Truncated Vif176 and Vif140 were cloned into pET21 vector

nce of varying concentrations of the unlabeled antibody on ice for 60 min. After washing, radioactivity bound to the cells was counted. Data were analyzed and the Kd was estimated using GraphPad Prism software. In an internalization assay, SY cells were preincubated in culture medium with 125I- or 111In-labeled antibody on ice for 60 min. After washing, collected cells were order Vatalanib further cultured at 37uC or on ice in fresh medium without radiolabeled antibodies. At various time points, the supernatant and the cells were separated by centrifugation. Trichloroacetic acid was added to the supernatant on ice and then separated by centrifugation to determine the non-protein-bound fraction and protein-bound fraction. The cells were washed with acidic buffer and then separated by centrifugation to determine both the membrane-bound and internalized fraction. Materials and Methods Cells A human SCLC cell line SY, Takasaki, Japan) that has high c-kit expression was maintained in RPMI1640 containing 5% fetal bovine serum in a humidified incubator maintained at 37uC with 5% CO2. Biodistribution of 111 In-labeled antibody Antibodies Two anti-c-kit mouse monoclonal antibodies were purchased from IBL. Each antibody binds to a different epitope on the extracellular portion of c-kit: 12A8 binds to the fifth domain nearest to the transmembrane domain and 67A2 binds to the second domain. The equilibrium dissociation constant of 12A8 and 67A2 is 1.06 and 0.26 nM, respectively, as measured by a surface plasmon resonance assay. 12A8 has strong neutralization and moderate antitumor activities but does not induce antibody-dependent cell cytotoxicity or complement-dependent cytotoxicity. 67A2 does not possess any of the above-mentioned activities. Radiolabeling of antibodies Antibodies were conjugated with p-SCN-BzCHX-A”-DTPA as previously described, and then DTPA-conjugated antibodies were purified using a Sephadex G-50 column. These DTPA-conjugated antibodies were mixed with 1.2 MBq of Cl3 or 9.25 MBq of Cl3 in 0.5 M acetate buffer, and the mixture was incubated for 30 min at room temperature. Radiolabeled antibodies were separated from free 111In or 90Y using a Sephadex G-50 column. The conjugation ratio of DTPA to both antibodies was estimated to be 1.1, as determined by cellulose acetate electrophoresis, and the specific activities of 12A8, BALB/c-nu/nu mice were inoculated subcutaneously with 26106 SY cells in the left thigh. Mice bearing SY tumors were intravenously injected with 37 kBq of 111In-labeled antibody. The injected protein dose was adjusted to 20 mg per mouse. At 1, 2, 4, 7 and 10 days after injection of 111In-labeled antibody, five mice at each time point were euthanized, and blood was obtained from the heart. The tumor and major organs were removed and weighed, and radioactivity counts were measured using a gamma counter. The data were expressed as the percentage of injected dose per gram of tissue decay-corrected and normalized to a 20-g body weight mouse. The tumor absorbed dose for 90Y-labeled antibodies was estimated from the biodistribution data of 111In-labeled antibodies. 90 Y emits a b-particle with 23446639 a mean emitted energy of 0.9331 MeV, thus the mean energy emitted per transition was calculated as 1.495610213 Gy kg 8234901 21 based on 1 eV is equal to1.60218610219 J. Tumor uptake at various time points was plotted against time, from which the area under the curve was calculated. The dose absorbed by the tumor up to 10 days after injection was esti