Background Although T cells have been a central focus of cancer immunotherapy studies, there is a growing appreciation for the role of B cells in anti-tumor immunity. We recently reported that increased B cell gene signature expression and B cell receptor diversity in pretreatment samples were associated with clinical response to immune checkpoint blockade (ICB) in triple negative breast cancer (TNBC).1 In murine models of TNBC, ICB response has been found to be dependent upon B cell responses.2 Beyond TNBC, intratumoral presence of tertiary lymphoid structures (TLS; enriched with B cells, T cells and dendritic cells (DCs)) have been associated with ICB response in various cancer types.3–7 Deeper understanding of B cell responses in the context of ICB is needed.
Methods We evaluated tumor antigen-specific antibody responses in patients with metastatic triple negative breast cancer treated with pembrolizumab following low dose cyclophosphamide therapy using custom peptide arrays (figure 1).
Results We found that a minority of predicted linear epitopes were associated with antibody signal, and antibody signal was associated with both mutated epitopes and self epitopes. No associations were observed between antibody signal and subcellular localization or RNA expression of parent proteins (figure 2). Patient-specific patterns of antibody signal boostability (e.g., antibody signal increase on-therapy) were observed and were independent of clinical response (figure 3). Intriguingly, measures of cumulative antibody signal intensity relative to immunotherapy treatment showed that the one complete responder in the trial had the greatest increase in total antibody signal (figure 3), which supports a putative association between ICB-dependent antibody boosting and clinical response. The antibody boost in the complete responder was largely driven by increased levels of IgG specific to a sequence of N-terminal residues in native Epidermal Growth Factor Receptor Pathway Substrate 8 (EPS8) protein (figure 4), a known oncogene in several cancer types including breast cancer. Structural protein prediction analysis showed that the antibody-targeted region of EPS8 was in a region of the protein with mixed linear/helical structure, and that this region was solvent-exposed and not predicted to bind to interacting macromolecules (figure 4).
Conclusions High-throughput peptide arrays can be used to map tumor antigen-specific antibody responses. This study highlights the potential importance of humoral immune responses recognizing neoantigen epitopes and unmutated self protein epitopes in anti-tumor immunity. Future studies will be needed to better elucidate the role of tumor antigen-specific antibodies in tumor growth inhibition and immunotherapy response.
Funding Merck Sharp & Dohme, a subsidiary of Merck & Co, Kenilworth, NJ, USA (MSD) provided financial support for the clinical trial under which samples from this study were acquired. This work was also supported by Susan G. Komen for the Cure (BV), V Foundation for Cancer Research (BV), and UNC University Cancer Research Fund (MW, JS, BV).
The authors thank Ken Fowler and Karen McKinnon with the Immune Monitoring and Genomics Facility (IMGF) at UNC-Chapel Hill for their assistance with this study. We also thank the patients in this study and their families, without whom this study would not have been possible.
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