Production and characterization of anti–CD20-mIFNα. (A) The heavy chain and light chain variable regions of anti-CD20 2B8 were cloned into human γ3 heavy chain and human κ light chain expression vectors. Mature murine IFNα was inserted downstream of a Gly₄Ser (GlySer) linker following the C_H3 domain of the constant region gene. (B) Flow cytometry using 38C13-huCD20 cells demonstrates that the fusion protein retains the ability to bind human CD20. Cells were incubated with anti–CD20-mIFNα (black peak), rituximab (solid line), isotype control for IgG1 (dashed line), or isotype control for IgG3 (shaded peak). Anti–human kappa-PE was used to detect cell-bound antibodies. (C) Comparative biologic activity of IFNα fusion protein and recombinant mIFNα as measured by inhibition of growth of wild-type 38C13 (hu-CD20 negative) cells. (D) Antiproliferative activity of fusion protein against 38C13-huCD20 cells. Cells were incubated with the indicated proteins for 48 hours. Cell proliferation was measured using the MTS assay and the data are represented as mean ± SD of triplicate values.