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.

A novel approach for targeted elimination of CSPG4‐positive triple‐negative breast cancer cells using a MAP tau‐based fusion protein

“…Briefly, CFPs were expressed in the periplasm of E. coli BL21(DE3) under osmotic stress in the presence of compatible solutes. The noncytolytic anti‐CSPG4(scFv)‐SNAP was expressed in HEK293T cells and purified as described previously …”

“…The noncytolytic anti-CSPG4(scFv)-SNAP was expressed in HEK293T cells and purified as described previously. 28,29 Two-step purification was performed using Ni-NTA superflow (Qiagen, Hilden, Germany) according to the manufacturer's instructions followed by FPLC purification using a Strep-TactinV R SuperflowV R column (IBA GmbH, Goettingen, Germany) on an € AKTApurifier System (GE Healthcare, Solingen, Germany). Subsequent preparative size exclusion chromatography was performed using a XK16-70 column packed with Superdex 75 (GE Healthcare) with a mobile phase of PBS (pH 7.4) at a flow rate of 1.0 ml/min.…”

The Fc‐alpha receptor is a new target antigen for immunotherapy of myeloid leukemia

“…The CD89(scFv)‐SNAP protein was produced by secretory expression in HEK293T cells, and coupled to BG‐Vista ® Green according to the manufacturer's instructions . Internalization was verified by incubating 1 × 10 6 HL‐60 (1,000 U/ml TNFα) and Ramos cells with 50 nM (∼2 µg) CD89(scFv)‐SNAP‐BG‐Vista ® Green in 100 µL PBS at 4°C or 37°C for 30 min.…”

Human Antibody Fusion Proteins/Antibody Drug Conjugates in Breast and Ovarian Cancer

“…Additionally, organic fluorophores can be conjugated to SNAP-tag fusion proteins to generate imaging probes for in vitro and in vivo diagnostics [96,97]. SNAP-tag imaging probes have advantages over autofluorescent protein-antibody probes.…”