Trifunctional conjugation reagents. Reagents that contain a biotin and a radiometal chelation moiety for application to extracorporeal affinity adsorption of radiolabeled antibodies.
A method of removing radiolabeled monoclonal antibodies (mAbs) from blood using a device external to the body, termed extracorporeal affinity-adsorption (EAA), is being evaluated as a means of decreasing irradiation of noncancerous tissues in therapy protocols. The EAA device uses an avidin column to capture biotinylated-radiolabeled mAbs from circulated blood. In this investigation, three trifunctional reagents have been developed to minimize the potential deleterious effect on antigen binding brought about by the combination of radiolabeling and biotinylation of mAbs required in the EAA approach. The studies focused on radiolabeling with (111)In and (90)Y, so the chelates CHX-A' '-DTPA and DOTA, which form stable attachments to these radionuclides, were incorporated in the trifunctional reagents. The first trifunctional reagent prepared did not incorporate a group to block the biotin cleaving enzyme biotinidase, but the two subsequent reagents coupled aspartic acid to the biotin carboxylate for that purpose. All three reagents used 4,7,10-trioxa-1,13-tridecanediamine as water-soluble spacers between an aminoisophthalate core and the biotin or chelation group. The mAb conjugates were radioiodinated to evaluate cell binding as a function of substitution. Radioiodination was used so that a direct comparison with unmodified mAb could be made. Evaluation of the number of conjugates per antibody versus cell binding immunoreactivities indicated that minimizing the number of conjugates was best. Interestingly, a decrease of radioiodination yield as a function of the number of isothiocyanate containing conjugates per mAb was noted. The decreased yields were presumably due to the presence of thiourea functionality formed in the conjugation reaction. Radiolabeling with (111)In and (90)Y was facile at room temperature for conjugates containing the CHX-A' ', but elevated temperature (e.g., 45 degrees C) was required to obtain good yields with the DOTA chelate. Stability of (90)Y labeled mAb in serum, and when challenged with 10 mM EDTA, was high. However, challenging the (90)Y labeled mAb with 10 mM DTPA demonstrated high stability for the DOTA containing conjugate, but low stability for the CHX-A' ' containing conjugate. Thus, the choice between these two chelating moieties might be made on requirements for facile and gentle labeling versus very high in vivo stability. Application of the trifunctional biotinylation reagents to the blood clearance of labeled antibodies in EAA is under investigation. The new reagents may also be useful for other applications.
Wilbur
D Scott
DS
Department of Radiation Oncology, University of Washington, Seattle, Washington 98195-9103, USA. dswilbur@u.washinton.edu
Chyan
Ming-Kuan
MK
Hamlin
Donald K
DK
Kegley
Brian B
BB
Nilsson
Rune
R
Sandberg
Bengt E B
BE
Brechbiel
Martin
M
D000345:Affinity Labels / Q000737:chemistry; D001360:Avidin / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004355:Drug Stability; D005112:Extracorporeal Circulation; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry / Q000493:pharmacokinetics*; D007205:Indium Radioisotopes; D004369:Pentetic Acid / Q000737:chemistry; D014407:Tumor Cells, Cultured; D015021:Yttrium Radioisotopes
D000345:Affinity Labels; D002614:Chelating Agents; D003432:Cross-Linking Reagents; D006573:Heterocyclic Compounds, 1-Ring; D018796:Immunoconjugates; D007205:Indium Radioisotopes; D015021:Yttrium Radioisotopes; D001360:Avidin; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; D001710:Biotin; D004369:Pentetic Acid