Nanobody–antigen conjugates elicit HPV-specific antitumor immune responses

High-risk human papillomavirus-associated cancers express viral oncoproteins (e.g., E6 and E7) that induce and maintain the malignant phenotype. The viral origin of these proteins makes them attractive targets for development of a therapeutic vaccine. Camelid-derived single-domain antibody fragments (nanobodies or VHHs) that recognize cell surface proteins on antigen-presenting cells (APC) can serve as targeted delivery vehicles for antigens attached to them. Such VHHs were shown to induce CD4 ^þ and CD8 ^þ T-cell responses against model antigens conjugated to them via sortase, but antitumor responses had not yet been investigated. Here, we tested the ability of an anti-CD11b VHH (VHH _CD11b ) to target APCs and serve as the basis for a therapeutic vaccine to induce CD8 ^þ T-cell responses against HPV ^þ tumors. Mice immunized with VHH _CD11b conjugated to an H-2D ^b -restricted immunodominant E7 epitope (E7 _49-57 ) had more E7-specific CD8 ^þ T cells compared with those immunized with E7 _49-57 peptide alone. These CD8 ^þ T cells acted prophylactically and conferred protection against a subsequent challenge with HPV E7-expressing tumor cells. In a therapeutic setting, VHH _CD11b -E7 _49-57 vaccination resulted in greater numbers of CD8 ^þ tumor–infiltrating lymphocytes compared with mice receiving E7 _49-57 peptide alone in HPV ^þ tumor-bearing mice, as measured by in vivo noninvasive VHH-based immune-positron emission tomography (immunoPET), which correlated with tumor regression and survival outcome. Together, these results demonstrate that VHHs can serve as a therapeutic cancer vaccine platform for HPV-induced cancers.