Molecular design, optimization and genomic integration of chimeric B cell receptors in murine B cells

Abstract

Immune cell therapies based on the integration of synthetic antigen receptors provide a powerful strategy for the treatment of diverse diseases, most notably retargeting T cells engineered to express chimeric antigen receptors (CAR) for cancer therapy. In addition to T lymphocytes, B lymphocytes may also represent valuable immune cells that can be engineered for therapeutic purposes such as protein replacement therapy or recombinant antibody production. In this article, we report a promising concept for the molecular design, optimization and genomic integration of a novel class of synthetic antigen receptors, chimeric B cell receptors (CBCR). We initially optimized CBCR expression and detection by modifying the extracellular surface tag, the transmembrane regions and intracellular signaling domains. For this purpose, we stably integrated a series of CBCR variants into immortalized B cell hybridomas using CRISPR-Cas9. Subsequently, we developed a reliable and consistent pipeline to precisely introduce cassettes of several kilobases size into the genome of primary murine B cells, again via CRISPR-Cas9 induced HDR. Finally, we were able to show the robust surface expression and antigen recognition of a synthetic CBCR in primary B cells. We anticipate that CBCRs and our approach for engineering primary B cells will be a valuable tool for the advancement of future B cell-based immune therapies.