Abstract LB-34: Targeted delivery of siRNAs to diverse solid cancers by a novel immunoconjugate comprising tetrameric human protamine linked to an internalizing anti-TROP-2 humanized antibody

Abstract

Abstract Despite the potential of RNA interference (RNAi) to silence specific genes, the full therapeutic potential of RNAi remains to be realized due to the lack of an effective delivery system to target cells in vivo. To address this critical need and prompted by the promising results obtained with fusion proteins comprising the nucleic-acid-binding human protamine and a cell-targeting Fab or scFv, we have developed novel immunoconjugates having multiple copies of human protamine tethered to a tumor-targeting, internalizing antibody for targeted delivery of siRNAs in vivo. The Dock-and-Lock (DNL) method developed by us provides the design and strategy for building such multifunctional agents. DNL enables site-specific, self-assembly of two modular components with each other, resulting in a stable covalent structure of defined composition with retained bioactivity. The basic strategy of DNL involves the generation of two types of modules, one containing the dimerization and docking domain (DDD) of cAMP-dependent protein kinase (PKA) and the other containing the anchoring domain (AD) of a related A-kinase anchoring protein (AKAP). Docking a DDD-containing module with an AD-containing module occurs spontaneously, and the resulting complex is locked with disulfide bonds to enhance in vivo stability. We generated DDD2-L-thP1, a DDD-module of truncated human protamine (thP1, residues 8 to 29 of full human protamine 1), in which the sequences of DDD2 and thP1 are fused to the light chain of a humanized antibody at the N- and C-termini, respectively. The reaction of DDD2-L-thP1 under mild redox conditions with hRS7-IgG-AD2, an AD-module derived from an anti-TROP-2, internalizing, humanized antibody, resulted in the facile formation of the desirable DNL complex, designated E1-L-thP1, which contains four copies of thP1 appended to the carboxyl termini of the heavy chains. The purity and molecular integrity of E1-L-thP1 following Protein A purification was shown by size-exclusion HPLC and SDS-PAGE. In addition, the ability of E1-L-thP1 to bind plasmid DNA or siRNAs was demonstrated by the gel shift assay. E1-L-thP1 was effective in protecting the bound DNA from digestion by nucleases added to the sample or present in serum, and its function to carry siRNAs into TROP-2 expressing cancer cells was confirmed by fluorescence microscopy using FITC-conjugated siRNAs and the human lung cancer cell line, Calu-3. Further evaluation of the in vitro and in vivo efficacy of E1-L-thP1 for delivery of CD74-specific siRNAs to treat TROP-2 expressing pancreatic cancer is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-34.