Further Studies on the Protein Conjugation of Hydroxamic Acid Bifunctional Chelating Agents:  Group-Specific Conjugation at Two Different Loci

Abstract

A procedure utilizing an activated ester approach for conjugation of unprotected hydroxamic acids to antibodies and peptides was recently reported. Here, an alternative method with advantages over the activated ester strategy is described. This protocol utilizes the hydrazone formation between a hydrazide derivative of the trihydroxamate ligand trisuccin and either a ketone derivative of antibody or the aldehyde groups, generated by oxidation of the carbohydrate residues. Thus, the trisuccin carboxylic acid (1) was derivatized with tert-butyl carbazate to the hydrazide 2, and the protecting groups were removed by catalytic hydrogenation and acidolysis with TFA to afford the hydroxamate hydrazide 4. Conjugation of 4 to monoclonal antibody CC49 was effected by two approaches: attachment through the amine (e.g., lysine) residues of the antibody or oxidation of the carbohydrate residues. The extent of conjugations were monitored by MALDI, through evaluation of the increases in molecular weights of the conjugates compared to the unconjugated antibody. The first approach utilizing a ketone linker (6-oxoheptanoic acid, OHA) which served as a hydrazide anchor, is being introduced in this report as a new technique for conjugation of hydrazide derivatives to proteins. The OHA approach proved to be a superior strategy over the aldehyde approach in the ease of the procedure and yield of protein recovery. It also had the advantage of yielding more control in adjusting the ligand-to-protein ratio and was therefore selected for protocol optimization. All conjugates resulting from both approaches were radiolabeled with 125I and screened for their immunoreactivity. Furthermore, the conjugates prepared through the optimized OHA protocol were radiolabeled with both 99mTc and 125I for which the radiolabeling yields and immunoreactivities are reported.

In Vitro and In Vivo Evaluation of a 64Cu-Labeled Polyethylene Glycol-Bombesin Conjugate

“…8 The progress of the synthesis was monitored by direct molecular weight (MW) measurements using MALDI-TOF MS, which has proved to be a valuable tool for direct MW monitoring in large-molecule proteins and polymeric material. 8,32 Under the controlled reaction conditions of this synthesis, the increases in MWs clearly indicated the formation of intermediate and final-product compounds, in a 1:1:1 molar ratio with respect to the DOTA, PEG, and BN(7-14) (Fig. 2).…”

Intraperitoneal Radioimmunotherapy with a Humanized Anti-TAG-72 (CC49) Antibody with a Deleted CH2 Region

“…Previous studies have demonstrated the specific localization of radiolabeled murine CC49 to LS174T tumors. 37 The results of our study indicate that there was greater tumor uptake of 177 Lu-PA-DOTA-HuCC49 compared to 177 Lu-PA-DOTA-HuCC49⌬CH2 over time, but that 177 Lu-PA-DOTA-HuCC49⌬CH2 (T 1/2el ϭ 2.7 hours) cleared the blood more rapidly than 177 Lu-PA-DOTA-HuCC49 (T 1/2el ϭ 61.2 hours). These results are similar to those described by Slavin-Chiorini et al in which the radioiodinated HuCC49 and HuCC49⌬CH2 had similar uptake in S.C. LS174T tumors at early time points after I.V.…”

Synthesis, antibody conjugation, radiolabeling, and biodistribution of the first diethylene triamine pentahydroxamate (DTPH) bifunctional chelating agent

“…Conjugation of this BCA to the MAb ?CH2HuCC49, which binds to the TAG72 antigen, widely expressed on adenocarcinomas, was carried out through our hydrazide conjugation technique (2) and produced a conjugate with DTPH:MAb of 2.2 as indicated by MALDI mass spectroscopy.…”

“…h4Ab Conjugation. The DTPH 1 was conjugated to the MAb ?CH2HuCC49 by our hydrazide procedure (2). Briefly, 6-oxoheptanoic acid was attached to the MAb to serve as the hydrazide anchor.…”