival-associated cytokines (IL2/7/15). We then used NSG mice to exclude uncontrolled Senza5TM CART5 growth and observed enhanced expansion and proliferation of Senza5TM CART5 with a xenogeneic GVHD rate similar to that of control mice with similar wild-type CART5 expansion. Since the CAR5 does not cross-react with murine CD5, we tested non-specific toxicity in tissues using an NSG xenograft model of Jurkat. On Day 10 post-CAR T injections, animals were euthanized, and a complete necropsy was performed on all animals. Senza5TM CART5 demonstrated tumor clearance with no detection of treatment-related lesions. Mice treated with untransduced T cells or CART19 demonstrated significant overall and organ-specific weight loss compared to Senza5TM CART5. Blood analyses determined no differences in their clinical chemistry profile or complete blood counts. Conclusion: In this study, we developed a novel dual-population, full-scale GMP-ready CD5 KO anti-CD5 CART product for T-cell lymphomas characterized by enhanced anti-tumor effect and a strategy to potentially mitigate toxicity. Autologous Senza5TM CART5 will be tested for CD5+ nodal T-cell lymphomas in a phase I clinical trial. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal