O12.04 A modular CAR T cell platform for targeted immunotherapy of acute myeloid leukemia

Background CAR T cell therapy has shown limited efficacy in acute myeloid leukemia (AML), largely due to genetic and molecular heterogeneity and the emergence of antigen-negative relapse. Adapter CAR T cells can overcome key limitations in AML. Anti-P329G adapter CAR T cells recognize the P329G mutation within the Fc region of Fc-silenced human IgG1 antibodies carrying L234A/L235A mutations and thereby represent a disease agnostic and modular cell therapy platform. The advantage of such P329G-containing antibodies lays in their clinical validation against multiple tumor antigens. These antibodies as adaptor modules are therefore readily available for combination with universal anti-P329G adapter CAR T cells.Methods ScFv-based 2nd generation anti-P329G CAR T cells were combined with P329G-Fc-mutated antibodies targeting the established AML-related target antigens CD33 and CD123 as well as the newly identified target CSF1R. Conventional, scFv-matched CAR T cells targeting the same antigens were used as positive controls. Untransduced or anti-CD19 CAR T cells served as negative control. Target antigen expression was analyzed on cell lines and primary patient samples (peripheral blood, bone marrow). Immunophenotyping and proliferation measurement of anti-P329G CAR T cells was performed using flow cytometry. Effector functions were evaluated against different wildtype and antigen knockout (KO) AML cell lines and primary AML blasts by ELISA-, flow cytometry- and luciferase-based analysis. Effector function in an antigen-escape setting was evaluated in vitro using IncuCyte live cell imaging. Confocal imaging of anti-P329G CAR T cell-tumor cell interaction was performed. In vivo functionality was confirmed in immunodeficient mice after injection of THP-1 and MV4-11 cells.Results AML cell lines and primary AML blasts could be successfully targeted by anti-P329G CAR T cells combined with P329G-Fc-mutated CAR-adapter molecules targeting CD33, CD123 and CSF1R. Effector functions of anti-P329G CAR T cells were equivalent to conventional CAR T cells. Differences in efficacy were depending on the expression profile of target antigens. The platform demonstrated efficient in vitro activation, proliferation and cytotoxicity. Co-culture assays using antigen KO cells and protein stimulation assays confirmed that anti-P329G CAR T cells operated in a modular, antigen-specific and reversible manner and enabled both combinatorial and sequential targeting of AML. Live-cell imaging based serial cytotoxicity assays employing re-challenge with antigen knockout cell lines showed that targeting antigen escape by switching of the binder antibody is feasible. In immunodeficient mice, growth of THP-1 or MV4-11 tumor cells could be successfully controlled and eradicated by anti-P329G CAR T cells.Conclusion Combination of anti-P329G CAR T cells with P329G-Fc-mutated CAR-adapter molecules mediated robust effector functions across multiple AML models when directed against CD33, CD123 and CSF1R. The platform enables modular, combinatorial and sequential targeting of distinct antigens, providing a flexible platform with the potential to overcome key limitations of CAR T cell therapy in AML. K. Gabriel: none S. Stock: none A. Hauptstein: none T. Strzalkowski: none L. Rohrbacher: none A. Gottschlich: Tabby Therapeutics, immuno-oncology patents (all significant) J. Dörr: none V. D. Menkhoff: none S. Nandi: none G. Hänel: none C. Carcopino: none D. Simnica: none M. Surowka: Roche previous employment and stock ownership (significant) P. Bruenker: Roche previous employment and stock and patent ownership (significant) D. Darowski: Roche employment and stock and patent ownership (significant) S. Endres: none M. von Bergwelt-Baildon: M. Subklewe: AbbVie, Amgen, Molecular Partners, Pierre Fabre, Roche (all significant) C. Klein: Roche previous employment and stock and patent ownership (significant) S. Kobold: Miltenyi, Galapagos, Cymab, Novartis, Plectonic, TCR2 Inc., BMS, GSK, Carina Biotech, Tabby Therapeutics, Catalym GmbH, Plectonic GmbH, and Arcus Bioscience, several immuno-oncology patents (all significant)