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Generation Using Phage-Display of pH-Dependent Antibodies Against the Tumor-Associated Antigen AXL
Tristan Mangeat1, Célestine Mairaville1, Myriam Chentouf1
1Institut de Recherche en Cancérologie de Montpellier (IRCM), Université Montpellier, Institut Régional du Cancer de Montpellier (ICM), INSERM, 34298 Montpellier, France.
Background/Objectives:
Tumor-associated antigens are not tumor-specific antigens but proteins that are overexpressed by tumor cells and also weakly expressed at the surface of healthy tissues. Therefore, some side effects are observed when targeted by therapeutic antibodies, a phenomenon named "on-target, off-tumor toxicity". As tumors generate an acidic microenvironment, we investigated whether we could generate pH-dependent antibodies to increase their tumor specificity. For this proof-of-concept study, we selected the tyrosine kinase receptor AXL because we already developed several antibodies against this target.
Methods:
To generate a pH-dependent anti-AXL antibody, we performed classical panning of a single-chain variable fragment (scFv) library using phage display at an acidic pH throughout the process.
Results:
After the third round of panning, 9 scFvs, among the 96 picked clones, bound to AXL at acidic pH and showed very low binding at a neutral pH. After reformatting them into IgG, two clones were selected for further study due to their strong pH-sensitive binding. Using molecular docking and alanine scanning, we found that their binding strongly depended on two histidine residues present on AXL at positions 61 and 116.
Conclusions:
To conclude, we set-up an easy process to generate pH-dependent antibodies that may increase their tumor-binding specificity and potentially decrease toxicity towards healthy tissues.
Insights
Researchers developed pH-dependent antibodies targeting tumor-associated antigens like AXL. This approach aims to enhance tumor specificity and reduce on-target, off-tumor toxicity in antibody therapies.
Area of Science:
- Immunology
- Biotechnology
- Oncology
Background:
- Tumor-associated antigens are overexpressed in cancer but also present in healthy tissues, leading to on-target, off-tumor toxicity with therapeutic antibodies.
- Tumor microenvironments are often acidic, presenting an opportunity to exploit pH differences for targeted therapies.
Purpose of the Study:
- To develop pH-dependent antibodies that specifically target tumor-associated antigens.
- To investigate if pH sensitivity can enhance antibody tumor specificity and reduce off-tumor side effects.
- To create a proof-of-concept for pH-dependent antibody generation using the tyrosine kinase receptor AXL as a target.
Main Methods:
- Utilized phage display with a single-chain variable fragment (scFv) library, performing panning exclusively under acidic pH conditions.
- Selected and reformatted promising pH-sensitive scFvs into immunoglobulin G (IgG) format for further evaluation.
- Employed molecular docking and alanine scanning to identify key amino acid residues involved in pH-dependent binding.
Main Results:
- Identified 9 scFvs that bound to AXL at acidic pH with minimal binding at neutral pH after three rounds of panning.
- Selected two IgG clones exhibiting strong pH-sensitive binding to AXL.
- Determined that binding of these antibodies is critically dependent on two histidine residues (positions 61 and 116) on the AXL receptor.
Conclusions:
- Established a straightforward method for generating pH-dependent antibodies.
- Demonstrated the potential of pH-dependent antibodies to improve tumor-specific binding.
- Indicated that this strategy may significantly decrease toxicity in healthy tissues for antibody-based cancer therapies.
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