ROLE OF CROSS-LINKING AGENTS IN DETERMINING THE BIOCHEMICAL AND PHARMACOKINETIC PROPERTIES OF MGR6-CLAVIN IMMUNOTOXINS

Several immunotoxins (ITs) were synthesized by the attachment of clavi n, a recombinant toxic protein derived from Aspergillus clavatus, to t he monoclonal antibody Mgr6 that recognizes an epitope of the gp185(HE R-2) extracellular domain expressed on breast and ovarian carcinoma ce lls. Conjugation and purification parameters were analyzed in an effor t 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 thioeth er linkages were obtained. Unhindered and hindered disulfide with a me thyl group linkage ethyl S-acetyl 3-mercaptopropionthioimidate ester h ydrochloride (AMPT) or ethyl S-acetyl 3-mercaptobutyrothioimidate este r hydrochloride (M-AMPT) were obtained by reaction with recombinant cl avin, while the monoclonal antibody Mgr6 was derivatized with ethyl 3- [(4-carboxamidophenyl)dithio]propionthioimidate ester hydrochloride (C DPT). To achieve higher hindrance (a disulfide bond with a geminal dim ethyl group), Mgr6 was derivatized with the N-hydroxysuccinimidyl 3-me thyl-3-(acetylthio)butanoate (SAMBA) and clavin with CDPT. To evaluate the relevance of the disulfide bond in the potency and pharmacokineti c behavior of the ITs, a conjugate consisting of a stable thioether bo nd was also prepared by derivatizing Mgr6 with the N-hydroxysuccinimid yl ester of iodoacetic acid (SIA) and clavin with AMPT. The immunotoxi ns were purified and characterized using a single-step chromatographic procedure. Specificity and cytotoxicity were assayed on target and un related cell lines. The data indicate that the introduction of a hinde red disulfide linkage into ITs has little or no effect on antitumor ac tivity 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 ste ric hindrance. Analysis of the pharmacokinetic behavior of ITs in Balb /c mice given intravenous bolus injections indicated that ITs with hig her in vitro stability were eliminated more slowly; i.e., the disulfid e 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 lin kage 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 d isulfide suggest clinical usefulness.