Journal: Clinical Cancer Research

Article Title: A Novel Engineered Anti-CD20 Tracer Enables Early Time PET Imaging in a Humanized Transgenic Mouse Model of B-cell Non-Hodgkins Lymphoma

doi: 10.1158/1078-0432.ccr-13-0626

Figure Lengend Snippet: Figure 1. A, Biacore 100 sensorgram analysis demonstrates FN3CD20 binds to CD20 peptide with nmol/L affinity. Analysis shows the interaction between 15 mL of the 0.5 to 1,000 nmol/L FN3 proteins and the biotin-CD20 peptide coated on sensor streptavidin chip (Biacore CM5 chip). FN3CD20 protein (0.4, 2, 10, 50, 250, and 750 nmol/L) was diluted in HEPES buffer saline (HBS) and injected into chip at a flow rate of 15 mL/min for 1 minute at 25C. The change in refractive index due to binding of FN3CD20 and CD20 peptide were measured (response units) at these concentrations and a 1:1 binding curve was fit, which yields a KD value of 22 nmol/L. B, antigen-binding activity of FN3CD20 and Do-FN3CD20. Ramos cells were incubated with the indicated concentration (0.5–80 nmol/L) of FITC-FN3CD20 for 1 hour at 4C. Cells were washed and analyzed by flow cytometry. The data are expressed as the mean SD of 3 independent experiments. Data were normalized with respect to saturated fluorescence intensity (plateau) observed at the highest target concentrations. A 1:1 binding curve fit to the data indicates KD ¼ 20 nmol/L (FN3CD20) and 23 nmol/L (Do-FN3CD20). Value and error bars represent the mean and SD of triplicate samples.

Article Snippet: The CD20 positive B-cell lymphoma cell line Ramos and CD20-negative Jurkat cells were obtained from the American Type Culture Collection (ATCC numbers: CRL-1555 and TIB-152, authenticated by short tandem repeat profiling) and were used shortly thereafter.

Techniques: Saline, Injection, Refractive Index, Binding Assay, Activity Assay, Incubation, Concentration Assay, Cytometry

Journal: Clinical Cancer Research

Article Title: A Novel Engineered Anti-CD20 Tracer Enables Early Time PET Imaging in a Humanized Transgenic Mouse Model of B-cell Non-Hodgkins Lymphoma

doi: 10.1158/1078-0432.ccr-13-0626

Figure Lengend Snippet: Figure 2. A, schematic diagram of 64Cu-Do-FN3 [FN3 domain depicted as the NMR solution structure of wild-type fibronectin domain (PDB code: 1TTG)]. Mutated residues for CD20 binding shown in black. B, 64Cu-Do- FN3CD20 tracer exhibits specific binding on B cells. Ramos: CD20- positive; Jurkat: CD20-negative; Block: Ramos cells blocked with 1,000 nmol/L of unlabeled FN3CD20 0.5 hours before 64Cu- FN3CD20. C, serum stability of 64Cu-Do-FN3CD20 assayed by cellulose acetate electrophoresis (CAE). Radioconjugate (25 mg) was mixed with 1 mL of human serum and incubated at 37C. At various time points (0, 4, and 24 hours), 10 mL samples were drawn and tested for stability on CAE. CAE was performed for 45 minutes with barbital buffer (0.05M, pH 8.6) at room temperature. Radioactivity (cpm) is decay corrected and normalized.

Article Snippet: The CD20 positive B-cell lymphoma cell line Ramos and CD20-negative Jurkat cells were obtained from the American Type Culture Collection (ATCC numbers: CRL-1555 and TIB-152, authenticated by short tandem repeat profiling) and were used shortly thereafter.

Techniques: Binding Assay, Blocking Assay, Electrophoresis, Incubation, Radioactivity

Journal: Clinical Cancer Research

Article Title: A Novel Engineered Anti-CD20 Tracer Enables Early Time PET Imaging in a Humanized Transgenic Mouse Model of B-cell Non-Hodgkins Lymphoma

doi: 10.1158/1078-0432.ccr-13-0626

Figure Lengend Snippet: Figure 5. A, 64Cu-Do-FN3CD20 tracer biodistribution in huCD20 mice (n ¼ 3). The huCD20 mice were injected with 3.7 MBq of 64Cu-Do-FN3CD20 tracer and euthanized at 24 hours postinjection. Organs were isolated and measured by the g counter. %ID/g of tissue were decay corrected. Organs uptake of tracer dose was measured (mean%ID/g SD) after 24 hours postinjection. Preblock mice received 8 mg/kg predose of unconjugated FN3CD20 2 hours before tracer injection (i.v., dose 3.7 MBq) and nonblock (NB) mice received no predose. B, comparison of 2 different lymphoma PET tracers tested against huCD20 target in huCD20 transgenic mice (n ¼ 3). Tracer uptake value (mean SD) of 2 tracer groups of nonblocking mice were measured by ROI and compared at 1 and 4 hours, respectively. The 64Cu-FN3CD20 tracer uptake value in spleen was significantly higher than 64Cu- rituximab. C, at 4 hours tracer uptake value (mean%ID/g SD) in spleen and blood of 2 tracer groups of nonblocking mice were measured by ex vivo and compared for spleen-to-blood ratio of 64Cu- rituximab and 64Cu-FN3 CD20.

Article Snippet: The CD20 positive B-cell lymphoma cell line Ramos and CD20-negative Jurkat cells were obtained from the American Type Culture Collection (ATCC numbers: CRL-1555 and TIB-152, authenticated by short tandem repeat profiling) and were used shortly thereafter.

Techniques: Injection, Isolation, Comparison, Transgenic Assay, Ex Vivo