Role of Cross-Linking Agents in Determining the Biochemical and Pharmacokinetic Properties of Mgr6−Clavin Immunotoxins

Abstract

Several immunotoxins (ITs) were synthesized by the attachment of clavin, a recombinant toxic protein derived from Aspergillus clavatus, to the monoclonal antibody Mgr6 that recognizes an epitope of the gp185(HER-2) extracellular domain expressed on breast and ovarian carcinoma cells. Conjugation and purification parameters were analyzed in an effort to optimize the antitumor activity and stability of the ITs in vivo. To modulate the in vitro and in vivo properties of the immunotoxins, different coupling procedures were used and both disulfide and thioether linkages were obtained. Unhindered and hindered disulfide with a methyl group linkage ethyl S-acetyl 3-mercaptopropionthioimidate ester hydrochloride (AMPT) or ethyl S-acetyl 3-mercaptobutyrothioimidate ester hydrochloride (M-AMPT) were obtained by reaction with recombinant clavin, while the monoclonal antibody Mgr6 was derivatized with ethyl 3-[(4-carboxamidophenyl)dithio]propionthioimidate ester hydrochloride (CDPT). To achieve higher hindrance (a disulfide bond with a geminal dimethyl group), Mgr6 was derivatized with the N-hydroxysuccinimidyl 3-methyl-3-(acetylthio)butanoate (SAMBA) and clavin with CDPT. To evaluate the relevance of the disulfide bond in the potency and pharmacokinetic behavior of the ITs, a conjugate consisting of a stable thioether bond was also prepared by derivatizing Mgr6 with the N-hydroxysuccinimidyl ester of iodoacetic acid (SIA) and clavin with AMPT. The immunotoxins were purified and characterized using a single-step chromatographic procedure. Specificity and cytotoxicity were assayed on target and unrelated cell lines. The data indicate that the introduction of a hindered disulfide linkage into ITs has little or no effect on antitumor activity and suggest that disulfide cleavage is essential for activity; indeed, the intracellularly unbreakable thioether linkage produced an inactive IT. Analysis of IT stability in vitro showed that the release of mAb by incubation with glutathione is proportional to the presence of methyl groups and increases exponentially with the increase in steric hindrance. Analysis of the pharmacokinetic behavior of ITs in Balb/c mice given intravenous bolus injections indicated that ITs with higher in vitro stability were eliminated more slowly; i.e., the disulfide bearing a methyl group doubled the beta-phase half-life (from 3.5 to 7.1 h) compared with that of the unhindered, while a geminal dimethyl protection increased the elimination phase to 24 h. The thioether linkage showed its intrinsic stability with a beta-phase half-life of 46 h. The thioether linkage also increased the distribution phase from 17 to 32 min. The in vitro characteristics and in vivo stability of Mgr6-clavin conjugates composed of a methyl and dimethyl steric hindered disulfide suggest clinical usefulness.

Tunable Degradation of Maleimide–Thiol Adducts in Reducing Environments

“…Indeed, in vivo blood circulation stability has been shown to be as little as 4hrs for disulfide-based immunotoxin-antibody conjugates, with circulation stability directly dependent on glutathione reduction kinetics. 7,33,34 Accordingly, the decreased rate of the retro reaction of the maleimide-thiol adduct with glutathione would increase compound circulation stability while maintaining cleavage sensitivity in highly reducing environments.…”

Poly(ethyleneimine)/arginine–glycine–aspartic acid conjugates prepared with N‐succinimidyl 3‐(2‐pyridyldithio)propionate: An investigation of peptide coupling and conjugate stability

“…The disulfide linkage between the peptides and the polymers is expected to be sterically hindered because of the presence of the bulky polymer chain and the amino acids adjacent to the cysteine moiety. The incorporation of bulky groups next to the disulfide linkages, such as the methyl and benzene ring instead of Hs, was shown to reduce the reaction rate with the thiolate anion and increase the linkage stability 35, 36. Accordingly, amino acids lacking bulky side groups (e.g., glycine) are expected to present a more favorable environment for the access of the thiolate anion to the disulfide linkage.…”

A disulfide conjugate between anti‐tetanus antibodies and HIV (37–72)Tat neutralizes tetanus toxin inside chromaffin cells

“…The disulfide bridge is reduced inside the cell, whereupon the effector domain of the molecule leaves the vesicle and exerts its action. The effect of the protein on cellular components is eliminated or weakened when the disulfide bridge is replaced with a thioether bridge (for instance, [23]). Similarly in our experiments, the reducible disulfide conjugate restored the transmitter release of TET‐intoxicated cells, while the non‐reducible thioether conjugate was inefficient.…”