Clay B. Siegall

Development of potent monoclonal antibody auristatin conjugates for cancer therapy

We describe the in vitro and in vivo properties of monoclonal antibody (mAb)-drug conjugates consisting of the potent synthetic dolastatin 10 analogs auristatin E (AE) and monomethylauristatin E (MMAE), linked to the chimeric mAbs cBR96 (specific to Lewis Y on carcinomas) and cAC10 (specific to CD30 on hematological malignancies). The linkers used for conjugate formation included an acid-labile hydrazone and protease-sensitive dipeptides, leading to uniformly substituted conjugates that efficiently released active drug in the lysosomes of antigen-positive (Ag+) tumor cells. The peptide-linked mAb-valine-citrulline-MMAE and mAb-phenylalanine-lysine-MMAE conjugates were much more stable in buffers and plasma than the conjugates of mAb and the hydrazone of 5-benzoylvaleric acid-AE ester (AEVB). As a result, the mAb-Val-Cit-MMAE conjugates exhibited greater in vitro specificity and lower in vivo toxicity than corresponding hydrazone conjugates. In vivo studies demonstrated that the peptide-linked conjugates induced regressions and cures of established tumor xenografts with therapeutic indices as high as 60-fold. These conjugates illustrate the importance of linker technology, drug potency and conjugation methodology in developing safe and efficacious mAb-drug conjugates for cancer therapy.We describe the in vitro and in vivo properties of monoclonal antibody (mAb)-drug conjugates consisting of the potent synthetic dolastatin 10 analogs auristatin E (AE) and monomethylauristatin E (MMAE), linked to the chimeric mAbs cBR96 (specific to Lewis Y on carcinomas) and cAC10 (specific to CD30 on hematological malignancies). The linkers used for conjugate formation included an acid-labile hydrazone and protease-sensitive dipeptides, leading to uniformly substituted conjugates that efficiently released active drug in the lysosomes of antigen-positive (Ag+) tumor cells. The peptide-linked mAb-valine-citrulline-MMAE and mAb-phenylalanine-lysine-MMAE conjugates were much more stable in buffers and plasma than the conjugates of mAb and the hydrazone of 5-benzoylvaleric acid-AE ester (AEVB). As a result, the mAb-Val-Cit-MMAE conjugates exhibited greater in vitro specificity and lower in vivo toxicity than corresponding hydrazone conjugates. In vivo studies demonstrated that the peptide-linked conjugates induced regressions and cures of established tumor xenografts with therapeutic indices as high as 60-fold. These conjugates illustrate the importance of linker technology, drug potency and conjugation methodology in developing safe and efficacious mAb-drug conjugates for cancer therapy.

Selective tumor sensitization to taxanes with the MAB-drug conjugate CBR96-doxorubicin

The chimeric monoclonal antibody cBR96 conjugated to doxorubicin (cBR96‐Dox) is selectively internalized by a wide variety of human carcinomas expressing an extended form of Lewis Y antigen (Ley). Endocytosis is followed by cleavage and release of free doxorubicin from the endocytic vesicles and subsequent cytotoxicity. Combination studies with standard anti‐cancer agents, undertaken to further increase the potency of this targeted therapy, identified significant synergistic anti‐tumor activity of cBR96‐Dox and either of the taxanes paclitaxel or docetaxel. Treatment with cBR96‐Dox 24 hr prior to paclitaxel resulted in a steady increase in the percentage of G2 tumor cells and corresponding increase in sensitivity to taxanes. Cell cycle analysis indicated the cBR96‐delivered doxorubicin was most effective against S‐phase cells, yet cells exposed to even subtoxic levels progressed to and arrested in G2, at a point of high sensitivity to the anti‐tubulin agent paclitaxel. The synergy obtained by staged combination of cBR96‐Dox and paclitaxel in vitro was reflected in significant anti‐tumor efficacy in vivo against xenograft models of human lung and breast tumors that could not be achieved by either agent alone. The staged combination elicited significant or complete regressions of established human Ley‐positive tumor xenografts using significantly reduced drug levels. Taken together, these data demonstrate a mechanistic approach to the selective elimination of Ley‐positive tumors by using targeted doxorubicin followed by taxane treatment.