Preparation, characterization, and biological evaluation of a streptavidin-chimeric t84.66 conjugate for antibody pretargeting.

In order to develop a new system for the antibody pretargeting of carcinoembryonic antigen (CEA)-positive cancers, a streptavidin (SA) conjugate of the monoclonal antibody (mAb) chimeric, T84.66, was synthesized and characterized. Antibody disulfide bonds were reduced with 1,4-dithiothreitol, and the resulting thiols were reacted with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate-derivatized streptavidin (SMCC-SA). The desired SA-cT84.66 conjugate was purified by iminobiotin affinity chromatography and size-exclusion high-performance liquid chromatography (HPLC). The molecular weight of the SA-cT84.66 conjugate (210 kDa) and immounoreactivity (100%) were confirmed by size-exclusion HPLC, and the conjugate bound three equivalents of (111)In-DOTA-biotin. SA-cT84.66-pretargeted (111)In-DOTA-biotin was evaluated in nude mice bearing LS174T human colon carcinoma xenografts. Tumor uptake of (111)In-DOTA-biotin peaked at 3.32% injected dose per gram after 15 minutes. Clearance from blood and normal organs was extremely rapid, and tumor-to-blood ratios were >20:1 after 24 hours. The specific tumor targeting and rapid whole-body clearance of SA-cT84.66-pretargeted (111)In-DOTA-biotin indicated that this system is promising for the imaging and therapy of CEA-positive cancers.

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.

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.

Radiolanthanide-labeled monoclonal antibody CC49 for radioimmunotherapy of cancer: biological comparison of DOTA conjugates and 149Pm, 166Ho, and 177Lu.

The radiolanthanides 149Pm, 166Ho, and 177Lu have decay characteristics suitable for radioimmunotherapy (RIT) of cancer. N-Hydroxysulfosuccinimidyl DOTA (DOTA-OSSu) and methoxy-DOTA (MeO-DOTA) were conjugated to the anti-TAG-72 monoclonal antibody CC49 for radiolabeling with 149Pm, 166Ho, and 177Lu. While both DOTA conjugates could be labeled to high specific activity with 177Lu, MeO-DOTA afforded superior conjugate stability, radiolabeling, and radiochemical purity. Pilot biodistributions in nude mice bearing LS174T human colon carcinoma xenografts demonstrated that MeO-DOTA afforded higher tumor uptake and lower kidney retention of 177Lu than DOTA-OSSu. The in vitro stability of 149Pm-, 166Ho-, and 177Lu-MeO-DOTA-CC49 was evaluated using serum and hydroxyapatite assays. Serum stability of radiolanthanide-labeled MeO-DOTA-CC49 followed a trend based on the coordination energies of the radiometals, with 177Lu showing the highest stability after 96 to 168 h at 37 C. In contrast, MeO-DOTA-CC49 labeled with all three radiolanthanides was >92% stable to hydroxyapatite challenge for 168 h at 37 C. Comprehensive biodistributions of 149Pm-, 166Ho-, and 177Lu-MeO-DOTA-CC49 were obtained in LS174T-bearing nude mice. Maximum tumor uptakes were 100.0% ID/g for 149Pm at 96 h, 69.5% ID/g for 166Ho at 96 h, and 132.4% ID/g for 177Lu at 168 h. Normal organ uptakes were generally low, except in the liver, spleen, and kidney at early time points. By 96 to 168 h postinjection, nontarget organ uptake decreased to approximately 7% ID/g (kidney), 12% ID/g (spleen), and 20% ID/g (liver) for each radiolanthanide. When labeled with 149Pm, 166Ho, and 177Lu, MeO-DOTA-CC49 has potential for RIT of colorectal cancer and other carcinomas.