Development of targeted imaging and therapeutic agents for cholecystokinin 2 receptor expressing tumors

Charity Wayua, Purdue University


The use of conventional systemic chemotherapeutics is limited by their indiscriminate accumulation in both cancer and healthy cells resulting in dose limiting toxicities in untargeted tissues. Ligand-targeted therapies that take advantage of receptor over-expression, can deliver cytotoxic agents selectively to malignant cells, avoiding uptake by healthy cells, and are attractive alternatives to non-targeted therapies. The cholecystokinin 2 receptor (gastrin / CCK2R/CCKBR) is reported to be over expressed in a variety of cancers, including medullary thyroid and hepatocellular carcinomas, and gastric, colorectal, pancreatic and small cell lung cancers. In normal tissues the expression of CCK2R is limited to the gastrointestinal mucosa and the central nervous system. Herein we exploit this selective expression of CCK2R in tumor cells to deliver attached war heads for imaging and therapy of CCK2R. We elected to use a small molecule antagonist, Z-360, that has a sub-nanomolar affinity to the CCK2R and excellent selectivity over CCK1R as a targeting ligand. A ligation site on the antagonist that could be chemically modified without altering its binding affinity to the receptor was then identified and several linkers attached. The optimal linker, a peptidoglycan spacer found to improve the conjugate's water solubility, was then attached to a 99m technetium chelating moiety. The radio imaging conjugate (CRL-99mTc) was found to bind CCK2R expressing cells with nanomolar affinity (KD =30 nM). Binding was quantitatively inhibited by competition with 100 fold excess of the unlabeled conjugate. We evaluated the specificity of the CRL-99mTc in vivo by intravenous injection into athymic nu/nu mice bearing CCK2R tumors. Imaging and biodistribution studies revealed that CRL-99mTc localizes primarily to HEK-293 CCK2R tumor cell xenografts in nu/nu mice (14.25% Injected dose/gram of tissue at 2h post injection; tumor: muscle ratio of 35:1). Similar specificity was observed with a CRL-LS288, targeted near infrared dye conjugated to this antagonist. Blockade of tumor targeting upon administration of excess unlabeled conjugate and the absence of targeting to CCK2R-negative tumors confirmed its specificity CCK2R. Finally, two CCK2R targeted chemotherapeutics (CRL3 DAVBLH and CRL3 TubH) were synthesized by attaching Z-360 to desacetyl vinblastine hydrazide and tubulysin b hydrazide respectively. Both conjugates exhibited high in vitro potency (IC50= 29 nM and 12 nM respectively) and eliminated xenografts expressing CCK2R without any detectable toxicity as evidenced by weight loss. The data from these studies are designed to lead to new targeted therapeutic and companion diagnostic agents for radioimaging, fluorescence-guided surgery and treatment of CCK2R expressing cancers. This tandem use of an imaging and therapeutic agent targeted to the same receptor could allow for detection, staging, monitoring and treatment of CCK2R cancers with improved accuracy and efficacy.




Low, Purdue University.

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