Trifunctional Conjugation Reagents. Reagents That Contain a Biotin and a Radiometal Chelation Moiety for Application to Extracorporeal Affinity Adsorption of Radiolabeled Antibodies

Abstract

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 111In and 90Y, 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 111In and 90Y was facile at room temperature for conjugates containing the CHX-A‘ ‘, but elevated temperature (e.g., 45 °C) was required to obtain good yields with the DOTA chelate. Stability of 90Y labeled mAb in serum, and when challenged with 10 mM EDTA, was high. However, challenging the 90Y 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.

Determining Maximal Tolerable Dose of the Monoclonal Antibody BR96 Labeled with 90Y or 177Lu in Rats: Establishment of a Syngeneic Tumor Model to Evaluate Means to Improve Radioimmunotherapy

“…BR96 was conjugated with the trifunctional chelator 1033 (MitraTag, Mitra Medical AB, Lund, Sweden), carrying a 1,4,7,10-tetraazacyclododecane-N ,NV ,N 00 ,N 00 V -tetraacetic acid moiety and a biotin moiety (8). Before conjugation, BR96 was transferred by dialysis into a 50 mmol/L HEPES, 1 mmol/L diethylenetriaminepentaacetic acid buffer (pH 8.5).…”

Preparation and testing of bevacizumab radioimmunoconjugates with Bismuth-213 and Bismuth-205 / Bismuth-206

“…The inability of cDTPA to hold the radiolabel in the product in vivo can be described as a big negative for this chelator. However, it needs to be kept in mind that 213 Bi with a half-life of just 46-minutes as the therapeutic agent, the blood half-life of four hours for this conjugate may still be more than adequate to achieve the desired therapeutic effects without the need of removing radiolabeled monoclonal antibodies from blood using extracorporeal affinity-adsorption (EAA) as suggested by Wilbur et al 39 (for CHX-A″ based conjugates). The pharmacokinetics of the conjugates showed a significantly higher renal uptake and retention of the cDTPA conjugate compared to CHX-A″ conjugate up to four hours (p-value 0.02).…”

Blood Pharmacokinetics of Various Monoclonal Antibodies Labeled with a New Trifunctional Chelating Reagent for Simultaneous Conjugation with 1,4,7,10-Tetraazacyclododecane-N,N′,N″,N‴-Tetraacetic Acid and Biotin before Radiolabeling

“…The trifunctional chelator 1033 was used to label the three antibodies-rituximab, BR96, and trastuzumab-with 111 In. The preparation of 1033 has previously been described by Wilbur et al (11). The fourth antibody, hMN14, was conjugated with the chelator DOTA at 4 to 6 DOTA/IgG (this conjugate was obtained from Immunomedics).…”

“…As a result, the heterogeneity of radiolabeled mAbs is reduced, as the same number of biotin molecules as chelates is always present and a minimum number of conjugation reagents or moieties can be used (11).…”