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.
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The authors thank Deborah Scollard and Teesha Komal at the STTARR Innovation Centre for technical support. The authors also thank Dr. Sachdev Sidhu at the Toronto Recombinant Antibody Centre at the University of Toronto for providing Fab 3913.
This study was supported by a grant from the Canadian Cancer Society to RMR (Grant #704660). VF and NA received scholarships from the STARS21 strategic training program in radiation research supported by the Terry Fox Foundation. VF is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) by the Polymer Nanoparticles for Drug Delivery (PoND) Training Program, MDS Nordion Graduate Scholarship in Radiopharmaceutical Sciences (OTSS), and William Knapp Buckley Award. VF and AK received scholarships from the Centre for Pharmaceutical Oncology at the University of Toronto. NA was supported by a fellowship with the Precision Medicine Initiative (PRiME) at the University of Toronto. AK is supported by the Queen Elizabeth II Graduate Scholarship in Science and Technology (QEII-GSST). Pertuzumab was provided by Genentech Inc. (South San Francisco, CA, USA) through a Materials Transfer Agreement with the University of Toronto.
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Facca, V.J., Al-saden, N., Ku, A. et al. Imaging of HER2-Positive Tumors in NOD/SCID Mice with Pertuzumab Fab-Hexahistidine Peptide Immunoconjugates Labeled with [99mTc]-(I)-Tricarbonyl Complex. Mol Imaging Biol (2021). https://doi.org/10.1007/s11307-020-01571-z
- Molecular imaging
- Ovarian cancer
- Breast cancer