Statistical significance was determined with an unpaired test or one-way ANOVA, and correlation coefficients were calculated with Pearson tests. instant thin-layer chromatography (ITLC) on a silica gel chromatography strip (Biodex, Shirley, NY, USA), using 0.1 M citrate buffer, pH 6.0 as the mobile phase. Purity exceeded 95% in all experiments. Cell Culture SK-RC-52 (clear cell renal cell carcinoma cell line which ubiquitously overexpresses CAIX independent of oxygenation status)32 was cultured in RPMI-1640 (GIBCO, Thermo-Fisher Scientific, Waltham, MA, USA), supplemented with 2 mmol/L glutamine (GIBCO) and 10%FCS (Sigma-Aldrich, Saint Louis, MS, USA) at 37 C in a humidified atmosphere with 5% CO2. Binding and Internalization Assay SK-RC-52 cells were grown to confluency (>90%) in 6-well plates and incubated with 1600 Bq [111In]In-DTPA-B9, [111In]In-DTPA-B9-ABDlow, or [111In]In-DTPA-B9-ABDhigh in 0.5% BSA in RPMI (binding buffer) for 1, 2, 24, and 48 h. The competition was performed by coincubation with 1 g of unlabeled B9 or 30 g of unlabeled girentuximab per well. Cells were washed twice with binding buffer, and receptor-bound VHH was collected by incubation with 0.1 M acetic acid and 154 mM NaCl, pH 2.6, for 10 min at 4 C. After washing with binding buffer, cells were collected with 1 mL of 0.1 M NaOH. Activity in both receptor-bound and internalized fractions were counted in a -counter (2480 Wizard 3, LKB/Wallace, PerkinElmer, Boston, MA, USA). IC50 SK-RC-52 cells were grown to confluency in 6-well plates and incubated with increasing concentrations (0.005C300 nM) of BAX either unlabeled DTPA-B9, DTPA-B9-ABDlow, or DTPA-B9-ABDhigh in binding buffer, in the presence of 1600 Bq of [111In]In-DTPA-B9 for 4 h on ice. Upon washing, cell fractions were collected using 1 mL of 0.1 M NaOH, and activity was counted in a -counter. The IC50 values (the concentrations that are required to inhibit binding of [111In]In-DTPA-B9 with 50%) were determined in GraphPad Prism. Scatchard Assay SK-RC-52 cells were grown to confluency in 6-well plates and incubated with increasing concentrations of [111In]In-DTPA-B9, [111In]In-DTPA-B9-ABDlow, or [111In]In-DTPA-B9-ABDhigh (3C3000 pM) in binding buffer for 4 h on ice. The specificity of binding was determined by coincubation with 1 g unlabeled B9 per well. After incubation, cells were washed twice with PBS and collected with 1 mL of 0.1 M NaOH. Activity was counted in a -counter. The dissociation constant (experiments. Six to eight week old female athymic BALB/c nu/nu mice (Janvier Laboratories, Le Genes-Saint-Ile, France) were implanted subcutaneously with a tumor (2 mm diameter) on the right hind leg. Animals were included when tumors were >80 mm3 (4C5 weeks after implantation). Allocation to the treatment groups was block-randomized by tumor size. The studies were approved by the Central Authority for Scientific Procedures on Animals (RU-DEC-2015-0071) and carried out under the supervision of the local Animal Welfare Body. Note that ethical approval for the mice experiments in this study was provided Emtricitabine on September 8, 2015 by the institutional Animal Welfare Committee of the Radboud University Emtricitabine Medical Center (application no. AVD103002015209), in accordance with the guidelines of the Revised Dutch Act on animal experimentation. Dose Escalation Tumor uptake of [111In]In-DTPA-B9 was determined in a dose-escalation study in mice bearing SCCNij153 xenografts. Mice (= 3C5/group) were injected intravenously with 1, 5, or 25 g of 1 1 MBq [111In]In-DTPA-B9 in PBS/0.5%BSA. A separate group of mice (= 3) was injected with 300 g unlabeled B9 at 3 h before injection of 5 g of 1 1 MBq [111In]In-DTPA-B9 to determine the specificity of uptake. Mice were euthanized by CO2/O2 asphyxation at 4 h after tracer Emtricitabine injection. Tumors and other tissue samples were harvested and weighed, and radioactivity in these samples was determined in a -counter. Radioactivity concentrations were calculated as a.