Bill McBride

Abstract 4445: Novel antibody-dendrimer conjugates efficiently complex plasmid DNA

Introduction: Cationic polymers, such as polylysine, polyethylenimine, or polyamidoamine (PAMAM)-based dendrimers, form complexes with nucleic acids. However, their potential applications in nanomedicne as non-viral vectors for delivering therapeutic genes or siRNAs remain a challenge. One approach to improve selectivity and potency of a dendrimeric nanoparticle may be achieved by conjugation with an antibody that internalizes upon binding to target cells. In this study, we describe the synthesis and initial characterization of a novel immunoconjugate, designated E1-G5/2, which was made by the modular Dock-and-Lock (DNL) method to comprise half of a generation 5 (G5) PAMAM dendrimer (G5/2) site-specifically linked to a stabilized dimer of Fab derived from hRS7, a humanized antibody that is rapidly internalized upon binding to the Trop-2 antigen expressed on various solid cancers. Methods: E1-G5/2 was prepared by combining two self-assembling modules, AD2-G5/2 and hRS7-Fab-DDD2, under mild redox conditions, followed by purification on a Protein L column. To make AD2-G5/2, we derivatized the AD2 peptide with a maleimide group to react with the single thiol generated from reducing a G5 PAMAM with a cystamine core and used reversed-phase HPLC to isolate AD2-G5/2. We produced hRS7-Fab-DDD2 as a fusion protein in myeloma cells. The molecular size, purity and composition of E1-G5/2 were analyzed by size-exclusion HPLC, SDS-PAGE, and Western blotting. The biological functions of E1-G5/2 were assessed by binding to an anti-idiotype antibody against hRS7, a gel retardation assay, and a DNase protection assay. Results: E1-G5/2 was shown by size-exclusion HPLC to consist of a major peak (>90%) flanked by several minor peaks. The three constituents of E1-G5/2 (Fd-DDD2, the light chain, and AD2-G5/2) were detected by reducing SDS-PAGE and confirmed by Western blotting. Anti-idiotype binding analysis revealed E1-G5/2 contains a population of antibody-dendrimer conjugates of different size, all of which are capable of recognizing the anti-idiotype antibody, thus suggesting structural imperfections in the commercial supply of the G5 dendrimer. Gel retardation assay showed E1-G5/2 was able to maximally condense plasmid DNA at a charge ratio of 6:1 (+/-), with the resulting dendriplexes completely protecting the complexed DNA from degradation by DNase I. Conclusion: The DNL method can be used to build dendrimer-based nanoparticles that are targetable with antibodies. Such agents may have improved properties as carriers of drugs, plasmids, or siRNAs for diverse applications in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4445. doi:10.1158/1538-7445.AM2011-4445