A comparison of the activity, lysosomal stability, and efficacy of legumain-cleavable and cathepsin-cleavable ADC linkers

Abstract

Over the past two decades, antibody-drug conjugates (ADCs) have emerged as a highly effective drug delivery technology. ADCs utilise a monoclonal antibody, a chemical linker, and a therapeutic payload to selectively deliver highly potent pharmaceutical agents to specific cell types.Challenges such as premature linker cleavage and clearance due to linker hydrophobicity have adversely impacted the stability and safety of ADCs. While there are various solutions to these challenges, our team has focused on replacement of hydrophobic ValCit linkers (cleaved by CatB) with Asn-containing linkers that are cleaved by lysosomal legumain.Legumain is abundantly present in lysosomes and is known to play a role in tumour microenvironment dynamics. Herein, we directly compare the lysosomal cleavage, cytotoxicity, plasma stability, and efficacy of a traditional cathepsin-cleavable ADC to a matched Asn-containing legumain-cleavable ADC.We demonstrate that Asn-containing linker sequences are specifically cleaved by lysosomal legumain and that Asn-linked MMAE ADCs are broadly active against a variety of tumours, even those with low legumain expression. Finally, we show that AsnAsn-linked ADCs exhibit comparable or improved efficacy to traditional ValCit-linked ADCs. Our study paves the way for replacement of the traditional ValCit linker technology with more hydrophilic Asn-containing peptide linker sequences.

Keywords: ADC; Antibody–drug conjugate; MMAE; cancer; cathepsin B; legumain; linker.

Figure 1.

Comparison of previous generation ValCit ADC linkers and the currently described AsnAsn linkers.

Figure 2.

A) Anti-HER2 ADCs employing the three linker variants exhibit equivalent cytotoxicity in a high HER2 expressing cell line, SKBR3; B) The design of the lysosomal stability assay; C-D) Release of MMAE in the presence and absence of a CatB or legumain inhibitor upon incubation in rat liver lysosomes (C) or human liver lysosomes (D). The release of MMAE as a function of time is plotted in the left three plots while the rate of release (initial rate) is summarized in the bar graphs on the right.

Figure 3.

Postulated cleavage linker cleavage mechanisms based on the lysosomal catabolism studies in the presence and absence of legumain and CatB inhibitors.

Figure 4.

Cytotoxicity against a panel of Trop2-expressing and non-expressing cell lines. The HER2, Trop2, and legumain expression (mRNA) expression are shown, as reported by Human Protein Atlas. (proteinatlas.org)

Figure 5.

A) Structure of two anti-CD79b ADCs; B) Western blot showing the presence and absence of legumain in Granta-519 and RL cells, respectively; C-D) Cytotoxicity of the two ADCs in Granta (C) and RL (D) cells.

Figure 6.

Incubation of anti-HER2-XX-PABC-MMAE ADCs in mouse plasma, evaluating A) mAb and ADC concentrations by immunoassay and B) cytotoxicity against SKBR3 (HER2+) cells.

Figure 7.

Mouse xenograft study using TNBC MDA-MB-468, as assessed by tumor volume. (n=5–8 per group)