Antibody–drug Conjugates (ADCs) are a powerful therapeutic modality for cancer treatment. ADCs are multi-functional biologics in which a disease-targeting antibody is conjugated to an effector payload molecule via a linker. The success of currently used ADCs has been largely attributed to the development of linker systems, which allow for the targeted release of cytocidal payload drugs inside cancer cells. Many lysosomal proteases are over expressed in human cancers. They can effectively cleave a variety of peptide sequences, which can be exploited for the design of ADC linker systems. As a well-established linker, valine-citrulline-p-aminobenzyl carbamate (ValCitPABC) is used in many ADCs that are already approved or under preclinical and clinical development. Although ValCitPABC and related linkers are readily cleaved by cathepsins in the lysosome while remaining reasonably stable in human plasma, many studies have shown that they are susceptible to carboxylesterase 1C (Ces1C) in mouse and rat plasma, which hinders the preclinical evaluation of ADCs. Furthermore, neutropenia and thrombocytopenia, two of the most commonly observed dose-limiting adverse effects of ADCs, are believed to result from the premature hydrolysis of ValCitPABC by human neutrophil elastase. In addition to ValCitPABC, the GGFG tetrapeptidyl-aminomethoxy linker is also cathepsin-cleavable and is used in the highly successful ADC drug, DS8201a. In addition to cathepsin-cleavable linkers, there is also growing interest in legumain-sensitive linkers for ADC development. Increasing plasma stability while maintaining lysosomal cleavability of ADC linkers is an objective of intensive current research. This review reports recent advances in the design and structure–activity relationship studies of various peptide/peptidomimetic linkers in this field.

Antibody-Drug Conjugates (ADCs) are a powerful therapeutic modality for cancer treatment. ADCs are multi-functional biologics in which a disease-targeting antibody is conjugated to an effector payload molecule through a linker. The success of currently used ADCs has been largely attributed to the development of linker systems which allow targeted release of cytocidal payload drugs inside cancer cells. Many lysosomal proteases are over-expressed in human cancers. They can effectively cleave a variety of peptide sequences, which can be exploited in the design of ADC linker systems. As a well-established linker, valine-citrulline-p-aminobenzyl carbamate (ValCitPABC) is used in many ADCs that are already approved or under preclinical and clinical development. Although ValCitPABC and related linkers are readily cleaved by cathepsins in the lysosome while remaining reasonably stable in human plasma, many studies have shown that they are susceptible to carboxylesterase 1C (Ces1C) in mouse and rat plasma, which hinders preclinical evaluation of the ADCs. Furthermore, neutropenia and thrombocytopenia, two of the most commonly observed dose-limiting adverse effects of ADCs, are believed to result from the premature hydrolysis of ValCitPABC by human neutrophil elastase. In addition to cathepsin-cleavable linkers, there is also growing interest in legumain-sensitive linkers for ADC development. Increasing plasma stability while maintaining lysosomal cleavability of ADC linkers is an objective of intensive current research. This review reports recent advances in the design and structure-activity relationship studies of various peptide/peptidomimetic linkers in this field.

Supplementary Figure 6. Mouse body weight change post-ADC treatment compared to vehicle control groups. (A-C) Mice were transplanted with HER2-expressing BT-474 cells and administered with a single intravenous dose of 1 mg/kg (A), 3 mg/kg (B), 10 mg/kg (C) of SGZ002-LC-MMAE, SGZ002-HC-MMAE or Kadcyla. (D-F) Mice were transplanted with HER2 (3+)-expressing PDX model, T226 and administered with a single intravenous dose of SGZ026 or Enhertu at 1 mg/kg (D), 3 mg/kg (E), or 7.5 mg/kg (F). (G-I) Mice were transplanted with HER2 (2+)-expressing PDX model, HBCx-1 and administered with a single intravenous dose of 10 mg/kg of SGZ022 or SGZ024 (G), SGZ230 or RC48 (Aidixi) (H), SGZ026 or Enhertu (I). Body weights were measured for up to 42 days (A-F) or 42 days (G-I).

Supplementary Figure 5. In vitro stability of SGZ230 vs Disitamab-MMAE in human plasma.

Supplementary Figure 2A. Supplementary Figure 2A. Analysis by HIC of Trastuzumab based antibody-drug conjugates (ADCs) with varying Drug-to-Antibody Ratios (DARs) and payloads. Supplementary Figure 2B. Analysis of Disitamab based antibody-drug conjugates (ADCs) with varying Drug-to-Antibody Ratios (DAR) and payload types by HIC.

Antibody–drug conjugates (ADC) are a rapidly expanding class of biopharmaceuticals for cancer-targeted therapy, with 15 ADCs approved for clinical use and more than a hundred candidates in clinical development. ADCs comprise a tumor-specific monoclonal antibody to which a cytotoxic payload is attached via a linker. Approved ADCs are heterogeneous products encompassing a range of drug-to-antibody ratios (DAR) due to the chemical methods employed for conjugation. Achieving precise DAR control is expected to improve product homogeneity, pharmacokinetics, safety, and clinical efficacy. In this study, we developed a rapid and efficient two-component bioconjugation system comprising a recombinant peptide asparaginyl ligase (PAL) and a human glutaminyl cyclase (hQC) to conjugate anti-HER2 antibodies. Trastuzumab and disitamab were conjugated with single or branched payloads, yielding ADCs with DAR values ranging from 2 to 8. These ADCs demonstrated cytotoxicity comparable with their chemically conjugated counterparts, trastuzumab deruxtecan (DXd, ENHERTU) and disitamab vedotin (Aidixi), against HER2-expressing breast cancer cell lines and Patient-Derived Xenograft (PDX) models. Notably, SGZ026, a PAL-conjugated trastuzumab DXd with a DAR of 3.9, exhibited better in vivo mouse plasma stability compared with ENHERTU (DAR 8). Despite carrying approximately half the cytotoxic payload per antibody, a single dose of SGZ026 effectively inhibited tumor growth in breast cancer PDX models with high or intermediate HER2 expression, achieving efficacy comparable with ENHERTU. These findings highlight a novel and robust enzyme-based conjugation technology for developing homogeneous ADCs with an improved therapeutic window.