Paratope mapping of tilvestamab, an anti-AXL function-blocking antibody, using high-throughput bacterial expression of secreted scFv-osmY fusion proteins.

Targeting AXL receptor kinase with a highly selective antibody presents a promising approach for inhibiting AXL and potentially improving cancer treatment. An essential step in antibody optimisation is the mapping of paratope residues to epitope residues. In the present study, we identify the residues of tilvestamab, a function-blocking anti-AXL monoclonal antibody, that are essential for its binding to the extracellular domain of AXL. A single-chain variable fragment (scFv) fused to osmotically inducible protein Y (osmY) was designed to enable the secretion of soluble scFv-osmY mutants, which could be directly subjected to high-throughput biolayer interferometry screening for binding to the AXL Ig1 domain. Each complementarity-determining region residue of scFv was mutated to Ala, while additional mutations were made on the basis of predicted contribution to binding. We generated AlphaFold3 predictions for the scFv (tilvestamab)-AXL Ig1 complex to gain insights into the molecular interactions of the essential residues, as determined by the experimental data. Our study reveals that tilvestamab binds to the Ig1 domain of AXL, with twelve residues on scFv (tilvestamab) contributing most to binding, likely being situated at the binding interface. Glu2 near the N-terminus of AXL is essential for binding. The data give a structural view into the AXL-tilvestamab complex and allow for further optimisation of the binding interface.