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Human angiogenin fused to human CD30 ligand (Ang-CD30L) exhibits specific cytotoxicity against CD30-positive lymphoma

: Huhn, M.; Sasse, S.; Tur, M.K.; Matthey, B.; Schinköthe, T.; Rybak, S.; Barth, S.; Engert, A.

Cancer research 61 (2001), Nr.24, S.8737-8742
ISSN: 0008-5472
ISSN: 1538-7445
ISSN: 0099-7013
ISSN: 0099-7374
Fraunhofer IUCT ( IME) ()

A number of different immunotoxins composed of cell-specific targeting structures coupled to plant or bacterial toxins have increasingly been evaluated for immunotherapy. Because these foreign proteins are highly immunogenic in humans, we have developed a new CD30 ligand-based fusion toxin (Ang-CD30L) using the human RNase angiogenin. The completely human fusion gene was inserted into a pET-based expression plasmid. Transformed Escherichia coli BL21(DE3) were grown under osmotic stress conditions in the presence of compatible solutes. After isopropyl beta -D-thiogalactoside induction, the M-r 37,000 His(10)-tagged Ang-CD30L was directed into the periplasmic space and functionally purified by a combination of metal ion affinity followed by enterokinase cleavage of the His(10)-Tag and molecular size chromatography. The characteristics of the recombinant protein were assessed by ELISA, flow cytometry, and toxicity assays showing specific activity against CD30(+) Hodgkin-derived cells. Specific binding activity of Ang-CD30L was verified by competition with anti-CD30 monoclonal antibody Ki-4 and commercially available CD30L-CD8 chimeric protein. Ang-CD30L showed RNase activity in vitro. The human recombinant immunotoxin showed significant toxicity toward several CD30-positive cell lines (HDLM-2, L1236, KM-H2, and L540Cy) and exhibited highest cytotoxicity against L540 cells (IC50 = 8 ng/ml) as determined by cell proliferation assays. CD30 specificity was confirmed by competitive toxicity assays. This is the first report on the specific cytotoxicity of a recombinant completely human fusion toxin with possibly largely reduced immunogenicity for the treatment of CD30-positive malignancies.