Proteomic-based evaluation of nuclear transport of NLS-tagged trastuzumab-emtansine with enhanced cytotoxic potency

Abstract

ABSTRACTNuclear transport receptors (NTRs) are the only proteins able to transport large molecular weight payloads into the nucleus. A dominant area of molecular therapeutic research is the extension of the use of NTRs to target the nucleus for the development of pharmaceuticals or as tools for investigating fundamental biological questions. Although several examples of synthesized peptides harbouring nuclear localization signal (NLS) sequences conjugated to various payloads exist in the literature, the assumption has been that transport occurs by classical nuclear localization via the NTRs importin-α and importin-β. This assumption is relevant to nuclear-targeted therapeutics that aim for full potential clinical impact. In addition, fundamental research can benefit from unbiased approaches to investigate the role of NTRs. Herein, we report the construction of a novel NLS-modified agent composed of trastuzumab-emtansine (T-DM1) coupled to cell accumulator (Accum), a technology that enables monoclonal antibodies to escape endosome entrapment and accumulate conjugated payloads in the nucleus without abrogating affinity or specificity to target antigens. Accum harbours a classical NLS sequence from SV-40 large T-antigen. We demonstrate that routing T-DM1 to the nucleus successfully increased cytotoxic potency in the HER2-positive cell line SKBR3. More importantly, through the development of a novel bait-prey proteomic approach, we show that the non-classical NTR importin 7 and not importin-α/importin-β was required for the cytotoxicity effect. This result was validated by siRNA knock down. Our findings also indicate that by discovering an unanticipated NTR regulator of an NLS-modified agent, this study demonstrates the utility of combining an unbiased proteomic approach to probe NTR function in mammalian cell system and, is a foresight for future NLS-based development initiatives.