Imaging of HER2-Positive Tumors in NOD/SCID Mice with Pertuzumab Fab-Hexahistidine Peptide Immunoconjugates Labeled with [99mTc]-(I)-Tricarbonyl Complex

Molecular imaging of tumor HER2 expression may allow patient selection for HER2-targeted therapies. Our aim was to introduce hexahistidine (His6) peptides into pertuzumab Fab to enable labeling with the [99mTc(CO)3(H2O)3]+ complex and study these radioimmunoconjugates for microSPECT/CT imaging of HER2-positive tumor xenografts in mice. Fab were produced by papain digestion of pertuzumab and reacted with sulfo-SMCC for conjugation to His6-containing peptides (CGYGGHHHHHH). His6-peptide conjugation was measured by a radiometric assay. His6-pertuzumab Fab were labeled at 0.4–1.0 MBq/μg with [99mTc(CO)3(H2O)3]+ for 1 h at 37 °C. HER2 immunoreactivity was assessed in a direct (saturation) binding assay using HER2-overexpressing SK-BR-3 human breast cancer (BC) cells. MicroSPECT/CT and biodistribution studies were performed in NOD/SCID mice with HER2-positive s.c. SK-OV-3 human ovarian cancer, or MDA-MB-361 or MDA-MB-231 human BC xenografts at 4 or 24 h post i.v. injection of [99mTc]His6-pertuzumab Fab (29–49 MBq, 70 μg). The specificity of tumor uptake was assessed by comparison to irrelevant [99mTc]Fab 3913 in SK-OV-3 tumor-bearing mice. SDS-PAGE analysis demonstrated cleavage of pertuzumab to produce Fab, which eluted as a single peak with a retention time of 13.8 min on SE-HPLC. Fab were conjugated to 2.1 ± 0.5 His6 peptides and labeled with [99mTc(CO)3(H2O)3]+ to a radiochemical purity of 92–97 % at 0.4–0.8 MBq/μg. [99mTc]His6-pertuzumab Fab exhibited saturable and specific binding to SK-BR-3 cells with a KD = 51.3 ± 5.2 × 10−9 M and Bmax = 3.5 ± 0.1 × 106 receptors/cell. SK-OV-3 tumors were imaged at 4 and 24 h p.i [99mTc]His6-pertuzumab Fab. Tumor uptake at 24 h p.i. was 4.1 ± 0.6 %ID/g, which was 13-fold significantly greater than [99mTc]Fab 3913 (0.3 ± 0.0 %ID/g; P < 0.01). MicroSPECT/CT imaged HER2-overexpressing MDA-MB-361 tumors but not MDA-MB-231 tumors with low HER2 expression. Tumor uptake was 5.2-fold significantly greater at 24 h p.i. in MDA-MB-361 than MDA-MB-231 tumors (3.2 ± 0.1 %ID/g vs. 0.8 ± 0.1 %ID/g; P < 0.05). MicroSPECT/CT with [99mTc]His6-pertuzumab Fab imaged tumors in NOD/SCID mice that exhibited intermediate or high HER2 expression, but not tumors with low HER2. [99mTc]His6-pertuzumab Fab is promising for SPECT imaging of tumor HER2 expression.