Immunotherapy with anti-GD₂ antibody (Ab) ch14.18/CHO is effective for treatment of high-risk neuroblastoma (NB) patients and is mainly based on GD₂-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⁺/GD₂⁺ 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 GD₂-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-GD₂ 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 cell-mediated 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 high-affinity 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.

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.

Immunotherapies against high-risk neuroblastoma (NB), using the anti-GD2 antibody (Ab) dinutuximab beta (DB), significantly improved patient survival. Ab-dependent cellular cytotoxicity (ADCC) is one of the main mechanisms of action and it is primarily mediated by NK cells. To further improve antitumor efficacy, we investigated here a combinatorial immunotherapy with DB and the double immune checkpoint blockade of T-cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) and programmed cell death ligand-1 (PD-L1). The effects of ADCC, mediated by DB against NB cells on NK-cell activity, and the expression of TIGIT and CD226 and their ligands CD112 and CD155, as well as of PD-1 and PD-L1 on NB and effector cells, were investigated using flow cytometry. ADCC was assessed with a calcein-AM-based cytotoxicity assay. The efficacy of a combinatorial immunotherapy with DB, given as a long-term treatment, and the double immune checkpoint blockade of TIGIT and PD-L1 was shown using a resistant murine model of NB, followed by an analysis of the tumor tissue. We detected both TIGIT ligands, CD112 and CD155, on all NB cell lines analyzed. Although ADCC by DB resulted in a strong activation of NK cells leading to an effective tumor cell lysis, a remarkable induction of PD-L1 expression on NB cells, and of TIGIT and PD-1 on effector cells, especially on NK cells, was observed. Additional anti-TIGIT or anti-PD-L1 treatments effectively inhibited tumor growth and improved survival of the mice treated with DB. The superior antitumor effects were observed in the “DB + double immune checkpoint blockade” group, showing an almost complete eradication of the tumors and the highest OS, even under resistant conditions. An analysis of tumor tissue revealed both TIGIT and TIGIT ligand expression on myeloid-derived suppressor cells (MDSCs), suggesting additional mechanisms of protumoral effects in NB. Our data show that the targeting of TIGIT and PD-L1 significantly improves the antitumor efficacy of anti-GD2 immunotherapy, with DB presenting a new effective combinatorial treatment strategy against high-risk tumors.

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 (T_regs), 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 T_reg 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 T_regs showed significantly improved PFS compared to those who had high levels. T_reg 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 T_reg induction that inversely correlated with IFN-γ levels and PFS.