Binding of CD20 mAbs is abolished by mutation of alanine-170 and proline-172.

(A) The human CD20 extracellular sequence was mutated toward the murine sequence to produce constructs 1 through 7. Mutated residues in constructs 1 through 7 are highlighted in bold. (B) Wild-type (WT) human CD20 and constructs 1 through 7 were expressed in HEK293 cells and equimolar amounts of cell lysates were tested for CD20 expression by immunoblot analysis using a polyclonal antibody generated against a cytoplasmic peptide (anti-CD20N). To confirm cell surface expression of construct 6, transfected cells were either untreated or treated with proteinase K before lysis (lanes 6 and 6*, far right). (C) Binding of anti-CD20 mAbs 1F5, 2H7, and B1 to transfected cells was monitored by flow cytometry (top); mean fluorescence values, with the isotype control values subtracted, are shown in the bar graph below. Results are representative of 6 independent experiments.