Directed Evolution of Stabilized Monomeric CD19 for Monovalent CAR Interaction Studies and Monitoring of CAR-T Cell Patients

CD19 is among the most relevant targets in cancer immunotherapy. However, its extracellular domain (ECD) is prone to aggregation and misfolding, representing a major obstacle for the development and analysis of CD19-targeted therapeutics. Here, we engineered stabilized CD19-ECD (termed SuperFolder) variants, which also showed improved expression rates and, in contrast to the wild type protein, they could be efficiently purified in their monomeric forms. Despite being considerably more stable, these engineered mutants largely preserved the wild type sequence (>98.8%). We demonstrate that the variant SF05 enabled the determination of the monovalent affinity between CD19 and a clinically approved FMC63-based CAR, as well as monitoring and phenotypic characterization of CD19-directed CAR-T cells in the blood of lymphoma patients. We anticipate that the SuperFolder mutants generated in this study will be highly valuable tools for a range of applications in basic immunology and CD19-targeted cancer immunotherapy.

Automated summary

Engineered stable CD19-ECD variants (SuperFolder) have been developed, with improved expression rates and efficient purification in monomeric forms, overcoming obstacles in the development and analysis of CD19-targeted therapeutics. These mutants preserve the wild type sequence (>98.8%) and hold promise for a range of applications in basic immunology and CD19-targeted cancer immunotherapy.