Identification of antigen-experienced human anti-HER2-specific B cells from peripheral blood 2256237

Introduction HER2 is a clinically validated oncogenic receptor and therapeutic antibody target; however, the natural occurrence and maturation of anti-HER2 antibodies in non-cancer individuals remain poorly characterized. Methods Peripheral blood mononuclear cells (PBMCs) from seven people with HIV with pre-diabetes and four people without HIV with diabetes were stained with fluorescently tagged double-barcoded HER2 ectodomain, and controls were stained with fluorescently tagged barcoded CMV gB (pre- and post-fusion). Anti-CD14, CD3, IgG, and CD19 antibodies were also included in the panel. Antigen-positive IgG+ B cells were sorted for paired B-cell receptor (BCR) and antigen barcode sequencing. The Linking B-cell Receptor to Antigen specificity through sequencing (LIBRA-seq) score for each antigen in the screening library was computed based on the unique molecular identifiers (UMIs) for the respective antigen barcode. Selected HER2-assigned antibodies were recombinantly expressed and assessed by ELISA. Results We profiled B cells from 160 million PBMCs, isolated 1,910 antigen- and IgG-positive B cells, obtained 936 paired heavy—light sequences, and retained 126 high-quality sequences. From donors without HIV, we had 29 million PBMCs from which we isolated 521, obtained 94 pairs, and retained 7. Antibody isotype profiles were IgG-skewed in PLWH, whereas HIV-negative donors showed more IgM. After filtering IgG sequences with strong read support (UMI > 10), we identified 13 antibody clones that bound HER2 (or HER2 plus CMV gB). Of these, 11/13 were from PLWH (10 IgG1, 1 IgG3), and 2/13 were from HIV-negative donors (both IgG2). All thirteen recombinant antibodies bound to HER2 with different strengths, compared to negative controls. Conclusion LIBRA-seq mapping of BCRs in non-cancer donors identified naturally occurring, antigen-experienced human anti-HER2 antibodies, showcasing its potential as a powerful tool for immune profiling and BCR sequencing with promising diagnostic and therapeutic applications.