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 V_HHs against human Axl using multiple complementary phage display selection strategies and characterized a subset of high-affinity V_HHs. The V_HHs 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_HHs cross-reacted with cynomolgus monkey Axl and none recognized mouse Axl. As fusions to human IgG1 Fc, V_HH-Fcs bound Axl⁺ tumor cell lines and mertansine-loaded V_HH-Fcs were cytotoxic in vitro against Axl⁺ cells in proportion to their binding affinities. Engineered biparatopic V_HH-V_HH heterodimers bound Axl avidly, and a subset of molecules showed dramatically enhanced association rates indicative of intramolecular binding. These V_HHs may have applications as modular elements of biologic drugs such as antibody-drug conjugates.