In vitro and in vivo evaluation of ¹⁷⁷Lu- and ⁹⁰Y-labeled E. coli heat-stable enterotoxin for specific targeting of uroguanylin receptors on human colon cancers

Abstract 

The human E. coli heat-stable enterotoxin (ST_h, amino acid sequence N¹SSNYCCELCCNPACTGCY¹⁹) binds specifically to the guanylate cyclase C (GC-C) receptor, which is present in high density on the apical surface of normal intestinal epithelial cells as well as on the surface of human colon cancer cells. Analogs of ST_h are currently being used as vectors targeting human colon cancers. Previous studies in our laboratory have focused on development of ¹¹¹Indium-labeled ST_h analogs for in vivo imaging applications. Here, we extend the scope of this work to include targeting of the therapeutic radionuclides ⁹⁰Y and ¹⁷⁷Lu. The peptide DOTA-F¹⁹-ST_h(1–19) was synthesized using conventional Fmoc-based solid-phase techniques and refolded in dilute aqueous solution. The peptide was purified by RP-HPLC and characterized by MALDI-TOF MS and in vitro receptor binding assay. The DOTA-conjugate was metallated with nonradioactive Lu(III)Cl₃ and Y(III)Cl₃, and IC₅₀ values of 2.6±0.1 and 4.2±0.9 nM were determined for the Lu- and Y-labeled peptides, respectively. ¹⁷⁷Lu(III)Cl₃ and ⁹⁰Y(III)Cl₃ labeling yielded tracer preparations that were inseparable by C18 RP-HPLC, indicating that putative differences between Lu-, Y- and In coordination spheres are not observed in the context of labeled ST_h peptides. In vivo biodistribution studies of the ¹⁷⁷Lu-labeled peptide in severe combined immunodeficient (SCID) mice bearing T-84 human cancer tumor xenografts showed rapid clearance from the bloodstream, with >90 %ID in the urine at 1 h pi. Localization of the tracer within tumor xenografts was 1.86±0.91 %ID/g at 1 h pi, a value higher than for all other tissues with the exception of kidney (2.74±0.24 %ID/g). At 24 h pi, >98 %ID was excreted into the urine, and 0.35±0.23 %ID/g remained in tumor, again higher than in all other tissues except kidney (0.91±0.46 %ID/g). Biodistribution results at 24 h pi for the ⁹⁰Y-labeled peptide mirrored those for the ¹⁷⁷Lu analog, in agreement with the identical behavior of the labeled analogs by C18 RP-HPLC. These results demonstrate the ability of ¹⁷⁷Lu- and ⁹⁰Y-labeled ST_h molecules to specifically target GC-C receptors expressed on T-84 human colon cancer cells.

Keywords: Colorectal cancer, E. coli heat-stable enterotoxin, Uroguanylin, Lutetium-177, Yttrium-90