Antibody labeling with copper-67 using the bifunctional macrocycle 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)methyl]benzoic acid

Abstract

The high kinetic stability of the Cu2+ complex of the chelator 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl]benzoic acid was demonstrated at physiological pH as well as under acidic conditions. The chelating agent was conjugated to AB35, a monoclonal antibody directed against CEA, without a significant loss of immunoreactivity. The conjugate could, under optimal labeling conditions, be labeled with 67Cu in acetate buffer with a full occupancy of ligands within 20 min. This radiolabeled conjugate showed no transfer of radiocopper to serum proteins in human serum over 7 days. The biodistribution in tumor-bearing mice was measured and compared to that of iodinated AB35. Tumor uptake was high with 15 +/- 3% ID (injected dose)/g after 24 h and 32 +/- 7% ID/g after 96 h for the 67Cu-labeled antibody and 13 +/- 4% ID/g after 24 h and 14 +/- 2% ID/g after 96 h for the 125I-labeled antibody. Whereas radioactivity in normal organs decreased with time after 24 h, increased residence time was shown up to 4 days with the 67Cu-labeled AB35.

Comparison of 64Cu-Complexing Bifunctional Chelators for Radioimmunoconjugation: Labeling Efficiency, Specific Activity, and in Vitro/in Vivo Stability

“…Numerous chelators have been reported for complexing copper [5][6][7][8][9][10][11][12][13][14][15][16] and several of these have been functionalized to allow attachment to antibodies [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] and are now commercially available. The choice of antibody/BFC combination will affect efficacy as an imaging or therapy agent.…”

Peptide Linkers Lead to Modification of Liver Metabolism and Improved Tumor Targeting of Copper-67-labeled Antibody Fragments

“…Additional advantages to these favorable physical characteristics of the 67 Cu nuclide include the availability of copper chelators with high in vivo stability and the existence of simple and convenient procedures for labeling anti-tumor antibodies. [1][2][3][4][5][6] During the course of preclinical and clinical studies with radiocopper labeled mAbs it was found as an additional benefit, that the biological properties of copper labeled Abs and their metabolites lead to higher tumor uptake and prolonged residence time at the tumor, compared with radioiodinated mAbs. 7,8 Clinical studies with 67 Cu-labeled antibodies have been performed with intact mAbs both at diagnostic and therapeutic levels and indicated that some of the advantages observed in preclinical work with tumor bearing mice, such as improved tumor/blood ratios and higher mean tumor uptake compared with the radioiodinated counterpart, translate to the patient situation.…”

Site-specific conjugation of bifunctional chelator BAT to mouse IgG1 Fab1 fragment

“…Current techniques for preparing Fab' fragments necessitate purification steps that may cause irreversible damage to the antigen-binding sites due to denaturation of the antibody [16,17] . For several conjugation methods of antibody fragments and other molecules, such as glutaraldehyde and periodate methods, some active groups or linking agents have to be introduced to the Fab' fragments before they are conjugated to the other molecules [9,[18][19][20] . These steps may further destroy the immunoreactivities of the Fab' fragments.…”