The Earlier Past

Mesothelioma is a malignancy of the mesothelium and current treatments are generally ineffective. One promising area of anticancer drug development is to explore tumor susceptibility to targeted therapy. To achieve efficient, targeted intracellular delivery of therapeutic agents to mesothelioma cells, we selected a naive human single-chain (scFv) phage antibody display library directly on the surface of live mesothelioma cells to identify internalizing antibodies that target mesothelioma-associated cell surface antigens. We have identified a panel of internalizing scFvs that bind to mesothelioma cell lines derived from both epithelioid (M28) and sarcomatous (VAMT-1) types of this disease. Most importantly, these antibodies stain mesothelioma cells in situ and therefore define a panel of clinically represented tumor antigens. We have further exploited the internalizing function of these scFvs to achieve targeted intracellular drug delivery to mesothelioma cells. We showed that scFv-targeted immunoliposomes were efficiently and specifically taken up by both epithelioid and sarcomatous mesothelioma cells, but not control cells, and immunoliposomes encapsulating the small-molecule drug topotecan caused targeted killing of both types of mesothelioma cells in vitro.


Unfortunately, very few mesothelioma cell surface markers are known (9). For example, mesothelin is a cell surface antigen of unknown function that has been reported to be expressed in epithelioid mesothelioma cells (10). Serologic tests and treatment studies based on mesothelin are under active development (11). Mesothelin, however, is also expressed on normal mesothelial cells and is not expressed on sarcomatous mesothelioma (12), a particularly recalcitrant subtype of mesothelioma. Other cell surface molecules such as CD44, the 67-kDa laminin receptor, and a subset of voltage-dependent anion channels have also been reported to be expressed at an elevated level in mesothelioma cells (13). Those molecules, however, are also found in many other types of tumor as well as in normal tissues. Thus, much work is needed to identify additional cell surface markers with more restricted patterns of tissue distribution and more specific associations with mesothelioma, of both epithelioid and sarcomatous subtypes.

Differential gene expression-based approaches have been widely used for discovery and identification of tumor markers, but they face important limitations, especially in analyzing tumor-associated post-translational modifications (14). In contrast, regardless of the exact chemical makeup, the tumor cell surface epitope space can be mapped by complementary monoclonal antibodies. Monoclonal antibodies are able to discern subtle differences in antigen structure and conformation and recognize antigenic determinants of diverse chemical composition with high affinity and specificity (15, 16). As such, the tumor cell surface epitope space can be efficiently mapped by high-affinity interactions with monoclonal antibodies (17). Isolating these epitopes enables the antibodies to achieve specific binding to neoplastic cells, an ability that could be used in applications such as induction of antibody-dependent cell cytotoxicity or inhibition of signaling pathways involved in tumor cell migration, growth, and survival. In addition, antibodies targeting internalizing tumor epitopes could be exploited to achieve specific intracellular delivery of chemotherapeutic drugs (7, 18, 19).

We report here the identification of a panel of internalizing monoclonal antibodies that target mesothelioma cell surface antigens. We have used a naive phage antibody display library to probe the cell surface epitope space of live mesothelioma cells and identified human single-chain Fv fragments (scFv) that target mesothelioma-associated internalizing cell surface epitopes present on both epithelial and sarcomatous cell lines. We have further showed that several of these scFvs are capable of mediating targeted intracellular drug delivery to mesothelioma cells, suggesting that they may be useful for developing targeted therapies against mesothelioma.