An anti-GD2 single chain Fv selected by phage display and fused to Pseudomonas exotoxin A develops specific cytotoxic activity against neuroblastoma derived cell lines.
Since the disialoganglioside GD2 is abundantly present on the surface of neuroblastoma cells, we constructed a new recombinant immunotoxin for possible clinical use in patients with neuroblastoma. A functional 14.18 scFv-phage was obtained by selection of an anti-GD2 hybridoma derived phage antibody mini-library on the neuroblastoma-derived, GD2-expressing cell line IMR5. By insertion into the bacterial expression vector pBM1.1 the selected scFv was fused to a deletion mutant of Pseudomonas exotoxin A (ETA'). Periplasmically expressed 14.18(scFv)-ETA' bound to the GD2 expressing cell line IMR5, but not to the GD2 negative Hodgkin-derived cell line L540Cy as documented by ELISA and flow cytometry. The recombinant immunotoxin (rIT) inhibited cell viability of IMR5 cells by 50% at concentrations (IC(50)) of 0.326 microg/ml. This recombinant immunotoxin will be further investigated in vivo for its value as a new immunotherapeutic agent for the treatment of patients with neuroblastoma.
Tur
M K
MK
Fraunhofer IUCT, Department of Molecular Biotechnology, Pharmaceutical Product Development, Aachen, Germany.
Sasse
S
S
Stöcker
M
M
Djabelkhir
K
K
Huhn
M
M
Matthey
B
B
Gottstein
C
C
Pfitzner
T
T
Engert
A
A
Barth
S
S
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000494:pharmacology*; D001427:Bacterial Toxins; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects / Q000276:immunology; D003001:Cloning, Molecular; D003602:Cytotoxicity, Immunologic; D004305:Dose-Response Relationship, Drug; D005098:Exotoxins / Q000235:genetics; D005732:Gangliosides / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D007135:Immunoglobulin Variable Region / Q000235:genetics; D000922:Immunotoxins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D009447:Neuroblastoma / Q000188:drug therapy* / Q000276:immunology; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000276:immunology; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D005098:Exotoxins; D005732:Gangliosides; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C055966:immunoglobulin Fv; C019403:ganglioside, GD2