Products Cited in Publications Worldwide Engineering unnatural amino acids in peptide linkers enables cathepsin-selective antibody-drug conjugates for HER2-positive breast cancer

Engineering unnatural amino acids in peptide linkers enables cathepsin-selective antibody-drug conjugates for HER2-positive breast cancer

Gorzeń, Oliwia; Łęcka, Maria; Ćwilichowska-Puślecka, Natalia; Majchrzak, Martyna; Horbach, Natalia; Wiśniewski, Jerzy; Jakimowicz, Piotr; Szpot, Paweł; Zawadzki, Marcin; Dołęga-Kozierowski, Bartosz

DOI: PMID:

Abstract

Antibody-drug conjugates (ADCs) are a rapidly evolving class of targeted cancer therapeutics that combine the specificity of monoclonal antibodies with the potent cytotoxicity of small-molecule payloads. Their clinical success has led to significant advances in oncology, positioning ADCs as a transformative modality in cancer treatment. Most clinically approved ADCs utilize a protease-cleavable valine-citrulline (Val-Cit) dipeptide linker designed to facilitate intracellular payload release upon proteolytic activation by the lysosomal cathepsins. However, the linker is susceptible to off-target cleavage by proteases expressed in non-malignant tissues, resulting in premature payload release and systemic toxicity. To address this limitation, we established a high-throughput peptide linker discovery platform using Hybrid Combinatorial Substrate Library (HyCoSuL) screening to comprehensively profile protease substrate preferences. By incorporating unnatural amino acids, we identified peptide sequences with high selectivity toward cancer-associated proteases, thereby overcoming the constraints of conventional linker design. As proof of concept, we engineered trastuzumab-based ADCs selectively activated by cathepsin B or cathepsin L and evaluated their cytotoxic efficacy in HER2-positive breast cancer models. Our HyCoSuL-guided linkers display higher selectivity toward cathepsins than and enable faster, protease-dependent activation of peptide prodrugs and ADCs in vitro, while also exhibiting enhanced stability in human plasma to minimize premature payload release. Furthermore, recognizing that efficient protease-activated function requires co-expression of both the target antigen and the activating protease, we performed single-cell mass cytometry analysis of patient-derived breast cancer samples to assess the correlation between and expression. This analysis revealed heterogeneous expression, underscoring that pairing the antibody target with the appropriate protease-selective linker might critical for robust, tumor-confined payload release. Our findings highlight the importance of patient stratification based on antigen and protease expression profiles, providing a foundation for developing ADCs with greater protease selectivity and minimized off-target activation.

Keywords

antibody-drug conjugates ; unnatural amino acids ; protease-activated prodrugs ; cathepsin L ; cathepsin B ; breast cancer ; mass cytometry ; CyTOF ; HER-2 ; ; monomethyl auristatin E

Purchased from AmBeed

Fmoc-Leu-OH

A13222435661-60-098%

Fmoc-Leu-OH

10.0/5g

Detail
Fmoc-Phe-OH

A25374035661-40-698%

Fmoc-Phe-OH

58.0/100g

Detail
Fmoc-Thr(tBu)-OH

A41574471989-35-097%

Fmoc-Thr(tBu)-OH

10.0/5g

Detail