MicroSPECT/CT Imaging of Cell-Line and Patient-Derived EGFR-Positive Tumor Xenografts in Mice with Panitumumab Fab Modified with Hexahistidine Peptides To Enable Labeling with ^99mTc(I) Tricarbonyl Complex

We aimed to investigate the feasibility of conjugating synthetic hexahistidine peptides (His 6 ) peptides to panitumumab Fab (PmFab) to enable labeling with [ 99m Tc(H 2 O) 3 (CO) 3 ] + complex and study these radioimmunoconjugates for imaging EGFR-overexpressing tumor xenografts in mice by microSPECT/CT. Fab were reacted with a 10-fold excess of sulfo-SMCC to introduce maleimide functional groups for reaction with the terminal thiol on peptides [CGYGGHHHHHH] that harbored the His 6 motif. Modification of Fab with His 6 peptides was assessed by SDS-PAGE/Western blot, and the number of His 6 peptides introduced was quantified by a radiometric assay incorporating 123 I-labeled peptides into the conjugation reaction. Radiolabeling was achieved by incubation of PmFab-His 6 in PBS, pH 7.0, with [ 99m Tc(H 2 O) 3 (CO) 3 ] + in a 1.4 MBq/μg ratio. The complex was prepared by adding [ 99m TcO 4 ] − to an Isolink kit (Paul Scherrer Institute). Immunoreactivity was assessed in a direct (saturation) binding assay using MDA-MB-468 human triple-negative breast cancer (TNBC) cells. Tumor and normal tissue uptake and imaging properties of 99m Tc-PmFab-His 6 (70 μg; 35–40 MBq) injected i.v. (tail vein) were compared to irrelevant 99m Tc-Fab 3913 in NOD/SCID mice engrafted subcutaneously (s.c.) with EGFR-overexpressing MDA-MB-468 or PANC-1 human pancreatic ductal carcinoma (PDCa) cell-line derived xenografts (CLX) at 4 and 24 h post injection (p.i.). In addition, tumor imaging studies were performed with 99m Tc-PmFab-His 6 in mice with patient-derived tumor xenografts (PDX) of TNBC, PDCa, and head and neck squamous cell carcinoma (HNSCC). Biodistribution studies in nontumor bearing Balb/c mice were performed to project the radiation absorbed doses for imaging studies in humans with 99m Tc-PmFab-His 6 . PmFab was derivatized with 0.80 ± 0.03 His 6 peptides. Western blot and SDS-PAGE confirmed the presence of His 6 peptides. 99m Tc-PmFab-His 6 was labeled to high radiochemical purity (≥95%), and the K d for binding to EGFR on MDA-MB-468 cells was 5.5 ± 0.4 × 10 –8 mol/L. Tumor uptake of 99m Tc-PmFab-His 6 at 24 h p.i. was significantly ( P < 0.05) higher than irrelevant 99m Tc-Fab 3913 in mice with MDA-MB-468 tumors (14.9 ± 3.1%ID/g vs 3.0 ± 0.9%ID/g) and in mice with PANC-1 tumors (5.6 ± 0.6 vs 0.5 ± 0.1%ID/g). In mice implanted orthotopically in the pancreas with the same PDCa PDX, tumor uptake at 24 h p.i. was 4.2 ± 0.2%ID/g. Locoregional metastases of these PDCa tumors in the peritoneum exhibited slightly and significantly lower uptake than the primary tumors (3.1 ± 0.3 vs 4.2 ± 0.3%ID/g; P = 0.02). In mice implanted with different TNBC or HNSCC PDX, tumor uptake at 24 h p.i. was variable and ranged from 3.7 to 11.4%ID/g and 3.8–14.5%ID/g, respectively. MicroSPECT/CT visualized all CLX and PDX tumor xenografts at 4 and 24 h p.i. Dosimetry estimates revealed that in humans, the whole body dose from administration of 740–1110 MBq of 99m Tc-PmFab-His 6 would be 2–3 mSv, which is less than for a 99m Tc-medronate bone scan (4 mSv).

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