# Generation Using Phage-Display of pH-Dependent Antibodies Against the Tumor-Associated Antigen AXL

## TL;DR

Researchers developed pH-sensitive antibodies against AXL, a tumor-associated antigen, to reduce toxicity in healthy tissues by exploiting the acidic tumor environment.

## Contribution

A novel method using phage display at acidic pH to generate pH-dependent antibodies targeting AXL is introduced.

## Key findings

- Nine scFvs showed strong acidic pH-dependent binding to AXL with minimal neutral pH binding.
- Two IgG antibodies demonstrated strong pH sensitivity due to histidine residues at positions 61 and 116 on AXL.
- The method offers a potential strategy to enhance tumor specificity and reduce off-tumor toxicity.

## Abstract

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.