Background Chimeric antigen receptor (CAR) T cell therapy has achieved encouraging results for patients suffering from B and plasma cell diseases. Clinical success in acute myeloid leukemia (AML) is however still impaired by therapy-associated toxicities, antigen heterogeneity and antigen escape. Adaptor CAR T cells which allow sequential, transient, or simultaneous targeting of multiple antigens have the potential to overcome these limitations. These CAR T cells were combined with tumor antigen-recognizing CAR-adaptor molecules for indirect tumor binding. We developed a modular anti-P329G CAR targeting the P329G mutation in the Fc part of antigen-targeting human IgG1 antibodies carrying P329G L234A/L235A (PGLALA) mutations. These mutations render the Fc part inactive for binding to Fc gamma receptors (FcgRs) and the complement system and are already clinically validated. Therefore, this approach does not rely on haptens or artificial tags fused to the targeting antibody facilitating the application.

Materials and Methods A scFv-based 2^nd generation CAR vector system was used in primary human T cells. Anti-P329G CAR T cells were combined with PGLALA-Fc-mutated antibodies targeting CD33, CD123 or CSF1R. Conventional anti-CD33 and anti-CSF1R CAR T cells have been used as positive controls, while anti-CD19 CAR T cells and untransduced T cells have been used as negative controls. Effector functions of CAR T cells were evaluated against different AML cell lines, the ALL cell line NALM-6 and primary human AML blasts by luciferase-based killing assays, ELISA- and flow-based analysis. THP-1-bearing immunodeficient mice were used for evaluation of in vivo functionality.

Results Anti-P329G CAR T cells revealed specific effector functions only when combined with antibodies carrying PGLALA-Fc-mutations. Anti-P329G CAR T cells combined with antibodies targeting CD33, CD123 und CSF1R achieved efficient in vitro activation, cytotoxicity and cytokine production against tested AML cell lines and primary AML blasts, while antigen-negative ALL cell line NALM-6 was not killed. Remarkably, effector functions of anti-P329G CAR T cells were similar to classical CAR T cells particularly when targeting CD33. Anti-P329G CAR T cells activated by recombinant protein showed no cytokine production in the presence of an antibody which was not recognizing the respective antigen. However, by switching the antibody after 24 h of stimulation towards the tumor antigen-targeting antibody, CAR T cell activation could be again achieved by the recombinant protein. Depleting the tumor antigen-targeting antibody after 24 h of stimulation decreased the cytokine production after 48 h and 72 h despite ongoing presence of the recombinant protein. This confirms the modularity and reversibility of this adaptor CAR T cell platform. CAR T cells combined with a CD33-targeting antibody showed efficient tumor clearance of THP-1-bearing immunodeficient mice.

Conclusions Taken together, anti-P329G CAR T cells combined with Fc-silenced tumor antigen-targeting IgG1 antibodies carrying the clinically validated PGLALA-mutations in the Fc part achieved profound effector functions against various human AML cell lines and primary AML blasts. The modular platform has the potential to overcome certain limitations of CAR T cell therapy in AML.

S. Stock: Other; Significant; Novartis (Award for young scientists). T. Strzalkowski: None. A. Gottschlich: C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Significant; Tabby Therapeutics. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; LMU University Hospital, LMU Munich. L. Rohrbacher: None. L. Fertig: None. V.D. Menkhoff: None. M. Surowka: A. Employment (full or part-time); Significant; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Roche. P. Bruenker: A. Employment (full or part-time); Significant; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Roche. D. Darowski: A. Employment (full or part-time); Significant; Roche. S. Endres: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; TCR2 Inc., Carina Biotech, Tabby Therapeutics, Plectonic GmbH, Arcus Bioscience. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; LMU University Hospital, LMU Munich. M. von Bergwelt-Baildon: None. M. Subklewe: 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. C. Klein: A. Employment (full or part-time); Significant; Roche/Genentech. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Roche/Genentech. S. Kobold: 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. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Significant; BMS, GSK, Novartis, TCR2 Inc, Miltenyi Biotech. Other; Significant; Carina Biotech.