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A yeast surface display platform for characterizing CAR T cell responses to cancer antigens

Nature Communications. 2025-11; 
Marcus Deichmann, Giovanni Schiesaro, Keerthana Ramanathan, Katrine Zeeberg, Nanna M T Koefoed, Maria Ormhøj, Rasmus U W Friis, Ryan T Gill, Sine R Hadrup, Emil D Jensen, Michael K Jensen Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark
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Peptide Synthesis cultures were diluted 50-fold by transfer to 200 µL volumes comprising a 10-fold dilution series of ɑ-factor (0–100 µM) (peptide-seq.: WHWLQLKPGQPMY) (GenScript) in SC + 1%DMSO for each replicate, 50,000 cells were transferred to 300 µL volumes comprising a 10-fold dilution series of ɑ-factor (0–100 µM) (GenScript) dissolved in SC + 1%DMSO DIX45 (Ste2) and DIX59 (No GPCR) was stimulated at pH=5 with 0 µM, 0.01 µM, and 1 µM ɑ-factor (GenScript), DIX60 (Mam2) at pH=5 with 0 µM, 1 µM, and 10 µM P-factor (peptide-seq.: TYADFLRAYQSWNTFVNPDRPNL) (GenScript) In addition, the 1.0x co-cultures were assessed with supplementation of 20 µM ɑ-factor (GenScript). cultures were diluted 50-fold by transfer to 200 µL volumes comprising a 10-fold dilution series of ɑ-factor (0–1 µM) (GenScript) in SC + 1%DMSO To establish different antigen-density conditions, each yeast strain was stimulated through the controlling GPCR differentially by 20-fold culture dilutions into a 10-fold dilution series of ɑ-factor (0-1 µM) (GenScript) in full SC. Get A Quote

Abstract

Chimeric antigen receptor (CAR) T cells have become an established immunotherapy with promising results for the treatment of hematological malignancies. However, modulation of the targeted antigen's surface level in cancer cells affects the quality and safety of CAR-T cell therapy. Here we present an engineered yeast-based antigen system for simulation of cancer cells with precise regulation of surface-antigen densities, providing a tool for controlled activation of CAR T cells and systematic assessment of antigen density effects. This Synthetic Cellular Advanced Signal Adapter (SCASA) system uses G protein-coupled receptor signaling to control cancer antigen densities on the yeast surface and provides a custom... More

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