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Introduction: 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. Non-invasive 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 single photon emission computed tomography (SPECT), but showed modest binding affinity and slow blood clearance in vivo. Materials and methods: Based on the crystal structure of cCPE, a series of smaller-sized cCPE194-319 mutants with putatively improved binding affinity for claudin-4 were generated by site-directed mutagenesis (SDM). All peptides were conjugated site-specifically on a C-terminal cysteine using maleimide- diethylenetriamine pentaacetate (DTPA) to enable radiolabelling 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/ computerised tomography (CT) imaging of BALB/c Nude mice bearing PSN-1 or HT1080 tumour 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 compared to HT1080 negative controls. All peptides showed a marked improvement in affinity for claudin-4 in vitro when compared to previously reported values (KD values of 2.2 ± 0.8, 3 ± 0.1, 4.2 ± 0.5 nM, 10 ± 0.9 and 9.7 ± 0.7 nM). Blood clearance of [111In]In-cCPE194-319, as measured by SPECT, was considerably faster when compared to that of [111In]In-cCPE.GST (t1/2<1 min). All radiopeptides showed significantly higher accumulation in PSN-1 xenografts than HT1080 tumours at 90 min post-injection of the tracer ([111In]In-cCPE194-319: 2.7 ± 0.8 vs 0.4 ± 0.1 %ID/g, P<0.001; [111In]In-S313A: 2.3 ± 0.9 vs 0.5 ± 0.1 %ID/g, P<0.01; [111In]In- S307A+N309A+S313A: 2 ± 0.4 vs 0.3 ± 0.1 %ID/g, P<0.01; [111In]In- D284A: 2 ± 0.2 vs 0.7 ± 0.1 %ID/g, P<0.05; [111In]In- L254F+K257D: 6.3 ± 0.9 vs 0.7 ± 0.2 %ID/g, P<0.001). Conclusion: These optimised cCPE-based SPECT imaging agents show great promise as claudin-4-targeting vectors for in vivo imaging of claudin-4 overexpression in pancreatic cancer.


Journal article


The Journal of Nuclear Medicine

Publication Date