MT3-KN035 Nanoparticles Based on PD-L1 Nanobodies Allow for Multiple Drug Conjugations that Promote Chemo- and...

Nanobody-based therapeutics that incorporate multifunctional drug molecules into nanobodies (nanobody-drug conjugates, NDCs) have gained substantial attention as promising therapeutic strategies in biomedicine due to their unique ability to precisely target specific cancer cells. However, the efficacy of NDCs depends on efficient internalization of the antibody-antigen complex to facilitate intracellular drug release in the lysosome, and not all surface antigens suffer from internalization upon antibody binding, posing a significant limitation to the effectiveness of NDCs. To address this gap, we rationally designed and constructed MT3 and anti-PD-L1 nanobody (Nb-PD-L1:KN035) fusion protein that enabled self-assembly into nanoparticles during the expression process. The MT3 domain of MT3-KN035 fusion protein nanoparticles did not interrupt the binding affinity between KN035 and PD-L1. In addition, MT3-KN035 protein nanoparticles not only migrated from the PD-L1-positive cell surface to intracellular lysosomes in vitro but also hold a huge targeting potential for tumor regions in vivo. Meanwhile, MT3-KN035 conjugated multiple aldoxorubicin (hereafter, these conjugates are referred to as MT3-KN035-DOX) via an acidic cleavable linker; MT3-KN035-DOX^High induced tumor apoptosis and exhibited significant antitumor efficacy via facilitating immune cell infiltrations. This work provides a novel therapeutic strategy via nanobody-mediated target engagement and internalization that achieves synergistic therapeutic efficacy between chemotherapy and immune checkpoint blockade therapy.