Department of Pediatrics, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.
The Center for Integrated Solutions to Infectious Diseases (CISID), The Broad Institute of MIT and Harvard, Cambridge, MA, USA.
Nat Biomed Eng. 2024 Apr;8(4):361-379. doi: 10.1038/s41551-024-01179-6. Epub 2024 Mar 14.
Mice adoptively transferred with mouse B cells edited via CRISPR to express human antibody variable chains could help evaluate candidate vaccines and develop better antibody therapies. However, current editing strategies disrupt the heavy-chain locus, resulting in inefficient somatic hypermutation without functional affinity maturation. Here we show that these key B-cell functions can be preserved by directly and simultaneously replacing recombined mouse heavy and kappa chains with those of human antibodies, using a single Cas12a-mediated cut at each locus and 5' homology arms complementary to distal V segments. Cells edited in this way to express the human immunodeficiency virus type 1 (HIV-1) broadly neutralizing antibody 10-1074 or VRC26.25-y robustly hypermutated and generated potent neutralizing plasma in vaccinated mice. The 10-1074 variants isolated from the mice neutralized a global panel of HIV-1 isolates more efficiently than wild-type 10-1074 while maintaining its low polyreactivity and long half-life. We also used the approach to improve the potency of anti-SARS-CoV-2 antibodies against recent Omicron strains. In vivo affinity maturation of B cells edited at their native loci may facilitate the development of broad, potent and bioavailable antibodies.
通过 CRISPR 编辑表达人抗体可变区的小鼠 B 细胞过继转移，可用于评估候选疫苗并开发更好的抗体疗法。然而，目前的编辑策略会破坏重链基因座，导致体细胞高频突变效率低下，而没有功能亲和力成熟。在这里，我们通过在每个基因座处用单个 Cas12a 介导的切割以及与远端 V 片段互补的 5'同源臂，直接且同时用人类抗体替换重组的小鼠重链和 κ 链，展示了这些关键 B 细胞功能可以被保留。以这种方式编辑以表达人类免疫缺陷病毒 1（HIV-1）广谱中和抗体 10-1074 或 VRC26.25-y 的细胞能够强烈高频突变，并在接种疫苗的小鼠中产生有效的中和性血浆。从小鼠中分离出的 10-1074 变体比野生型 10-1074 更有效地中和了更广泛的 HIV-1 分离株，同时保持其低多反应性和长半衰期。我们还使用该方法提高了针对最近的 Omicron 株的抗 SARS-CoV-2 抗体的效力。在天然基因座编辑的 B 细胞的体内亲和力成熟可能有助于开发广泛、有效和可生物利用的抗体。
