The extent of the impaired growth of radiolabeled rituximab differed both between the two cell lines and the two radio-nuclides

The extent of the impaired growth of radiolabeled rituximab differed both between the two cell lines and the two radio-nuclides. CD44v6 expression. Biodistribution experiments demonstrated a greater uptake Thrombin Inhibitor 2 of 177Lu-AbN44v6 in the liver, spleen and bone, compared to 125I-AbN44v6, whereas 125I-AbN44v6 demonstrated a longer circulation time. In dosimetric calculations, the critical organs for 177Lu-AbN44v6 were the liver and spleen, whereas the kidneys and red marrow were considered the critical organs for 131I-AbN44v6. The effective dose was in the order of 0.1 mSv/MBq for both labels. In conclusion, AbN44v6 bound specifically and with high affinity to CD44v6. Furthermore, RIT demonstrated growth inhibition in a CD44v6-specific activity-dependent manner for both radioconjugates, demonstrating that both 177Lu-AbN44v6 and 131I-AbN44v6 may be promising RIT candidates. Furthermore, biodistribution and dosimetric analysis supported the applicability of both conjugates Rabbit polyclonal to MEK3 for RIT. The CD44v6-specific therapeutic effects observed with radiolabeled AbN44v6 in the 3D tumor models (0.25 and 0.4 mm, respectively). Both emitters also emit gamma radiation, where the dominant energies are 208 keV (11%) and 113 keV (5%) for 177Lu and 364 keV (82%) and 637 keV (7%) for 131I. The gamma emissions of both nuclides offer the possibility of using SPECT to monitor the response to treatment, although the higher gamma energies of 131I are less favorable. Furthermore, the slightly shorter range of 177Lu makes it more suitable for micrometastases and minimizes the risk of toxicity to adjacent tissue (29). We have recently developed a fully human recombinant monoclonal antibody targeting CD44v6, AbN44v6. It was derived from a Fab (“type”:”entrez-protein”,”attrs”:”text”:”AbD15179″,”term_id”:”86769743″,”term_text”:”ABD15179″AbD15179), previously developed by our group for use within the field of molecular imaging, shown to bind selectively and with high affinity to CD44v6 (24). The full-length variant AbN44v6 was developed for the purpose of targeted therapy and has not previously been evaluated or characterized. The aim of this study was to characterize AbN44v6 and examine the possibility of utilizing the antibody for RIT, using 177Lu or 131I as therapeutic nuclides. The affinity and specificity of the conjugates were first characterized 3D tumor model was then assessed, comparing the therapeutic effects of 177Lu- and 131I-labeled AbN44v6 in two models with different CD44v6 expression. Finally, a dual nuclide biodistribution and dosimetric analysis of 177Lu-AbN44v6 and 125I-AbN44v6 (as a model for 131I-AbN44v6) was performed in normal tissues using a mouse model. Materials and methods Cell lines The human colorectal carcinoma cell line, HCT116, previously Thrombin Inhibitor 2 demonstrated to express moderate amounts of CD44v6 (16) was purchased from ATCC (Manassas, VA, USA) and cultured in McCoy’s modified Eagle’s medium (Biochrom Kg, Berlin, Germany) with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 1% L-glutamine and 1% antibiotics (100 IU penicillin and 100 experiments, the free 177Lu was purified from labeled antibody using a NAP5 size exclusion chromatography column equilibrated with serum-free medium. For experiments, the 177Lu-labeled AbN44v6 was purified using Nanosep 3K (Pall Norden AB, Lund, Sweden); the sample was added to four different filters and centrifuged for 35 min at 14,000 g before washing twice with 200 specificity tests of the radioconjugates were performed. Approximately 2104 UM-SCC-74B or 3104 HCT116 cells/well were seeded in 48-well plates and incubated for 24 h prior to the addition of 20 kBq (25C30 nM) of 177Lu-AbN44v6 or 125I-AbN44v6 to each well. In selected wells, 20-fold molar excess of unlabeled AbN44v6 was added to the wells to block specific binding. The plates were incubated at 37C for 24 h before Thrombin Inhibitor 2 the cells were washed and collected and the binding measured using a Thrombin Inhibitor 2 1480 WIZARD gamma well-counter (Wallac Oy, Turku, Finland). LigandTracer In order to assess cellular uptake and the retention of the radiolabeled conjugates, LigandTracer experiments were performed. The cells (5C10105) were seeded in 10-cm dishes and incubated for at least 24 h before running LigandTracer Yellow for 177Lu/131I or LigandTracer Grey for 125I (Ridgeview Instruments, V?nge, Sweden). Two different concentrations (3 and 10 nM) of radiolabeled antibodies were added, and the uptake was measured for 90 min for each concentration before removing the radiolabeled antibodies and replacing the medium to measure the dissociation rate. Analysis was performed using TraceDrawer, version 1.7 (Ridgeview Instruments). 3D cell culture assays In order to assess the potential therapeutic effects of the radioconjugates, therapy experiments were performed on 3D multicellular.

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