Figure 6. Naïve IGHV4-39-derived antibodies from naïve B cells.

(a) Schematics of naïve IGHV4-39 antibody phage library generation. CD19+ CD27− IgM+ naïve B cells were isolated individually by FACS from 36 donors. Total Ig RNA was converted into cDNA using an IgM specific reverse primers, and then uniquely barcoded IGHV4-39 primers for each donor were used to selectively amplify the IGHV4-39 VH gene from the cDNA. The amplified VH genes were then pooled together and paired with the light chain variable genes from IGKV families 1–4 amplified from the same set of donors to generate the single-chain Fv library. Antibody libraries were then displayed on phage and 4 rounds of panning against recombinant IsdB NEAT1 were performed. (b) Binding characterization of IGHV4-39 encoded naïve NEAT1-binding antibodies from seven different donors. Heavy and light chain germlines usage, CDR-H3 sequence identities and number of variable heavy chain framework nucleotide mutation of the seven NEAT1 binders are shown. The observed single framework mutation in selected clones may have been introduced by the amplification process during library generation. The binding of the parental antibodies and their Y52A/Y53A variants to full-length IsdB was determined by SPR-based biosensor binding analysis at 37 °C. The binding of the parental antibodies to the full-length IsdB Y165R variant was determined by ELISA (n=2).