Rituximab-Drug Conjugate Incorporating Auristatin E via A Quaternary Ammonium Linker Inducing Potent Antitumor Activity against Non-Hodgkin’s B-Cells

Background: Rituximab represents a drug used for standard Non-Hodgkin’s B-cell lymphoma therapy; however, it displays limited clinical efficacy. Antibody-drug conjugate (ADC) is one of the potential strategies to increase the antitumor activity of an antibody, with improved cytotoxicity directly resulting from the delivery of a molecular warhead. Currently, the warhead monomethyl auristatin E (MMAE) has been widely applied in the study of ADCs, conjugated to a carbamate-based linker (MCVC- PABC). However, the hydrophobic drug-linker (MC-VC-PABC-MMAE) may lead to ADC aggregation, ultimately resulting in decreased activity. Objective: In this study, we developed a hydrophilic drug-linker MC-VC-PABQ-AE linked to rituximab. If the replacement of the tertiary amine in AE for a secondary amine in MMAE represents a characteristic modification, the change of antitumor activity of two corresponding anti-CD20 ADC is unknown, requiring further verification. Method: The structural elucidation of MC-VC-PAB-AE was displayed by high-resolution mass spectra. The average drug antibody ratio (DAR) of rituximab-VC-AE/MMAE ADCs was performed by HICHPLC. The cell cycle arrest analysis of two ADCs was detected by flow cytometry and the antitumor activity of two ADCs was evaluated in vitro against Ramos and Daudi cells. Results: The average drug antibody ratio (DAR) of two ADCs was approximately 4.0. The activities of rituximab-VC-AE could be increased in CD20 positive B-lymphoma cell lines, most notably due to the higher cell viability inhibitory rates and apoptosis rates compared to rituximab-VC-MMAE. Conclusions: A hydrophilicity linker of ADC was developed and studied. Rituximab-VC-AE may potentially be used against CD20-positive cells, and the therapeutic efficacy and safety bring about further investigations.