The paper entitled “Glutamic acid–valine–citrulline linkers ensure stability and efficacy of antibody–drug conjugates in mice” can be found here: https://www.nature.com/articles/s41467-018-04982-3

Cancer chemotherapy is seeing the beginning of a new era with the emergence of antibody–drug conjugates (ADCs), a novel class of drug delivery systems. Their clinical potential has undoubtedly been demonstrated; 4 ADCs have been clinically approved and more than 60 are in clinical trials.

ADC chemical linkers connect therapeutic antibodies and highly potent antitumor drug molecules. The linker component is important because its chemical and physicochemical properties affect ADC potency, in vivo stability, and tolerability. Among the many ADC linker types in use, the valine-citrulline (Val-Cit or VCit) dipeptide and similar linkers are most common (e.g., Adcetris). Previous studies have shown that, in the human body, VCit linkers are stable in circulation but are immediately cleaved by lysosomal enzymes inside target tumor cells. This feature enables pinpoint delivery and intracellular release of toxic payloads. However, a recent study by Dorywalska and co-workers cautioned ADC researchers that VCit is unstable in mouse plasma because of the carboxylesterase Ces1c (ref. 1). Depending on the linker length and attachment site, VCit can undergo premature drug release in mouse circulation before delivering payloads to tumors.

Most initial preclinical safety and efficacy studies of drug candidates are performed in mouse models. It appears to be acceptable to use VCit linkers in later stages with other animal models, such as monkeys. However, advancing to other animal models is difficult without first seeing positive outcomes in mouse models. Thus, the issue of linker instability is a big headache for ADC researchers who want to create and evaluate novel ADCs using VCit-type linkers. Linker length and attachment site could be carefully selected, but such a task is not trivial. We faced this problem in the process of developing branched cleavable linkers for installing multiple drugs (ref. 2). Incorporating an adequate spacer within the linker scaffold is prerequisite to ensuring rapid drug release upon enzymatic cleavage. However, we found that the long spacer left the VCit moieties exposed and vulnerable to degradation in mouse plasma.

Fig. 1 EVCit ensures high ADC stability and efficacy in mouse models (figures from our paper with modifications). a chemical structure of EVCit linker. This linker is cleaved by cathepsins in lysosome but not by mouse Ces1c, which can degrade conventional VCit linkers in circulation. b Circulation stability of VCit, SVCit, and EVCit ADCs. c Treatment efficacy of VCit and EVCit ADCs in a xenograft mouse model.

Inspired by the report by Dorywalska and co-workers, and thanks to tireless efforts by my team, we finally found a simple solution: addition of a glutamic acid residue next to the valine (Fig 1a). Despite the high linker exposure to proteases, the glutamic acid-appended VCit (EVCit) tripeptide dramatically improved the ADC half-life in mouse models from 2 days to 12 days (Fig. 1b). Twelve days is close to the half-life of therapeutic human IgGs. As anticipated, this improvement was associated with a remarkable treatment effect on tumors in mouse models (Fig. 1c). Another bonus is the increased hydrophilicity of EVCit, which may reduce the risk of ADC aggregation and fast clearance. This simple cleavable linker system does not rely on a unique conjugation method or site. Therefore, this technology may allow researchers to revisit previous ADCs that have unfortunately failed due to poor stability and efficacy in mouse models. We hope many researchers will enjoy expanded flexibility in designing ADCs and other drug delivery systems with this new chemical linker.

If you want to learn more about our findings, read our paper: https://www.nature.com/articles/s41467-018-04982-3

If you want to know about us, visit our website: https://www.tsuchikamalab.org

References

1. Dorywalska, M. et al. Molecular basis of valine-citrulline-PABC linker instability in site-specific ADCs and its mitigation by linker design. Mol. Cancer Ther. 15, 958–970 (2016).
2. Anami, Y. et al. Enzymatic conjugation using branched linkers for constructing homogeneous antibody-drug conjugates with high potency. Org. Biomol. Chem. 15, 5635–5642 (2017).