Abstract 5745: Synthesis and characterization of a folate receptor alpha-specific ligand for distinguishing cancer tissue from sites of inflammation
Authors: Vaitilingam Balasubramaniam , Chelvam Venkatesh , Kularatne Sumith A. , Poh Scott , Ayala-Lopez Wilfredo , Low Philip S. 
Published: August 2012

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. References 1.Low, P. S.; Henne, W. A.; Doorneweerd D. D. Acc. Chem. Res. 2008;41:120-129. 2.Kularatne, S. K.; Low, P. S.; Current opinion Chem Biol. 2009;13:256-262. 3. Low, P. S.; Antony, C. A. Adv Drug Deliv Rev. 2004;56:1055-1058. 4. Ayala-Lopez, W.; Xia, W.; Varghese, B.; Low, P. S. J Nucl Med. 2010;51:768-774. 5. Leamon, P. C.; Parker, M. A.; Vlahov, I. R. Bioconjugate Chem. 2002;13:1200-1210.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5745. doi:1538-7445.AM2012-5745