224. Development of Safer & Optimized CAR-T Cells Using Lentiviral Vectors

Abstract

Chimeric antigen receptors (CAR) modified T cells have shown very promising clinical results in hematological malignancies. CAR are usually composed of a scFv antigen binding domain linked to a spacer and intracellular signaling domains. CAR introduction into T cells leads to cells with optimal effector functions and with a new antigen binding ability. This strategy is well suited to skirt immune escape mechanisms.Despite their efficacy, CAR-T cells still show toxicity due to a cytokine storm, even if it is manageable. Improving the safety while maintaining or even improving the anti-tumor efficacy of CAR-T cells is therefore a crucial therapeutic challenge.Based on our proprietary lentiviral vector technology, we are developing a CAR-T cell platform aiming at producing innovative CAR-T cells. The development conducted by Theravectys are: -Development of proprietary lentiviral vectors coding for CAR of the second (containing the CD3ζ and the 4-1BB cosignaling domains) and third generations (containing the CD3ζ, the CD28 and the 4-1BB domains) directed against CD19 (for CD19+ leukemias and lymphomas), LMP-1 and -2 (for EBV-induced leukemias). Our lentiviral vectors will allow optimal expression of CAR therefore leading to a better efficacy of CAR-T cells. Hematological malignancies will be used as a benchmark.-Development of an inducible RUSH (Retention Using Selective Hook) system which is based on the streptavidin anchored to the membrane of the endoplasmic reticulum (ER) through a hook, and on the SBP (streptavidin binding protein) introduced into the CAR structure. The interaction between the streptavidin-hook and the SBP-CAR allows the CAR retention inside the ER. The addition of biotin displaces the equilibrium of binding of streptavidin towards biotin instead of SBP, thus leading to the release of the CAR from the ER and its expression to the cytoplasmic membrane. View Large Image | Download PowerPoint SlideThe release of the CAR will stop with drug exhaustion (or antagonist) and remaining cells can be easily reactivated through reintroduction of biotin. This system will increase safety of CAR-T cells. A CD19-CAR RUSH will be used as a proof-of-concept of the RUSH system.Most of the scFv used to date are of murine origin. Neutralizing antibodies against these murine scFv can limit the efficacy of CAR. As an alternative, we are screening and engineering specific humanized camelid nanobodies to be used as binding domains since they are highly homologous to the human VH domain of antibodies and they display high antigen binding capacities. The proof-of-concept will be made with a second generation CAR containing a nanobody directed against Her2 as a binding domain.Theravectys’ technological platforms allows flexibility and reactivity in the CAR design, production and evaluation, thus leading to the fast generation of safe and optimized CAR-T cells.