Although many antibody drug conjugates (ADCs) have been currently approved, they still face the problem of difficulty in having sufficient therapeutic index due to off-target toxicity caused by linker instability. To solve this problem, we developed a novel UBE’s versatile hydrophilic (UVH) linker and auristatin E (AE) prodrugs. UVH linker contains a novel, highly hydrophilic amino acid derivative enables high stability in the blood with the maximum drug to antibody ratio (DAR) of 16. This linker is cleaved by Cathepsin B. AE prodrug is significantly less toxic than AE due to its lower cell membrane permeability. On the other hand, AE prodrugs released from ADCs taken up by cancer cells are converted to AE in the cancer cells and exhibit potent cytotoxic activity. Using this state-of-the-art linker and prodrug, we generated UD-086, a next generation HER2-directed ADC consisting of trastuzumab, UVH linker, and AE prodrugs with a DAR of eight and evaluated in vitro and in vivo efficacy and the safety profile.

The drug-linkers were conjugated to trastuzumab via their maleimide groups to prepare UD-086. Linker stability of UD-086 in human serum was evaluated for up to 168 h of incubation. The amounts of released payloads were measured by LC-MS/MS. In vitro cytotoxicity of AE prodrug and AE was tested in NCI-N87 cells. In vitro bystander killing effect of UD-086 was evaluated in a co-culture system of NCI-N87 (HER2-positive) and MDA-MB-231 (HER2-negative). The pharmacokinetic (PK) profile of UD-086 was determined in BALB/c mice. In vivo efficacy of UD-086 was evaluated in NCI-N87, DXd-resistant NCI-N87 and JIMT-1 cells xenograft mouse models. Toxicity study of UD-086 was conducted in cynomolgus monkeys.

UD-086 showed much higher linker stability in human serum in vitro than trastuzumab deruxtecan (DS-8201a). AE prodrug was stable in human serum, and it was approximately 300 times less toxic than AE in NCI-N87 cells. UD-086 indicated in vitro bystander killing effect as well as DS-8201a. UD-086 demonstrated a comparable total antibody PK profile to trastuzumab. In NCI-N87 xenograft model, UD-086 showed three to10-fold potent efficacy than DS-8201a and 10-fold potent efficacy than disitamab vedotin, respectively. In NCI-N87 xenograft model, the minimum effective dose of UD-086 was 1 mg/kg. In DXd-resistant NCI-N87 and JIMT-1 xenograft models, DS-8201a resistance was exhibited, and UD-086 showed more than 30-fold potent effecacy than DS-8201a. The maximum tolerated dose of UD-086 was 6 mg/kg in cynomolgus monkey (Q3W×3, non-GLP).

The therapeutic index of UD-086 is six. UD-086 shows strong efficacy in the DS-8201a resistance tumor models. Despite the conjugating AE with DAR of eight, UD-086 shows very high blood stability and has a significantly improved best in class therapeutic index compared to previous MMAE-based ADCs.