Monoclonal antibody conjugates of doxorubicin prepared with branched linkers: A novel method for increasing the potency of doxorubicin immunoconjugates.

Immunoconjugates of monoclonal antibody BR96 and Doxorubicin have been prepared using a novel series of branched hydrazone linkers. Since each linker bound to the mAb carries two DOX molecules, the DOX/mAb molar ratios of these conjugates were approximately 16, twice that of those previously prepared with single-chain hydrazone linkers. The conjugates were stable at a physiological pH of 7, but released DOX rapidly at lysosomal pH 5. The branched series of BR96 conjugates demonstrated antigen-specific cytotoxicity, and were more potent in vitro than the single-chain conjugate on both a DOX (4-14-fold) and mAb (7-23-fold) basis. The results suggest that, by using the branched linker methodology, it is possible to significantly reduce the amount of mAb required to achieve antigen-specific cytotoxic activity. In this paper, the synthesis and in vitro biology of branched chain immunoconjugates are described.

Effect of linker variation on the stability, potency, and efficacy of carcinoma-reactive BR64-doxorubicin immunoconjugates.

The internalizing anti-Le(y) monoclonal antibody (MAb) BR64 was conjugated to the anticancer drug doxorubicin (DOX) using an acid-labile hydrazone bond to the DOX and either a disulfide or thioether bond to the MAb. The resulting disulfide (BR64-SS-DOX) and thioether (BR64-S-DOX) conjugates were evaluated for stability, potency, and antigen-specific activity in both in vitro and in vivo model systems. The BR64-SS-DOX conjugates demonstrated antigen-specific activity both in vitro and when evaluated against antigen-expressing, DOX-sensitive human carcinoma xenografts. However, the stability and potency of disulfide conjugates were poor, and in vivo activity superior to unconjugated DOX was seen only at doses approaching the maximum tolerated dose. Furthermore, BR64-SS-DOX conjugates were not active against antigen-expressing, DOX-insensitive colon tumor xenografts. In contrast, the BR64-S-DOX conjugates demonstrated good stability both in vitro and in vivo. The increased stability of the BR64-S-DOX conjugates resulted in the delivery of more biologically active DOX to tumors with a concomitant increase in potency and efficacy over that which could be achieved with either unconjugated DOX or BR64-SS-DOX conjugates. Delivery of DOX by BR64-SS-DOX conjugates resulted in complete regressions and cures of both DOX-sensitive lung xenografts and DOX-intensitive colon tumor xenografts. These results demonstrate the importance of linker stability when delivering drugs such as DOX to carcinomas via internalizing antibodies and are likely to have direct relevance to the clinical utility of MAb-directed delivery.

(6-Maleimidocaproyl)hydrazone of doxorubicin--a new derivative for the preparation of immunoconjugates of doxorubicin.

The (6-maleimidocaproyl)hydrazone of doxorubicin was synthesized and conjugated to several mAbs, including chimeric BR96, via a Michael addition reaction to thiol-containing mAbs. DTT reduction of disulfides present in the mAb was a reliable and general method for generating a consistent number of reactive SH groups. The conjugates, after purification by Bio-Beads, were free of unreacted linker and/or doxorubicin. All conjugates released doxorubicin under acidic conditions that mimic the lysosomal environment, while they were relatively stable at neutral pH. BR96 conjugates showed antigen-specific cytotoxicity.

Cure of xenografted human carcinomas by BR96-doxorubicin immunoconjugates.

Immunoconjugates (BR96-DOX) were prepared between chimeric monoclonal antibody BR96 and the anticancer drug doxorubicin. The monoclonal antibody binds an antigen related to Lewis Y that is abundantly expressed at the surface of cells from many human carcinomas; it has a high degree of tumor selectivity and is internalized after binding. BR96-DOX induced complete regressions and cures of xenografted human lung, breast, and colon carcinomas growing subcutaneously in athymic mice and cured 70 percent of mice bearing extensive metastases of a human lung carcinoma. Also, BR96-DOX cured 94 percent of athymic rats with subcutaneous human lung carcinoma, even though the rats, like humans and in contrast to mice, expressed the BR96 target antigen in normal tissues.

Antigen-specific activity of carcinoma-reactive BR64-doxorubicin conjugates evaluated in vitro and in human tumor xenograft models.

The anticarcinoma antibody BR64 was conjugated to a doxorubicin derivative, doxorubicin 13-[3-(2-pyridyldithio)propionyl]hydrazone, and the resulting conjugates (BR64-DOX) were evaluated for activity and immunological specificity in vitro and in human tumor xenograft models. The BR64-DOX immunoconjugates retained immunoreactivity and cytotoxicity and demonstrated antigen-specific cytotoxicity in vitro. The potency of BR64-DOX immunoconjugates in vitro was related to the drug:monoclonal antibody mole ratio of the conjugates. The antitumor activity of BR64-DOX conjugates was consistently superior to the maximal activity obtained with the parent drug, doxorubicin (DOX), in established human lung and human breast carcinoma xenograft models. The superior antitumor activity of BR64-DOX conjugates was reflected both in tumor growth inhibition and in regressions and cures of established tumors following the administration of tolerated doses of BR64-DOX. The antitumor activity of BR64-DOX conjugates was not the result of synergism between monoclonal antibody BR64 and DOX, because mixtures consisting of monoclonal antibody and optimized DOX were not more active than an equivalent dose of DOX administered alone. The antitumor activity of BR64-DOX conjugates was antigen specific; equivalent doses of nonbinding isotype-matched conjugates were not active against established tumor xenografts.