Anti-P329g-Car-T Cells As A Novel Universal Car-T Cell Platform

Introduction: In the rapidly growing field of chimeric antigen receptor (CAR) engineered T cells new approaches aim to make CAR-T cell therapy safer and more effective. Recent, designs aim towards universal or modular CARs that do not directly recognize the target antigen itself, but instead facilitate contact to CAR-adaptor molecules, which in turn bind to the target antigen. Current CAR-adaptor molecules include human antibodies of the IgG1 isotype binding to CD16 or antibodies modified by peptide or hapten tags. We developed a modular CAR-T approach that recognizes the P329G mutation clinically used to silence the Fc effector function of therapeutic antibodies. These modular anti-P329G-CAR-T cells are only functional in the presence of antibodies that possess the P329G mutation. Methods: The anti-P329G interaction with P329G-containing Fc fragment was analyzed using surface plasmon resonance and co-crystallography. Lentivirus transfected anti-P329G CAR-T cells were characterized in vitro for their selectivity and potential to mediate antigen specific tumor cell lysis, cytokine secretion and proliferation. Immunological synapse formation was investigated using confocal microscopy. Results: Anti-P329G-CAR-T cells allow the precise recognition of the P329G mutation present in therapeutic IgG1 based adaptor-molecules. Crystal structure- and SPR-analysis revealed a 1:1 binding stoichiometry with low nanomolar affinity of the P329G-Fab fragment applied in the CAR for P329G-containing IgG1 antibodies. Potent tumor cell lysis was demonstrated for multiple tumor antigens e.g. CD20, HER2, FOLR1, EpCAM, FAP and others. For all tested antigens, a huIgG1 dose-dependent activation of anti-P329G-CAR-T cells as well as dose-dependent tumor cell lysis was observed. For selected antigens P329G-CAR-T activity was found comparable to the activity mediated by T cell bispecific antibodies recognizing the respective tumor antigen. Finally, the immunological synapse formed by P329-CAR-T cells was compared to the one formed by T cell bispecific antibodies in a 2+1 format. Conclusions: P329G-CAR-T cells mediate potent and specific tumor cell killing using various tumor targeted antibodies as adaptor molecules. Based on these data in vivo studies to investigate efficacy and safety of the approach are foreseen. Notably, this approach allows control of CAR-T activity and potential side effects by titrating the adaptor molecule, as well as the simultaneous targeting of more than one antigen at the same time with the goal to prevent tumor escape mechanisms. Combining the P329G-CAR with allogenic T-cells may provide a truly off-the-shelf P329G-CAR-T cell therapy approach. Citation Format: Diana Darowski, Christian Jost, Zeno Riester, Mohamed Benmebarek, Kay Stubenrauch, Anne Freimoser-Grundschober, Uwe Wessels, Jorg Benz, Ekkehard Mossner, Renier Myburgh, Floriana Cremasco, Mario Perro, Pablo Umana, Sebastian Kobold, Christian Klein. Anti-P329G-CAR-T cells as a novel universal CAR-T cell platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4229.