Monoclonal antibody generation by controlled immunoglobulin gene rearrangements

Monoclonal antibodies (mAbs) are essential for various applications including experimental reagents, diagnostics, and therapeutics. Thus, the platform technologies that stably generate antigen-specific mAbs are increasingly crucial. We previously developed a method to generate mAbs, termed the "ADLib system", utilizing the avian-derived B cell line DT40. Avian immunoglobulin (Ig) genes diversify principally through gene conversion-a kind of homologous recombination. The ADLib system isolates antigen-specific clones from libraries constructed using DT40 cells treated with Trichostatin A (TSA), a histone deacetylase inhibitor that enhances gene conversion frequencies. The obtained antigen-specific clones are cultured without TSA to minimize further diversification. However, low-frequency spontaneous gene conversion still occurs, potentially leading to gradual changes in the specificity of the clones. To address this, we engineered conditional mutants of activation-induced deaminase (AID), the initiator of gene conversion, using auxin-inducible degron system which enables targeted protein degradation via the auxin-dependent ubiquitin-proteasome pathway. The addition of the phytohormone auxin led to the degradation of degron-tagged AID proteins, effectively halting gene conversion. Subsequently, we carried out the ADLib system using these clones and successfully isolated antigen-specific mAbs. These suggest that our AID conditional mutants provide a powerful tool for generating and stabilizing antigen-specific clones isolated by the ADLib system.