Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy

Abstract

The efficacy of an antibody-drug conjugate (ADC) is dependent on the properties of its linker-payload which must remain stable while in systemic circulation but undergo efficient processing upon internalization into target cells. Here, we examine the stability of a non-cleavable Amino-PEG6-based linker bearing the monomethyl auristatin D (MMAD) payload site-specifically conjugated at multiple positions on an antibody. Enzymatic conjugation with transglutaminase allows us to create a stable amide linkage that remains intact across all tested conjugation sites on the antibody, and provides us with an opportunity to examine the stability of the auristatin payload itself. We report a position-dependent degradation of the C terminus of MMAD in rodent plasma that has a detrimental effect on its potency. The MMAD cleavage can be eliminated by either modifying the C terminus of the toxin, or by selection of conjugation site. Both approaches result in improved stability and potency in vitro and in vivo. Furthermore, we show that the MMAD metabolism in mouse plasma is likely mediated by a serine-based hydrolase, appears much less pronounced in rat, and was not detected in cynomolgus monkey or human plasma. Clarifying these species differences and controlling toxin degradation to optimize ADC stability in rodents is essential to make the best ADC selection from preclinical models. The data presented here demonstrate that site selection and toxin susceptibility to mouse plasma degradation are important considerations in the design of non-cleavable ADCs, and further highlight the benefits of site-specific conjugation methods.

Highly Potent, Anthracycline-based Antibody–Drug Conjugates Generated by Enzymatic, Site-specific Conjugation

“…However, this observation seems to be specific to this type of cleavable linker and can be mitigated by changes in the linker design (49). Accordingly, no stability issues have been observed upon C-terminal conjugation using noncleavable linkers (23,50). In agreement with these observations, the C-terminally conjugated ADCs described here, employing noncleavable peptide linkers, are highly stable in serum, both in vitro (Figs.…”

Molecular Basis of Valine-Citrulline-PABC Linker Instability in Site-Specific ADCs and Its Mitigation by Linker Design

“…Conjugates were tested for stability in vitro in whole plasma, whole serum, or fractionated serum following described protocols (9,14). Typically, 0.125 mg/mL ADC was incubated in 62.5% (v/v) plasma diluted in 1Â PBS.…”

“…While it was unclear whether the C6-VC-PABC linker cleavage and the degradation of MMAD payload might be due to the activity of the same 1 Rinat Laboratories, Pfizer Inc., South San Francisco, California. 2 Worldwide Medicinal Chemistry, Pfizer Inc., Groton, Connecticut. or different enzyme(s), both processes were sensitive to the same protease inhibitors that suggested a serine-dependent hydrolase mechanism (9,14).…”

“…Interestingly, when we examined the stability of noncleavable site-specific conjugates in rodent plasma, we observed an enzymatic degradation of the C-terminal portion of the amino-PEG6-propionyl-MMAD (PEG6-C2-MMAD) linker-payload, showing a similar dependence on conjugation position (9). While it was unclear whether the C6-VC-PABC linker cleavage and the degradation of MMAD payload might be due to the activity of the same 1 Rinat Laboratories, Pfizer Inc., South San Francisco, California.…”

“…Various degrees of instability in the systemic circulation have been observed for many ADCs bearing commonly utilized linkers, such as the decoupling of the maleimide-based linker payloads resulting in reduced ADC exposure, the premature loss of disulfide-linked payloads leading to shortened pharmacokinetic profiles, as well as the degradation of the payload moiety itself (7)(8)(9).…”