O-10.04 Adapter P329G-directed CAR T cells for modular targeting of lung cancer

<h3>Background</h3> Chimeric antigen receptor (CAR) T cell therapy has shown unconvincing response rates in treating solid tumors including lung cancer. Insufficient efficacy is due to several challenges such as antigen heterogeneity, antigen loss, immunosuppressive tumor microenvironment and therapy-associated toxicities. The anti-P329G CAR T cell adapter platform offers a modular and adaptable approach to address some of these limitations. Anti-P329G CAR T cells specifically recognize the P329G mutation in the Fc region of human IgG1 antibodies, allowing them to pair with these P329G-Fc-mutated antibodies targeting diverse antigens. This work evaluates the efficacy of anti-P329G CAR T cells in different lung cancer models targeting the cancer-associated antigens HER2/neu, mesothelin (MSLN) and EGFR. <h3>Materials and Methods</h3> Human second-generation anti-P329G CAR T cells were combined with P329G-Fc-mutated antibodies targeting HER2/neu, MSLN or EGFR. Effector functions were compared with conventional CAR T cells which directly target the same antigens. Anti-CD19 CAR T cells and untransduced T cells were used as controls<i>. In vitro</i> functional efficacy against recombinant protein and lung cancer cell lines was assessed with impedance-based cytotoxicity assays, ELISA- and FACS-based readouts. <i>In vivo</i> functionality was tested in immunodeficient mice bearing different lung cancer xenografts. Healthy precision cut lung slices (PCLS) with the addition of GFP-labeled A549 cells (GFP-A549-PCLS) or primary patient tumor-derived PCLS (tPCLS) were used as <i>ex vivo</i> models. <h3>Results</h3> Anti-P329G adapter CAR T cells combined with HER2/neu-, MSLN-, or EGFR-targeting antibodies demonstrated sufficient and specific activation, cytokine release, and cytotoxic capacity <i>in vitro</i> when co-incubated with recombinant protein or lung cancer cell lines. The platform demonstrated specificity, modularity, and reversibility. <i>In vivo,</i> anti-P329G CAR T cells combined with EGFR-directed P329G-Fc-mutated antibodies induced remissions in lung cancer-xenograft bearing mice. Increased numbers of anti-P329G CAR T cells were detectable one week after T cell plus antibody injection compared to injection of conventional anti-EGFR CAR T cells. <i>In vitro</i> and <i>in vivo</i> effector functions of anti-P329G CAR T cells were comparable to conventional tumor antigen-targeting CAR T cells. Anti-P329G adapter CAR T cells combined with EGFR-targeting antibodies were efficient in the <i>ex vivo</i> GFP-A549-PCLS model without harming healthy lung tissue as well as in the tPCLS model. In these models, higher apoptosis and reduced proliferation of tumor cells was only achieved in presence of the PG-mutated antibodies, corroborating the specificity of the approach. <h3>Conclusions</h3> The anti-P329G CAR T cell adapter platform provides a versatile and effective strategy for lung cancer treatment. Anti-tumor activity could be showcased <i>in vitro</i>, in immunodeficient mouse models and patient-derived <i>ex vivo</i> models. The ability of the platform to be combined with multiple target-specific antibodies makes it a promising tool to overcome some of the essential challenges of CAR T cell therapy in solid tumors. <b>S. Stock:</b> A. Employment (full or part-time); Modest; 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>V.D. Menkhoff:</b> None. <b>J. Nabhanizadeh:</b> A. Employment (full or part-time); Significant; Justus Liebig University. <b>L. Fertig:</b> None. <b>J. Dörr:</b> A. Employment (full or part-time); Significant; LMU University Hospital. <b>A. Hauptstein:</b> None. <b>L. Gregor:</b> A. Employment (full or part-time); Significant; LMU University Hospital. <b>K. Großhauser:</b> A. Employment (full or part-time); Significant; LMU University Hospital. <b>M. Henrich:</b> A. Employment (full or part-time); Significant; LMU University Hospital. <b>G.V. Hoffmann:</b> None. <b>R. Müller:</b> None. <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>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. 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>M. von Bergwelt-Baildon:</b> A. Employment (full or part-time); Significant; LMU University Hospital. <b>R. Savai:</b> A. Employment (full or part-time); Significant; Justus Liebig University. <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.