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 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.
Kuan
C T
CT
Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA.
Wikstrand
C J
CJ
Archer
G
G
Beers
R
R
Pastan
I
I
Zalutsky
M R
MR
Bigner
D D
DD
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D002462:Cell Membrane / Q000378:metabolism; D066246:ErbB Receptors / Q000276:immunology / Q000378:metabolism*; D005260:Female; D005910:Glioma / Q000276:immunology / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000276:immunology / Q000378:metabolism*; D000922:Immunotoxins / Q000276:immunology / Q000378:metabolism* / Q000493:pharmacokinetics; D007457:Iodine Radioisotopes / Q000378:metabolism / Q000493:pharmacokinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D014018:Tissue Distribution; D014183:Transplantation, Heterologous
D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D007457:Iodine Radioisotopes; C410723:epidermal growth factor receptor VIII; D066246:ErbB Receptors