CD30L-ETA': a new recombinant immunotoxin based on the CD30 ligand for possible use against human lymphoma.
Recombinant DNA technology makes it possible to genetically fuse V genes or cytokines to toxin domains, resulting in immunotherapeutics for selective destruction of tumor cells. Since recombinant immunotoxins can be easily manipulated in terms of affinity or cytotoxic potency and produced in large quantities, we have developed a new CD30 ligand-based fusion toxin (CD30L-ETA'). Human CD30L cDNA was ligated into a pET-based expression plasmid and thereby fused to a modified Pseudomonas aeruginosa exotoxin A (ETA') lacking its cell-binding domain I. After IPTG-indiced expression in E. coli strain BL21(DE3), the 60 kDa His-tagged fusion protein (CD30L-ETA') was isolated from inclusion bodies. Denatured protein was renatured in the presence of 0.4 M arginine and a glutathione redox system. Refolded protein was purified and concentrated by ion-exchange chromatography on a HiTrap Q column. The binding properties of CD30L-ETA' were evaluated by competitive ELISA, immunohistochemical staining, and FACS analysis on CD30-expressing cells. The in vitro toxicity of the fusion protein was then tested on the CD30+ Hodgkin-derived cell line L540cy and the Burkitt's lymphoma cell line BL38. CD30L-ETA' exhibited specific cytotoxicity against L540cy cells (IC50 = 24 ng/ml) as determined by [3H]leucine uptake assays. This is the first report on the specificity and cytotoxic potency of a chimeric CD30L fusion toxin against Hodgkin's disease-derived cells.
Barth
S
S
Medizinische Klinik I de Universität zu Köln, Cologne, Germany.
Matthey
B
B
Huhn
M
M
Diehl
V
V
Engert
A
A
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D001427:Bacterial Toxins; D001483:Base Sequence; D002051:Burkitt Lymphoma / Q000188:drug therapy* / Q000378:metabolism; D053285:CD30 Ligand; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D005098:Exotoxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D006689:Hodgkin Disease / Q000188:drug therapy* / Q000378:metabolism; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D017730:Ki-1 Antigen; D008024:Ligands; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D053285:CD30 Ligand; D017931:DNA Primers; D004274:DNA, Recombinant; D005098:Exotoxins; D000922:Immunotoxins; D017730:Ki-1 Antigen; D008024:Ligands; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C505774:TNFSF8 protein, human