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. 2010:2010:187621.
doi: 10.1155/2010/187621. Epub 2010 Feb 24.

A human recombinant autoantibody-based immunotoxin specific for the fetal acetylcholine receptor inhibits rhabdomyosarcoma growth in vitro and in a murine transplantation model

S Gattenlöhner  1 , H Jörissen, M Huhn, A Vincent, D Beeson, S Tzartos, A Mamalaki, B Etschmann, H K Muller-Hermelink, E Koscielniak, S Barth, A Marx
Affiliations

A human recombinant autoantibody-based immunotoxin specific for the fetal acetylcholine receptor inhibits rhabdomyosarcoma growth in vitro and in a murine transplantation model

S Gattenlöhner et al. J Biomed Biotechnol. 2010.

Abstract

Rhabdomyosarcoma (RMS) is the most common malignant soft tissue tumor in children and is highly resistant to all forms of treatment currently available once metastasis or relapse has commenced. As it has recently been determined that the acetylcholine receptor (AChR) gamma-subunit, which defines the fetal AChR (fAChR) isoform, is almost exclusively expressed in RMS post partum, we recombinantly fused a single chain variable fragment (scFv) derived from a fully human anti-fAChR Fab-fragment to Pseudomonas exotoxin A to generate an anti-fAChR immunotoxin (scFv35-ETA). While scFv35-ETA had no damaging effect on fAChR-negative control cell lines, it killed human embryonic and alveolar RMS cell lines in vitro and delayed RMS development in a murine transplantation model. These results indicate that scFv35-ETA may be a valuable new therapeutic tool as well as a relevant step towards the development of a fully human immunotoxin directed against RMS. Moreover, as approximately 20% of metastatic malignant melanomas (MMs) display rhabdoid features including the expression of fAChR, the immunotoxin we developed may also prove to be of significant use in the treatment of these more common and most often fatal neoplasms.

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Figures

Figure 1
Figure 1
Schematic presentation of the synthesis of scFv fragments based on the recombinant Fab fragments Fab35 and Fab38 [21] using overlap extension PCR.
Figure 2
Figure 2
FACS Analysis of scFv35-ETA.
Figure 3
Figure 3
Colorimetric XTT cytotoxicity assays with various immunotoxin concentrations (n = 3 parallel cell cultures per dilution) showing strong dose-dependent toxicity of the immunotoxin scFv35-ETA directed against the acetylcholine receptor (AChR) γ-subunit on positive RMS cell lines TE671, RD, and FL-OH-1, RH-30 and Ax-OH-1 even at higher dilutions (a). Lower to no toxicity of the immunotoxin were observed towards the control cell lines A-204 and U937 not expressing fAChR (b). Competition of toxicity could be achieved with 1–100-fold molar excess (C 1.5–150 nM) of scFv35 added to cells incubated with 1.5 nM scFv35-ETA, whereas scFv35 alone does not inhibit cell growth even at 100 fold higher concentration (c). Cell viability is expressed as percentage inhibition of cell proliferation compared to non-treated cells.
Figure 4
Figure 4
Annexin V apoptosis assay on RD cells incubated with either scFv35-ETA or PBScontrol for 12, 14 and 48 hours. Quadrants distinguish viable cells (lower left), early apoptotic cells (lower right), late apoptotic cells (upper right) and necrotic cells (upper left). Numbers indicate percentage of early and late apoptotic cells, respectively.
Figure 5
Figure 5
In vivo effect of scFv35-ETA on RD embryonal RMS cells (5 × 106 RD cells per mouse) transplanted subcutaneously into SCID mice. The first intraperitoneal immunotoxin injection (10 μg twice daily) was carried out 2 days after the injection of tumor cells and repeated daily until day 10.
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