Evaluation of an anti-p185HER2 (scFv-CH2-CH3)2 fragment following radioiodination using two different residualizing labels: SGMIB and IB-Mal-d-GEEEK

IntroductionA 105-kDa double mutant single-chain Fv-Fc fragment (scFv-Fc DM) derived from the anti-p185HER2 hu4D5v8 antibody (trastuzumab; Herceptin) has been described recently. The goal of this study was to investigate whether improved tumor targeting could be achieved with this fragment through the use of residualizing radioiodination methods.MethodsThe scFv-Fc DM fragment was radioiodinated using N-succinimidyl 4-guanidinomethyl 3-[131I]iodobenzoate ([131I]SGMIB) and Nɛ-(3-[131I]iodobenzoyl)-Lys5-Nα- maleimido-Gly1-GEEEK ([131I]IB-Mal-d-GEEEK), two residualizing radioiodination agents that have been used successfully with intact antibodies. Paired-label internalization assays of the labeled fragments were performed in vitro using MCF7 human breast cancer cells transfected to express HER2 (MCF7-HER2); comparisons were made to scFv-Fc DM directly radioiodinated using Iodogen. The tissue distribution of the scFv-Fc DM labeled with [125I]IB-Mal-d-GEEEK and [131I]SGMIB was compared in athymic mice bearing MCF7-HER2 xenografts.ResultsThe scFv-Fc DM fragment was labeled with [131I]SGMIB and [131I]IB-Mal-d-GEEEK in conjugation yields of 53% and 25%, respectively, with preservation of immunoreactivity for HER2. Internalization assays indicated that labeling via SGMIB resulted in a 1.6- to 3.5-fold higher (P<.05) retention of radioactivity, compared to that from the directly labeled fragment, in HER2-expressing cells during a 24-h observation period. Likewise, the amount of radioactivity retained in cells from the IB-Mal-d-GEEEK-labeled fragment was 1.4- to 3.3-fold higher (P<.05). Tumor uptake of radioiodine activity in athymic mice bearing MCF7-HER2 xenografts in vivo was significantly higher for the [125I]IB-Mal-d-GEEEK-labeled scFv-Fc DM fragment compared with that of the [131I]SGMIB-labeled fragment, particularly at later time points. The uptake of 125I was threefold (3.6±1.1 %ID/g vs. 1.2±0.4 %ID/g) and fourfold (3.1±1.7 %ID/g vs. 0.8±0.4 %ID/g) higher than that for 131I at 24 and 48 h, respectively. However, the [125I]IB-Mal-d-GEEEK-labeled scFv-Fc DM fragment also exhibited considerably higher levels of radioiodine activity in liver, spleen and kidney.ConclusionsThe overall results further demonstrate the potential utility of these two prosthetic groups for the radiohalogenation of internalizing monoclonal antibodies and their fragments. Specifically, the trastuzumab-derived double mutant fragment in combination with these residualizing agents warrants further evaluation for imaging and possibly treatment of HER2 expressing malignancies.