New bifunctional ligands for radioimmunoimaging and radioimmunotherapy

Syntheses of one bifunctional ethylenediaminetetraacetic acid (EDTA) and of five bifunctional diethylenetriaminepentaacetic acid (DTPA) ligands were performed. The bifunctional EDTA ligand and two bifunctional DTPA ligands were synthesized by direct aminolysis of an amino acid ester followed by reduction, alkylation, and functional group modification to introduce bifunctionality. The reactive substituent chosen for protein conjugation was the isothiocyanate group. The generality of this approach was demonstrated with 9 different amino acids to produce the respective substituted diethylenetriamines. The remaining three bifunctional DTPA ligands were synthesized via classical peptide methodology producing a dipeptide amide which, after deprotection, was reduced to the triamine and alkylated to produce the ligand. This route successfully provided an alternative entry to mono- or disubstituted DTPA ligands with potential stereocontrol of the substituents. Biodistribution studies of the ligands conjugated to monoclonal antibody B72.3 and labelled with the gamma emitter Indium-111 revealed that superior retention of Indium-111 was attained and the dose to the liver was minimized when a full intact octadentate bifunctional DTPA chelate was used, e.g. DTPA $>$ EDTA $>$ DTTA (diethylenetritetraacetic acid from use of DTPA dianhydride (CA-DTPA)). The best scintigraphic images were obtained after 72 hours when a DTPA ligand was used to complex the Indium-111. Biodistribution studies using the gamma emitter Yttrium-88 revealed that the disubstituted bifunctional DTPA was necessary to minimize the bone dose from the Yttrium while maintaining a high dose to the tumor, e.g. disub. DTPA $>$ monosub. DTPA $\gg$ EDTA $\sim$ CA-DTPA. Use of either the bifunctional EDTA ligand or the CA-DTPA reagent resulted in the %I.D./g at the tumor to decrease to less than 11.0% over 168 hours while the %I.D./g at the femur increased to greater than 17.0%. The disubstituted DTPA ligands maintained the %I.D./g at the femur at less than 3.5%. The %I.D./g in the femur using the best DPTA ligand for Yttrium was still greater than the %I.D./g in the femur for Indium indicating that yet more improvement is possible.