An Engineered Fc with a Single Glycosylation Site Enables the Precise Conjugation of an Antibody with One Payload.

Although ADCs have demonstrated great potential in tumor treatment, acquired resistance to ADCs has been reported, related to both the targets and the payloads. One strategy is to increase the diversity of the payloads. The cytotoxicity and other inherent properties of the payloads are important factors for the choice of the appropriate DAR value in the ADCs design. Due to the symmetrical structure of antibodies, the DAR values of ADCs prepared by classical conjugation strategies are basically between 2 and 8. With continuous advancement in their development, ultrapotent payloads require lower DAR values to achieve better pharmacokinetics and biodistribution. In this study, we provide a methodology for preparing DAR1 ADCs that utilizes engineered Fc domains to generate antibodies with a single N-glycosylation site and employs glycosite-specific chemoenzymatic conjugation technology to conjugate a single payload to the N-glycosylation site. The obtained ADCs have acceptable homogeneity and stability, as well as effective in vitro and in vivo biological activities. Additionally, our research indicates that while maintaining equivalent payload dosages, DAR1 ADCs have higher tumor penetration in both in vitro and in vivo models.