1. Toward experimental assessment of receptor occupancy: TGN1412 revisited 
Zoe Waibler, PhD, Linda Y. Sender, Christel Kamp, PhD, Jan Müller-Berghaus, MD, Bernd Liedert, PhD, Christian K. Schneider, MD, Johannes Löwer, MD, PhD, Ulrich Kalinke, PhD 
Journal of Allergy and Clinical Immunology 
Volume 122, Issue 5, Pages (November 2008)
DOI: /j.jaci
Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2. Fig 1
Determination of receptor occupancy by using a flow cytometry–based method. A, Human blood samples were stained with TGN1412–Alexa 488 at the indicated concentrations in micrograms per milliliter and an anti-CD3 antibody in a total volume of 50 μL. FACS analysis shows TGN1412–Alexa 488 staining gated on CD3+ cells (black curves; anti-CD3–allophycocyanin antibody was purchased from BD, Franklin Lakes, NJ). CD3 single-stained samples served as a negative control (gray-shaded curves). Alexa 488 labeling of TGN1412 was performed with an antibody-labeling kit (Molecular Probes, Eugene, Ore), according to the manufacturer's instructions. The red line indicates the peak of specific staining maximum. No TGN1412–Alexa 488 binding of CD3− cells was observed (data not shown). Note that among individual donors, the subset of CD3+CD28− T cells varied between 2.7% and 27.9% (data not shown). B, The median of TGN1412–Alexa 488 staining gated on CD3+ cells was determined in 3 independent experiments, and the percentage of specific binding was calculated by using the following formula (renormalized to the saturation levels): %Specificbinding=(Mediansample−MedianofCD3+singlestain)/(Maximummedian−MedianofCD3+singlestain)×100. The results of 3 individual experiments are indicated by open squares, open diamonds, and open circles, respectively. Diamonds and squares indicate 2 separate experiments performed on different days with the same lot of TN1412–Alexa 488, whereas circles indicate 1 experiment performed with another lot of TGN1412–Alexa 488. Error bars show SDs from 2 to 3 individual donors. Three of 8 similar experiments are shown. As a negative control, CD3+ T cells were stained with the CD20-specific antibody Rituximab–Alexa 488 (black squares; CD20 is not expressed on T cells). The red bar represents a range of TGN1412 concentrations that theoretically was present in the blood of a 70-kg man with a plasma volume between 2.5 and 3.99 L at a dose of 0.1 mg/kg TGN1412. Note that according to this analysis, a putative receptor occupancy between 45% and 80% was obtained in the probands.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions