RECOMBINANT IMMUNOTOXINS SPECIFIC FOR A MUTANT EPIDERMAL GROWTH-FACTOR RECEPTOR - TARGETING WITH A SINGLE-CHAIN ANTIBODY VARIABLE DOMAIN ISOLATED BY PHAGE DISPLAY

EGFRvIII is a mutant epidermal growth factor receptor found in gliobla stoma, and in carcinoma of the breast, ovary, and lung, The mutant rec eptor has a deletion in its extracellular domain that results in the f ormation of a new, tumor-specific extracellular sequence, Mice were im munized with a synthetic peptide corresponding to this sequence and pu rified EGFRvIII, A single chain antibody variable domain (scFv) phage display library of 8 x 10(6) members was made from the spleen of one i mmunized mouse. A scFv specific for EGFRvIII was isolated from this li brary by panning with successively decreasing amounts of synthetic pep tide, This was used to make an immunotoxin by fusing the scFv DNA sequ ence to sequences coding for domains II and III of Pseudomonas exotoxi n A. Purified immunotoxin had a K-d of 22 nM for peptide and a K-d of 11 nM for cell-surface EGFRvIII. The immunotoxin was very cytotoxic to cells expressing EGFRvIII, with an IC50 of 1 ng/ml (16 pM) on mouse f ibroblasts transfected with EGFRvIII and an IC50 of 7-10 ng/ml (110-16 0 pM) on transfected glioblastoma cells, There was no cytotoxic activi ty at 1000 ng/ml on the untransfected parent glioblastoma cell line, T he immunotoxin was completely stable upon incubation at 37 degrees C f or 24 h in human serum, The combination of good affinity, cytotoxicity and stability make this immunotoxin a candidate for further preclinic al evaluation.