A simple spectrophotometric method was developed to quantitate micromolar concentrations of a bifunctional DTPA ligand in DTPA monoclonal antibody (mAb) conjugates. Titration of a brightly colored 1:2 yttrium (III) complex of arsenazo III with the ligand 1B4M-DTPA obeyed Beer's law over the concentration range 0-2.0 microM 1B4M-DTPA at 652 nm. From a calibration plot of absorbance versus 1B4M molarity, concentrations of 1B4M-DTPA conjugated to mAb were determined. Mole ratios of 1B4M-DTPA to mAb agreed satisfactorily with the ratios obtained by a radioanalytical technique using carbon-14-labeled 1B4M-DTPA and a binding assay using 111In. The spectrophotometric method was applied successfully to the preparation of 1B4M-DTPA mAb anti-TAC, a mAb conjugate used in clinical trials of 90Y radioimmunotherapy.

Monoclonal antibodies (MAbs) labeled with radiometallonuclides via metal chelators are being investigated in the laboratory for use in clinical trials. The biodistribution of 111In- and 88Y-labeled antibody (MAb B72.3) using two isomeric forms (CHX-A and CHX-B) of the 2-(p-isothiocyanatobenzyl)-cyclohexyl-DTPA was compared in athymic mice bearing LS-174T tumors, human colon carcinoma xenografts. CHX-(A or B)-125I-DTPA-B72.3 was co-injected in all athymic mice to assess if the chelate conjugation altered the properties of MAb B72.3. In vitro studies demonstrated maintenance of integrity and immunoreactivity for both radioimmunoconjugates. The in vivo analysis, however, indicated major differences between the two isomer forms. In fact, the 88Y-CHX-A-DTPA radioimmunoconjugate demonstrated over the 7-day study period, a more efficient and stable tumor localization as well as a slower blood clearance rate than the CHX-B-DTPA chelate conjugate, suggesting a greater in vivo stability. Differences were also evident in critical normal organ uptake: no significant increase in liver- and spleen- or bone-to-blood ratios was observed when the CHX-A-DTPA chelate was labeled with indium or yttrium. The results described here demonstrate that the CHX-A-DTPA chelate conjugate can be considered more suitable than the CHX-B-DTPA isomer form when radiometallonuclides are coupled to an MAb.