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

Abstract

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 factor receptor-vIII (EGFRvIII) scFv MR1. One mutant, MR1-1(scFv), had increased specific binding affinity for EGFRvIII. It was produced and radiolabeled, 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 (131)I or (125)I was performed, and the immunoreactive fraction of the labeled MR1-1(scFv) was 50% to 55%. After incubation at 37 degrees C for 4 days, the binding affinity was maintained at 60% of initial levels. The specificity of MR1-1 for EGFRvIII was demonstrated in vitro by flow cytometry and incubation of FITC-labeled scFv with the EGFRvIII-expressing U87MG. DeltaEGFR cell line or with the EGFRvIII-negative U87MG cell line in the presence or absence of competing unlabeled MR1-1(scFv). We also investigated the internalization and processing of MR1-1 compared with MR1; MR1-1 exhibited levels of both cell surface retention and internalization up to 5 times higher than those by MR1. In biodistribution studies performed in athymic mice bearing s.c. U87MG. DeltaEGFR tumor xenografts, animals received paired-label intratumoral infusions of (131)I-labeled MR1-1(scFv) and (125)I-labeled MR1(scFv). Our results showed an up to 244% +/- 77% increase in tumor uptake for MR1-1 compared with that for MR1. The improved tumor 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 impact in vivo glioma-specific targeting and immunotherapy.

• Authors: C T Kuan, C J Wikstrand, G Archer, R Beers, I Pastan, M R Zalutsky, D D Bigner
• Journal: International journal of cancer (Int J Cancer) Vol. 88 Issue 6 Pg. 962-9 (Dec 15 2000) ISSN: 0020-7136 [Print] United States
• PMID: 11093822 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)
• Copyright: Copyright 2000 Wiley-Liss, Inc.

Chemical References

• Immunoglobulin Variable Region
• Immunotoxins
• Iodine Radioisotopes
• epidermal growth factor receptor VIII
• ErbB Receptors

Topics

• Amino Acid Sequence
• Animals
• Antibody Affinity
• Cell Membrane (metabolism)
• ErbB Receptors (immunology, metabolism)
• Female
• Glioma (immunology, metabolism)
• Immunoglobulin Variable Region (immunology, metabolism)
• Immunotoxins (immunology, metabolism, pharmacokinetics)
• Iodine Radioisotopes (metabolism, pharmacokinetics)
• Mice
• Mice, Inbred BALB C
• Mice, Nude
• Tissue Distribution
• Transplantation, Heterologous