Real Time Analysis of Binding between Rituximab (Anti-CD20 Antibody) and B Lymphoma Cells

Abstract

CD20, expressed on greater than 90% of B-lymphocytic lymphomas, is an attractive target for antibody therapy. Rituximab is a chimeric murine/human-engineered monoclonal antibody and can selectively deplete CD20-expressing cells in peripheral blood and lymphoid tissues. The immobilization of B-lymphoblast-like Burkitt's lymphoma Raji cells on the quartz crystal microbalance (QCM) gold electrode surface using RGD tripeptide was electrochemically confirmed. The real-time processes of attachment of Raji cells on the gold electrode and the subsequent binding of Rituximab to the cells were studied using QCM biosensor. The interaction between Rituximab and Raji cells led to the increased resonant frequency shifts (Δf0) in the studied antibody concentration range from 5 to 250 µg mL−1 following the Langmuir adsorption model. From these observations, the apparent binding constant between a single-layer of Rituximab and Raji cells was calculated to be 1.6×106 M−1. Control experiments using other therapeutic antibodies (i.e., Trastuzumab and Bevacizumab) and different cells (i.e., T cells and endothelial cells) proved the specific interaction between Rituximab and B cells. The effects of Ca2+ and Mn2+ ions on the Rituximab-Raji cell interaction were also studied providing the enhanced QCM signals, in particular, further indicating that CD20 is a calcium ion channel that can transport these metal ions into the cells and accelerate the cell lysis induced by Rituximab. Thus the real time capability of QCM and its simplicity of operation are highly suitable for multipurpose studies on living cells including cell-immobilization, cytotoxicity of drugs, and the cell action mechanisms.

Enzyme‐mediated assembly of chimeric membrane proteins at phospholipid bilayers

“…The measured dissociation constant K D of RtxAB to the EGFR/CD20 fusion protein is significantly (two orders of magnitude) weaker than that measured using affinity assays based on radioactive I 195 at the surface of the Raji cells (4.2 nM) and other sources (8 nM) (https://www.accessdata.fda.gov/drugsatfda_docs/label/2010/103705s5311lbl.pdf rituximab drug description). On the other hand, the K D determined here is in better agreement with data obtained on the same Raji cells measured using a quartz microbalance technique (160 nM) . The fast dissociation rates measured here also contradict previous data, which describe a slower dissociation rate.…”

“…The extracellular domain of the protein contains an unstructured loop of about 182 residues and forms oligomers at the cell surface. For immunotherapy, CD20 can be targeted with the monoclonal antibody rituximab (RtxAB) . Although RtxAB was approved by the FDA more than 20 years ago and is a widely used therapy, there remain many open questions regarding resistance to the drug and its mechanism of action …”

Real-time Characterization of Antibody Binding to Receptors on Living Immune Cells

“…The affinities derived from cell-based assays are much higher than those obtained by biophysical systems like SPR on isolated proteins (160 nM) (51) or QCM on fixated cells (650 nM) (52). The affinity of 0.18 nM obtained for Trastuzumab is similar to reported values independent of the technology [0.23 nM for ELISA (53), 0.13 nM for SPR (53)] or the fixation of cells [0.5–5.5 nM by SPR (28) and QCM (30), depending on cell line].…”

Concanavalin A–Polysaccharides binding affinity analysis using a quartz crystal microbalance

“…QCMs have been proven to be reliable in the field of electrochemistry (Paulo Tde et al, 2013; Sun et al, 2013a, 2013b), in petroleum characterization (Pejcic et al, 2011; Ueyama et al, 2002), in environmental chemistry (Nicolini et al, 2012), material science (Pei et al, 2010) and more interestingly in the development of biosensors (Bouchet-Spinelli et al, 2013; Eom et al, 2013; Guntupalli et al, 2013; Wangchareansak et al, 2013) which are valuable for disease diagnosis. QCMs also found a great interest in the monitoring of the affinity constants defining a given substrate-ligand interaction (Lebed et al, 2006; Li et al, 2013; Mori et al, 2009; Tan et al, 2013). Besides the fact that it does not require any calibration, one of the most exciting features of the QCM is its ability to quantify a mass change at the nano-gram level.…”