Nature Communications | LDC Technology with Ring-Opening Linker Enhances HER2-Targeting ADC Stability and Safety

This study significantly improves the stability and safety of HER2-targeting antibody-drug conjugates (ADCs) using site-specific ligase-dependent conjugation (LDC) technology combined with stable ring-opening linkers. GQ1001 and GQ1005 demonstrate superior antitumor activity and reduced off-target toxicity across multiple preclinical models, particularly maintaining activity in drug-resistant models with high ABCG2 expression. Additionally, combination with tyrosine kinase inhibitors or chemotherapy enhances therapeutic efficacy.

 

Literature Overview
The article 'Site-specific ligase-dependent conjugation with ring-opening linker improves safety and stability of HER2-targeting ADCs,' published in Nature Communications, reviews and summarizes the heterogeneity and off-target toxicity issues in traditional antibody-drug conjugates (ADCs) caused by random conjugation and unstable linkers. The study introduces a novel stable ring-opening linker technology based on site-specific conjugation to optimize the stability and safety of HER2-targeting ADCs, enhancing their potential for application in drug-resistant and HER2-low expressing tumors.

Background Knowledge
Antibody-drug conjugates (ADCs) are a class of targeted therapeutic agents formed by conjugating monoclonal antibodies with cytotoxic drugs via linkers. HER2-targeting ADCs (such as T-DM1 and T-DXd) have been approved for the treatment of HER2-positive breast and gastric cancers. However, clinical applications still face challenges including off-target toxicity, unstable pharmacokinetics, and drug resistance. Major toxicities include interstitial lung disease, thrombocytopenia, neutropenia, and neuropathy, some of which are associated with free drugs in plasma. Traditional conjugation methods, such as maleimide-based Michael addition, are unstable under physiological conditions and prone to reverse Michael reactions or thiol exchange, leading to premature drug release. Recent studies have shown that ring-opening linker designs can enhance linker stability; however, this strategy still faces limitations such as complex engineering and difficulties in purification. This study introduces an immobilized enzyme-mediated LDC technology combined with ring-opening linker design to address stability and safety issues in conventional ADCs.

 

 

Research Methods and Experiments
This study employed site-specific LDC technology by introducing short recognition peptides at the C-terminus of the antibody light chain, enabling site-directed conjugation of DM1 or DXd to HER2 antibodies. An immobilized enzyme-mediated conjugation system based on Sortase A was used to synthesize ring-opening linker precursors under weakly alkaline conditions, followed by further purification to ensure conjugate homogeneity. Drug-to-antibody ratio (DAR) and purity were evaluated using HIC-HPLC and size exclusion chromatography. In vitro cytotoxicity, cell cycle arrest, and apoptosis-inducing capacity were tested across multiple HER2-positive and HER2-negative cell lines and animal models. In vivo antitumor activity and enhanced effects of combination with TKI or chemotherapy were assessed in PDX and CDX models. Furthermore, pharmacokinetics and toxicity profiles of GQ1001 and GQ1005 were evaluated in cynomolgus monkeys, with DM1 or DXd plasma release levels measured.

Key Conclusions and Perspectives

• GQ1001 and GQ1005 demonstrate HER2 expression-dependent antitumor activity in vitro and in animal models, with significantly reduced off-target toxicity compared to traditional ADCs.
• GQ1001 exhibits slightly lower antitumor activity than T-DM1 in HER2-positive cell lines but shows almost no cytotoxicity in HER2-negative cells.
• The ring-opening linker design results in higher plasma stability of GQ1001 and GQ1005, with significantly lower DM1 or DXd exposure in plasma compared to T-DM1 or T-DXd.
• In cynomolgus monkeys, the HNSTD (highest non-severely toxic dose) of GQ1001 reaches 45 mg/kg, significantly higher than the 10 mg/kg of T-DM1.
• Combination of GQ1001 with TKIs or chemotherapy significantly enhances antitumor effects and remains effective in drug-resistant models.
• In cancer models with high ABCG2 expression resistant to T-DXd, GQ1001 maintains effective antitumor activity.

Research Significance and Prospects
This study presents an innovative LDC conjugation platform combined with stable ring-opening linker design, significantly improving the stability, homogeneity, and safety of HER2-targeting ADCs while maintaining antitumor efficacy. This technology holds promise for developing next-generation ADCs targeting HER2-low or drug-resistant tumors, expanding their clinical applicability. Future studies can further evaluate its potential across other target-toxin combinations and advance GQ1001 and GQ1005 into clinical trials.

 

 

Conclusion
This study successfully developed GQ1001 and GQ1005, two HER2-targeting ADCs based on LDC technology and ring-opening linker design. Compared to traditional ADCs, they exhibit higher homogeneity, stability, and HER2 specificity in vitro and in animal models, with significantly reduced off-target toxicity. In cynomolgus monkeys, GQ1001 achieves an HNSTD of 45 mg/kg, far exceeding T-DM1's 10 mg/kg. When combined with TKIs or chemotherapy, GQ1001's antitumor efficacy is further enhanced and remains effective in drug-resistant models with high ABCG2 expression. These results indicate that the new conjugation technology offers a safer and more effective treatment strategy for HER2-positive refractory cancers, holding significant clinical translational value.

 

Lei Huang, Gang Qin, Chengcheng Gong, Bo Yang, and Biyun Wang. Site-specific ligase-dependent conjugation with ring-opening linker improves safety and stability of HER2-targeting ADCs. Nature Communications.