Novel anti-human Axl monoclonal antibodies for improved patient biomarker studies

Abstract

Background: Axl receptor tyrosine kinase has been associated with poor clinical outcome in a wide variety of malignancies including breast, lung, prostate, pancreatic, brain and myeloid cancers. Axl is up-regulated during tumour epithelial-to-mesenchymal transition (EMT) and is associated with metastasis, immune evasion and drug resistance. Methods: The aim of this study was to develop improved assays for detection of Axl protein in patient samples. We developed a panel of mouse monoclonal antibodies directed against the extracellular domain of human Axl, from mice immunized with a recombinant antigen comprising the extracellular domains of human Axl fused to human IgG1 Fc domain. Anti-Axl monoclonal antibodies (MAbs) were assessed for sensitivity, specificity and utility using surface plasmon resonance (SPR) analysis, sandwich enzyme-linked immunosorbent assay (ELISA), Western blot analysis and immunohistochemistry (IHC) staining. Results: One of the anti-human Axl MAb,1H12, performed particularly well, and SPR analysis confirmed both high affinity (50 pM) and high selectivity for Axl versus other Axl-family receptors. In IHC applications on selected human carcinomas and cancer cell line pellets, MAb 1H12 showed improved sensitivity and reduced background staining compared to other members of the MAb panel. A second MAb 5F11 showed very high affinity (5.8 pM) at a non-overlapping epitope and was selected as a partner for 1H12 in sandwich ELISA, producing a highly sensitive and specific assay. Conclusion: MAbs 1H12 and 5F11 represent new anti-human Axl monoclonal antibodies for improved patient biomarker studies.