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

Abstract

Abstract 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. Funding Source Vanderbilt Institute for Clinical and Translational Research (VICTR) Topic Categories Immune Mechanisms of Human Disease (HUM)