Epidermal growth factor receptor (EGFR) is a rational target for cancer therapy, because its overexpression plays an important oncogenic role in a variety of solid tumors; however, EGFR‐targeted antibody–drug conjugate (ADC) therapy for esophageal squamous cell carcinoma (ESCC) is exceedingly rare. LR004 is a novel anti‐EGFR antibody with the advantages of improved safety and fewer hypersensitivity reactions. It may be of great value as a carrier in ADCs with high binding affinity and internalization ability. Here, we prepared an EGFR‐targeting ADC, LR004‐VC‐MMAE, and evaluated its antitumor activities against ESCC and EGFR‐positive cells. LR004 was covalently conjugated with monomethyl auristatin E (MMAE) via a VC linker by antibody interchain disulfide bond reduction. VC‐MMAE was conjugated with LR004 with approximately 4.0 MMAE molecules per ADC. LR004‐VC‐MMAE showed a potent antitumor effect against ESCC and other EGFR‐positive cells with IC
50 values of nM concentrations in vitro. The in vivo antitumor effects of LR004‐VC‐MMAE were investigated in ESCC KYSE520 and A431 xenograft nude mice models. Significant activity was seen at 5 mg·kg−1, and complete tumor regression was observed at 15 mg·kg−1 in the KYSE520 xenograft nude mice after four injections, while the naked antibody LR004 had little effect on inhibiting tumor growth. Similar promising results were obtained in the A431 models. In addition, the tumors also remained responsive to LR004‐VC‐MMAE for large tumor experiments (tumor volume 400–500 mm3). The study results demonstrated that LR004‐VC‐MMAE could be a potential therapeutic agent for ESCC and other EGFR‐expressing malignancies. We also evaluated PK profile of LR004‐VC‐MMAE ADC in the mice model, which would provide qualitative guiding significance for the further research.
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 (MC-VC-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 HIC-HPLC. 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.
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