The anticancer drug doxorubicin (DOX) has been linked to chimeric BR96, an internalizing monoclonal antibody that binds to a Lewis(y)-related, tumor-associated antigen, through two lysosomally cleavable dipeptides, Phe-Lys and Val-Cit, giving immunoconjugates 72 and 73. A self-immolative p-aminobenzyloxycarbonyl (PABC) spacer between the dipeptides and the DOX was required for rapid and quantitative generation of free drug. DOX release from model substrate Z-Phe-Lys-PABC-DOX 49 was 30-fold faster than from Z-Val-Cit-PABC-DOX 42 with the cysteine protease cathepsin B alone, but rates were identical in a rat liver lysosomal preparation suggesting the participation of more than one enzyme. Conjugates 72 and 73 showed rapid and near quantitative drug release with cathepsin B and in a lysosomal preparation, while demonstrating excellent stability in human plasma. Against tumor cell lines with varying levels of BR96 expression, both conjugates showed potent, antigen-specific cytotoxic activity, suggesting that they will be effective in delivering DOX selectively to antigen-expressing carcinomas.

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.

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.

New N-substituted hydrazine linkers were synthesized and their hydrazone derivatives of adriamycin were prepared. These functionalized adriamycin derivatives were conjugated with a monoclonal antibody, 5E9. The release rate of adriamycin from the hydrazones and from some of the conjugates was studied, and their relationship to the IC50's of the conjugate against 5E9-positive Daudi cells was investigated.

High mole ratio BR96 immunoconjugates were synthesized using branched peptide-doxorubicin linkers designed to liberate doxorubicin following antigen-specific internalization into lysosomes. However, these immunoconjugates are highly prone to noncovalent, dimeric aggregation. We hypothesize that this is due to (1) the hydrophobic nature of the peptides, (2) the loss of positive charge upon amide formation at the 3'-amino group of doxorubicin, and (3) the proximity of the peptide hydrophobic residues to form efficient intermolecular stacking interactions. By introducing a hydrophilic methoxytriethylene glycol chain onto the doxorubicin portion of the branched peptide linkers, aggregation has been eliminated or greatly reduced in the immunoconjugate products. The methoxytriethylene glycol chain was linked to the doxorubicin moiety of the linker via a hydrazone bond that is stable at pH 7 but hydrolyzes rapidly at pH 5 to release free drug. BR96 immunoconjugates synthesized from methoxytriethylene glycol-modified branched peptide-doxorubicin linkers are highly potent and immunospecific in vitro. The data suggest that the methoxytriethylene glycol chain hydrolyzes as designed upon antigen-specific internalization into tumor lysosomes in vitro, where enzymatic degradation of the peptide linker releases free doxorubicin.