Thienopyridines and other agents target the platelet P2Y(12) receptor and inhibit several platelet activities mediated by adenosine diphosphate (ADP). The measurement of vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation, expressed as platelet reactivity index (PRI), mirrors the degree of P2Y(12) receptor inhibition and can detect the well-known variable response to clopidogrel. The commercially available VASP assay uses flow cytometry (FC) and requires that the test be run within 48 hours of blood collection. A new ELISA VASP assay offers the advantages of using more widely available technology and the potential to freeze and store samples before analysis. The objectives of the present study were to compare the performance of the ELISA and FC methods and to describe the relative flexibility of the ELISA-based assay. Human blood samples encompassing a wide range of levels of P2Y(12) blockade achieved in vitro by preincubation with P2Y(12) antagonists or in vivo from patients treated with clopidogrel were included, reflecting the wide spread of values reported in clinical studies. The correlation between the PRI measured by ELISA and FC was highly significant (r=0.95, p<0.001), (n=80). After the initial activation, samples were stable for at least four weeks when frozen (-20°C) prior to analysis by ELISA. Frozen samples from patients treated with clopidogrel appeared stable for up to nine weeks. Based on these results, the ELISA-based assay appears to provide a reliable and more flexible alternative to the FC method to determine P2Y(12) receptor blockade and may enable more extensive utilisation of the VASP assay in clinical studies.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.