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.