Representative data are presented. cytotoxicity in several target-negative cell lines compared with non-stressed ADCs. Notably, ADC aggregates with FcR-activation properties showed dramatically enhanced cytotoxicity in FcR-expressing cells. The FcR-mediated off-target cytotoxicity of ADC aggregates was reduced by using a FcR-blocking antibody or Fc-engineering for silencing Fc-mediated effector functions. Conclusions These results indicated that FcRs play an important role for internalization of ADC aggregates into non-target cells, and the aggregation of ADCs increases the potential risk for off-target toxicity. Supplementary Information The online version contains supplementary material available at 10.1007/s11095-021-03158-x. strong class=”kwd-title” Key Words: Antibody-drug conjugates, aggregation, off-target toxicity, Fc receptors Introduction Antibody-drug conjugates (ADCs), which are monoclonal antibodies (mAbs) conjugated with highly toxic small molecules (payloads) via linkers, are one of the fastest growing classes of next generation mAbs. ADCs combine the advantages of the target-specificity of mAbs with the high tumor killing efficacy of payloads. Namely, ADCs are specifically transported to the cells expressing their target antigens in accordance with the function of mAbs, and the ADCs are internalized and subsequently release the payloads to kill the target cells. DPP-IV-IN-2 Therefore, it is expected that ADCs will reduce the systemic exposure Slc16a3 of cytotoxic small molecules while providing a wider therapeutic window compared with traditional chemotherapy. The development and commercial application of ADCs have been progressing in recent years. Six of ten FDA-approved ADCs were approved since the start of 2019, and 85 candidates are at the clinical development stage in various countries (1). Though ADCs have great advantages for cancer therapy, there are some ADC-specific problems resulting from particular characteristics of ADCs. One of the problems is the increase in hydrophobicity due to the conjugation of the hydrophobic payload to mAbs. Though mAbs naturally have a hydrophilic character, most of the payloads are too hydrophobic, and conjugation of payloads to mAbs often increases the hydrophobicity. The hydrophobicity of ADCs is affected by the drug antibody ratio (DAR) and characteristics of the linker and payload, and it is well known that the hydrophobicity of ADCs affects the plasma clearance and therapeutic index (2C4). In addition, the increase of surface hydrophobicity induced by conjugation of hydrophobic payloads promotes the aggregation of ADCs followed by enhancement of nonspecific protein interactions in the drug products (5). Thus, the aggregation rate of ADCs was often higher than that of the native mAbs (5, 6). In biopharmaceuticals, including ADCs, protein aggregates DPP-IV-IN-2 are believed to be key risk factors for immunogenicity (7). Therefore, aggregation of ADCs via an increase of hydrophobicity has been DPP-IV-IN-2 well studied in the development of ADC formulations. In therapeutic mAbs, some reports have indicated that the mAb aggregates could enhance immunogenicity through the activation of immune cells via Fc receptors (FcRs) (8C10). In addition, it was reported that mAb DPP-IV-IN-2 aggregates showed higher internalization properties compared with native mAbs, and quickly accumulated at the degradation pathways involving late endosomes in mouse dendritic cells (11). Thus, mAb aggregates could not only activate immune cells via the receptors on the cell surface but could also be internalized into the cells which did not express the target antigen. Considering the mechanism of action of ADCs, unintended cellular uptake and accumulation at the degradation pathway of ADCs in non-target cells may cause the off-target toxicity (12, DPP-IV-IN-2 13). However, the impact of aggregation on the safety of ADCs, especially off-target toxicities induced by unintended internalization into non-target cells, has been unclear. In this study, we evaluated the impact of ADC aggregation on cytotoxicity in -negative and target-positive cells using two commercially obtainable ADCs, and revealed which the aggregation of ADCs decreased.