Design and Characterization of an Anti-Clec9a Antibody Armed with CpG Oligonucleotides To Enable Targeted Delivery of Adjuvant to Cross-Presenting Dendritic Cells.

C-type lectin receptor, Clec9a, is a highly specific receptor expressed on cross-presenting conventional dendritic cells (cDC1). This receptor specificity for this rare population of dendritic cells (DCs), combined with their inherent ability to internalize and localize to the endocytic compartment, presents a unique opportunity for targeted delivery of innate immune agonists. By leveraging an anti-Clec9a antibody, we can specifically deliver these agonists to cross-presenting cDCs, thereby enhancing the cross-priming and expansion of tumor-specific cytotoxic T lymphocytes (CTLs). In this study, we detail the design and characterization of innovative bioconjugates composed of a Clec9a-targeting antibody and CpG oligodeoxynucleotides (ODNs), which activate Toll-like receptor 9 (TLR9) expressed by cDC1. These immunomodulatory bioconjugates exhibited significant cellular activity in cross-presentation assays within a coculture system of in vitro generated CD103+ DCs (iCD103 DCs), tumor cells, and antigen specific CTLs, driving robust cross priming. Furthermore, the targeted delivery of ODNs to cDC1 in vivo demonstrated target-dependent activity in a murine vaccination assay. Our findings underscore the potential of Clec9a-targeted bioconjugates as a powerful strategy to enhance antitumor immunity by precisely delivering adjuvants to cross-presenting dendritic cells.