Enzyme-Agnostic Lysosomal Screen Identifies New Legumain-Cleavable ADC Linkers

Over the past two decades, antibody
drug conjugates (ADCs) and
small molecule drug conjugates (SMDCs) have widely employed valine-citruline
and related cathepsin-cleavable linkers due to their stability in
plasma and their rapid cleavage by lysosomal cathepsins. However,
a number of recent studies have illustrated that these linkers are
subject to cleavage by exogenous enzymes such as Ces1C and neutrophil
elastase, thus resulting in off-target release of drug. As such, there
is a need to diversify the portfolio of ADC linkers in order to overcome
nonspecific drug release. Rather than targeting cathepsins, we began
with an “enzyme agnostic” screen in which a panel of
75 peptide FRET pairs were screened for cleavage in lysosomal extracts
and in plasma. Unexpectedly, a series of Asn-containing peptides emerged
from this screen as being cleaved far more quickly than traditional
ValCit-type linkers while retaining excellent stability in plasma.
Catabolism studies demonstrated that these linkers were cleaved by
legumain, an asparaginyl endopeptidase that is overexpressed in a
variety of cancers and is known to be present in the lysosome. MMAE-containing
ADCs that incorporated these new linkers were shown to exhibit highly
potent and selective cytotoxicity, comparable to analogous ValCit
ADCs. Importantly, the Asn-containing linkers were shown to be completely
stable to human neutrophil elastase, an enzyme thought to be responsible
for the neutropenia and thrombocytopenia associated with ValCitPABC-MMAE
ADCs. The legumain-cleavable ADCs were shown to have excellent stability
in both mouse and human serum, retaining >85% of the drug after
1
week of incubation. Moreover, the corresponding small molecule FRET
pairs exhibited <10% cleavage after 18 h in mouse and human serum.
On the basis of these results, we believe that these new linkers (AsnAsn
in particular) have significant potential in both ADC and SMDC drug
delivery applications.