Abstract
The aim of this article was to evaluate the use of a novel engineered anti-CD20 protein based on the 10 kDa human fibronectin type 3 domain (FN3) and subsequently compare with (64)Cu-rituximab for positron emission tomography (PET) imaging of CD20. The engineered FN3(CD20) and FN3(WT) were produced in Escherichia coli cells at 2 to 5 mg/L, conjugated to DOTA, labeled with (64)Cu, and used for PET imaging of huCD20 expression in B cells. Humanized transgenic mice and subcutaneously xenografted mice each received intravenous (64)Cu-FN3(CD20) or FN3(WT) (3.7 MBq/4 μg Do-FN3 in 200 μL PBS). Control group received a blocking dose (50-fold excess) of unconjugated FN3(CD20) two hours before radiotracer injection. PET imaging was carried out at 1 to 24 hours postinjections. In vitro assay demonstrated FN3 binds CD20 with 20 nmol/L affinity on CD20-expressing cells. (64)Cu-FN3(CD20) showed clear, high-contrast visualization of huCD20-expressing B cells in the spleen of transgenic mice as early as 1 hour postinjection [38 ± 3% injected dose (ID)/g] and exhibited a spleen-to-blood ratio of 13 by 4 hours. This is higher uptake (P = 0.04) and 10-fold greater signal-to-background (P = 0.04) than the (64)Cu-rituximab antibody radiotracer. Tumor uptake (16.8 ± 1.6 vs. 5.6 ± 1.4%ID/g) and tumor:background ratios were superior for FN3CD20 relative to rituximab in xenograft studies as well. The (64)Cu-Do-FN3(CD20) radiotracer represents a novel small, high-affinity binder for imaging human CD20, which may be well suited for B-cell non-Hodgkin's lymphoma imaging in patients at early time points.
Highlights
The CD20 antigen is present in greater than 90% of B-cell lymphomas and is neither degraded nor internalized after antibody binding, making it an effective target for immunotherapeutic removal of malignant B cells [1,2,3]
Tumor uptake (16.8 Æ 1.6 vs. 5.6 Æ 1.4%injected dose (ID)/g) and tumor:background ratios were superior for FN3CD20 relative to rituximab in xenograft studies as well
Engineering and characterization of FN3CD20 After 6 iterations of selection and maturation, 4 dominant clones were identified by sequence analysis (Table 1)
Summary
The CD20 antigen is present in greater than 90% of B-cell lymphomas and is neither degraded nor internalized after antibody binding, making it an effective target for immunotherapeutic removal of malignant B cells [1,2,3]. Immunotherapies using monoclonal antibodies targeting B-cell surface antigens have been widely accepted for the treatment of B-cell non-Hodgkin lymphomas (NHL), with antiCD20 antibodies being most commonly used [1]. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). For imaging, radiolabeled antibodies provide high tumor signal (8% injected dose [ID]/g) and modestly high liver uptake (2–11% ID/g), and slow clearance yields low tumor-to-blood ratios For imaging, radiolabeled antibodies provide high tumor signal (8% injected dose [ID]/g) and modestly high liver uptake (2–11% ID/g), and slow clearance yields low tumor-to-blood ratios ( 0.5; refs. 6, 7) and necessitates late imaging time points (days)
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