Recombinant Concanavalin a as a proximity-activated glycan-binding module for nanobody-mediated intracellular delivery

Tumor cells display aberrant glycosylation patterns, making these altered glycans promising targets for selective oncology therapies. Lectins, which specifically recognize distinct glycan structures, have been widely investigated for their anticancer efficacy and intracellular delivery potential. However, clinical application is severely hindered by off-target toxicity, as glycans are ubiquitously expressed on healthy cells. To overcome this limitation, we developed a targeted drug delivery platform by fusing an antigen-specific nanobody with Concanavalin A (ConA), a mannose/glucose-binding lectin. Nanobodies offer high stability and ease of production, but their therapeutic efficacy depends on efficient receptor-mediated internalization. To enhance this internalizing capability, we engineered a fusion protein connecting a nanobody to a recombinant ConA (hereafter rConA) expressed in Escherichia coli. Characterization of the rConA revealed diminished cell binding and internalization compared to plant-derived ConA, which concurrently resulted in reduced cytotoxicity. Utilizing a green fluorescent protein (GFP)-targeting nanobody, the linker length was optimized to alleviate steric hindrance and maximize structural flexibility, identifying an 8 × G4S linker as the configuration that gave the highest binding and internalization among the lengths tested. This optimized framework was subsequently applied to the HER2-targeting nanobody, 2rs15d. The rConA-HER2 nanobody retained its HER2 binding affinity while significantly enhancing HER2 internalization in HER2-positive cells. Finally, conjugating the cytotoxic payload DM1 to the rConA-HER2 nanobody maintained HER2 specificity, exerting potent cytotoxicity against HER2-positive cells while exhibiting minimal toxicity toward HER2-low cells. These findings demonstrate that rConA can serve as a functional module to boost intracellular delivery, highlighting a promising strategy to enhance nanobody-based therapeutics.