A single chain immunotoxin, targeting the melanoma-associated chondroitin sulfate proteoglycan, is a potent inducer of apoptosis in cultured human melanoma cells.
A recombinant immunotoxin was constructed by fusing a single chain fragment variable antibody fragment, specific for the melanoma-associated chondroitin sulfate proteoglycan (MCSP), to a truncated variant of Pseudomonas exotoxin A (ETA'), carrying a C-terminal KDEL-peptide for improved retrograde intracellular transport. The resulting immunotoxin MCSP-ETA' was periplasmatically expressed in Escherichia coli and purified under native conditions by affinity chromatography resulting in a yield of approximately 30 mug/l bacterial culture. This immunotoxin induced antigen-specific apoptosis in the cultured human melanoma-derived cell lines A2058 and A375M, and treatment with a single dose of the agent eliminated up to 80% of these cells within 72 h. The dose needed for half-maximum killing (EC50) was approximately 1 nmol/l for both cell lines. MCSP-ETA' also displayed cytotoxic activity against cultured primary melanoma cells from patients with advanced disease (pathologic stages IIIC and IV), with net cell death reaching up to 70% within 96 h after treatment with a single dose of 14 nmol/l. MCSP-ETA' induced cell death synergistically with cyclosporin A, both in established human melanoma cell lines and cultured primary melanoma cells. The distinctive antigen-restricted induction of apoptosis and the synergy with cyclosporin A justify further evaluation of this novel agent with regard to its potential application for the treatment of malignant melanoma.
Schwenkert
Michael
M
University of Erlangen-Nuremberg, Erlangen, Germany.
Birkholz
Katrin
K
Schwemmlein
Michael
M
Kellner
Christian
C
Kügler
Markus
M
Peipp
Matthias
M
Nettelbeck
Dirk M
DM
Schuler-Thurner
Beatrice
B
Schaft
Niels
N
Dörrie
Jan
J
Ferrone
Soldano
S
Kämpgen
Eckhart
E
Fey
Georg H
GH
D036002:ADP Ribose Transferases / Q000276:immunology / Q000378:metabolism; D017209:Apoptosis; D001427:Bacterial Toxins / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D011508:Chondroitin Sulfate Proteoglycans / Q000276:immunology* / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology; D003602:Cytotoxicity, Immunologic; D005098:Exotoxins / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007167:Immunotherapy; D000922:Immunotoxins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D008545:Melanoma / Q000276:immunology / Q000378:metabolism / Q000473:pathology* / Q000628:therapy; D011993:Recombinant Fusion Proteins / Q000276:immunology; D014407:Tumor Cells, Cultured; D037521:Virulence Factors / Q000276:immunology / Q000378:metabolism; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D011508:Chondroitin Sulfate Proteoglycans; D016572:Cyclosporine; D005098:Exotoxins; D007166:Immunosuppressive Agents; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A