Monomethyl Auristatin E (MMAE), a Payload for Multiple Antibody Drug Conjugates (ADCs), Demonstrates Differential Red Blood Cell Partitioning Across Human and Animal Species

Abstract

Background: Monomethyl auristatin E (MMAE) has been used as a payload for several Food and Drug Administration (FDA) approved antibody-drug conjugates (ADCs). It is known that MMAE is released from the ADC following binding, internalization and proteolytic degradation in target tissues. A striking discrepancy in systemic MMAE levels has been observed across species with 50-fold higher MMAE levels in human than that in rodents when normalized by ADC dose with unknown mechanism. Hypothesis and purpose: Multiple factors could affect systemic MMAE levels such as production and elimination of unconjugated MMAE following ADC dosing. In this study, we have explored whether MMAE displays differential red blood cell (RBC) partitioning across species that may contribute to the different MMAE levels seen between human and animals. Experiments: To determine MMAE RBC partitioning, tritium labeled MMAE ([3H]-MMAE) was incubated in whole blood from mice, rats, monkeys and humans in vitro, then RBC partitioning was determined and compared across species. To test whether MMAE released from the ADC would show any difference in RBC partitioning, pinatuzumab vedotin or polatuzumab vedotin was administered to mice, rats, and monkeys. MMAE levels were measured in both blood and plasma, and the ratios of MMAE levels were calculated as blood-to-plasma ratio (in vivo RBC partitioning). Results: Our in vitro data showed that unconjugated MMAE has a species-dependent RBC partitioning with strong RBC partitioning in mouse, rat, followed by monkey blood, whereas minimal RBC partitioning was seen in human blood. Incubation of 2 nM of MMAE in mouse blood resulted in a blood-to-plasma ratio of 11.8 ± 0.291, followed by rat, monkey, and human at 2.36 ± 0.0825, 1.57 ± 0.0250, and 0.976 ± 0.0620, respectively. MMAE RBC partitioning is also concentration-dependent, with an inverse relationship between RBC partitioning and MMAE concentration (higher RBC partitioning at lower concentration). In vivo dosing of pinatuzumab vedotin in mouse displayed systemic MMAE at about a 5-fold higher blood concentration compared to plasma concentration once MMAE reached a pseudo-equilibrium, while systemic MMAE from blood and plasma concentration showed a 1.65-fold difference in rat. Implication and conclusion: These data demonstrated that MMAE has a distinct RBC partitioning across different species, which may contribute to, at least in part, to the differential in the systemic MMAE levels observed in vivo between preclinical and clinical studies. These findings highlight the importance of fully characterizing the ADME properties of both the ADC and its payload, to enable better translation from animals to human for ADC development.