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Background In recent years, Antibody-drug conjugates (ADCs) are one of the fastest growing therapeutic modalities in the field of cancer treatment. A high drug-to-antibody ratio (DAR) is a desirable characteristic because it increases the efficacy. However, preparing high DAR ADCs often lead to issues such as aggregation and decreased antibody stability in the blood due to hydrophobicity of the drugs. One approach to solving this issue is the use of hydrophilic linkers. We developed a pendant type PEG linker which have two PEG chains between different functional groups. In this study, we prepared ADCs using cleavable pendant type PEG linkers incorporating a ValCit dipeptide for drug release and varying the PEG chain length to 4, 8, or 12 (hereafter referred to as PEG4, PEG8, and PEG12, respectively), and compared their efficacy.Methods We prepared DAR8-ADCs using Trastuzumab, cleavable pendant-type PEG and auristatin E (MMAE). As a control, we prepared DAR4-ADCs using a linker without PEG. First, we evaluated the hydrophobicity of the DAR8-ADCs by HIC analysis. Then, we performed stability studies at 40°C in formulation buffer. Aggregates content was monitored by native size-exclusion chromatography. The performance of the ADCs was evaluated by pharmacokinetics studies, in vitro cytotoxicity studies, in vivo anti-tumor activity studies and in vivo tolerability.Results It was confirmed by HIC analysis that increasing the PEG chain length led to a decrease in the overall hydrophobicity of the conjugates. Stability studies also showed that the aggregates content decreases as the PEG length increases. In pharmacokinetic studies, DAR8-ADCs with PEG8 and PEG12 showed better PK profile than DAR8-ADCs with PEG4 and DAR4-ADCs without PEG. In vitro cytotoxicity studies, all ADCs showed a IC50 in the nanomolar range in HER2+ cell lines, suggesting that the pendant type PEG linkers does not interfere with the release of MMAE by lysosomal enzymes. In vivo anti-tumor activity studies demonstrated that DAR8-ADCs with PEG8 and PEG12 had the stronger activity. This tendency was the same as PK profiles. Furthermore, DAR8-ADC with PEG12 was no weight loss and higher tolerability.Conclusions We developed proprietary cleavable pendant type PEG linkers and successfully synthesized DAR8-ADCs. In vitro and in vivo studies demonstrated that DAR8-ADCs, especially those with PEG8 and PEG12, showed higher efficacy than conventional DAR4-ADC.