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 Tc-99m(I) Tricarbonyl Complex

摘要

We aimed to investigate the feasibility of conjugating synthetic hexahistidine peptides (His(6)) peptides to panitumumab Fab (PmFab) to enable labeling with [Tc-99m(H2O3(CO)(3)](+) complex and study these radio-immunoconjugates 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 I-123-labeled peptides into the conjugation reaction. Radiolabeling was achieved by incubation of PmFab-His(6) in PBS, pH 7.0, with [Tc-99m(H2O3(CO)(3)](+) in a 1.4 MBq/mu g ratio. The complex was prepared by adding [(TcO4)-Tc-99m](-) 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 Tc-99m-PmFab-His(6) (70 mu g; 35-40 MBq) injected i.v. (tail vein) were compared to irrelevant Tc-99m-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 Tc-99m-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 Tc-99m-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. Tc-99m-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 x 10(-8) mol/L. Tumor uptake of (99m)TcPmFab-His(6) at 24 h p.i. was significantly (P < 0.05) higher than irrelevant Tc-99m-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 Tc-99m-PmFab-His(6) would be 2-3 mSv, which is less than for a Tc-99m-medronate bone scan (4 mSv).