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.

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.

10014 Background: We determined the role of scIL-2 combined with long term infusion (LTI) of DB in patients (pts) with high-risk relapsed/refractory neuroblastoma. Methods: 160 pts were enrolled into an open label SIOPEN Phase II clinical trial (EudraCT 2009-018077-31). Pts were randomly assigned to receive up to 5 cycles of 100 mg/m2 DB-LTI (d8-17) and 160 mg/m2 oral isotretinoin (d19-32) (81 pts) with and without 6x106IU/m2 scIL-2 (d1-5; 8-12) (79 pts). Endpoints were toxicity, response rates and 2yrs-event free and -overall survival. Results: Between 07/2014 and 07/2017, 160 pts from 11 countries were randomised. Median follow-up is 2.6 years. Pts were well balanced between arms according to stage, age, MYCN amplification, patients with relapse and remission status. The 2yrs-EFS and -OS for DB (81 pts) vs. DB combined with scIL-2 (79 pts) was 59%±6% vs 65%±6% (p = 0.721) and 79%±5% vs 84%±4% (p = 0.904). In 97 pts with evaluable disease, a response rate of 49% (9% CR, 40% PR) vs 52% (26% CR, 26% PR) after treatment with DB vs DB and scIL-2 was observed. Grade 3&4 fever (16% vs 46%, P = 0.000), allergic reaction (1% vs 14%, P = 0.004), hematological toxicity (46% vs 66%, P = 0.013) and neurotoxicity (0% vs 8%, p = 0.003) were significantly worse in the combination arm, but no difference was seen for capillary leak, gastrointestinal, liver enzyme elevation and pain. Paraplegia possibly related to the treatment was observed in 2 pts in the combination arm, none in the arm without scIL-2, and one resolved to baseline. A subgroup of 34 pts who had a relapse and measurable disease at treatment start, showed a 2yrs-EFS and -OS in DB (17 pts) vs DB combined with scIL-2 (17 pts) of 35%±12% vs 69%±12% (p = 0.116) and 59%±12% vs 81%±10% (p = 0.167). However, this trend was statistically not significant. Pharmacokinetic and HACA response between both arms was not different with overlapping antibody concentration-time curves and a HACA response of 15/81 (19%) (DB) vs 16/79 (20%) (DB and scIL-2). Conclusions: No significant difference in efficacy of DB combined with scIL-2 and increased toxicity in this arm suggests that this schedule of scIL-2 is of no additional benefit. Clinical trial information: 2009-018077-31.

Immunotherapy with the anti-GD2 antibody (Ab) ch14.18/CHO in combination with interleukin 2 (IL-2) has improved survival of high-risk neuroblastoma (NB) patients. Here, we report immunotherapy-related effects on circulating NK cells, regulatory T cells (Tregs), granulocytes as well as on Ab-dependent cell-mediated cytotoxicity (ADCC) and cytokines IFN-γ, IL-6, IL-10, IL-18 and CCL2 and their association with progression-free survival (PFS).In a closed single-center program, 53 patients received five cycles of 6 × 106 IU/m2 subcutaneous IL-2 (d1-5; 8–12) combined with long-term infusion (LTI) of 100 mg/m2 ch14.18/CHO (d8-18). Immune cells and cytokines were analyzed by flow cytometry and ADCC by calcein-AM-based cytotoxicity assay.IL-2 administration increased cytotoxic NK cell-, eosinophil- and Treg counts in cycle 1 (2.9-, 3.1- and 20.7-fold, respectively) followed by further increase in subsequent cycles, whereas neutrophil levels were elevated only after the ch14.18/CHO infusion (2.4-fold change). Serum concentrations of IFN-γ, IL-6, IL-10, IL-18 and CCL2 in cycle 1 were increased during the combinatorial therapy (peak levels of 3,656 ± 655 pg/ml, 162 ± 38 pg/ml, 20.91 ± 4.74 pg/ml, 1,584 ± 196 pg/ml and 2,159 ± 252 pg/ml, respectively). Surprisingly, we did not observe any correlation between NK-, eosinophil- or neutrophil levels and PFS. In contrast, patients with low Tregs showed significantly improved PFS compared to those who had high levels. Treg counts negatively correlated with INF-γ serum concentrations and patients with high INF-γ and IL-18 had significantly improved survival compared to those with low levels.In conclusion, LTI of ch14.18/CHO in combination with IL-2 resulted in Treg induction that inversely correlated with IFN-γ levels and PFS.

GD2-directed immunotherapies improve survival of high-risk neuroblastoma (NB) patients (pts). Treatment with chimeric anti-GD2 antibodies (Ab), such as ch14.18, can induce development of human anti-chimeric Ab (HACA). Here, we report HACA effects on ch14.18/CHO pharmacokinetics, pharmacodynamics and pain intensity in pts treated by long-term infusion (LTI) of ch14.18/CHO combined with IL-2. 124 pts received up to 5 cycles of ch14.18/CHO 10 days (d) infusion (10 mg/m2/d; d8–18) combined with s.c. IL-2 (6 × 106 IU/m2/d; d1–5, d8–12). HACA, treatment toxicity, ch14.18/CHO levels, Ab-dependent cellular- (ADCC) and complement-dependent cytotoxicity (CDC) were assessed using respective validated assays. HACA-negative pts showed a steadily decreased pain in cycle 1 (74% pts without morphine by d5 of LTI) with further decrease in subsequent cycles. Ch14.18/CHO peak concentrations of 11.26 ± 0.50 µg/mL found in cycle 1 were further elevated in subsequent cycles and resulted in robust GD2-specific CDC and ADCC. Development of HACA (21% of pts) resulted in strong reduction of ch14.18/CHO levels, abrogated CDC and ADCC. Surprisingly, no difference in pain toxicity between HACA-positive and -negative pts was found. In conclusion, ch14.18/CHO LTI combined with IL-2 results in strong activation of Ab effector functions. Importantly, HACA response abrogated CDC but did not affect pain intensity indicating CDC-independent pain induction.