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Increased binding affinity enhances targeting of glioma xenografts by EGFRvIII‐specific scFv
Author(s) -
Kuan ChienTsun,
Wikstrand Carol J.,
Archer Gary,
Beers Richard,
Pastan Ira,
Zalutsky Michael R.,
Bigner Darell D.
Publication year - 2000
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/1097-0215(20001215)88:6<962::aid-ijc20>3.0.co;2-u
Subject(s) - internalization , flow cytometry , microbiology and biotechnology , biodistribution , chemistry , glioma , mutant , cell , cell culture , receptor , in vitro , cancer research , biology , biochemistry , genetics , gene
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 × 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°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.ΔEGFR 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.ΔEGFR 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. Int. J. Cancer 88:962–969, 2000. © 2000 Wiley‐Liss, Inc.