Targeting DNGR‐1 with Fangchinoline Elevates Dendritic Cell Antigen Cross‐Presentation‐Mediated Antitumor Immunity in Melanoma

ABSTRACT Cross‐presentation of exogenous antigens by dendritic cells (DCs) is essential for initiating CD8 ⁺ T cell–mediated anti‐tumor immunity. DNGR‐1 (CLEC9A), a C‐type lectin receptor on conventional type 1 DCs (cDC1s), facilitates uptake and cytosolic delivery of dead cell–associated antigens. However, no small‐molecule modulators of DNGR‐1 have been reported. Here, we identify fangchinoline—a natural bisbenzylisoquinoline alkaloid—as a small‐molecule modulator that enhances antigen cross‐presentation in a DNGR‐1‐dependent manner. Functional screening shows fangchinoline promotes MHC‐I–restricted cross‐presentation and CD8 ⁺ T cell priming. It binds a DNGR‐1 pocket (Trp209, Glu225, Arg226), confirmed by surface plasmon resonance, thermal shift, probe labeling, and mutagenesis. DNGR‐1 engagement activates the Syk–Nox2 pathway, increasing phagosomal reactive oxygen species and membrane permeabilization to enable cytosolic antigen access. Fangchinoline exerts subset‐dependent effects: in GM‐CSF/IL‐4–derived DCs and endogenous cDC1‐associated settings in vivo, it enhances cross‐presentation‐associated function, whereas in Flt3L‐induced DCs with high basal DNGR‐1 expression it promotes DNGR‐1‐dependent cell death. In melanoma models, fangchinoline synergizes with DC transfer and PD‐1 blockade to promote CD8 ⁺ T cell–mediated tumor control. It enhances antigen‐specific CD8 ⁺ T cell activation in human DC–T cell co‐cultures. Together, these findings establish fangchinoline as a DNGR‐1‐targeting modulator for context‐dependent enhancement of anti‐tumor immunity.