Digestion of T-mavg-MMAU DAR8 with recombinant cathepsin B.

Digestion of lintuzumab-mavg-MMAU DAR8 by cynomolgus monkey liver microsomes at pH 4.5.

Digestion of lintuzumab-mcvc-MMAE DAR4 (lintuzumab vedotin) by cynomolgus monkey liver microsomes at pH 4.5.

Effect of cysteine protease inhibitor E-64 on lysosomal degradation of T-mavg-MMAU DAR8.

Individual and mean serum MMAE toxicokinetic parameters following three i.v. bolus doses of T-mavg-MMAU DAR4 in male cynomolgus monkeys.

Digestion of T-mavg-MMAU DAR8 with bovine liver β-glucuronidase.

Individual and mean serum T-MMAU toxicokinetic parameters following three i.v. bolus doses of T-mavg-MMAU DAR4 in male cynomolgus monkeys.

Mean plasma MMAE toxicokinetic estimates following weekly i.v. doses of free MMAU to Sprague-Dawley rats.

Lysosomal degradation of T-mcvc-MMAE DAR4 and T-mavg-MMAU DAR8.

Comparison of published toxicokinetic parameters of approved vedotin ADCs with T-mavg-MMAU DAR4.

Digestion of T-mcvc-MMAE DAR4 with recombinant cathepsin B.

Lysosomal degradation of T-mavg-MMAU DAR8.

Characterization of T-mavg-MMAU DAR8.

Mean plasma MMAE toxicokinetic estimates following weekly i.v. doses of free MMAE to Sprague-Dawley rats.

Individual and mean serum MMAU toxicokinetic parameters following three i.v. bolus doses of T-mavg-MMAU DAR4 in male cynomolgus monkeys.

Lysosomal degradation of non-hydrolyzed Cys-mavg-MMAU linker-payload molecule.

ADC in vitro activity against cancer cell lines with different HER2 expression levels.

Effect of cysteine protease inhibitor E-64 on lysosomal degradation of T-mavg-MMAU DAR8 and T-mcvc-MMAE DAR4.

Lysosomal degradation of T-mavg-MMAU DAR8 at pH 4.5.

Antibody-drug conjugates (ADC) have shown impressive clinical activity with approval of many agents in hematologic and solid tumors. However, challenges remain with both efficacy and safety of ADCs. This study describes novel trastuzumab-auristatin conjugates with the hydrophilic monomethylauristatin E (MMAE) prodrug MMAU, and optimization of a glycopeptide linker leading to a wider therapeutic window. Trastuzumab was conjugated with auristatin payloads via a series of linkers using a stabilized maleimide handle. The ADCs were characterized in vitro and their relative in vivo antitumor efficacies were assessed in HER2+ xenograft models. Relative linker stabilities and the mechanism of linker cleavage were studied using in vitro assays. Toxicity and toxicokinetics of the best performing ADC were evaluated in cynomolgus monkey (cyno). The trastuzumab-MMAU ADC with stabilized glycopeptide linker showed maleimide stabilization and higher resistance to cleavage by serum and lysosomal enzymes compared with a valine-citrulline conjugated trastuzumab ADC (trastuzumab-vc-MMAE). A single dose of 1 or 2 mg/kg of trastuzumab-MMAU at drug-to-antibody ratios (DAR) of eight and four respectively resulted in xenograft tumor growth inhibition, with superior efficacy to trastuzumab-vc-MMAE. Trastuzumab-MMAUDAR4 was tolerated at doses up to 12 mg/kg in cyno, which represents 2- to 4-fold higher dose than that observed with vedotin ADCs, and had increased terminal half-life and exposure. The optimized trastuzumab-MMAU ADC showed potent antitumor activity and was well tolerated with excellent pharmacokinetics in nonhuman primates, leading to a superior preclinical therapeutic window. The data support potential utility of trastuzumab-MMAU for treatment of HER2+ tumors.