Figure 5.

Determining antigen-specificity sequence landscapes by deep mutational scanning (DMS). Heat maps and their corresponding sequence logo plots from the results of DMS of all variable heavy chain CDRs in both the HEL23 (A) and HEL24 (B) variants reveals positional variance for antigen-binding is clone specific. Libraries of single point-mutations across all heavy chain CDRs were integrated into hybridomas by HDM. NGS was then performed on libraries pre- and post-screening for antigen specificity (Supplementary Figure S9 and Table S3). Clonal (CDRH3) frequencies of variants post-screening were divided by clonal frequencies of variants pre-screening to calculate ERs. Sequence logo plots are generated by normalizing all variants with an ER > 1 (or log[ER] > 0).