Human angiogenin fused to human CD30 ligand (Ang-CD30L) exhibits specific cytotoxicity against CD30-positive lymphoma.
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 (IC(50) = 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.
Huhn
M
M
Fraunhofer IME, Department of Pharmaceutical Product Development, 52074 Aachen, Germany.
Sasse
S
S
Tur
M K
MK
Matthey
B
B
Schinköthe
T
T
Rybak
S M
SM
Barth
S
S
Engert
A
A
D053285:CD30 Ligand; D003001:Cloning, Molecular; D006689:Hodgkin Disease / Q000188:drug therapy* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D017730:Ki-1 Antigen / Q000276:immunology / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D012259:Ribonuclease, Pancreatic / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D012260:Ribonucleases / Q000378:metabolism; D014407:Tumor Cells, Cultured
D053285:CD30 Ligand; D000922:Immunotoxins; D017730:Ki-1 Antigen; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C505774:TNFSF8 protein, human; D012260:Ribonucleases; C046366:angiogenin; D012259:Ribonuclease, Pancreatic