One well established markermesothelin, a 40kDa cell surface glycoprotein, has been reported to be expressed by EM cells (8), but not SM (9). studied (liver, 2.62%ID/g; other organs or tissues <1.7%ID/g), except the kidneys (130.7%ID/g), giving tumor-to-blood ratios of 5:1 and 7:1 and tumor-to-muscle ratios of 45:1 and Azamethiphos 60:1, for M28 and VAMT-1 respectively. The target-mediated uptake was confirmed by a nearly 70% reduction in tumor activity following administration of 10-fold excess of unlabeled scFv. Azamethiphos Taken together, these results indicate that M40 can rapidly and specifically target epithelioid and sarcomatoid tumor cells, demonstrating the potential of this agent as a versatile targeting ligand for imaging and therapy of all subtypes of mesothelioma. == Introduction == Malignant mesothelioma (MM), caused primarily Azamethiphos by exposure to asbestos, is a highly aggressive tumor arising from serosal surfaces of the pleura, peritoneum and pericardium (1,2). MM has three major subtypes; epithelioid (EM) that is more likely to respond to treatment and accounts for 5070% Azamethiphos of all cases, sarcomatoid (SM) that rarely responds to any treatment and represents 720%, and mixed/biphasic for the remaining 2030 %. MM has a median survival time of 814 months (3). There is an urgent unmet need to develop new diagnostics and therapeutics for MM (4) as the disease has a long latency period with past and ongoing exposure to asbestos contributing to the development of new cases worldwide. One approach to detect and treat cancer is to conjugate imaging and/or therapeutics to molecules which can recognize internalizing antigens, receptors or cell surface markers that are over-expressed on tumor cells, leading to efficient localization and tumor cell killing (5,6). However, presently there are very few MM-associated cell surface antigens that are over-expressed by all subtypes of MM, especially the SM (7). One well established markermesothelin, a 40kDa cell surface glycoprotein, has been reported to be expressed by EM cells (8), but not SM (9). Recently we have identified a panel of internalizing human scFv antibodies by phage display selection that target cell surface antigens associated with both EM and Azamethiphos SM (6). The selected scFvs bind to human mesothelioma cellsin situ, thereby recognizing clinically represented tumor antigens (6) and thus offer the potential to deliver high levels of imaging probes to tumor cells but not to nontarget normal tissues based on intracellular delivery strategies. By screening the yeast surface human cDNA display library with mesothelioma targeting phage antibody, we have further identified MCAM/CD146/MUC18 as one of the target antigens for MM cells that was over-expressed in >80% of EM and SM tissues, but not other tissues (10). In the present study, we investigated both thein vitroandin vivotumor targeting and imaging potential of an additional scFv (M40) for both EM (M28) and SM (VAMT-1) subtypes. == Materials and Methods == == Expression and Purification of M40 == The M40 was produced as previously reported (6,1113). == 99mTc Radiolabeling of M40 == The scFv was radiolabeled as previously reported (14,16). The carbonyl-kit (IsoLinkTyco/Mallinckrodt) was used to prepare the [99mTc(CO)3] moiety. An aliquot IRAK3 (4060 g) of M40 was mixed with 100500 L of [99mTc(CO)3(OH2)3]+solution and the mixture was heated at 37C for 60 min. The product was purified using a PD-10 column. Labeling efficiency and purity were determined by size-exclusion- HPLC and thin-layer-chromatography (TLC). == Fluorescence labeling of M40 (Cy5.5-M40) == The M40 was labeled with Cy5.5 by incubation with 3:1 molar excess of Cy5.5-NHS ester in a carbonate/bicarbonate buffer (pH 7.28.5) for 1 h followed by purification using PD-10 column. == In vitrocell culture assay == Internalization experiments were performed as described previously (12,15,16). Briefly, 1 million VAMT-1, M-28 or BPH-1 cells (control) were incubated with 0.052 Ci99mTc-M40 in various concentrations for 1 h or 3 h. All cell lines had been tested for mycoplasma contamination and characterized by cell proliferation and morphology evaluation (6). The cells were washed to determine cell surfacebound (acid releasable) and internalized (acid.