P09.10 Competitive modulation of anti-P329G adapter CAR T cell activity using an untargeted CAR-adapter molecule

Background Chimeric antigen receptor (CAR) T cell therapy has demonstrated limited efficacy in treating solid tumors like lung cancer. This insufficient therapeutic response is mainly attributed to challenges such as antigen loss, antigen heterogeneity, and treatment-associated toxicities. The anti-P329G CAR T cell adapter platform combined with P329G-Fc-mutated antibodies provides a modular and flexible strategy to overcome these obstacles. Toxicity control can be achieved by interruption of the administration of the CAR-adapter molecule or by step-up dosing. Although antibody dosing enables partial control over therapy-associated toxicity, the capacity to modulate CAR T cell activity becomes limited once the antibody is present in the system. In this project, we aim to explore whether the activity of anti-P329G adapter CAR T cells can be attenuated through the competitive administration of a non–antigen-binding, inert P329G-mutated CAR-adapter molecule.Methods Second generation anti-P329G CAR T cells were activated with an EGFR-recognizing P329G-Fc-mutated antibody to evaluate the impact of varying concentrations and administration time points of a non–antigen-binding DP47-based antibody. Controls included CAR T cells exposed to a single antibody, unstimulated CAR T cells, and untransduced cells. In vitro activation following stimulation with recombinant protein or co-culture with EGFR-expressing lung cancer cells was assessed by measuring cytokine production using ELISA or FACS-based assays. Cytotoxic efficacy against tumor cells was measured with impedance-based cytotoxicity assays.Results Anti-P329G adapter CAR T cells combined with the EGFR-targeting antibody demonstrated antigen-specific activation, cytokine release, and cytotoxic activity in vitro. Additional administration of the untargeted antibody led to a trend toward reduced cytokine production after stimulation with recombinant protein or co-culture with EGFR-expressing lung cancer cells. Despite this reduction, CAR T cells maintained full cytotoxic capacity in the presence of the untargeted antibody. Stepwise increase of the non–antigen-binding to antigen-binding antibody up to 1000x did not fully inhibit anti-P329G CAR T cell activity. Altering the sequence of antibody administration did not further enhance the reduction of activation. Whereas removal of the anti-EGFR antibody led to a relevant reduction of CAR T cell activation. Compared to discontinuing the administration of the EGFR antibody, the untargeted antibody did not provide additional benefit.Conclusion Administration of an untargeted P329G-Fc-mutated antibody resulted in a slight trend toward reduced activation of anti-P329G adapter CAR T cells. However, this intervention did not provide additional benefit beyond that achieved by simple withdrawal of the anti-EGFR antibody administration. These findings indicate that step-up dosing and scheduled discontinuation remain the most effective strategies for toxicity control in adapter CAR T cell therapy. This approach further underscores the inherent advantages of adapter CAR T cells, particularly their modularity and potential for improved safety in immunotherapy. M. Surowka: Previous employment and patents (significant): Roche M. Subklewe: Research Grant (significant): Amgen, BMS/Celgene, Gilead/Kite, Janssen, Miltenyi Biotec, Novartis, Roche, Seattle Genetics, Takeda. Speakers Bureau/Honoraria (significant): Amgen, BMS/Celgene, Gilead/Kite, Novartis. Consultant/Advisory Board (significant): Janssen, Takeda, AstraZeneca, Pfizer, Ichnos Sciences, AvenCell, Incyte. M. von Bergwelt Baildon: Consultancy, Research Funding, and Honoraria (significant): MSD Sharp & Dohme, Novartis, Roche, Kite/Gilead, Bristol Myers Squibb, Astellas, Mologen, and Miltenyi C. Klein: Previous employment, stock ownership, patents (significant): Roche S. Kobold: Research Grant (significant): TCR2 Inc., Arcus Biosciences, Tabby Therapeutics, Plectonic, Catalym GmbH. Speakers Bureau/Honoraria (significant): BMS, GSK, Novartis, TCR2 Inc., Miltenyi Biotech. Ownership Interest/patents (significant): LMU University Hospital. Other (Significant): Carina Biotech, TCR2 Inc. The remaining authors have nothing relevant for this submission to declare.