Sulfatase-cleavable linkers for antibody-drug conjugates

Abstract

Antibody-drug conjugates (ADCs) are a class of targeted drug delivery agents combining the cell-selectivity of monoclonal antibodies (mAbs) and the cytotoxicity of small molecules. These two components are joined by a covalent linker, whose nature is critical to the efficacy and safety of the ADC. Enzyme-cleavable dipeptidic linkers have emerged as a particularly effective ADC linker type due to their ability to selectively release the payload in the lysosomes of target cells. However, these linkers have a number of drawbacks, including instability in rodent plasma and their inherently high hydrophobicity. Here we show that arylsulfate-containing ADC linkers are cleaved by lysosomal sulfatase enzymes to tracelessly release their payload, while circumventing the instability problems associated with dipeptide-linkers. When incorporated with trastuzumab and the highly potent monomethyl auristatin E (MMAE) payload, the arylsulfate-containing ADC 2 and ADC 3 were more cytotoxic than the non-cleavable ADC 4 against HER2-positive cells, while maintaining selectivity over HER2-negative cells. We propose that the stability, solubility and synthetic tractability of our arylsulfate linkers make them an attractive new motif for cleavable ADC linkers, with clear benefits over the widely used dipeptidic linkers.

Fig. 1. (a) Previously developed dipeptide linkers, cleaved by lysosomal cathepsins and mouse plasma enzyme Ces1C; (b) arylsulfate linkers for use in ADCs are cleaved by lysosomal sulfatases.

Fig. 2. Design of linker-AMC model compounds. It was envisaged that the linker-payloads could be joined to the antibody through an ortho-amide (blue route) or a benzyl-alkyl (red route).

Fig. 3. (a) Synthesis of linker-AMC 7 and (b) synthesis of linker-AMC 12. Np = neopentyl.

Fig. 4. (a) Structures of model linker-AMC constructs including arylsulfates 7 and 12 and Val–Ala 13 and Val–Cit 14. Upon hydrolysis and subsequent 1,6-elimination, the AMC payload is released and fluoresces. (b) Comparison of enzymolysis rates of 7versus12 when incubated with sulfatase from Helix pomatia. (c) Stability comparison of arylsulfates 7 and 12versus dipeptides 13 and 14 in mouse plasma over 8 hours. (d) Stability comparison in mouse plasma over 7 days. The t = 0 reading was taken after adding plasma to all the samples, by which time 13 and 14 were already partially hydrolysed. (e) Stability comparison in human plasma over 7 days.

Fig. 5. Bioconjugation of linker-payloads 15, 16a, 16b, 17 and 18 to trastuzumab to afford ADCs 1–5.

Fig. 6. In vitro biological evaluation of ADCs 1–5 in (a) HER2-positive BT474 cells and (b) HER2-negative MCF7 cells. Viability data shows the mean of three independent experiments and error bars represent standard error of the mean. For BT474 cells, IC₅₀ values are as follows: ADC 1 = N/A; ADC 2 = 111 pM; ADC 3 = 61 pM; ADC 4 = 609 pM; ADC 5 = 92 pM.