P-07.01 Modular targeting of acute myeloid leukemia with anti-P329G adapter CAR T cells

<h3>Background</h3> Chimeric antigen receptor-modified (CAR) T cell therapy has so far failed in demonstrating efficacy in acute myeloid leukemia (AML). High heterogeneity at genetic and molecular levels within and between AML patients is a major obstacle. In AML, different surface antigens are currently targeted with CAR T cells including CD33 and CD123. In a recent scRNA-seq approach, CSF1R was identified as a potential target with broad expression on AML blasts and minimal expression on healthy tissue. Adapter CAR T cells have the potential to address certain limitations in AML. Anti-P329G adapter CAR T cells specifically target the P329G mutation in the Fc region of human IgG1 antibodies, which carry P329G L234A/L235A (PGLALA) mutations for Fc-silencing. These mutations disrupt interactions with Fc gamma receptors and the complement system. Fc-silenced antibodies containing the P329G mutation have already been developed and clinically validated for various tumor antigens. They are thus readily available for combination therapy in conjunction with the suitable adapter CAR T cells. <h3>Materials and Methods</h3> Human scFv-based 2nd generation anti-P329G CAR T cells were combined with PGLALA-Fc-mutated antibodies targeting CD33, CD123 or CSF1R. Conventional CAR T cells with a CAR containing the same scFv as the mutated antibodies were used as positive controls. Anti-CD19 CAR T cells served as negative control. Effector functions were evaluated against different AML cell lines and primary AML blasts by ELISA-, flow cytometry- and luciferase-based analysis. Functionality was confirmed <i>in vivo</i> in immunodeficient mice after injection of THP-1 and MV4-11 cells. <h3>Results</h3> Anti-P329G CAR T cells targeting CD33, CD123 und CSF1R via the CAR-adapter antibodies achieved efficient <i>in vitro</i> activation, cytokine production, proliferation and cytotoxicity against AML cell lines and primary AML blasts depending on the expression profile of the target antigens compared to anti-P329G CAR T cells alone. Effector functions were comparable to conventional CAR T cells. Anti-P329G adapter CAR T cells performed in a modular, specific and reversible fashion and when combined with the CAR-adapter antibodies could be used for combinatorial and sequential targeting. CD33 and CD123 knockout AML cell lines could still be killed by anti-P329G CAR T cells co-administered with antibodies targeting CSF1R. Anti-P329G CAR T cells controlled AML in immunodeficient mice bearing THP-1 or MV4-11 tumor cells. <h3>Conclusions</h3> Anti-P329G CAR T cells combined with P329G-Fc-mutated CAR-adapter antibodies achieved sufficient effector functions against various AML models when targeting CD33, CD123 and CSF1R. Anti-P329G CAR T cells allow modular, combinatorial and sequential targeting of multiple antigens. This platform has the potential to overcome limitations of CAR T cells such as tumor heterogeneity or antigen escape in AML. <b>S. Stock:</b> A. Employment (full or part-time); Significant; LMU University Hospital. B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Novartis InCa Förderpreis 2022 for young researchers. <b>A. Hauptstein:</b> None. <b>T. Strzalkowski:</b> None. <b>V.D. Menkhoff:</b> None. <b>A. Gottschlich:</b> A. Employment (full or part-time); Significant; LMU University Hospital. B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Tabby Therapeutics. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; LMU University Hospital. <b>L. Rohrbacher:</b> A. Employment (full or part-time); Significant; LMU University Hospital. <b>J. Dörr:</b> A. Employment (full or part-time); Significant; LMU University Hospital. <b>C. Carcopino:</b> A. Employment (full or part-time); Significant; LMU University Hospital. <b>L. Fertig:</b> None. <b>K. Großhauser:</b> A. Employment (full or part-time); Significant; LMU University Hospital. <b>S. Nandi:</b> A. Employment (full or part-time); Significant; LMU University Hospital. <b>M. Surowka:</b> A. Employment (full or part-time); Significant; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Roche. <b>P. Brünker:</b> A. Employment (full or part-time); Significant; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Roche. <b>D. Darowski:</b> A. Employment (full or part-time); Significant; Roche. <b>S. Endres:</b> A. Employment (full or part-time); Significant; LMU University Hospital. B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Tabby Therapeutics, Plectonic GmbH, Arcus Bioscience, TCR2 Inc. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; LMU University Hospital. Other; Significant; TCR2 Inc., Carina Biotech. <b>M. von Bergwelt-Baildon:</b> A. Employment (full or part-time); Significant; LMU University Hospital. <b>M. Subklewe:</b> A. Employment (full or part-time); Significant; LMU University Hospital. B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Amgen, BMS/Celgene, Gilead/Kite, Janssen, Miltenyi Biotec, Novartis, Roche, Seattle Genetics, Takeda. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Significant; Amgen, BMS/Celgene, Gilead/Kite, Novartis. F. Consultant/Advisory Board; Significant; Janssen, Takeda, AstraZeneca, Pfizer, Ichnos Sciences, AvenCell, Incyte. <b>C. Klein:</b> A. Employment (full or part-time); Significant; Roche/Genentech. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Roche/Genentech. <b>S. Kobold:</b> A. Employment (full or part-time); Significant; LMU University Hospital. B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; TCR2 Inc., Arcus Biosciences, Tabby Therapeutics, Plectonic, Catalym GmbH. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Significant; BMS, GSK, Novartis, TCR2 Inc., Miltenyi Biotech. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; LMU University Hospital. Other; Significant; Carina Biotech, TCR2 Inc.