Biparatopic Single-Domain Antibodies Against Axl Achieve Ultra-High Affinity Through Intramolecular Engagement

Overexpression of Axl, a TAM-family receptor tyrosine kinase, plays key roles in the formation, growth, and spread of tumors as well as resistance to targeted therapies and chemotherapies. We identified novel llama VHHs against human Axl using multiple complementary phage display selection strategies and characterized a subset of high-affinity V(H)Hs. The V(H)Hs targeted multiple sites in Ig-like domains 1 and 2 of the Axl extracellular domain, including an immunodominant epitope overlapping the site of Gas6 interaction and two additional non-Gas6 competitive epitopes recognized by murine monoclonal antibodies. Only a subset of V(H)Hs cross-reacted with cynomolgus monkey Axl and none recognized mouse Axl. As fusions to human IgG1 Fc, VHH-Fcs bound Axl thorn tumor cell lines and mertansine-loaded VHH-Fcs were cytotoxic in vitro against Axl thorn cells in proportion to their binding affinities. Engineered biparatopic VHH-VHH heterodimers bound Axl avidly, and a subset of molecules showed dramatically enhanced association rates indicative of intramolecular binding. These V(H)Hs may have applications as modular elements of biologic drugs such as antibody-drug conjugates. Crown Copyright (C) 2021 Published by Elsevier Inc. All rights reserved.