Multimodality imaging of coiled-coil mediated self-assembly in a "drug-free" therapeutic system.

Abstract

Two complementary coiled-coil peptides CCE/CCK are used to develop a "drug free" therapeutic system, which can specifically kill cancer cells without a drug. CCE is attached to the Fab' fragment of anti-CD20 1F5 antibody (Fab'-CCE), and CCK is conjugated in multiple grafts to poly[N-(2-hydroxypropyl)methacrylamide] (P-(CCK)x ). Two conjugates are consecutively administered: First, Fab'-CCE coats peptide CCE at CD20 antigen of lymphoma cell surface; second, CCE/CCK biorecognition between Fab'-CCE and P-(CCK)x leads to coiled-coil formation, CD20 crosslinking, membrane reorganization, and ultimately cell apoptosis. To prove that two conjugates can assemble at cell surface, multiple fluorescence imaging studies are performed, including 2-channel FMT, 3D confocal microscopy, and 4-color FACS. Confocal microscopy shows colocalization of two fluorescently labeled conjugates on non-Hodgkin's lymphoma (NHL) Raji cell surface, indicating "two-step" targeting specificity. The fluorescent images also reveal that these two conjugates can disrupt normal membrane lipid distribution and form lipid raft clusters, leading to cancer cell apoptosis. This "two-step" biorecognition capacity is further demonstrated in a NHL xenograft model, using fluorescent images at whole-body, tissue and cell levels. It is also found that delaying injection of P-(CCK)x can significantly enhance targeting efficacy. This high-specificity therapeutics provide a safe option to treat NHL and other B cell malignancies.