A novel engineered anti-CD20 tracer enables early time PET imaging in a humanized transgenic mouse model of b-cell non-hodgkins lymphoma

Purpose: 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 ⁶⁴Cu-rituximab for positron emission tomography (PET) imaging of CD20. Experimental Design: The engineered FN3CD20 and FN3WT were produced in Escherichia coli cells at 2 to 5 mg/L, conjugated to DOTA, labeled with ⁶⁴Cu, and used for PET imaging of huCD20 expression in B cells. Humanized transgenic mice and subcutaneously xenografted mice each received intravenous ⁶⁴Cu-FN3CD20 or FN3WT (3.7 MBq/4 mg Do-FN3 in 200 mL PBS). Control group received a blocking dose (50-fold excess) of unconjugated FN3CD20 two hours before radiotracer injection. PET imaging was carried out at 1 to 24 hours postinjections. Results: In vitro assay demonstrated FN3 binds CD20 with 20 nmol/L affinity on CD20-expressing cells. ⁶⁴Cu-FN3CD20 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-toblood ratio of 13 by 4 hours. This is higher uptake (P0.04) and 10-fold greater signal-to-background (P 0.04) than the ⁶⁴Cu-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. Conclusions: The ⁶⁴Cu-Do-FN3CD20 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.