Molecular pharmaceutics

Positron-Emission Tomography of HER2-Positive Breast Cancer Xenografts in Mice with 89Zr-Labeled Trastuzumab-DM1: A Comparison with 89Zr-Labeled Trastuzumab.

PMID 29957952


Our aim was to synthesize 89Zr-labeled trastuzumab-emtansine (89Zr-DFO-T-DM1) to probe the delivery of trastuzumab-emtansine (T-DM1) to HER2-positive breast cancer (BC) by positron emission tomography (PET). We further aimed to compare the tumor and normal tissue uptake of 89Zr-DFO-T-DM1 with 89Zr-DFO-trastuzumab. T-DM1 was modified with 3.0 ± 0.2 desferrioxamine (DFO) chelators for complexing 89Zr by reaction with a 14-fold molar excess of p-NCS-Bz-DFO. The number of DFO chelators per T-DM1 molecule was quantified spectrophotometrically at 430 nm after the reaction with FeCl3. SDS-PAGE and SE-HPLC demonstrated a pure and homogeneous immunoconjugate. DFO-T-DM1 and DFO-trastuzumab were labeled to high efficiency (>97%) with 89Zr at a specific activity of 0.55 MBq/μg in a 2 M Na2CO3/0.5 M HEPES buffer, pH 7.0, at RT for 60-90 min. The labeling efficiency was measured by instant thin layer-silica gel chromatography (ITLC-SG) and SE-HPLC. HER2 immunoreactivity was measured in a saturation binding assay using SK-BR-3 human BC cells. 89Zr-DFO-T-DM1 exhibited high affinity HER2 binding ( Kd = 3.7 ± 0.4 nM) that was not significantly different than 89Zr-DFO-trastuzumab (4.4 ± 0.5 nM; P = 0.06). The optimal time for tumor imaging with 89Zr-DFO-T-DM1 was 96 h post-injection in NOD-scid mice with s.c. HER2 overexpressing (HER2 3+) BT-474 human BC xenografts. Tumor uptake was dependent on the level of HER2 expression in mice with s.c. BT-474 (HER2 3+), MDA-MB-231/H2N (HER2 2+), MDA-MB-231 (HER2 0-1+), or MDA-MB-468 (HER2 0) human BC xenografts injected with 89Zr-DFO-T-DM1 (10 μg, 5.2 MBq). All tumors were visualized by microPET/CT, but the tumor intensity was greatest for BT-474 and MDA-MB-231/H2N xenografts. The tumor uptake of 89Zr-DFO-T-DM1 was 4.1-fold significantly higher than 89Zr-DFO-trastuzumab in mice with s.c. BT-474 (HER2 3+) xenografts (43.5 ± 4.3%ID/g vs 10.6 ± 5.4%ID/g, respectively; P < 0.001). Tumor uptake of 89Zr-DFO-T-DM1 in MDA-MB-231/H2N xenografts (HER2 2+) was 3.7-fold significantly higher than 89Zr-DFO-trastuzumab (10.1 ± 3.6%ID/g vs 2.7 ± 0.5%ID/g; P < 0.001). The higher tumor uptake of 89Zr-DFO-T-DM1 compared to 89Zr-DFO-trastuzumab was not due to a higher HER2 binding affinity or to differences in the residence time in the blood or tumor size. We conclude that 89Zr-DFO-T-DM1 is a useful probe to assess the delivery of T-DM1 to HER2-positive BC. PET with 89Zr-DFO-trastuzumab has been studied clinically to predict response to T-DM1, but our results suggest that 89Zr-DFO-T-DM1 may be more accurate due to the differences in the tumor uptake observed in the preclinical BC xenograft mouse models.

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P9523 Plasma, from human