Efficient reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire

Abstract

Abstract B cells have been engineered ex vivo to express an HIV-1 broadly neutralizing antibody (bNAb). B-cell reprograming may be scientifically and therapeutically useful, but current approaches limit B-cell repertoire diversity and disrupt the organization of the heavy-chain locus. A more diverse and physiologic B-cell repertoire targeting a key HIV-1 epitope could facilitate evaluation of vaccines designed to elicit bNAbs, help identity more potent and bioavailable bNAb variants, or directly enhance viral control in vivo . Here we address the challenges of generating such a repertoire by replacing the heavy-chain CDR3 (HCDR3) regions of primary human B cells. To do so, we identified and utilized an uncharacterized Cas12a ortholog that recognizes PAM motifs present in human JH genes. We also optimized the design of 200 nucleotide homology-directed repair templates (HDRT) by minimizing the required 3’-5’ deletion of the HDRT-complementary strand. Using these techniques, we edited primary human B cells to express a hemagglutinin epitope tag and the HCDR3 regions of the bNAbs PG9 and CH01. Those edited with bNAb HCDR3 efficiently bound trimeric HIV-1 antigens, implying they could affinity mature in vivo in response to the same antigens. This approach generates diverse B-cell repertoires recognizing a key HIV-1 neutralizing epitope.