Validated detection of anti-GD2 antibody ch14.18/CHO in serum of neuroblastoma patients using anti-idiotype antibody ganglidiomab.
Human/mouse chimeric monoclonal antibody (mAb) ch14.18 is directed against disialoganglioside GD2 and has demonstrated activity and efficacy in high-risk neuroblastoma (NB). For the purpose of industrial production, ch14.18 was manufactured in Chinese hamster ovarian cells (ch14.18/CHO) in order to facilitate clinical trials in Europe. To determine immunopharmacological effects of ch14.18 in preclinical models and clinical trials, a validated method of quantitative detection of ch14.18/CHO in serum is an important tool. We recently described the generation and characterization of ganglidiomab, a monoclonal anti-idiotype Ab (AIT) of ch14.18 (Lode et al., 2013), which was used to establish quantitative and validated enzyme-linked immunosorbent assay (ELISA) methods using ganglidiomab as a capture mAb. With these ELISA methods, we first demonstrated binding of ch14.18/CHO to ganglidiomab to a similar extent as to the nominal antigen GD2 and in contrast to GD1b and GM2 precursor and metabolite gangliosides, used as negative controls. In order to determine both low (0.5-3.1 μg/ml) and high levels of ch14.18/CHO (1.0-25 μg/ml) in the serum of NB patients treated with ch14.18/CHO, we established two ELISA methods with high and low sensitivity using 1/1001, and 1/5126 sample dilutions, respectively. For validation, we used a set of tailored quality controls (QC) containing distinct concentrations of ch14.18/CHO (1.0, 2.0, 7.0, and 20.0 μg/ml). We determined the limit of detection (LOD) for both ELISA methods to be 0.50 μg/ml for the high sensitivity and 1.02 μg/ml for low sensitivity ELISA. The within-assay precision was 12% for high and 4% for low sensitivity ELISA, and the coefficients of variation (CV) were under 20% for all assays (3% for QC-1.0, 5% for QC-2.0, 7% for QC-7, and 3% for QC-20). With this method, we showed that neither eight freeze-thaw cycles nor storage at room temperature for up to 168 h affected ch14.18/CHO stability in serum. Finally, we analyzed ch14.18 Ab serum levels in selected NB patients receiving ch14.18/CHO as a continuous or bolus infusion with a peak concentration at the last day of Ab application (17.14 ± 7.20mg/ml with continuous and 19.78 ± 2.26 mg/ml with bolus infusion). In summary, we describe validated ELISA methods using ganglidiomab as a capture mAb suitable for the pharmacological evaluation of ch14.18/CHO in NB patients.
Department of Pediatric Oncology and Hematology, University Medicine Greifswald, 17475 Greifswald, Germany. Electronic address: nikolai.siebert@uni-greifswald.de.
Nikolai
N
Siebert
Diana
D
Seidel
Christin
C
Eger
Diana
D
Brackrock
Daniel
D
Reker
Manuela
M
Schmidt
Holger N
HN
Lode
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry* / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000276:immunology / Q000493:pharmacokinetics*; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry* / Q000276:immunology; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D005732:Gangliosides; D006801:Humans; D009447:Neuroblastoma / Q000097:blood* / Q000188:drug therapy / Q000276:immunology; D012680:Sensitivity and Specificity
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D005732:Gangliosides; C019403:ganglioside, GD2; C112746:dinutuximab
3,3′,5,5′-tetramethylbenzidine; AIT; BSA; CV; DMEM; ELISA; GD(2); Ganglidiomab; HRP; IL-2; LOD; LTI; NB; Neuroblastoma; QC; RT; SD; TMB; Validation; anti-idiotype; bovine serum albumin; ch14.18; coefficient of variation; enzyme-linked immunosorbent assay; horseradish peroxidase; interleukin-2; limit of detection; long term infusion.; mAb; modified Eagle's medium; monoclonal antibody; neuroblastoma; quality controls; room temperature; standard deviation