Imaging a functional tumorigenic biomarker in the transformed epithelium

Abstract

Proteases responsible for the increased peritumoral proteolysis associated with cancer represent functional biomarkers for monitoring tumorigenesis. One attractive extracellular biomarker is the transmembrane serine protease matriptase. Found on the surface of epithelial cells, the activity of matriptase is regulated by its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1). Quantitative mass spectrometry allowed us to show that, in selected cancers, HAI-1 expression decreases, leading to active matriptase. A preclinical probe specific for the measurement of emergent active matriptase was developed. Using an active-site-specific, recombinant human antibody for matriptase, we found that the selective targeting of active matriptase can be used to visualize the tumorigenic epithelium. Live-cell fluorescence imaging validated the selectivity of the antibody in vitro by showing that the probe localized only to cancer cell lines with active matriptase on the surface. Immunofluorescence with the antibody documented significant levels of active matriptase in 68% of primary and metastatic colon cancer sections from tissue microarrays. Labeling of the active form of matriptase in vivo was measured in human colon cancer xenografts and in a patient-derived xenograft model using near-infrared and single-photon emission computed tomography imaging. Tumor uptake of the radiolabeled antibody, 111 In-A11, by active matriptase was high in xenografts (28% injected dose per gram) and was blocked in vivo by the addition of a matriptase-specific variant of ecotin. These findings suggest, through a HAI-1-dependent mechanism, that emergent active matriptase is a functional biomarker of the transformed epithelium and that its proteolytic activity can be exploited to noninvasively evaluate tumorigenesis in vivo.cancer biomarker | molecular imaging

Tumor-Specific Activation of an EGFR-Targeting Probody Enhances Therapeutic Index

“…Indeed, across a combination of stage I, II, and III colon cancer samples, 90% (n = 30) of the samples showed evidence of ex vivo Probody activation. This high fraction of protease-positive samples compares favorably with a recent study (25), which found that 68% (n = 152) of colon tumors expressed active matriptase. We speculate that the higher fraction of positive results in our experiment may reflect the ability of PB1 to be cleaved by uPA and legumain, in addition to matriptase.…”

“…In particular, the clinically validated anti-EGFR antibody cetuximab was converted into a prodrug form, PB1, using a masking peptide that was linked recombinantly to the light chain of the antibody through a proteasecleavable linker peptide. In this case, our choice of protease selectivity was based on literature reports of uPA, matriptase, and legumain as proteases that are widely expressed in solid tumors of epithelial origin (23)(24)(25)(26)(27)(28). We confirmed that some combinations of these proteases were active in tumors by using the Probody itself as a probe of proteolytic activity, both via analysis of frozen tissue sections and by in vivo imaging in mice.…”

Probody therapeutics for targeting antibodies to diseased tissue

“…We have demonstrated this approach using an EGFR Probody [3] and with a method --termed IHZÔ analysis --a zymography-like approach using the Probody itself as a probe of proteolytic activity on frozen tissue sections. In a survey of tumor sections from colorectal and lung cancer patients, a majority of the tumors contained Probody-activating protease activity, while healthy tissues lacked this activity, consistent with previously reported results using an active-site antibody to detect active-site protease in situ [4]. As in the case of cleavable (and non-cleavable) linkers used for the conjugation of payload-linkers in conventional antibody--drug conjugates (ADCs), Probody substrate linkers appear to be relatively portable between Probody constructs, allowing a range of Probodies with different antigen specificities to be constructed with the same set of cleavable substrate linkers.…”

Imaging Active Urokinase Plasminogen Activator in Prostate Cancer

“…uPA was detected with antibody sc-14019 (Santa Cruz) (1:100) following the manufacturer’s recommendation using an anti-rabbit AlexaFluor 488 conjugated secondary. The protocol for antigen retrieval and staining for e-cadherin was previously published (29). …”