title: Functional bioassays for immune monitoring of high-risk neuroblastoma patients treated with ch14.18/CHO anti-GD2 antibody.
abstract: Effective treatment of high-risk neuroblastoma (NB) remains a major challenge in pediatric oncology. Human/mouse chimeric monoclonal anti-GD2 antibody (mAb) ch14.18 is emerging as a treatment option to improve outcome. After establishing a production process in Chinese hamster ovary (CHO) cells, ch14.18/CHO was made available in Europe for clinical trials. Here, we describe validated functional bioassays for the purpose of immune monitoring of these trials and demonstrate GD2-specific immune effector functions of ch14.18/CHO in treated patients. Two calcein-based 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 identified 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 20:1 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 40:1. Optimal results for CDC were found with a serum dilution at 1:8. For validation, both within-assay and inter-assay precision were determined and coefficients of variation (CV) were below 20%. Sample quality following storage at room temperature (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/m(2)) revealed GD2-specific increases in CDC (4.5-9.4 fold) and ADCC (4.6-6.0 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.
authors.affiliation: Department of Pediatric Oncology and Hematology, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Nikolai
authors.initials: N
authors.lastname: Siebert
authors.affiliation: Department of Pediatric Oncology and Hematology, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Diana
authors.initials: D
authors.lastname: Seidel
authors.affiliation: Department of Pediatric Oncology and Hematology, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Christin
authors.initials: C
authors.lastname: Eger
authors.affiliation: Department of Pediatric Oncology and Hematology, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Madlen
authors.initials: M
authors.lastname: Jüttner
authors.affiliation: Department of Pediatric Oncology and Hematology, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Holger N
authors.initials: HN
authors.lastname: Lode
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology / Q000627:therapeutic use; D000920:Antibody-Dependent Cell Cytotoxicity; D000954:Antigens, Surface / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use*; D045744:Cell Line, Tumor; D003165:Complement System Proteins / Q000276:immunology; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods / Q000592:standards; D003602:Cytotoxicity, Immunologic; D005732:Gangliosides / Q000037:antagonists & inhibitors; D006801:Humans; D016130:Immunophenotyping; D007958:Leukocyte Count; D015166:Monitoring, Immunologic / Q000379:methods / Q000592:standards; D009447:Neuroblastoma / Q000188:drug therapy / Q000235:genetics / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D010641:Phenotype; D011786:Quality Control; D015203:Reproducibility of Results
chemical_list: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D000970:Antineoplastic Agents; D005732:Gangliosides; C019403:ganglioside, GD2; C112746:dinutuximab; D003165:Complement System Proteins