Generation of Allogeneic CAR T Cells through Specific Degradation of the T Cell Antigen Receptor by E3 Ubiquitin Ligase Fusion Proteins

• DOI: 10.1021/acssynbio.1c00397
• Publication Date: 2022-05-13T15:17:45Z
• Authors: Michael J. Harris; Hao Chen; Tianyu Cai; Yuting Yi; Qiaowen Deng; Yi Yao; Tianle Lan; Yanfeng Guo; Xiufang Xu; Xian Wen; Joshua E. McGee; Daniella Tatang; James Brock; Feng Shi; Li Zhou
• Keywords: Gene Editing; 0301 basic medicine; 03 medical and health sciences; T-Lymphocytes; Ubiquitin-Protein Ligases; Hematopoietic Stem Cell Transplantation; Receptors, Antigen, T-Cell

Receptor downregulation is instrumental for many therapeutic interventions. Receptor knockout through gene-editing technologies is efficient but can introduce off-target mutations and chromothripsis. Regulation of gene expression at the protein level is a promising alternative. Here, we present results showing the targeted T cell antigen receptor (TCR) degradation using chimeric E3 fusion proteins that we call Receptor Targeting Chimeras (ReceptorTAC). We show that TCR degradation is dependent on enzymatically active, membrane-anchored E3 ligase variants. TCR specificity was achieved by direct fusion of an E3 domain to the CD3ζ transmembrane sequence. Jurkat and primary T cells stably expressing the ReceptorTAC constructs showed significantly reduced responses to TCR stimulation. We also used our ReceptorTAC technology to generate TCR-deficient, claudin18.2-specific CAR T cells, where the activity of the CAR was unaffected by the expression of the ReceptorTAC. These data indicate that our ReceptorTAC molecule can be used to generate allogeneic CAR T cells.