Radiolabeled Ccpe Peptides For Spect Imaging Of Claudin-4 Overexpression In Pancreatic Cancer

Overexpression of tight-junction protein claudin-4 has been detected in primary and metastatic pancreatic cancer tissue and is associated with better prognosis in patients. Noninvasive measurement of claudin-4 expression by imaging methods could provide a means for accelerating detection and stratifying patients into risk groups. Clostridium perfringens enterotoxin (CPE) is a natural ligand for claudin-4 and holds potential as a targeting vector for molecular imaging of claudin-4 overexpression. A glutathione S-transferases (GST)-tagged version of the C terminus of CPE (cCPE) was previously used to delineate claudin-4 overexpression by SPECT but showed modest binding affinity and slow blood clearance in vivo. Methods: On the basis of the crystal structure of cCPE, a series of smaller cCPE194-319 mutants with putatively improved binding affinity for claudin-4 was generated by site-directed mutagenesis. All peptides were conjugated site-specifically on a C-terminal cysteine using maleimide-diethylenetriamine pentaacetate to enable radio labeling with 111In. The binding affinity of all radioconjugates was evaluated in claudin-4-expressing PSN-1 cells and HT1080-negative controls. The specificity of all cCPE mutants to claudin-4 was assessed in HT1080 cells stably transfected with claudin-4. SPECT/CT imaging of BALB/c nude mice bearing PSN-1 or HT1080 tumor xenografts was performed to determine the claudin-4-targeting ability of these peptides in vivo. Results: Uptake of all cCPE-based radioconjugates was significantly higher in PSN-1 cells than in HT1080-negative controls. All peptides showed a marked improvement in affinity for claudin-4 in vitro when compared with previously reported values (dissociation constant: 2.2 +/- 0.8, 3 +/- 0.1, 4.2 +/- 0.5, 10 +/- 0.9, and 9.7 +/- 0.7 nM). Blood clearance of [In-111]In-cCPE194-319, as measured by SPECT, was considerably faster than that of [111In]IncCPE.GST (half-life, <1 min). All radiopeptides showed significantly higher accumulation in PSN-1 xenografts than in HT1080 tumors at 90 min after injection of the tracer ([In-111]In-cCPE(194-319), 2.7 +/- 0.8 vs. 0.4 +/- 0.1 percentage injected dose per gram [%ID/g], P < 0.001; [In-111]In-S313A, 2.3 +/- 0.9 vs. 0.5 +/- 0.1 %ID/g, P < 0.01; [In-111]In-S307A + N309A + S313A, 2 +/- 0.4 vs. 0.3 +/- 0.1 %ID/g, P < 0.01; [In-111]In-D284A, 2 +/- 0.2 vs. 0.7 +/- 0.1 %ID/g, P < 0.05; [In-111]In L254F + K257D, 6.3 +/- 0.9 vs. 0.7 +/- 0.2 %ID/g, P < 0.001). Conclusion: These optimized cCPE-based SPECT imaging agents show great promise as claudin-4-targeting vectors for in vivo imaging of claudin-4 overexpression in pancreatic cancer.