A Novel Proteomic Method Reveals NLS Tagging of T-DM1 Contravenes Classical Nuclear Transport in a Model of HER2-Positive Breast Cancer

Abstract

The next breakthrough for protein therapeutics is effective intracellular delivery and accumulation within target cells. Nuclear localization signal (NLS)-tagged therapeutics have been hindered by the lack of efficient nuclear localization due to endosome entrapment. Although development of strategies for tagging therapeutics with technologies capable of increased membrane penetration has resulted in proportional increased potency, nonspecific membrane penetration limits target specificity and, hence, widespread clinical success. There is a long-standing idea that nuclear localization of NLS-tagged agents occurs exclusively via classical nuclear transport. In the present study, we modified the antibody-drug conjugate trastuzumab-emtansine (T-DM1) with a classical NLS linked to cholic acid (cell accumulator [Accum]) that enables modified antibodies to escape endosome entrapment and increase nuclear localization efficiency without abrogating receptor targeting. In parallel, we developed a proteomics-based method to evaluate nuclear transport. Accum-modified T-DM1 significantly enhanced cytotoxic efficacy in the human epidermal growth factor receptor 2 (HER2)-positive SKBR3 breast cancer system. We discovered that efficacy was dependent on the nonclassical importin-7. Our evaluation reveals that when multiple classical NLS tagging occurs, cationic charge build-up as opposed to sequence dominates and becomes a substrate for importin-7. This study results in an effective target cell-specific NLS therapeutic and a general approach to guide future NLS-based development initiatives.

Accum™ Technology: A Novel Conjugable Primer for Onco-Immunotherapy

“…limitations of CPP-and NLS-equipped conjugates [10] and highly adaptable for function-alizing different conjugates for different therapeutic purposes [10][11][12][13][14][15]. For instance, Beaudoin et al [12] designed 7G3-Accum-Accum coupled to 7G3-a mAb specifically targeting interleukin-3 receptor-α (IL-3Rα), a cell-surface antigen expressed by leukemic cells.…”

“…In the context of muscle invasive bladder cancer (MIBC), the same team [13,14] designed A14-Accum, where A14 is a mAb specific for interleukin-5 receptor α-subunit (IL-5Rα). In another therapeutic context, Accum was coupled to the clinically approved ADC trastuzumab-emtansine (T-DM1) [10]. Recently, Bikorimana et al [15] introduced Accum to the setting of cellular vaccines, coupling ChAcNLS to the xenoantigen ovalbumin (OVA) with the rationale of triggering potent antigen presentation by DCs.…”

“…However, this transport system faces a backlash from cancer cell mechanisms, including receptor downregulation/recycling and increased expression of multidrug resistance pumps, that either entrap drug payloads in endosomes or counteract their intracellular accumulation [45,46]. On another note, several new ADC modifications, including ADCs coupled to synthetic CPPs or pH-sensitive polymers, have been effective in Accum can be likened to a polymath primer: it is well-informed about the biological limitations of CPP-and NLS-equipped conjugates [10] and highly adaptable for functionalizing different conjugates for different therapeutic purposes [10][11][12][13][14][15]. For instance, Beaudoin et al [12] designed 7G3-Accum-Accum coupled to 7G3-a mAb specifically targeting interleukin-3 receptor-α (IL-3Rα), a cell-surface antigen expressed by leukemic cells.…”

“…Molecules 2022, 27, 3807 2 of 13 Accum™ is a novel biotechnology exemplifying, in its initial concept, the need to circumvent the biological challenges of ADCs [10][11][12][13][14][15]. Despite their targeted therapy nature (a receptor-specific mAb linked to a cytotoxic payload), ADCs become captives of the cellular transport mechanisms they were designed to exploit to deposit their cargo in a trojan horse-like fashion [9].…”

“…On the other hand, the NLS moiety of Accum™ redirects trafficking of the conjugate to the nucleus; ultimately, both Accum moieties increase intracellular retention in target cells [12]. The concept of the ChAc-NLS fusion compound serving as a cell accumulator (Accum for short) has been shown to be pliable, with the potential for coupling to different molecular conjugates for use in different cancer settings [10][11][12][13][14][15]. In this review article, we discuss the genesis of Accum™ and highlight its docility in producing context-specific therapeutic strategies.…”