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Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies

Proteases are involved in the control of numerous physiological processes, and their dysregulation has been identified in a wide range of pathologies, including cancer. Protease activity is normally tightly regulated post-translationally and therefore cannot be accurately estimated based on mRNA or...

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Autores principales: Howng, Bruce, Winter, Michael B., LePage, Carol, Popova, Irina, Krimm, Michael, Vasiljeva, Olga
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8471274/
https://www.ncbi.nlm.nih.gov/pubmed/34575469
http://dx.doi.org/10.3390/pharmaceutics13091390
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author Howng, Bruce
Winter, Michael B.
LePage, Carol
Popova, Irina
Krimm, Michael
Vasiljeva, Olga
author_facet Howng, Bruce
Winter, Michael B.
LePage, Carol
Popova, Irina
Krimm, Michael
Vasiljeva, Olga
author_sort Howng, Bruce
collection PubMed
description Proteases are involved in the control of numerous physiological processes, and their dysregulation has been identified in a wide range of pathologies, including cancer. Protease activity is normally tightly regulated post-translationally and therefore cannot be accurately estimated based on mRNA or protein expression alone. While several types of zymography approaches to estimate protease activity exist, there remains a need for a robust and reliable technique to measure protease activity in biological tissues. We present a novel quantitative ex vivo zymography (QZ) technology based on Probody(®) therapeutics (Pb-Tx), a novel class of protease-activated cancer therapeutics that contain a substrate linker cleavable by tumor-associated proteases. This approach enables the measurement and comparison of protease activity in biological tissues via the detection of Pb-Tx activation. By exploiting substrate specificity and selectivity, cataloguing and differentiating protease activities is possible, with further refinement achieved using protease-specific inhibitors. Using the QZ assay and human tumor xenografts, patient tumor tissues, and patient plasma, we characterized protease activity in preclinical and clinical samples. The QZ assay offers the potential to increase our understanding of protease activity in tissues and inform diagnostic and therapeutic development for diseases, such as cancer, that are characterized by dysregulated proteolysis.
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spelling pubmed-84712742021-09-27 Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies Howng, Bruce Winter, Michael B. LePage, Carol Popova, Irina Krimm, Michael Vasiljeva, Olga Pharmaceutics Article Proteases are involved in the control of numerous physiological processes, and their dysregulation has been identified in a wide range of pathologies, including cancer. Protease activity is normally tightly regulated post-translationally and therefore cannot be accurately estimated based on mRNA or protein expression alone. While several types of zymography approaches to estimate protease activity exist, there remains a need for a robust and reliable technique to measure protease activity in biological tissues. We present a novel quantitative ex vivo zymography (QZ) technology based on Probody(®) therapeutics (Pb-Tx), a novel class of protease-activated cancer therapeutics that contain a substrate linker cleavable by tumor-associated proteases. This approach enables the measurement and comparison of protease activity in biological tissues via the detection of Pb-Tx activation. By exploiting substrate specificity and selectivity, cataloguing and differentiating protease activities is possible, with further refinement achieved using protease-specific inhibitors. Using the QZ assay and human tumor xenografts, patient tumor tissues, and patient plasma, we characterized protease activity in preclinical and clinical samples. The QZ assay offers the potential to increase our understanding of protease activity in tissues and inform diagnostic and therapeutic development for diseases, such as cancer, that are characterized by dysregulated proteolysis. MDPI 2021-09-02 /pmc/articles/PMC8471274/ /pubmed/34575469 http://dx.doi.org/10.3390/pharmaceutics13091390 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Howng, Bruce
Winter, Michael B.
LePage, Carol
Popova, Irina
Krimm, Michael
Vasiljeva, Olga
Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies
title Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies
title_full Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies
title_fullStr Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies
title_full_unstemmed Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies
title_short Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies
title_sort novel ex vivo zymography approach for assessment of protease activity in tissues with activatable antibodies
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8471274/
https://www.ncbi.nlm.nih.gov/pubmed/34575469
http://dx.doi.org/10.3390/pharmaceutics13091390
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