NEW COUPLING REAGENTS FOR THE PREPARATION OF DISULFIDE CROSS-LINKED CONJUGATES WITH INCREASED STABILITY

To improve the in vivo stability of disulfide-linked immunotoxins (ITs ), a series of sterically hindered cross-linking reagents were designe d and synthesized. These ligands are characterized by a thioimidate gr oup linked to an S-acetyl thiol or a substituted aryldithio group. To select the reagent of choice, several aryldithio thioimidates, substit uted with a methyl or a phenyl group adjacent to the disulfide, were a nalyzed in thiol-disulfide exchange reactions. Also analyzed were the following: (i) the stability and solubility of the linkers in aqueous solution, (ii) the rate of protein derivatization, and (iii) the steri c hindrance due to methyl or phenyl group substituents toward cleavage of the disulfide bond by glutathione. Ethyl S-acetyl 3-mercaptobutyro thioimidate (M-AMPT) was chosed as reagent to prepare two types of sta ble disulfide-containing AR-3-gelonin conjugates (IT2 and IT3). IT2 wa s prepared by a 3-(4-carboxamidophenyldithio)propionthioimidate (CDPT) -derivatized antibody coupled to the M-AMPT-derivatized gelonin to aff ord a conjugate characterized by the presence of a methyl group adjace nt to the sulfide bond. In the IT3 conjugate, an M-AMPT-derivatized to xin was coupled to the antibody thiolated with M-AMPT and then activat ed with Ellman's reagent (DNTB). The in vitro and in vivo stabilities of the three immunoconjugates were assayed, respectively, (i) by addin g an excess of glutathione and monitoring protein release and (ii) by studying their pharmacokinetic behaviors. The specificity and cytotoxi city of all ITs were analyzed on target and unrelated cell lines, and no significant differences in activity were observed. IT3, consisting of a symmetrical dimethylsubstituted disulfide bond, was substantially more stable in vivo (t(1/2 beta) = 88.3 h) than the corresponding IT2 , characterized by a disulfide-protected monomethyl substituent bond ( t(1/2 beta) = 60.2 h) compared to the unhindered conjugate IT1 (t(1/2 beta) = 27.9 h). This family of cross-linking reagents therefore offer s advantages, such as minimal perturbation of the protein structure an d controlled reactivity due to the thioimidate moiety, as well as the capacity to yield immunotoxins possessing substantial stability in viv o.