Enhancing rituximab therapy: Analyzing the interaction between rituximab and the human complement pathway

Rituximab (RTX) is a chimeric monoclonal antibody (mAb) used for treatment of B cell lymphomas as well as for several autoimmune diseases. In vitro, RTX opsonization of CD20+ cells in the presence of serum promotes covalent deposition of large amounts of C3b(i) (C3 activation fragments) ultimately leading to cell lysis by the membrane attack complex. RTX-mediated C3b(i) deposition and cell killing are enhanced with the anti-C3b mAb 3E7; this enhancement does not occur with other anti-C3b(i) mAbs and killing is directed at B cells. Fluorescence microscopy experiments suggest that C3b(i) is co-localized with bound RTX on B cells. However, <12% of deposited C3b(i) co-precipitates with RTX, suggesting that most of the deposited C3b(i) is located in close proximity with RTX.;Intravenous infusion of RTX, in non-human primates and CLL patients, leads to binding of RTX to B cells, rapid deposition of C3b(i), and clearance of CD20+ cells from the circulation. Infusion of RTX in patients with high circulating tumor burdens leads to depletion of serum complement activity, by consumption of component C2. RTX-mediated lysis of tumor cells requires all proteins of the classical complement pathway, and supplementing CLL patient sera (sera low in complement activity but containing RTX) with normal human serum or purified human C2 replenishes in vitro killing activity.;High dose RTX therapy in CLL patients is followed by recrudescence of malignant cells (CD45+CD19+) with an altered CD20 phenotype. After RTX infusion, CD20 is "shaved" from the B cell surface and CD20 is reduced (>90-95%) as ascertained by flow cytometry and Western blot analysis. The level of expression of several other protein markers remains approximately constant after RTX infusion. Thrice weekly low dose RTX therapy (20 mg/m2) leads to progressive decreases in malignant cells in the bloodstream and preserves the CD20 antigen for a longer period of time compared to standard RTX therapy (375 mg/m2). The "saving" mechanism proposed here utilizes the mononuclear phagocytic system; THP-1 cells remove CD20 from RTX-opsonized cells. The shaving mechanism as well as C3b(i) deposition on RTX-opsonized cells, are being studied in a SCID mouse model.