Fig. 2: Alemtuzumab neutralizing antibody assay with plasma of patient with endogenous neutralizing anti-alemtuzumab antibodies.

Flow cytometry was performed on healthy donor peripheral blood mononuclear cells (PBMC) incubated with Alexa Fluor 488-labeled alemtuzumab in the presence of healthy donor (no neutralizing antibodies) or CTCL patient plasma. Top panels: In the absence of plasma or with healthy donor plasma, >98% of CD3+ cells bind fluorescently labeled alemtuzumab. In the presence of positive control plasma with known neutralizing anti-alemtuzumab antibodies, 96.7% of CD3+ cells remain unbound by labeled alemtuzumab. Bottom panels: In the presence of increasing concentrations of test patient plasma (10–50%), CD3+ cells display decreasing binding of labeled alemtuzumab, indicating increasing concentration of anti-alemtuzumab neutralizing antibodies in the patient plasma.