Recognition of Mesothelin by the Therapeutic Antibody MORAB-009: Structural and Mechanistic Insights

Abstract

Mesothelin is a cell surface protein that is normally found in mesothelial cells lining the pleura, pericardium and peritoneum, but is aberrantly expressed at high level in a variety of cancers including mesothelioma, ovarian, pancreatic and lung cancers. Although the physiological function of mesothelin is unclear, studies have shown that it is capable of binding to the tumor antigen CA-125 (also known as MUC16), leading to cell adhesion and tumor metastasis. Since mesothelin is specifically expressed at a significantly higher level in malignant tumors, development of an antibody against mesothelin is therefore of major importance in the field of cancer therapy. MORAb-009 is a humanized monoclonal antibody against mesothelin currently under clinical trials. Animal experiments have shown that application of MORAb-009 in combination with chemotherapy leads to a marked reduction in tumor growth of mesothelin-expressing tumors. We show here that MORAb-009 recognizes a non-linear epitope that is contained in the first 64-residue fragment of the mesothelin. We further demonstrate that the recognition is sensitive to the loss of a disulfide bond linking residues Cys7 and Cys31. The crystal structure of the complex between the mesothelin N-terminal fragment and Fab of MORAb-009 at 2.6 Å resolution reveals an epitope encompassing multiple secondary structural elements of the mesothelin, The mesothelin fragment has a compact, right-handed superhelix structure consisting of five short helices and connecting loops. A residue essential for complex formation has been identified as Phe22, which projects its side chain into a hydrophobic niche formed on the antibody's recognition surface upon antigen-antibody contact. The overlapping binding footprints of both the monoclonal antibody and the cancer antigen CA125 explains the therapeutic effect and provides a basis for further antibody improvement.