Linker substitution influences succinimide ring hydrolysis equilibrium impacting the stability of attachment to antibody-drug conjugates.

ABSTRACT

Maleimide chemistry is widely used in antibody-drug conjugate (ADC) generation to connect drugs to antibodies through a succinimide linker. The resulting ADC is prone to payload loss via a reverse Michael reaction, leading to premature drug release in vivo. Complete succinimide hydrolysis is an effective strategy to overcome the instability of ADC. However, we discovered through previous work that hydrolysed succinimide rings can close again in a liquid formulation during storage and under thermal stress conditions. In this work, a set of maleimide linkers with hydrolysis-prone groups were designed. The corresponding ADCs were prepared and subjected to thermal stress conditions. The extent of succinimide hydrolysis and drug release was measured, and ADC properties such as SEC, DAR, pI and clog P of linkers were calculated. Our results demonstrated that even though all these groups increased the hydrolysis rate, they have different impacts on maintaining the hydrolysed succinimide ring in an open conformation and ADC stability in a liquid formulation.