Immunotoxins with increased activity against epidermal growth factor receptor vIII-expressing cells produced by antibody phage display

Recombinant immunotoxins are fusion proteins composed of Fv regions of anti bodies and bacterial or plant toxins that are being developed for the targe ted therapy of cancer, MR1(Fv)-PE38 is a single-chain recombinant immunotox in that targets a mutant form of the epidermal growth factor receptor (EGFR ), EGFRvIII, that is frequently overexpressed in malignant glioblastomas. W e have used random complementarity determining region (CDR) mutagenesis to obtain mutants of MR1(Fv) with an increased affinity for EGFRvIII and an in creased activity when converted to a recombinant immunotoxin. Initially, ni ne residues of heavy chain CDR3 were randomly mutagenized, and several muta nts with increased binding affinity were isolated. All mutations were locat ed at amino acids 98 and 99, which correspond to a DNA hot spot, a DNA sequ ence that mutates at high frequency during natural antibody maturation. A s pecific region of variable region of antibody light chain CDR3 was mutageni zed that corresponded to a hot spot and a mutant (MR1-1) with an additional increase in affinity, and cytotoxic activity was isolated. These studies s how that targeting hot spots in the CDRs of Fvs is an effective approach to obtaining Fvs with increased affinity. The increased affinity of MR1-1(FV) makes it an attractive candidate for the targeted therapy of glioblastomas .