10.3389/fimmu.2019.02630.s001 Theresa Pesch Theresa Pesch Lucia Bonati Lucia Bonati William Kelton William Kelton Cristina Parola Cristina Parola Roy A. Ehling Roy A. Ehling Lucia Csepregi Lucia Csepregi Daisuke Kitamura Daisuke Kitamura Sai T. Reddy Sai T. Reddy Data_Sheet_1_Molecular Design, Optimization, and Genomic Integration of Chimeric B Cell Receptors in Murine B Cells.docx Frontiers 2019 B cells synthetic antigen receptor cellular engineering genome editing cellular immunotherapy CRISPR-Cas9 2019-11-14 04:33:44 Dataset https://frontiersin.figshare.com/articles/dataset/Data_Sheet_1_Molecular_Design_Optimization_and_Genomic_Integration_of_Chimeric_B_Cell_Receptors_in_Murine_B_Cells_docx/10302434 <p>Immune cell therapies based on the integration of synthetic antigen receptors comprise a powerful strategy for the treatment of diverse diseases, most notably T cells engineered to express chimeric antigen receptors (CAR) for targeted 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 using CRISPR-Cas9 into immortalized B cell hybridomas. Subsequently, we developed a reliable and consistent pipeline to precisely introduce cassettes of several kb size into the genome of primary murine B cells also using 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 CBCRs and our approach for engineering primary B cells will be a valuable tool for the advancement of future B cell- based immune cell therapies.</p>