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/m2) 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.

Neuroblastoma patients with high-affinity FCGR2A, -3A and stimulatory KIR 2DS2 treated by long-term infusion of anti-GD2 antibody ch14.18/CHO show higher ADCC levels and improved event-free survival

“…27 Additionally, the level of ADCC increase achieved by ch14.18/CHO on day 8 of Ab infusion in cycle 1 was used to analyze correlations of ADCC levels with distinct FCGR polymorphisms. For this purpose, cytotoxicity levels of individual patients detected on day 1 prior to ch14.18/CHO application was subtracted from the absolute ADCC level determined on day 8.…”

“…ADCC was analyzed with Calcein-AM-based cytotoxicity assay using GD 2 -positive neuroblastoma cell line LA-N-1, patient-specific leukocytes and heat inactivated serum. 27 ADCC in patients with low-(n D 15) and high-affinity (n D 38) polymorphisms of FCGR2A (8.05 § 4% vs. …”

“…Following treatment protocol was used: cytokine IL-2 was given for 5 d (s.c., day 1-5, 6 £ 10 6 IU/m 2 /day), followed by a combined application of IL-2 (s.c., day 8-12, 6 £ 10 6 IU/m 2 / day) with ch14.18/CHO (i.v., day 8-18, 10 mg/m 2 /day) and 13-cis-retinoic acid (p.o., day [22][23][24][25][26][27][28][29][30][31][32][33][34][35]. At initial diagnosis, 46 patients had stage 4, four stage 3, one stage 2 and two stage 1 disease.…”

“…Cytotoxicity was analyzed using a calcein-acetoxymethyl ester (AM)-based cytotoxicity assay as previously described. 27 Briefly, patient leukocytes and heat-inactivated serum (12.5% final concentration) were incubated with 5,000 calcein-AMlabeled human GD 2 -positive NB cells LA-N-1 for 4 h (effectorto-target cell ratio: 40:1). Finally, supernatants of each well were transferred to black 96-well plates for determination of fluorescence at 495 nm excitation and 515 nm emission wavelengths using Synergy HT multimode microplate reader (BioTek Germany).…”

Anti-GD2 MABS and Next-Generation mAb-Based Agents for Cancer Therapy

“…When such an endogenous antibody is directed against the mAb's antigen binding region (idiotype or ID) it is termed anti-ID. In tumor-reactive mAb therapy, high levels of anti-ID mAbs are a concerning development as they can decrease the detectable level of mAb in serum and interfere with desired mechanisms of antitumor action of the mAb [138,139].…”