Radiolabeled cCPE Peptides for SPECT Imaging of Claudin-4 Overexpression in Pancreatic Cancer

Abstract

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. <i>Clostridium perfringens</i> 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. <b>Methods:</b> On the basis of the crystal structure of cCPE, a series of smaller cCPE_194–319 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 radiolabeling 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. <b>Results:</b> 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 [¹¹¹In]In-cCPE_194–319, as measured by SPECT, was considerably faster than that of [¹¹¹In]In-cCPE.GST (half-life, &lt;1 min). All radiopeptides showed significantly higher accumulation in PSN-1 xenografts than in HT1080 tumors at 90 min after injection of the tracer ([¹¹¹In]In-cCPE_194–319, 2.7 ± 0.8 vs. 0.4 ± 0.1 percentage injected dose per gram [%ID/g], <i>P</i> &lt; 0.001; [¹¹¹In]In-S313A, 2.3 ± 0.9 vs. 0.5 ± 0.1 %ID/g, <i>P</i> &lt; 0.01; [¹¹¹In]In-S307A + N309A + S313A, 2 ± 0.4 vs. 0.3 ± 0.1 %ID/g, <i>P</i> &lt; 0.01; [¹¹¹In]In-D284A, 2 ± 0.2 vs. 0.7 ± 0.1 %ID/g, <i>P</i> &lt; 0.05; [¹¹¹In]In-L254F + K257D, 6.3 ± 0.9 vs. 0.7 ± 0.2 %ID/g, <i>P</i> &lt; 0.001). <b>Conclusion:</b> 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.

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