Single-hit genome editing in B cells to redirect their specificity toward tumor antigens

Abstract T-cell-based adoptive immunotherapy is a new pillar of cancer care. Despite recent breakthroughs, B cells remain a challenging target in terms of survival after in vitro manipulation and functional expression of rewired immunoglobulin (Ig) genes. To address these limitations, we designed a single-chain Ig-encoding cassette (“scFull-Ig”) that, when inserted at a single genomic position, redirects antigen specificity but preserves all the Ig functional domains and the intrinsic regulatory mechanisms that modulate Ig expression in activated cells from the IgM B-cell receptor (BCR) expression to Ig secretion and/or class switching. Single-locus editing may then provide an efficient and safe strategy to both disrupt endogenous Ig expression and encode a new Ig paratope. As a proof of concept, the functionality of scFull BCR and/or secreted Ig was validated against two different classical tumor antigens, HER2 and hCD20. Once validated in cell lines, the strategy was extended to primary B cells, and successful engineering of BCR and Ig expression was confirmed, as the ability of scFull-Ig to undergo further class switching was confirmed. These results pave the way for future B-cell-based adoptive immunotherapy and strategies to express a therapeutic mAb with a variety of switched H-chains that provide complementary functions.