T-cell-based adoptive immunotherapy is a new pillar of cancer care. Tumor-redirected B cells could also contribute to therapy if their manipulation to rewire immunoglobulin (Ig) genes is mastered. We designed a single-chain Ig-encoding cassette (“scFull-Ig”) that redirects antigen specificity when inserted at a single position of the IgH locus. This design, which places combined IgH and IgL variable genes downstream of a pVH promoter, nevertheless preserves all Ig functional domains and the intrinsic mechanisms that regulate expression from the IgM B cell receptor (BCR) expression to Ig secretion, somatic hypermutation and class switching. This single-locus editing provides 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 human tumor antigens, HER2 and hCD20. Once validated in cell lines, the strategy was extended to primary B cells, confirming the successful engineering of BCR and Ig expression and the ability of scFull-Ig to undergo further class switching. These results further 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.

ABSTRACT Lymphocytes have become attractive agents for adoptive immunotherapy but only the reformatting of T cells is efficiently mastered. Despite some recent breakthroughs, B cells remain challenging targets, with regard to both their long-term survival after in vitro manipulation and the rewiring of immunoglobulin (Ig) expression. Working on these two aspects, we have designed a new format of single-chain Ig (“scFull-Ig”) coding cassette, the insertion of which at a single genomic position can redirect B cells toward a new antigen specificity, while preserving all functional domains of the B cell receptor. Precise genomic edition at a single locus then provides the most efficient and safe strategy to both disrupt endogenous Ig expression while encoding a new Ig paratope. As proofs of concept, functionality of such scFull BCR was validated by checking specific binding of two different classical targets of tumor immunotherapy, HER2 and CD20. Once the strategy validated in cell lines, it was also validated in primary B cells, again showing successful engineering of BCR expression. These results contribute to pave the way for future B cell-based adoptive cell therapy.

Since the early days of vaccination, targeted immunotherapy has gone through multiple conceptual changes and challenges. It now provides the most efficient and up-to-date strategies for either preventing or treating infections and cancer. Its most recent and successful weapons are autologous T cells carrying chimeric antigen receptors, engineered purposely for binding cancer-specific antigens and therefore used for so-called adoptive immunotherapy. We now face the merger of such achievements in cell therapy: using lymphocytes redirected on purpose to bind specific antigens and the  clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) revolution, which conferred genome-editing methodologies with both safety and efficacy. This unique affiliation will soon and considerably expand the scope of diseases susceptible to adoptive immunotherapy and of immune cells available for being reshaped as therapeutic tools, including B cells. Following the monumental success story of passive immunotherapy with monoclonal antibodies (mAbs), we are thus entering into a new era, where a combination of gene therapy/cell therapy will enable reprogramming of the patient's immune system and notably endow his B cells with the ability to produce therapeutic mAbs on their own.

L'utilisation des anticorps monoclonaux a révolutionné la fabrication de réactifs d'hématologie. Des anticorps sont disponibles pour la plupart des antigènes sanguins, cependant l'isotype de la chaîne lourde des immunoglobulines restreint leur application en tant que réactif. Pour lever cette limitation et valoriser un patrimoine existant, la modification d'hybridomes établis sécréteurs d'IgG humaines a été réalisée à l'aide des outils CRISPR Cas9 pour l'obtention d'anticorps de type IgM. Trois hétéro-hybridomes humains sécréteur d'anticorps anti-FY1, -KEL1 et MNS3 ont été co-transfectés avec un complexe gRNA-Cas9 et un ADN donneur double brin contenant le gène codant pour la chaîne mu des immunoglobulines. Après sélection des clones modifiés par clonage, les surnageants de culture ont été analysés par cytométrie afin d'isoler les clones ayant intégrés la cassette d'intérêt. La stabilité des clones obtenus, ainsi que leurs utilisations pour la fabrication de réactifs d'immuno-hématologie est actuellement en cours. Cependant, l'utilisation d'outils CRISPR pour la modification d'hybridome, en particulier le « rétro-switch », a été validé dans cette étude. D'autres modifications permettant d'améliorer des clones existants sont à l'étude et devrait permettre d'enrichir la source de matières premières disponible pour les industriels du domaine du diagnostic.