A Novel FACS Assay for Absolute Quantification of CD20 on B-Cells and Determination of CD20 Occupancy in Whole Blood Samples Shows a Higher Binding Strength of Obinutuzumab (GA101) to CD20 on B-Lymphocytes
Authors: Beroual Drifa , Boulay-Moine Danièle , Badoil Carole , Klein Christian , Moulard Maxime 
Published: October 2019

Abstract
Abstract 4611

Introduction.
The absolute quantification of CD20 on malignant B-lymphocytes on patients who already received anti-CD20 MAbs is technically challenging, but may play a role in identifying patients who will respond to subsequent treatment or help define patients that are rituximab-refractory due to decreased expression of CD20. We have developed a new quantitative flow cytometry assay to scrutinize the absolute number of receptors on cell lines and B-lymphocytes in whole blood (WB) samples as well as target occupancy. This assay allows us to accurately determine the number of human MAbs bound per target cell on various cell subsets. CD20 density on B-lymphocytes in WB and binding properties of the type II CD20 antibody obinutuzumab (GA101) and the classical Type I CD20 antibody Rituximab were tested on samples from healthy volunteers. Acidic striping of bound human anti-CD20 MAbs was explored to enable the set up of a trustworthy CD20 occupancy assay.

Methods.
Binding of anti-CD20 mAbs rituximab (RTX) and GA101 to CD20 on normal and malignant B-cells was assessed using the Human IgG Calibrator (BioCytex, F) on EDTA anticoagulated WB samples and cell lines to determine the CD20 density. In parallel, the murine MAb (Clone B9E9) was tested using Cellquant Calibrator (BioCytex, F). CD20 density and apparent Kd, a relative measure of affinity, for each MAb was calculated from the data obtained with whole blood samples. Both the total number of CD20 molecules and the CD20 bound fraction were appraised by spiking the sample with anti-CD20 MAbs at saturation and acid striping.

Results.
The absolute number of anti-CD20 MAbs bound to CD20 on B-cells in WB samples was between 58,000 to 123,000 molecules per cell as determined by GA101 and RTX, respectively. The apparent CD20 quantitation on cell lines was approx. 2- to 3-fold lower for GA101 which is a characteristic and reported property of type II CD20 antibodies. The apparent Kd of GA101 (4.1 to 9.9 nM) was approximately 2-fold lower than that of RTX (10.5 to 22.7 nM) on several different lymphoma-derived cell lines (Daudi, Raji, Ramos and MEC-1). On B-lymphocytes from healthy volunteers the Kd were 1.2 and 3.5 nM for GA101 and RTX, respectively. Furthermore, CD20-bound GA101 could only be detached from its target CD20 using acid-striping assay at pH < 2.0 and appropriate ionic strength whereas RTX could be stripped at pH 2.5. Binding of anti-CD20 MAbs to acid-treated B-lymphocytes was found unaffected.

Conclusion.
The density of RTX bound to CD20 on B-lymphocytes in whole blood is similar to previous published data obtained from radioimmunoassays and using 125I-MAb (Teeling et al, Blood, 2006). In contrast the number of the Type II CD20 antibody GA101 bound to B-lymphocytes in whole blood was approximately half in comparison to RTX as previously reported (Moessner et al, Blood, 2010). The data further confirmed a higher apparent binding affinity of GA101 for CD20 with Kd values comparable to those reported previously using 125I-MAb. In summary, the new Human IgG Calibrator is a fast and accurate technique that can be used to evaluate the CD20 density and occupancy using human MAbs in complex matrix including whole blood. The test can be implemented in pharmacodynamic clinical trials and may allow for further understanding of complex pharacodynamic relationships.

Disclosures:
Beroual: BioCytex: Employment. Boulay-Moine:BioCytex: Employment. Badoil:BioCytex: Employment. Klein:Roche: Employment, Equity Ownership, Patents & Royalties. Moulard:BioCytex: Consultancy, Employment.