Reconstituting the immune killing functions and improving the pharmacokinetics of nanobodies by rhamnolipid conjugation.

Nanobodies (Nbs) hold great promise as next-generation cancer immunotherapies, but their efficacy is hindered by their poor pharmacokinetics and the inability to trigger Fc-mediated immune killing functions. To address these limitations, we designed and synthesized rhamnolipid-modified Nbs as a type of antibody-recruiting molecule by site-specifically conjugating EGFR-targeting Nb 7D12 to a series of rhamnolipid derivatives, and their biological profiles were evaluated in vitro and in vivo. Investigation of the structure-activity relationship revealed that the number of rhamnose (Rha) units and the length of the PEG linker in the conjugates affected anti-tumor activities. Conjugate R5, which contained two Rha units and a PEG linker, exhibited the most potent antibody-dependent cell-mediated phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) activities. In vivo, R5 had a significantly longer half-life because of its ability to bind to serum albumin and endogenous anti-Rha antibodies, and it demonstrated potent in vivo antitumor activity in a xenograft mouse model of A431 tumor. Our findings highlight the potential of rhamnolipidation as a strategy to enhance the efficacy of Nbs in cancer immunotherapy and provide a cost-effective platform for improving the therapeutic efficiency of Nbs.