| Abstract
| - We have synthesized two bifunctional derivatives of the macrocyclicchelating agent 1,4,7,10-tetraazacyclododecane-N,N‘,N‘ ‘,N‘ ‘‘-tetraaceticacid (DOTA) equipped with maleimide groups forconjugation to reduced disulfide bonds of monoclonal antibodies.Using water-soluble carbodiimidechemistry, DOTA was coupled to l-cysteine to incorporateboth a “pendant-type” carboxyl group formetal coordination and an orthogonal thiol group for proteinattachment. The homobifunctionalreagent 1,6-bis(maleimido)hexane was then used to introducethe maleimide functionality via a sulfidelinkage to the macrocycle, and alternatively, the sulfide group wasconverted to a sulfone side chain.Both maleimide derivatives were conjugated to theanticarcinoembryonic antigen chimeric monoclonalantibody cT84.66 after light reduction of the mAb with dithiothreitol.In this manner, antibodyconjugates were prepared which afforded near-quantitative labeling withthe radiometals 111In(III)and 90Y(III) as well as quantitative immunoreactivity.Radioimmunoconjugates prepared with thesulfide and sulfone compounds exhibited relatively rapidlinker-dependent radiometal loss whenincubated in human serum and aqueous solutions at physiologicaltemperature and pH. Theunconjugated maleimidocysteineamido-DOTA derivatives and their Y(III)complexes were incubatedin aqueous solution at 37 °C, and the resulting decompositionproducts were analyzed by HPLC andmass spectrometry. These studies revealed that the twobifunctional chelating agents underwentlinker-specific cleavage reactions which were considerably faster at pH7.4 than at pH 5.4. Thechemically labile linker systems are expected to release chelatedradiometal from mAb conjugates ina pH-dependent manner. This property may impart favorable tumoruptake and normal tissueclearance on radioimmunoconjugates prepared with these reagents, on thebasis of the observationthat many solid tumors are significantly more acidic than normaltissues.
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