Immunotherapy with anti-GD antibody (Ab) ch14.18/CHO is effective for treatment of high-risk neuroblastoma (NB) patients and is mainly based on GD-specific Ab-dependent cellular cytotoxicity (ADCC). Strategies to further enhance the efficacy are important and currently explored in prospective clinical trials randomizing ch14.18/CHO ± IL-2. Recently, expression of programmed death 1 (PD-1) inhibitory receptor by effector cells and its ligand (PD-L1) by tumor cells has been shown. Here, we report for the first time effects of PD-1 blockade on ch14.18/CHO-based immunotherapy and mechanisms involved. Expression of PD-1 and PD-L1 on NB and effector cells was analyzed by RT-PCR and flow cytometry in the presence of ch14.18/CHO and/or IL-2. The effect of PD-1 blockade on ch14.18/CHO-mediated anti-NB immune response was evaluated using anti-PD-1 Ab both in vitro (Nivolumab) and in a syngeneic PD-L1/GD NB mouse model (anti-mouse PD-1). Culture of NB cells LA-N-1 (low PD-L1 baseline expression) with leukocytes and subtherapeutic ch14.18/CHO concentrations for 24 h induced strong upregulation of PD-L1, which was further increased by IL-2 resulting in complete inhibition of ch14.18/CHO-mediated ADCC. Importantly, blockade with Nivolumab reversed the PD-L1-dependent inhibition of ADCC. Similarly, co-incubation with anti-CD11b Ab abrogated the PD-L1 upregulation and restored ADCC. Mice treated with ch14.18/CHO in combination with PD-1 blockade showed a strong reduction of tumor growth, prolonged survival and the highest cytotoxicity against NB cells. In conclusion, ch14.18/CHO-mediated effects upregulate the inhibitory immune checkpoint PD-1/PD-L1, and combination of ch14.18/CHO with PD-1 blockade results in synergistic treatment effects in mice representing a new effective treatment strategy against GD-positive cancers.

(2016) Pharmacokinetics and pharmacodynamics of ch14.18/CHO in relapsed/refractory high-risk neuroblastoma patients treated by long-term infusion in combination with IL-2, mAbs, 8:3, 604-616, DOI: 10.1080/19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 .8 mg£d/ml, respectively, were not significantly different. Importantly, we detected ch14.18/CHO trough concentration of 1 mg/ml at time points preceding subsequent antibody infusions after cycle 1, allowing a persistent activation of antibody effector mechanisms over the entire treatment period of 6 months. HACA responses were observed in 10/ 53 (19%) patients, similar to STI (21%), indicating LTI had no effect on the immunogenicity of ch14.18/ CHO. In conclusion, LTI of ch14.18/CHO induced effector mechanisms over the entire treatment period, and may therefore emerge as the preferred delivery method of anti-GD2 immunotherapy to NB patients.

Polymorphisms in Fc-gamma-receptor (FCGR) genes as well as killer cell immunoglobulin-like receptor (KIR) and KIR ligand (KIRL) repertoires may influence antitumor effects of monoclonal antibodies (mAb). Here, we systematically analyzed high-and low-affinity FCGR2A and -3A genotypes as well as stimulating and inhibitory KIR/KIRL combinations in 53 neuroblastoma (NB) patients treated by long-term infusion (LTI) of anti-GD 2 IgG1 Ab ch14.18/CHO using validated real-time PCR methods.Patients with high-affinity FCGR2A and -3A genotypes showed a higher level of Ab-dependent cellmediated cytotoxicity (ADCC) on day 8 after the start of ch14.18/CHO and superior event-free survival (EFS) compared to patients with low FCGR genotypes. Similar observations were made for patients with stimulatory KIR/KIRL haplotype B (combination of KIR genes including activating receptor genes) compared to inhibitory haplotype A (a fixed set of genes encoding for inhibitory receptors, except 2DS4) and stronger effects were found in patients when haplotype B and high-affinity FCGRs were combined. Surprisingly, independent analysis of KIRs showed a major role of activating KIR 2DS2 for high ADCC levels and prolongation of EFS. The greatest effect was observed in 2DS2-positive patients that also had highaffinity FCGR2A and -3A genotypes.In summary, the presence of the activating KIR 2DS2 has a major effect on ADCC levels and survival in NB patients treated by LTI of ch14.18/CHO and may therefore be a useful biomarker in combination with FCGR polymorphisms for Ab-based immunotherapies.

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.

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