Development of biologic devices for mesothelin immunotargeting

BACKGROUND: Mesothelin is a tumor differentiation antigen (Ag) that is normally present on the mesothelial cells lining the pleura, peritoneum and pericardium. It is, however, highly expressed in several human cancers including malignant mesothelioma, pancreatic, ovarian and lung adenocarcinoma. The normal biologic function of mesothelin is unknown but recent studies have shown that it binds to CA-125 and may play a role in the peritoneal spread of ovarian cancer. The limited mesothelin expression in normal tissues and high expression in many cancers makes it an attractive candidate for cancer immunotherapy. RESULTS: In this study we have developed a monoclonal antibody (mAb) that is specific for the Ag mesothelin. It was produced by hybridomas technologies and we have performed Fluorescence Activated Cell Sorting (FACS) analysis to evaluate the specificity and affinity of this antibody for the Ag of interest. The mAb binds mesothelin-positive cell lines expressing the Ag constitutively (OVCAR-3) and transfected cells (HEK293-mesothelin). The same mAb not recognizes mesothelin-negative cell lines demonstrating an high specificity in vitro. Binding studies have demonstrated that our mAb has a better affinity with respect to mAb K1, a commercially available anti-mesothelin mAb. Our anti-mesothelin mAb was chemically linked to ricin A chain (RTA) toxin obtaining a powerful immunodelivered drug (immunotoxin, IT) with specific cytotoxic activity on mesothelin positive cells; in a cytotoxic assay on HEK293-mesothelin transfected cells the anti-mesothelin mAb-RTA IT shows an IC50 of 0.03 nM and 0,09 nM after 36 hrs and 72 hrs of incubation respectively; no cytotoxic activity was observed against mock-not transfected one and other mesothelin negative cells. As a further proof of specificity we observed that the cytotoxic activity of 0,03 nM of the above-mentionated IT on HEK293-mesothelin cells, is fully prevented by addition of whole molecule mesothelin-specific antibody at a concentration 1000 fold over IT. CONCLUSION: Our mAb holds great potential to be used as a research reagent and diagnostic tool in research laboratories and in the clinics because of its high quality and versatility. This antibody is also a strong candidate to be investigated for further in vivo passive immunotherapy studies. Moreover, the discovered of this new mAb anti-mesothelin enable us to develop in vivo diagnostic approaches using radionuclide, fluorescence trackers or nanoparticles. Its conjugation with therapeutic molecules allows a better distribution of the drug to the tumor sites; this ability increase the antitumor efficacy and reduce the specific toxicity at the same time.