Abstract 5745: Synthesis and characterization of a folate receptor alpha-specific ligand for distinguishing cancer tissue from sites of inflammation

Abstract

Abstract The vitamin folic acid has been extensively utilized to target chemotherapeutic and imaging agents to cancer cells that over-express a folate receptor (FR).1-2 Importantly, both folic acid and conjugates of folic acid bind with similar affinity to the two major isoforms of FR; i.e. FR-α which is primarily expressed on tumor cells and FR-β that is upregulated on activated macrophages. Since some cancer patients may simultaneously suffer from an inflammatory disease that is characterized by accumulation of activated macrophages, folate targeting of therapeutic/imaging agents to either pathology is not currently possible.3 We have, therefore, undertaken to identify a targeting ligand that can deliver attached imaging and therapeutic agents selectively to FR-α expressing tumor cells while avoiding uptake by FR-β expressing activated macrophages. In this poster, we demonstrate that a reduced and alkylated form of folic acid, N5, N10-dimethyl tetrahydrofolate (DMTHF), exhibits the desired selectivity for FR-α. Preliminary in vitro analysis of the affinity of DMTHF for FR-α was performed by incubating increasing concentrations of a radioactive conjugate of DMTHF, termed 99mTc-DMTHF-chelate, with KB cells (a human nasopharyngeal cancer cell line known to express FR-α). The 9mTc-DMTHF-chelate was found to associate with FR expressing KB cells with an apparent Kd of 38 nM in a manner that was quantitatively inhibited by 100-fold molar excess of free folic acid. The negligible affinity of 9mTc-DMTHF-chelate for FR-β was further established by demonstrating its inability to image sites of inflammation4 in animals induced to develop ulcerative colitis, atherosclerosis, rheumatoid arthritis, or muscle trauma. Selectivity for FR-α over FR-β was also tested in mice bearing both an FR-α expressing tumor and dextran sulfate-induced ulcerative colitis. Thus, comparison of the uptake of equivalent doses of 99mTc-DMTHF-chelate and 99mTc-folate-chelate (a radioimaging agent5 that binds FR-α and FR-β equally) revealed that 99mTc-DMTHF-chelate was selectively enriched in FR-α tumors, whereas 99mTc-folate-chelate was concentrated in both FR-α+ tumors and FR-β+ sites of inflammation. Similar FR-α selectivity was further confirmed with multiple DMTHF-targeted fluorescent dyes. Taken together, the above experiments show that DMTHF will bind selectively to malignant cells expressing FR-α in the presence of inflammatory macrophages expressing FR-β.