Complete primer set for amplification and expression of full-length recombinant human monoclonal antibodies from single human B cells

Abstract

Human monoclonal antibodies (mAbs) are an important segment in precision therapeutics. Various methodologies are available for generating them. Recombinant human mAbs expression from sorted single B cells is preferred for its rapid expression using mammalian vectors while maintaining in vivo immunoglobulin (Ig) pairing. The success rate of generating recombinant mAbs from single sorted human B cells directly relies on Ig heavy (IgH) and light (IgL) gene coverage of the PCR primers. Existing primer sets fail to cover all functional human Ig gene rearrangements, exhibit high degeneracy leading to non-specific amplifications and mutations arising from primer mismatch/degeneracy, and require high amplification cycles. Some existing primer sets have high coverage but are not designed for expression as recombinant mAbs. Here, we have designed a primer set to amplify all functional V(D)J transcripts in human B cell repertoire using a nested RT-PCR approach. The resultant amplicons can be cloned into mammalian vectors for expression of recombinant mAb. Non-specific amplifications were minimized using isotype-specific primers for cDNA synthesis and limiting primer degeneracy. We validated the designed primers on single sorted B cells, bulk sorted B cells and peripheral blood mononuclear cells. We were successfully able to amplify paired heavy and light chain transcripts in 38.46 % (80/208) from naive, memory and B1 B cell subsets sorted as single B cells. Paired Ig transcripts from five single B cells were cloned into expression vectors and purified from mammalian cells as recombinant mAbs. Thus, our new primer set offers significant advantages over existing primers as it allows amplification of all functional V(D)J rearrangements, facilitating rapid generation of antigen-specific recombinant antibodies from diverse human B cell repertoires following vaccinations and infections previously inaccessible due to primer limitations.