Dataset Information

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 which 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 arginine-glycine-aspartic acid (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 a QCM biosensor. The interaction between Rituximab and Raji cells led to the increased resonant frequency shifts (?f0) in the studied antibody concentration range from 5 to 250 ?g 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 × 10(6) 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 Ca(2+) and Mn(2+) ions on the Rituximab-Raji cell interaction were also studied providing the enhanced QCM signals, in particular with Ca(2+), 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 shown to be highly suitable for multipurpose studies on living cells including cell-immobilization, cytotoxicity of drugs, and the cell action mechanisms.

SUBMITTER: Tan L

PROVIDER: S-EPMC3874876 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Real time analysis of binding between Rituximab (anti-CD20 antibody) and B lymphoma cells.

Tan Liang L Lin Peiling P Chisti Mohammad M MM Rehman Abdul A Zeng Xiangqun X

Analytical chemistry 20130826 18

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 which 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 arginine-glycine-aspartic acid (RGD) tripeptide was electrochemically confirmed. The re ...[more]

PMID: 23926879

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Project description:Rituximab, a murine-human chimera, is the first monoclonal antibody (mAb) developed as a therapeutic agent to target CD20 protein. Its Fab domain and its interaction with CD20 have been extensively studied and high-resolution atomic models obtained by X-ray diffraction or cryo-electron microscopy are available. However, the structure of the full-length antibody is still missing as the inherent protein flexibility hampers the formation of well-diffracting crystals and the reconstruction of 3D microscope images. The global structure of rituximab from its dilute solution is here elucidated by small-angle X-ray scattering (SAXS). The limited data resolution achievable by this technique has been compensated by intensive computational modelling that led to develop a new and effective procedure to characterize the average mAb conformation as well as that of the single domains. SAXS data indicated that rituximab adopts an asymmetric average conformation in solution, with a radius of gyration and a maximum linear dimension of 52 Å and 197 Å, respectively. The asymmetry is mainly due to an uneven arrangement of the two Fab units with respect to the central stem (the Fc domain) and reflects in a different conformation of the individual units. As a result, the Fab elbow angle, which is a crucial determinant for antigen recognition and binding, was found to be larger (169°) in the more distant Fab unit than that in the less distant one (143°). The whole flexibility of the antibody has been found to strongly depend on the relative inter-domain orientations, with one of the Fab arms playing a major role. The average structure and the amount of flexibility has been studied in the presence of different buffers and additives, and monitored at increasing temperature, up to the complete unfolding of the antibody. Overall, the structural characterization of rituximab can help in designing next-generation anti-CD20 antibodies and finding more efficient routes for rituximab production at industrial level.

Project description:OBJECTIVE:This study was designed to determine the safety, pharmacokinetics and biologic effects of a human-mouse chimeric anti-CD20 monoclonal antibody (SCT400) in Chinese patients with CD20-positive B-cell non-Hodgkin's lymphoma (CD20(+) B-cell NHL). SCT400 has an identical amino acid sequence as rituximab, with the exception of one amino acid in the CH1 domain of the heavy chain, which is common in Asians. METHODS:Fifteen patients with CD20(+) B-cell NHL received dose-escalating SCT400 infusions (250 mg/m(2): n=3; 375 mg/m(2): n=9; 500 mg/m(2): n=3) once weekly for 4 consecutive weeks with a 24-week follow-up period. The data of all patients were collected for pharmacokinetics and pharmacodynamics analyses. RESULTS:No dose-limiting toxicities were observed. Most drug-related adverse events were grade 1 or 2. Two patients had grade 3 or 4 neutropenia. Under premedication, the drug-related infusion reaction was mild. A rapid, profound and durable depletion of circulating B cells was observed in all dose groups without significant effects on T cell count, natural killer (NK) cell count or immunoglobulin levels. No patient developed anti-SCT400 antibodies during the course of the study. SCT400 serum half-life (T1/2), maximum concentration (Cmax) and area under the curve (AUC) generally increased between the first and fourth infusions (P<0.05). At the 375 mg/m(2) dose, the T1/2 was 122.5±46.7 h vs. 197.0±75.0 h, respectively, and the Cmax was 200.6±20.2 g/mL vs. 339.1±71.0 g/mL, respectively. From 250 mg/m(2) to 500 mg/m(2), the Cmax and AUC increased significantly in a dose-dependent manner (P<0.05). Patients with a high tumor burden had markedly lower serum SCT400 concentrations compared with those without or with a low tumor burden. Of the 9 assessable patients, 1 achieved complete response and 2 achieved partial responses. CONCLUSIONS:SCT400 is well-tolerated and has encouraging preliminary efficacy in Chinese patients with CD20(+) B-cell NHL.

Dataset Information

Real time analysis of binding between Rituximab (anti-CD20 antibody) and B lymphoma cells.

Publications

Real time analysis of binding between Rituximab (anti-CD20 antibody) and B lymphoma cells.

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