An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis

Abstract

Aberrant signaling through the Axl receptor tyrosine kinase has been associated with a myriad of human diseases, most notably metastatic cancer, identifying Axl and its ligand Gas6 as important therapeutic targets. Using rational and combinatorial approaches, we engineered an Axl ‘decoy receptor’ that binds Gas6 with high affinity and inhibits its function, offering an alternative approach from drug discovery efforts that directly target Axl. Four mutations within this high affinity Axl variant caused structural alterations in side chains across the Gas6/Axl binding interface, stabilizing a conformational change on Gas6. When reformatted as an Fc-fusion, the engineered decoy receptor bound to Gas6 with femtomolar affinity, an 80-fold improvement compared to the wild-type Axl receptor, allowing effective sequestration of Gas6 and specific abrogation of Axl signaling. Moreover, increased Gas6 binding affinity was critical and correlative with the ability of decoy receptors to potently inhibit metastasis and disease progression in vivo.

Inhibition of the GAS6/AXL pathway augments the efficacy of chemotherapies

“…Three mutations were found to occur in at least 20% of these unique variants: A72V, D87G, and V92A, the latter 2 to GAS6. The characterization of MYD1 revealed a strong correlation between GAS6-binding affinity and therapeutic efficacy of the AXL decoy receptor in preclinical models of cancer metastasis (17).…”

“…In our original work, an error-prone library was created using the WT AXL Ig1 domain as a template and placed into the yeast display system (23). After 6 rounds of flow cytometric sorting, the library was enriched for 3 AXL variants with improved binding to GAS6 (17). While only 3 variants were present after the sixth and final round of sorting, substantial diversity was retained in earlier sort products.…”

“…To ensure that potential improvements could be quantified, a dose of 0.5 mg/kg was used, which is the in vivo IC 50 of MYD1 Fc, as previously determined using the same assay (17). Mice were administered MYD1 Fc or MYD1-72 Fc, and serum samples were obtained at time points up to 36 hours after injection.…”

“…The AXL receptor contains 2 distinct GAS6-binding epitopes: a high affinity site on its N-terminal Ig-like domain and a low affinity site on the second Ig domain (22). Previously, we engineered the major site on AXL Ig1 using a combination of rational and combinatorial protein-engineering methods (17). The result of these efforts was MYD1, a high-affinity AXL variant containing 4 mutations that conferred improved binding…”

“…Furthermore, AXL has a single ligand, growth arrest-specific 6 (GAS6) (15,16), and constitutive activation is rarely observed in tumors, leaving GAS6-mediated signaling as the primary driver of pathogenesis. Nevertheless, an unusually strong binding affinity between GAS6 and AXL of approximately 30 pM (17) has made the development of competitive antagonists challenging.…”