Nε-(3-[*I]iodobenzoyl)-Lys5-Nα-maleimido-Gly1-GEEEK ([*I]IB-Mal-D-GEEEK):: A radioiodinated prosthetic group containing negatively charged D-glutamates for labeling internalizing monoclonal antibodies

Novel methods are needed for the radiohalogenation of cell-internalizing proteins and peptides because rapid loss of label occurs after lysosomal processing when these molecules are labeled using conventional radioiodination methodologies. We have developed a radiolabeled prosthetic group that contains multiple negatively charged D-amino acids to facilitate trapping of the radioactivity in the cell after proteolysis of the labeled protein. N-epsilon-(3-[I-125]iodobenzoyl)-Lys(5)-N-alpha-maleimido-Gly(1)-GEEEK ([I-125]IB-Mal-D-GEEEK) was synthesized via iodo-destannylation in 90.3 +/- 3.9% radiochemical yields. This radioiodinated agent was conjugated to iminothiolane-treated L8A4, an anti-epidermal growth factor receptor variant III (EGFRvIII) specific monoclonal antibody (mAb) in 54.3 +/- 17.7% conjugation yields. In vitro assays with the EGFRvIII-expressing U87MG Delta EGFR glioma cell line demonstrated that the internalized radioactivity for the [I-125]IB-Mal-D-GEEEK-L8A4 conjugate increased from 14.1% at 1 h to 44.7% at 24 h and was about 15-fold higher than that of directly radioiodinated L8A4 at 24 h. A commensurately increased tumor uptake in vivo in athymic mice bearing subcutaneous U87MG Delta EGFR xenografts (52.6 +/- 14.3% injected dose per gram versus 17.4 +/- 3.5% ID/g at 72 h) also was observed. These results suggest that [I-125]IB-Mal-D-GEEEK is a promising reagent for the radioiodination of internalizing mAbs.