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Human Angiogenin Fused to Human CD30 Ligand (Ang-CD30L) Exhibits Specific Cytotoxicity against CD30-positive Lymphoma¹

Fraunhofer IME, Department of Pharmaceutical Product Development, 52074 Aachen, Germany [M. K. T., M. H., S. B.]; Laboratory of Immunotherapy, Department I of Internal Medicine, University Hospital Cologne, 50931 Cologne, Germany [S. S., B. M., T. S., A. E.]; Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702 [S. M. R.]

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 ß-D-thiogalactoside induction, the M_r 37,000 His₁₀-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 His₁₀-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.

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