Increased binding affinity enhances targeting of glioma xenografts by EGFRVIII-specific scFv

Combinatorial variation of CDR3 of V-H and V-L, followed by phage display, was used to select affinity mutants of the parental anti-epidermal growth f actor receptor-vIII (EGFR-vIII) scFv MR1, One mutant, MR1-1(scFv), had incr eased specific binding affinity for EGFRvIII. It was produced and radiolabe led, and its biodistribution was evaluated in human glioma-bearing athymic mice, MR1-1 targeted the same EGFRvIII epitope as MR1 with an approximately 15-fold higher affinity (K-d 1.5 x 10(-9) M) measured by surface resonance analysis. Labeling with I-131 or I-125 was performed, and the immunoreacti ve fraction of the labeled MR1-1(scFv) was 50% to 55%, After incubation at 37 degreesC for 4 days, the binding affinity was maintained at 60% of initi al levels. The specificity of MR1-1 for EGFRvIII was demonstrated in vitro by flow cytometry and incubation of FITC-labeled scFv with the EGFRvIII-exp ressing U87MG.Delta EGFR cell line or with the EGFRvIII-negative U87MG cell line in the presence or absence of competing unlabeled MR1-1(scFv), We als o investigated the internalization and processing of MR1-1 compared with MR 1; MR1-1 exhibited levels of both cell surface retention and internalizatio n up to 5 times higher than those by MR1, In biodistribution studies perfor med in athymic mice bearing s.c. U87MG.Delta EGFR tumor xenografts, animals received paired-label intratumoral infusions of I-131-labeled MR1-1 (scFv) and I-125-labeled MR1 (scFv), Our results showed an up to 244% +/- 77% inc rease in tumor uptake for MR1-1 compared with that for MR1, The improved tu mor retention of MR1-1(scFv) combined with its rapid clearance from normal tissues also resulted in sustained higher tumor:normal organ ratios. These results suggest that the improved affinity of MR1-1 can significantly impac t in vivo glioma-specific targeting and immunotherapy, Int. J, Cancer 88:96 2-969, 2000, (C) 2000 Wiley-Liss, Inc.