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

Abstract

15Immune cell therapies based on the integration of synthetic antigen receptors provide 16 a powerful strategy for the treatment of diverse diseases, most notably retargeting 17 T cells engineered to express chimeric antigen receptors (CAR) for cancer therapy. In 18 addition to T lymphocytes, B lymphocytes may also represent valuable immune cells 19 that can be engineered for therapeutic purposes such as protein replacement therapy 20 or recombinant antibody production. In this article, we report a promising concept for 21 the molecular design, optimization and genomic integration of a novel class of 22 synthetic antigen receptors, chimeric B cell receptors (CBCR). We initially optimized 23 CBCR expression and detection by modifying the extracellular surface tag, the 24 transmembrane regions and intracellular signaling domains. For this purpose, we 25 stably integrated a series of CBCR variants into immortalized B cell hybridomas using 26 CRISPR-Cas9. Subsequently, we developed a reliable and consistent pipeline to 27 precisely introduce cassettes of several kilobases size into the genome of primary 28 murine B cells, again via CRISPR-Cas9 induced HDR. Finally, we were able to show 29 the robust surface expression and antigen recognition of a synthetic CBCR in primary 30 B cells. We anticipate that CBCRs and our approach for engineering primary B cells 31 will be a valuable tool for the advancement of future B cell-based immune therapies. 32 33 42 extracellular antigen-binding domain, typically an antibody fragment (e.g., a single-43 chain variable fragment [scFv]), linked by a spacer peptide to a transmembrane 44 domain, which is further fused to an intracellular T cell activation domain, such as 45 CD3ζ 9,10,11 . A broad range of extracellular binding domains and intracellular 46 costimulatory domains (e.g., CD28 and 4-1BB) have been incorporated into CARs to 47 65 has been severely compromised by technical challenges in their in vitro culture, 66

Vector Strategies to Actualize B Cell–Based Gene Therapies

“…Hung et al (18) inserted therapeutic protein transgenes under the control of exogenous promoters into the CCR5 safe harbor locus as this locus is not transcriptionally active in human B cells, it is not required for plasma cell differentiation, and null mutations are innocuous to humans. In a unique BCR editing approach, Pesch et al (19) modified murine B cells for the expression of a novel chimeric BCR (CBCR), at the Rosa26 safe harbor locus. A single-chain variable fragment acting as the extracellular Ag binding domain was joined to the CD28 transmembrane domain, and the cytoplasmic domain of the endogenous murine BCR was fused to the CD79b intracellular signaling domain via a spacer encoding a Strep tag.…”

“…A single-chain variable fragment acting as the extracellular Ag binding domain was joined to the CD28 transmembrane domain, and the cytoplasmic domain of the endogenous murine BCR was fused to the CD79b intracellular signaling domain via a spacer encoding a Strep tag. CBCRs could potentially offer a way to activate engineered B cells in an Ag-controllable manner (by vaccination), independent of the endogenous BCR (19). Luo et al (20) targeted an antiPD-1 Ab cassette to the GAPDH locus in mouse primary B cells for coexpression of the transgene along with the GAPDH enzyme.…”

Immune Literacy: Reading, Writing, and Editing Adaptive Immunity

“…Although integration was achieved, editing efficiencies for this strategy remained extremely low at less than 0.1%. Although not directly integrating antibodies, another study attempted to engineer murine B cells to express chimeric BCRs to sense antigen and in response secrete a protein of interest ( Pesch et al., 2019 ). Similar to other studies, it was found that HDR editing rates using the conventional B cell ex vivo culture protocol and dsDNA transfection was very low at less than 1%.…”

Development of Cellular Models to Study Efficiency and Safety of Gene Edition by Homologous Directed Recombination Using the CRISPR/Cas9 System

“…HDR is currently considered the most accurate GE strategy and therefore, it is the strategy of choice for several groups. HDR has been studied for the development of gene therapy treatments for cancer [ 17 , 18 ] and several genetic diseases such as beta-haemoglobinopathies [ 19 , 20 ], primary immunodeficiencies (PID) [ 21 , 22 ], and metabolic storage disorders [ 23 , 24 ]. However, there are several limitations in the HDR strategies that preclude its clinical translation, including low efficiency in some relevant target cells, as HDR only occurs in late S/G2 phase [ 25 , 26 , 27 , 28 , 29 ].…”