Development of Aptamer-based Targeted Therapy: Aptamer Multi-drug Conjugate and its Advanced Effects

Abstract

Aptamer-drug conjugate (ApDC) is one of the targeted therapeutics that has the advantage of reduced toxicity and improved effects. This study reports on the multiple-conjugate of cytotoxic drugs to an aptamer, the HER2 targeting aptamer HB5. So, the developed platform of Aptamer Multi-Drug Conjugate (ApMDC) can overcome limitations of original drug conjugates Aptamer-drug conjugate (ApDC) and even Antibody-drug conjugate (ADC). After characterizing the sequence of HB5, conjugated site and number of drugs, the cytotoxicity of the final products was verified by cell viability assay, showing its advanced drug effects of ApMDC compared to other conjugates (ApDC, ADC): 2.1 times higher cytotoxicity on the same concentration of ApDC (10 nM) and 6.8 times lower value of IC50 (26.9 ng/ml) than ADC (T-DM1). These results suggest not only the development of aptamer-drug conjugate but also the possibility of good competitor or substitute for antibody-based therapeutics in the field of targeted therapy. This aptamer drug conjugates were developed as a treatment for HER2-positive breast cancer that needs a new type of therapeutics overcoming some challenges: difficulty of general hormone therapy and side effects killing normal cells. Aptamer drug conjugates can resolve the problems as a targeted therapeutics using HER2 targeting DNA aptamer, HB5, conjugated with microtubule-disturbing agent, maytansine (DM1) by non-cleavable linker, Succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). After optimizing aptamer sequence from original aptamer (HB5, 86mer) to trimmed one (tHB5, 57mer), drugs were conjugated with different sites and numbers to tHB5. Each drug conjugate was all synthesized and verified by High Performance Liquid Chromatography (HPLC) and Electrospray Ionization-Mass Spectrometry (ESI-MS). To determine optimized sites and number of drugs, each efficacy was confirmed and compared in cell viability assay (WST assay) and cell cycle analysis in a cellular level. For multiple-drug conjugate, drugs were not only conjugated at 5’ or 3’ end of sequence but also internal thymine site (16T, 24T or 55T), then finally characterized as an Aptamer Multi-Drug Conjugate (ApMDC), tHB5(5’+55T+3’)-DM1. In addition, efficacy was increased as more drugs were conjugated compared to single, double and triple conjugates and their mechanism of action was verified by confirming G2/M phase arrest in cell cycle analysis by DM1 disturbing microtubule polymerization. Development of Aptamer multi-drug conjugate (ApMDC) has a significant meaning of more improved efficacy than original aptamer-drug conjugate (ApDC), the single-drug conjugated form of aptamer therapeutics, without adding special methods or modifications. Furthermore, much more drug efficacy than ADC (T-DM1) suggests the possibility to replace the antibody-based targeted therapeutics with aptamer-based targeted therapeutics, overcoming the limitations of immunogenicity and heterogeneity.