title: SNAP-tag based agents for preclinical in vitro imaging in malignant diseases.
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 O(6)-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.
authors: Department of Pharmaceutical Product Development, Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstr. 6, 52074 Aachen, Germany.
authors: Manal
authors: M
authors: Amoury
authors: Tobias
authors: T
authors: Blume
authors: Hannes
authors: H
authors: Brehm
authors: Judith
authors: J
authors: Niesen
authors: Niklas
authors: N
authors: Tenhaef
authors: Stefan
authors: S
authors: Barth
authors: Stefan
authors: S
authors: Gattenlöhner
authors: Wijnand
authors: W
authors: Helfrich
authors: Jenny
authors: J
authors: Fitting
authors: Thomas
authors: T
authors: Nachreiner
authors: Alessa
authors: A
authors: Pardo
mesh_terms: D000818:Animals; D000906:Antibodies; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000494:pharmacology*; D003001:Cloning, Molecular; D015972:Gene Expression Regulation, Neoplastic; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D000906:Antibodies; D000970:Antineoplastic Agents