Complete surgical resection of malignant disease is the only reliable method to cure cancer. Unfortunately, quantitative tumor resection is often limited by a surgeon's ability to locate all malignant disease and distinguish it from healthy tissue. Fluorescence-guided surgery has emerged as a tool to aid surgeons in the identification and removal of malignant lesions. While nontargeted fluorescent dyes have been shown to passively accumulate in some tumors, the resulting tumor-to-background ratios are often poor, and the boundaries between malignant and healthy tissues can be difficult to define. To circumvent these problems, our laboratory has developed high affinity tumor targeting ligands that bind to receptors that are overexpressed on cancer cells and deliver attached molecules selectively into these cells. In this study, we explore the use of two tumor-specific targeting ligands (i.e., folic acid that targets the folate receptor (FR) and DUPA that targets prostate specific membrane antigen (PSMA)) to deliver near-infrared (NIR) fluorescent dyes specifically to FR and PSMA expressing cancers, thereby rendering only the malignant cells highly fluorescent. We report here that all FR- and PSMA-targeted NIR probes examined bind cultured cancer cells in the low nanomolar range. Moreover, upon intravenous injection into tumor-bearing mice with metastatic disease, these same ligand-NIR dye conjugates render receptor-expressing tumor tissues fluorescent, enabling their facile resection with minimal contamination from healthy tissues.

Surgical resection of malignant disease remains one of the most effective tools for treating cancer. Tumor-targeted near-infrared dyes have the potential to improve contrast between normal and malignant tissues, thereby enabling surgeons to more quantitatively resect malignant disease. Because the cholecystokinin 2 receptor (CCK2R and its tumor-specific splice variant CCK2i4svR) is overexpressed in cancers of the lungs, colon, thyroid, pancreas, and stomach, but absent or inaccessible to parenterally administered drugs in most normal tissues, we have undertaken to design a targeting ligand that can deliver attached near-infrared dyes to CCK2R+ tumors. We report here the synthesis and biological characterization of a CCK2R-targeted conjugate of the near-infrared dye, LS-288 (CRL-LS288). We demonstrate that CRL-LS288 binds selectively to CCK2R+ cancer cells with low nanomolar affinity (Kd = 7 × 10(-9) M). We further show that CRL-LS288 localizes primarily to CCK2R-expressing HEK 293 murine tumor xenografts and that dye uptake in these xenografts is significantly reduced when CCK2R are blocked by preinjection of excess ligand (CRL) or when mice are implanted with CCK2R-negative tumors. Because CRL-LS288 is also found to reveal the locations of distant tumor metastases, we suggest that CRL-LS288 has the potential to facilitate intraoperative identification of malignant disease during a variety of cancer debulking surgeries.

As the delivery of selectively targeted cytotoxic agents via antibodies or small molecule ligands to malignancies has begun to show promise in the clinic, the need to identify and validate additional cellular targets for specific therapeutic delivery is critical. Although a multitude of cancers have been targeted using the folate receptor, PSMA, bombesin receptor, somatostatin receptor, LHRH, and αvβ3, there is a notable lack of specific small molecule ligand/receptor pairs to cellular targets found within cancers of the GI tract. Because of the selective GI tract expression of the cholecystokinin 2 receptor (CCK2R), we undertook the creation of conjugates that would deliver microtubule-disrupting drugs to malignancies through the specific targeting of CCK2R via a high affinity small molecule ligand. The cytotoxic activity of these conjugates were shown to be receptor mediated in vitro and in vivo with xenograft mouse models exhibiting delayed growth or regression of tumors that expressed CCK2R. Overall, this work demonstrates that ligands to CCK2R can be used to create selectively targeted therapeutic conjugates.

Tumor-specific targeting ligands were recently exploited to deliver both imaging and therapeutic agents selectively to cancer tissues in vivo. Because the cholecystokinin 2 receptor (CCK2R) is overexpressed in various human cancers (e.g., lung, medullary thyroid, pancreatic, colon, and gastrointestinal stromal tumors) but displays limited expression in normal tissues, natural ligands of CCK2R were recently explored for use in the imaging of CCK2R-expressing cancers. Unfortunately, the results from these studies revealed not only that the peptidic CCK2R ligands were unstable in vivo but also that the ligands that mediated good uptake by tumor tissues also promoted a high level of retention of the radioimaging agent in the kidneys, probably because of capture of the conjugates by peptidescavenging receptors. In an effort to reduce the normal organ retention of CCK2R-targeted drugs, we synthesized a nonpeptidic ligand of CCK2R and examined its specificity for CCK2R both in vitro and in vivo. Methods: Nonpeptidic agonists and antagonists of CCK2R described in the literature were evaluated for their affinities and specificities for CCK2R. Z-360, a benzodiazepine-derived CCK2R antagonist with subnanomolar affinity, was selected for complexation to 99m Tc via multiple spacers. After synthesis and purification, 4 complexes with different physicochemical properties were evaluated for binding to CCK2R-transfected HEK 293 cells. The best conjugate, termed CRL-3-99m Tc, was injected into mice bearing CCK2R tumor xenografts and examined by γ scintigraphy and SPECT/CT. The uptake of the conjugate in various organs was also quantified by tissue resection and γ counting. Results: CRL-3-99m Tc was shown to bind with low nanomolar affinity to CCK2R in vitro and was localized to tumor tissues in athymic nu/nu mice implanted with CCK2R-expressing tumors. At 4 h after injection, tumor uptake was measured at 12.0 ± 2.0 percentage injected dose per gram of tissue. Conclusion: Because the uptake of CRL-3-99m Tc by nonmalignant tissues was negligible and retention in the kidneys was only transient, we suggest that CRL-3-99m Tc may be a useful radioimaging agent for the detection, sizing, and monitoring of CCK2R-expressing tumors.

No abstract