In Vivo Engineered B Cells Retain Memory and Secrete High Titers of Anti-HIV Antibodies in Mice
2Departement of Hematology, St. Jude Children's Research Hospital, USA
3Department of Clinical Microbiology and Immunology, Tel Aviv University, Israel
4Department of Immunology and Microbiology, The Scripps Research Institute, USA
5The Shmunis School of Biomedicine and Cancer Research, Tel Aviv University, Israel
A potential single-shot HIV therapy may be transplanted engineered B cells allowing strong secretion of broadly neutralizing antibodies (bNAbs). However, extensive, and expensive ex-vivo manipulations performed in specialized facilities hinder the clinical potential of this approach. Furthermore, allogeneic B cell therapy necessitates MHC-II compatibility to receive T-cell help.
To overcome these limitations, we engineer B cells in-vivo. In particular, we demonstrate that a single, systemic dose of dual AAV, one coding for CRISPR/Cas9 and another coding for a bNAb donor cassette, allows for site specific integration in B cells. Following immunizations, we show memory retention and bNAb secretion at high titers. The engineered antibody coding genes underwent somatic hypermutation and positive selection occurred in a few positions, as indicated by high dn/ds values. Antibodies secreted by the engineered B cells were found to be of multiple isotypes and IgGs could neutralize autologous and heterologous pseudoviruses. Biodistribution of the donor AAV over time, indicated CRIPSR-dependent expansion of engineered B cells only in lymphatic tissues. We establish diminished CRISPR/Cas9 off-target cleavage, using unbiased, highly sensitive, CHANGE-Seq analysis, and on-target cleavage at undesired tissues is reduced by expressing Cas9 from a B cell specific promoter.
Eliciting a specific, neutralizing serological response to hypervariable viruses is a long-standing challenge in medicine. B cell engineering provides an opportunity to express therapeutic antibodies and to generate an adaptive and evolving immunity.