Natural Product Splicing Inhibitors: A New Class of Antibody–Drug Conjugate (ADC) Payloads

Abstract

There is a considerable ongoing work to identify new cytotoxic payloads that are appropriate for antibody-based delivery, acting via mechanisms beyond DNA damage and microtubule disruption, highlighting their importance to the field of cancer therapeutics. New modes of action will allow a more diverse set of tumor types to be targeted and will allow for possible mechanisms to evade the drug resistance that will invariably develop to existing payloads. Spliceosome inhibitors are known to be potent antiproliferative agents capable of targeting both actively dividing and quiescent cells. A series of thailanstatin-antibody conjugates were prepared in order to evaluate their potential utility in the treatment of cancer. After exploring a variety of linkers, we found that the most potent antibody-drug conjugates (ADCs) were derived from direct conjugation of the carboxylic acid-containing payload to surface lysines of the antibody (a "linker-less" conjugate). Activity of these lysine conjugates was correlated to drug-loading, a feature not typically observed for other payload classes. The thailanstatin-conjugates were potent in high target expressing cells, including multidrug-resistant lines, and inactive in nontarget expressing cells. Moreover, these ADCs were shown to promote altered splicing products in N87 cells in vitro, consistent with their putative mechanism of action. In addition, the exposure of the ADCs was sufficient to result in excellent potency in a gastric cancer xenograft model at doses as low as 1.5 mg/kg that was superior to the clinically approved ADC T-DM1. The results presented herein therefore open the door to further exploring splicing inhibition as a potential new mode-of-action for novel ADCs.

Multivalent peptidic linker enables identification of preferred sites of conjugation for a potent thialanstatin antibody drug conjugate

“…Eight randomized animals were dosed intravenously at 3mg/kg on days 0, 4, 8 and 12 (q4d x 4) when the tumor size reached approximately 350 mm 3 . In contrast to the excellent potency previously reported for DAR4 lysine thialanstatin conjugates [19], only a modest reduction in tumor growth rate was observed in comparison to a vehicle control (Fig 1C). …”

“…The cytotoxicity and selectivity of these ADCs ( ADC1-3 ) were assessed against several cancer cell lines with varying levels of Her2 expression (Fig 1B). Similar to our earlier published results [19], all the hinge cysteine conjugates of thailanstatin displayed potent activity (IC 50 ~1 nM) against the high Her2 (>600,000 receptors/cell) expressing N87 gastric cancer cell line but were not potent against moderate antigen expressing MDA-MB-361-DYT2 (361) breast cancer cell line (~150,000 receptors/cell). As expected, all ADCs were inactive against a non-antigen expressing cell lines, MDA-MB-468 (468).…”

“…As we have previously shown that the potency of lysine thailanstain ADCs [19] are highly dependent upon DAR, we wanted to evaluate if increasing the DAR at the engineered cysteine sites also improved the cytotoxicity of the thailanstain ADCs toward the 361 cell line. We envisioned increasing loading to an engineered cysteine by utilizing click chemistry to conjugate a small, multiple-payload carrying peptide in a two-step conjugation method.…”

“…Thailanstatin A, a natural product analog of spliceostatin, is a potent inhibitor of eukaryotic RNA splicing by virtue of its affinity to the SF3b subunit of the U2 snRNA subcomplex of the spliceosome [16, 17]. As the ADC field is actively looking for cytotoxic payloads beyond the microtubule inhibitor class [1, 18], we recently introduced thailanstatin as a potent payload for lysine conjugated ADCs [19]. In this report, we show that there is a high dependence of the potency of thailanstatin ADCs with the site of conjugation.…”

Expanding the Reach of Antibody–Drug Conjugates

“…Recent publications on new payload and linkers, although still sparse, along with understanding of the mechanism of action of ADCs, are encouraging signs that chemistry efforts to optimize ADCs are being prioritized and that some progress is now being made. 11,12 ■ AUTHOR INFORMATION Corresponding Author *E-mail: ravi.chari@immunogen.com. …”