Broad targeting of HPV E proteins by intratumoral and systemic B cell responses in HPV+ head and neck cancer.

B cells can account for a substantial proportion of tumor-infiltrating lymphocytes (TILs) and have been associated with improved outcomes in several malignancies. Yet, their antigen specificity, a key factor for ascribing a distinct function, is mostly unknown. Here, we demonstrate that in tumors of patients with human papillomavirus-positive head and neck cancer (HPV+ HNC) a substantial proportion of intratumoral antibody-secreting cells (ASCs) are specific for HPV E proteins. Using IgG ELISpots on TILs, we show that HPV E protein-specific ASCs are detectable in virtually all patients and can account for up to almost a third of all intratumoral IgG+ ASCs. ASC responses against the viral proteins E1 and E2 consistently outnumbered those against the classic oncoproteins E6 and E7. HPV-specific intratumoral ASCs correlated strongly between primary tumor and metastatic lymph nodes as well as with plasma IgG titers, indicating that intratumoral tumor-specific ASCs, at least partially, contribute to systemic anti-tumor responses. To further resolve the makeup of tumor-specific antibody responses, we developed a novel Multiplex Antigen Flow-based ImmunoAssay (MAFIA) enabling simultaneous and standardized quantification of HPV E-specific antibodies across all major Ig isotypes and IgG subclasses. Applying MAFIA to plasma samples of patients with HPV+ HNC revealed that HPV-specific plasma antibodies mainly consisted of IgG1, IgG3, and IgA, with the highest median responses directed against E1 and E2. Overall, our study delineates the antigenic hierarchy and makeup of HPV-specific humoral responses in HPV+ HNC, both intratumorally and systemically, thus providing a framework for future biomarker as well as next-generation therapeutics development.