Abstract
Although current cancer treatment strategies are highly aggressive, they are often not effective enough to destroy the collectivity of malignant cells. The residual tumor cells that survived the first-line treatment may continue to proliferate or even metastasize. Therefore, the development of novel more effective strategies to specifically eliminate also single cancer cells is urgently needed. In this respect, the development of antibody-based therapeutics, in particular example immunotoxins, has attracted broad interest. Since the internalization of immunotoxins is essential for their cytotoxic effectivity, it is of crucial importance to study their internalization behavior to assess the potential for their therapeutic use. In this study, we determined the internalization behavior of four different single-chain fragments variable (scFv) when binding to the corresponding target antigen as expressed on solid or non-solid tumor cell lines. The scFvs were recombinantly fused to the SNAP-tag, an engineered variant of the human repair enzyme O6-alkylguanine-DNA alkyltransferase that covalently reacts with benzylguanine derivatives. Since a large number of highly sensitive organic fluorescent dyes are already available or can easily be derivatized to react with the self-labeling SNAP-tag, this system provides versatile applications for imaging of intraand extracellular compartments of living cells. The fusion proteins were coupled to SNAP-surface® Alexa Fluor® 488 or SNAP-surface® Alexa Fluor® 647 and binding as well as internalization was monitored by flow cytometry and confocal microscopy, respectively. Depending on the respective target antigen, we could distinguish between slow and rapid internalization behavior. Moreover, we detected increased internalization rate for bivalent scFv constructs. Our approach allows for rapid and early stage evaluation of the internalization characteristics of new antibodies designated for further therapeutic development.
Keywords: Single-chain fragment variable (scFv), SNAP-tag technology, receptor-mediated internalization, protein therapeutics, molecular imaging.
Current Pharmaceutical Design
Title:SNAP-tag based Agents for Preclinical In Vitro Imaging in Malignant Diseases
Volume: 19 Issue: 30
Author(s): Manal Amoury, Tobias Blume, Hannes Brehm, Judith Niesen, Niklas Tenhaef, Stefan Barth, Stefan Gattenlohner, Wijnand Helfrich, Jenny Fitting, Thomas Nachreiner and Alessa Pardo
Affiliation:
Keywords: Single-chain fragment variable (scFv), SNAP-tag technology, receptor-mediated internalization, protein therapeutics, molecular imaging.
Abstract: Although current cancer treatment strategies are highly aggressive, they are often not effective enough to destroy the collectivity of malignant cells. The residual tumor cells that survived the first-line treatment may continue to proliferate or even metastasize. Therefore, the development of novel more effective strategies to specifically eliminate also single cancer cells is urgently needed. In this respect, the development of antibody-based therapeutics, in particular example immunotoxins, has attracted broad interest. Since the internalization of immunotoxins is essential for their cytotoxic effectivity, it is of crucial importance to study their internalization behavior to assess the potential for their therapeutic use. In this study, we determined the internalization behavior of four different single-chain fragments variable (scFv) when binding to the corresponding target antigen as expressed on solid or non-solid tumor cell lines. The scFvs were recombinantly fused to the SNAP-tag, an engineered variant of the human repair enzyme O6-alkylguanine-DNA alkyltransferase that covalently reacts with benzylguanine derivatives. Since a large number of highly sensitive organic fluorescent dyes are already available or can easily be derivatized to react with the self-labeling SNAP-tag, this system provides versatile applications for imaging of intraand extracellular compartments of living cells. The fusion proteins were coupled to SNAP-surface® Alexa Fluor® 488 or SNAP-surface® Alexa Fluor® 647 and binding as well as internalization was monitored by flow cytometry and confocal microscopy, respectively. Depending on the respective target antigen, we could distinguish between slow and rapid internalization behavior. Moreover, we detected increased internalization rate for bivalent scFv constructs. Our approach allows for rapid and early stage evaluation of the internalization characteristics of new antibodies designated for further therapeutic development.
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Cite this article as:
Amoury Manal, Blume Tobias, Brehm Hannes, Niesen Judith, Tenhaef Niklas, Barth Stefan, Gattenlohner Stefan, Helfrich Wijnand, Fitting Jenny, Nachreiner Thomas and Pardo Alessa, SNAP-tag based Agents for Preclinical In Vitro Imaging in Malignant Diseases, Current Pharmaceutical Design 2013; 19 (30) . https://dx.doi.org/10.2174/13816128113199990405
DOI https://dx.doi.org/10.2174/13816128113199990405 |
Print ISSN 1381-6128 |
Publisher Name Bentham Science Publisher |
Online ISSN 1873-4286 |
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