Effective tumor targeting by auristatin antibody-drug conjugates Free

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Antibody-drug conjugates (ADCs) are designed to deliver drugs to tumors in an antigen-dependent fashion. We have developed ADCs that utilize a protease-sensitive valine-citrulline-p-aminobenzyloxycarbamyl (vc-PAB) linker to release drug once internalized into the lysosomes of antigen positive cells. The potent antimitotic agents monomethyl auristatin E and F (MMAE and MMAF) were linked to antibodies using the drug linkers vc-PAB-MMAE and vc-PAB-MMAF. MMAF carries an extra negative change and was designed to have an impaired ability to cross cell membranes, therefore potentially persisting in cells longer than MMAE. ADCs made with these drug linkers have demonstrated high in vitro and in vivo potencies. To address the effectiveness of targeted delivery, we have developed methods to quantitate the amount of released drug in vivo by mass spectrometry. ADCs made from vc-PAB-MMAE and vc-PAB-MMAF have been demonstrated to release exclusively MMAE and MMAF when incubated with antigen positive tumor cells. With binding ADCs, significant quantities of released drug are observed in subcutaneous tumor xenografts, yielding intratumoral concentrations as high as 1 μM. This represents as much as 20% of the injected dose of drug carried by the ADC. Concentrations were well in excess of the IC₅₀ values measured for MMAE and MMAF in cell culture experiments and were much greater than those achieved using non-binding control ADCs. The intratumoral concentrations of MMAF were higher than for MMAE, with the differences consistent with slower escape of MMAF from the tumor, as would be expected from its potentially poorer ability to cross cell membranes. Serum concentrations were extremely low, between 0.01 and 1 nM, yielding tumor to serum values as high as 25,000:1. These results demonstrate that vc-PAB-MMAE and vc-PAB-MMAF ADCs deliver active drug to tumors through an antigen-mediated process and concentrate the released drug in the tumor while avoiding drug release in the serum.