Radioiodination via D-amino acid peptide enhances cellular retention and tumor xenograft targeting of an internalizing anti-epidermal growth factor receptor variant III monoclonal antibody

The mutant epidermal growth factor receptor variant III (EGFRvIII) has been found on gliomas and other tumors but not on normal tissues, including tho se that express the wild-type receptor. Monoclonal antibodies (mAbs) specif ic for EGFRvIII are rapidly internalized and degraded after binding to EGFR vIII-expressing cells. If anti-EGFRvIII mAbs are to be useful for radioimmu notherapy, then methods for trapping radionuclides in target cells after mA b processing are required, Because lysosomes are known to retain positively charged molecules, we have evaluated a new reagent for this purpose that u ses a polycationinc peptide composed of D-amino acids (D-Lys-D-Arg-D-Tyr-D- Arg-D-Arg; D-KRYRR). D-KRYRR was first labeled using Iodogen and then coupl ed to the murine anti-EGFRvIII mAb L8A4 via maleimido bond formation in 60% yield. In vitro assays with the U87 Delta EGFR cell line indicated that in ternalized and total cell-associated activity for the I-125-labeled D-KRYRR -L8A4 conjugate were up to 4 and 5 times higher, respectively, than for L8A 4 labeled with I-131 using Iodogen, Paired-label comparisons in athymic mic e with s,c, U87 Delta EGFR xenografts demonstrated up to 5-fold higher tumo r uptake for mAb labeled using D-KRYRR. Higher levels of radioiodine activi ty also were observed in kidney when L8A4 was labeled using KRYRR. Another paired-label study directly compared L8A4 labeled using radioiodinated D-KR YRR and L-KRYRR, and confirmed the role of D-amino acids in enhancing tumor uptake. These results suggest that D-KRYRR is a promising reagent for the radioiodination of internalizing mAbs, such as the anti-EGFRvIII mAb L8A4.