Mesothelioma is an intractable tumor with no curative treatment to date. In a first step toward developing targeted therapeutics against mesothelioma, we sought to identify internalizing antibodies that target mesothelioma-associated cell surface antigens. Taking a functional approach, we have used a nonimmune phage antibody library as an unbiased random-shaped affinity repertoire to select for tumor-targeting scFvs on live mesothelioma cells. The selection methodology was optimized to enrich for scFvs that efficiently target internalizing epitopes (17
), providing a means of efficient intracellular payload delivery to mesothelioma cells. We identified 95 unique mesothelioma-targeting scFvs, 21 of which were further characterized by FACS profiling on tumor cell lines, immunohistochemistry on mesothelioma tissue samples, and in vitro
internalization/payload delivery assays. All 21 scFvs bind to both epithelioid and sarcomatoid type mesothelioma cell lines. In addition, all 21 scFvs stain mesothelioma cells in situ
and therefore recognize clinically represented tumor antigens expressed on all mesothelioma subtypes. Two of the scFvs, M1 and M25, were shown to be capable of targeted intracellular payload delivery into mesothelioma cells. Cluster analysis and competition experiments indicate that the 21 scFvs bind to 17 unique epitopes and two pairs of overlapping epitopes. These properties make this panel of scFvs attractive candidates for therapeutic development.
A novel feature of this panel of scFvs is that they recognize all subtypes of mesothelioma. Many previously identified markers, such as mesothelin, recognize only the epithelioid mesothelioma, but not the sarcomatoid subtype, a particularly recalcitrant form of this disease (39
). Because we selected mesothelioma-targeting antibodies from an antibody library, selection conditions could be manipulated to enrich for scFvs with desired properties. By alternating the selection target between epithelioid and sarcomatoid cell lines, we were able to select for scFvs targeting both subtypes, therefore broadening therapeutic applicability.
Our study also shows that although mesothelioma is notorious for resistance to conventional chemotherapy (40
), it may nonetheless be susceptible to targeted therapy. Immunoliposomes encapsulating the small-molecule drug topotecan and targeted by the M1 or M25 scFvs showed efficient and selective killing of mesothelioma but not control cells. Topotecan, an inhibitor of the nuclear enzyme topoisomerase I, exists in two forms. At acidic pH, topotecan is mainly in the active ring-closed lactone form (42
). At neutral (physiologic) or alkaline pH, the drug is converted to a ring-open carboxylate form, which has poor membrane permeability, and thus poor cellular uptake and cytotoxicity (43
). Therefore, the use of a liposome carrier for topotecan is particularly relevant to its therapeutic effects (45
). Immunoliposomes can be constructed to have a long circulating half-life and to be nonimmunogenic (46
). As such, immunoliposomes represent one form of targeted therapy that can be used to exploit the internalizing function of this panel of scFvs.
For therapeutic development, it is important to identify antibodies binding to clinically represented tumor antigens. In this study, we selected the phage antibody library on mesothelioma cell lines and further studied their binding patterns to mesothelioma tissues to identify scFvs that target tumor cells in situ
. A very high percentage of our scFvs selected on mesothelioma cell lines were found to bind to mesothelioma tissues. This is rather surprising as our previous studies on other tumors such as prostate cancer have indicated that selection on tumor cell lines often generates antibodies that do not bind to tumor cells in situ
, and novel selection methods such as laser capture microdissection are required for identification of antibodies binding to tumor cells in situ
. There are several possible explanations for this discrepancy. (a
) The mesothelioma cell lines used in this study were obtained relatively recently (22
), whereas the prostate cancer lines have been cultured for nearly 30 years (47
). As such, the mesothelioma cell lines may have fewer culture artifacts and resemble more closely mesothelioma cells in situ
) We focused our study on scFvs that bind to both epithelioid and sarcomatoid cell lines, further reducing the chance of selecting for scFvs binding to artifacts caused by culture conditions. Regardless of the exact cause, we have taken advantage of the cell surface antigen similarity between mesothelioma cell lines and mesothelioma cells in tissues and identified a panel of scFvs that targets clinically relevant tumor markers.
We are identifying mesothelioma antigens bound by our panel of internalizing scFvs using a combination of approaches, including screening a novel yeast cDNA surface display library that we have developed (48
), and analyzing immunoprecipitation products by mass spectrometry (50
). Some of the selected scFvs, such as M1, target several other tumor cell lines such as breast and prostate cancer lines in addition to mesothelioma lines, suggesting that they bind to antigens commonly expressed by tumor cells. Mesothelioma thus shares cell surface markers with other tumors; therefore, treatments developed for other neoplastic diseases may also be effective against mesothelioma. Other scFvs, such as M25, target mesothelioma cells with very high specificity. Thus, the identification of these mesothelioma-specific antigens may reveal etiologies and pathogenic pathways unique to this disease and facilitate the development of early diagnostics and therapeutic strategies against mesothelioma.