For this purpose, we identified LA-N-1 NB cells as best suited within a panel of cell lines

For this purpose, we identified LA-N-1 NB cells as best suited within a panel of cell lines. bioassays for complement-dependent- (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were set up based on patient serum and immune cells tested against NB cells. For this purpose, we recognized LA-N-1 NB cells as best suited within a panel of cell lines. Assay conditions were first established using serum and cells of healthy donors. We found an effector-to-target (E:T) cell ratio of 201 for PBMC preparations as best suited for GD2-specific ADCC analysis. A simplified method of effector cell preparation by lysis of erythrocytes was evaluated revealing equivalent results at an E:T ratio of 401. Optimal results for CDC were found with a serum dilution at 18. For validation, both within-assay and inter-assay precision were decided and coefficients of variance (CV) were below 20%. Sample quality following storage at room heat (RT) showed that sodium-heparin-anticoagulated blood and serum are stable for 48 h and 96 h, respectively. Application of these bioassays to blood samples of three selected high-risk NB patients treated with ch14.18/CHO (100 mg/m2) revealed GD2-specific increases in CDC (4.5C9.4 fold) and ADCC (4.6C6.0 fold) on day 8 compared to baseline, indicating assay applicability for the monitoring of multicenter clinical trials requiring sample shipment at RT for central lab analysis. Introduction Monoclonal antibodies targeting disialoganglioside GD2 emerge as an important treatment option for NB, a dismal pediatric malignancy characterized by high expression of GD2 on tumor cells [1], [2]. Ganglioside GD2 is usually a glycolipid antigen devoid of an intracellular transmission transduction domain name. Therefore the mechanism of action of anti-GD2 monoclonal Ab mostly rely on immune effector functions mediated by mAbs, which are Cefaclor more and more recognized as the key features of this class of malignancy therapeutics [3]. These features include the activation of CDC and ADCC. CDC is usually induced through binding of a serine protease complex C1 to the Fc domains of two or more mAbs binding to antigens expressed on tumor cells. This classical complement pathway results in an activation cascade resulting in the membrane attack complex disrupting the target cell. ADCC is a result of Fc-gamma receptor (FcR) mediated conversation with effector immune cells such as natural killer (NK) cells, macrophages and granulocytes [3]. The binding of FcR to Fc domain name induces both release of granzymes and perforin from effector cells leading to a target cell lysis and Fc-dependent tumor cell phagocytosis. The clinical development of anti-GD2 monoclonal antibodies for NB patients originated from the discovery of two unique murine anti-GD2 antibodies designated 3F8 [4] and 14.18 [5], respectively. High-risk NB patients were successfully treated within clinical trials with both antibodies mostly conducted by cooperating academic groups of pediatric oncologists. In a more multi center and international approach, the human/mouse chimeric version of 14.18 (ch14.18) has demonstrated activity and efficacy as a monotherapy [6], [7] and in combination with cytokines [8]. In Europe, ch14.18 antibody was made available for clinical trials following the recloning of the antibody genes into CHO cells which was designated as ch14.18/CHO. This is important, as ch14.18/CHO revealed superior activity in mediating ADCC compared to ch14.18 antibody produced in other cell lines [9]. Subsequently, a validated industrial production process was established. This development was initiated by SIOPEN, a group of international clinical leaders in the field of neuroblastoma and funded by charities throughout Europe. Four European clinical trials with different treatment schedules of ch14.18/CHO are being conducted to investigate the influence of Cefaclor a combined immunotherapy of ch14.18/CHO, interleukin-2 (IL-2) and 13-cis-retinoid acid on the outcome of patients with high-risk NB in the absence or presence of haploidentical blood stem cell transplantation. The first trial established the safety profile of ch14.18/CHO in children with high risk NB Cefaclor [10]. The European phase III clinical trial (HR-NBL 1.5/ESIOP, Eudra CT: 2006-001489-17) and the trial in ITGA8 the context of haploidentical stem cell transplantation (Eudra CT: 2009-015936-14) are based on a short term infusion of 20 mg/m2/d ch14.18 over 8 h on five subsequent days. To Cefaclor reduce side effects including neuropathic pain, a Phase Cefaclor I/II clinical trial was initiated based on the same cumulative dose of ch14.18/CHO (100 mg/m2/cycle) infused over a longer time period (ten days) (Eudra CT: 2009-018077-3). Within these trial protocols, a set of immune monitoring assays including the detection of ch14.18/CHO serum levels [11] and human anti-ch14.18/CHO immune responses [12], are implemented with the aim to identify immune biomarkers correlating with clinical response to ch14.18/CHO therapy. For a comprehensive assessment, validated bioassays to determine effector functions of ch14.18/CHO namely patient specific ADCC and CDC are of critical importance. For analysis of patient-specific CDC and ADCC, we established and validated two non-radioactive and non-toxic cytotoxicity assays based on release of acetomethoxy derivate of calcein (calcein-AM), which is a membrane-permeable live-cell labeling dye. With these assays, we demonstrate GD2 specific CDC and.