Evaluation of an anti-p185(HER2) (scFv-C(H)2-C(H)3)2 fragment following radioiodination using two different residualizing labels: SGMIB and IB-Mal-D-GEEEK

Abstract

Introduction: A 105-kDa double mutant single-chain Fv-Fc fragment (scFv-Fc DM) derived from the anti-p185(HER2) 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.

Methods: The scFv-Fc DM fragment was radioiodinated using N-succinimidyl 4-guanidinomethyl 3-[(131)I]iodobenzoate ([(131)I]SGMIB) and N(epsilon)-(3-[(131)I]iodobenzoyl)-Lys(5)-N(alpha)- maleimido-Gly(1)-GEEEK ([(131)I]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 [(125)I]IB-Mal-d-GEEEK and [(131)I]SGMIB was compared in athymic mice bearing MCF7-HER2 xenografts.

Results: The scFv-Fc DM fragment was labeled with [(131)I]SGMIB and [(131)I]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 [(125)I]IB-Mal-d-GEEEK-labeled scFv-Fc DM fragment compared with that of the [(131)I]SGMIB-labeled fragment, particularly at later time points. The uptake of (125)I 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 (131)I at 24 and 48 h, respectively. However, the [(125)I]IB-Mal-d-GEEEK-labeled scFv-Fc DM fragment also exhibited considerably higher levels of radioiodine activity in liver, spleen and kidney.

Conclusions: The 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.

Figure 1

Size exclusion chromatography of scFv-Fc DM fragment following radioiodination (open circles) and after incubation with 10- to 20-fold excess of ECD at 37°C for 30 min (filled circles). Left: ¹²⁵I-scFv-Fc DM (Iodogen method); Right = [¹³¹I]SGMIB-scFv-Fc DM

Figure 2

Size exclusion chromatography of scFv-Fc DM fragment following radioiodination (open circles) and after incubation with 10- to 20-fold excess of ECD at 37°C for 30 min (filled circles). Left: ¹²⁵I-scFv-Fc DM (Iodogen method); Right = [¹³¹I]IB-Mal-D-GEEEK-scFv-Fc DM

Figure 3

Paired-label internalization of scFv-Fc DM fragment radiolabeled with ¹²⁵I using Iodogen (circles) and with ¹³¹I either by [¹³¹I]SGMIB (triangles; panel A) or by [¹³¹I]IB-Mal-D-GEEEK (squares; panel B) in MCF7-HER2 cells. Cells were incubated with the tracer pair at 4°C for 1 h, and after removal of the unbound radioactivity, brought to 37°C and processed at various time periods. Data shown are the percent of initially bound radioactivity that was trapped intracellularly.

Figure 4

Paired-label internalization of scFv-Fc DM fragment radiolabeled with [¹²⁵I]IB-Mal-D-GEEEK (squares) and [¹³¹I]SGMIB (triangles) in MCF7-HER2 cells. Cells were incubated with the tracer pair at 4°C for 1 h, and after removal of the unbound radioactivity, brought to 37°C and processed at various time periods. Data shown are the percent of initially bound radioactivity that was trapped intracellularly.

Figure 5

Tumor-to-tissue ratios observed in athymic mice bearing MCF7-HER2 xenografts and injected with [¹³¹I]SGMIB-scFv-Fc DM (triangles) and [¹²⁵I]IB-Mal-D-GEEEK-scFv-Fc DM (circles).