Rapid Generation of pH-Dependent Antibodies Targeting the Tumor-Associated Antigen AXL

This study employs phage-display technology to isolate pH-dependent single-chain antibodies (scFvs) against tumor-associated antigen AXL under acidic pH conditions, further evaluating their binding characteristics across varying pH environments. Through molecular docking and alanine scanning, histidine residues were identified as critical mediators of antibody binding, providing a novel strategy to enhance tumor specificity and reduce off-tumor toxicity in healthy tissues.

 

Literature Overview
The article 'Generation Using Phage-Display of pH-Dependent Antibodies Against the Tumor-Associated Antigen AXL', published in Antibodies, reviews methodologies for utilizing phage-display technology to select AXL-targeting antibodies under acidic pH conditions. This approach improves specificity in the tumor microenvironment while minimizing nonspecific binding in normal tissues, addressing the clinical challenge of 'on-target, off-tumor' toxicity in cancer immunotherapy.

Background Knowledge
AXL, a receptor tyrosine kinase overexpressed in multiple cancers, is associated with chemoresistance and recruitment of immunosuppressive macrophages. Conventional tumor-targeting antibodies often exhibit cross-reactivity in normal tissues, causing 'on-target, off-tumor' toxicity. Leveraging the acidic tumor microenvironment (pH 6.0–6.5), the research team proposed selecting antibodies with pH-dependent binding profiles—strong affinity at acidic pH but reduced binding at neutral pH. This study successfully obtained multiple pH-dependent scFv clones through phage-display screening at acidic pH and validated their binding characteristics correlated with histidine residues, establishing a theoretical foundation and technical framework for developing pH-dependent antibodies.

 

 

Research Methods and Experiments
The team utilized the HuscI human-derived scFv phage-display library, conducting three rounds of selection at pH 5.5. TYRO3-ECD-Fc and Fc proteins were used in depletion steps to eliminate nonspecific binders. Selected scFv clones binding AXL-ECD-Fc-6His were validated in IgG format for pH-dependent binding. Surface plasmon resonance (SPR) analyzed antibody-AXL kinetics, while molecular docking and alanine scanning identified critical binding residues.

Key Conclusions and Perspectives

• Nine scFv clones with strong pH dependence were successfully isolated at acidic pH (5.5) and retained partial pH-dependent binding in IgG format.
• The IgG antibodies D4apH and G10apH demonstrated high-affinity binding at pH 6 but significantly reduced binding at pH 7.4, with EC50 increases of 28- and 18-fold, respectively.
• Mutation analysis revealed histidine 61 (His61) and histidine 116 (His116) on AXL as essential binding sites influencing pH-dependent interactions in D4apH and G10apH.
• Molecular docking indicated that acidic pH enhances binding through hydrogen bonds and π-stacking interactions with His61 and His116, whereas neutral pH diminishes these interactions.
• The study confirms histidine residues' central role in pH-dependent antibody binding, providing theoretical support for developing similar antibodies targeting acidic tumor microenvironments.

Research Significance and Prospects
This work introduces a streamlined, efficient screening strategy for pH-dependent antibodies that activate specifically in acidic tumor microenvironments, reducing toxicity to normal tissues. Future optimization may focus on enhancing acid-specific binding and evaluating applicability in platforms like antibody-drug conjugates (ADCs) and T-cell engagers (TCEs) to improve safety and efficacy of tumor-targeted therapies.

 

 

Conclusion
This study pioneers the isolation of pH-dependent anti-AXL single-chain antibodies via phage display under acidic pH conditions, subsequently converted to IgG format. Structural mutation analysis and molecular docking confirmed histidine residues 61 and 116 as critical determinants for pH-sensitive binding, establishing a foundation for developing tumor microenvironment-specific antibodies. This strategy holds promise for broader application across tumor antigens, enhancing antibody-drug specificity in tumors while minimizing nonspecific toxicity in healthy tissues. Future work may optimize binding properties and explore applications in ADCs, CAR-T, or T-cell engager therapies to advance safer, more effective cancer immunotherapy.

 

Tristan Mangeat, Célestine Mairaville, Myriam Chentouf, Pierre Martineau, and Bruno Robert. Generation Using Phage-Display of pH-Dependent Antibodies Against the Tumor-Associated Antigen AXL. Antibodies.