GD2 targeting by dinutuximab beta is a promising immunotherapeutic approach against malignant glioma.

PURPOSE: Disialoganglioside GD2 is expressed by glioblastoma multiforme (GBM) cells representing a promising target for anti-GD2 immunotherapeutic approaches. The aim of the present study was to investigate anti-tumor efficacy of the chimeric anti-GD2 antibody (Ab) dinutuximab beta against GBM. METHODS: Expression levels of GD2 and complement regulatory proteins (CRP; CD46, CD55 and CD59) on well-known and newly established primary tumor originated GBM cell lines were analyzed by flow cytometry. Ab-dependent cellular (ADCC) and complement-dependent cytotoxicity (CDC) mediated by dinutuximab beta against GBM cells were determined by a non-radioactive calcein-AM-based assay. RESULTS: Analysis of primary GBM cells revealed a heterogeneous GD2 expression that varied between the cell lines analyzed with higher expression levels in the tumor surface compared to the core originated cells. Both GD2-positive and -negative tumor cells were detected in every cell line analyzed. In contrast to CDC, ADCC mediated by dinutuximab beta was observed against the majority of GBM cells. Importantly, CDC-resistant cells showed high expression of the CRP CD46, CD55 and CD59. CONCLUSION: Our present data show anti-tumor effects mediated by dinutuximab beta against GBM cells providing a rationale for a GD2-directed immunotherapy against GBM. Due to high CRP expression, a combining of GD2-targeting with CRP blockade might be a further treatment option for GBM.

Reduction of CD11b+ myeloid suppressive cells augments anti-neuroblastoma immune response induced by the anti-GD2 antibody ch14.18/CHO.

Neuroblastoma (NB) still remains a major challenge in pediatric oncology. We recently showed CD11b+-dependent upregulation of the PD-1/PD-L1 checkpoint on NB cells treated with the chimeric anti-GD2 antibody (Ab) ch14.18/CHO. Here, we report effects of reduction of CD11b+ myeloid suppressive cells on ch14.18/CHO immunotherapy against NB. Flow cytometry, immunohistochemistry and RT-PCR were used to assess tumor infiltrating leukocytes and expression of myeloid suppressive cell-associated genes. XTT assay was used to show impact of 5-FU on tumor and effector cells. Antitumor effects of the combined treatment with ch14.18/CHO and reduction of myeloid suppressive cells were evaluated in a syngeneic NB mouse model. Tumor tissue of untreated mice showed a strong infiltration by CD11b+ cells (53% of all tumor infiltrating leukocytes). RT-PCR analysis of tumors revealed strong expression of the myeloid suppressive cell-associated genes analyzed with the strongest induction of M-CSFr, CCL2, IL-1ß, IL-4, IL-6 r, IL-8, Arg1, and NOS2. Compared to controls, application of anti-CD11b Ab resulted in reduction of both CD11b+ cells in tumors and expression of myeloid suppressive cell-associated genes as well as delayed tumor growth and prolonged survival. These effects could be further improved by 5-FU. Importantly, the combinatorial immunotherapy with ch14.18/CHO and 5-FU showed the strongest antitumor effects and superior survival rates. In conclusion, reduction of immune suppressive myeloid cells augments anti-NB efficacy of a ch14.18/CHO-based immunotherapy representing a new effective treatment strategy against GD2-positive cancers.

Low CD4⁺/CD25⁺/CD127⁻ regulatory T cell- and high INF-γ levels are associated with improved survival of neuroblastoma patients treated with long-term infusion of ch14.18/CHO combined with interleukin-2.

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.

Co-expression of IL-15 enhances anti-neuroblastoma effectivity of a tyrosine hydroxylase-directed DNA vaccination in mice.

Long-term survival of high-risk neuroblastoma (NB) patients still remains under 50%. Here, we report the generation, in vitro characterization and anti-tumor effectivity of a new bicistronic xenogenic DNA vaccine encoding tyrosine hydroxylase (TH) that is highly expressed in NB tumors, and the immune stimulating cytokine interleukin 15 (IL-15) that induces cytotoxic but not regulatory T cells. The DNA sequences of TH linked to ubiquitin and of IL-15 were integrated into the bicistronic expression vector pIRES. Successful production and bioactivity of the vaccine-derived IL-15- and TH protein were shown by ELISA, bioactivity assay and western blot analysis. Further, DNA vaccine-driven gene transfer to the antigen presenting cells of Peyer's patches using attenuated Salmonella typhimurium that served as oral delivery system was shown by immunofluorescence analysis. The anti-tumor effect of the generated vaccine was evaluated in a syngeneic mouse model (A/J mice, n = 12) after immunization with S. typhimurium (3× prior and 3× after tumor implantation). Importantly, TH-/IL-15-based DNA vaccination resulted in an enhanced tumor remission in 45.5% of mice compared to controls (TH (16.7%), IL-15 (0%)) and reduced spontaneous metastasis (30.0%) compared to controls (TH (63.6%), IL-15 (70.0%)). Interestingly, similar levels of tumor infiltrating CD8+ T cells were observed among all experimental groups. Finally, co-expression of IL-15 did not result in elevated regulatory T cell levels in tumor environment measured by flow cytometry. In conclusion, co-expression of the stimulatory cytokine IL-15 enhanced the NB-specific anti-tumor effectivity of a TH-directed vaccination in mice and may provide a novel immunological approach for NB patients.

Impact of HACA on Immunomodulation and Treatment Toxicity Following ch14.18/CHO Long-Term Infusion with Interleukin-2: Results from a SIOPEN Phase 2 Trial.

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.

Platelet activation parameters and platelet-leucocyte-conjugate formation in glioblastoma multiforme patients.

Patients with glioblastoma multiforme (GBM) suffer from an increased incidence of vascular thrombotic events. However, key influencing factors of the primary hemostasis have not been characterized in GBM patients to date. Thus, the present study determines the activation level of circulating platelets in GBM patients, in-vitro reactivity to agonist-induced platelet stimulation and the formation of circulating platelet-leucocyte conjugates as well as the plasma levels of the proinflammatory lipid mediator sphingosine-1-phosphate (S1P). The endogenous thrombin potential (ETP) was determined as global marker for hemostasis. The 21 GBM patients and 21 gender and age matched healthy individuals enrolled in this study did not differ in mean total platelet count. Basal surface expression of platelet CD63 determined by flow cytometry was significantly increased in GBM patients compared to controls as was observed for the concentration of soluble P-selectin in the plasma of GBM patients. While the ETP was not affected, the immunomodulatory lipid S1P was significantly decreased in peripheral blood in GBM. Interestingly, monocyte expression of PSGL-1 (CD162) was decreased in GBM patient blood, possibly explaining the rather decreased formation of platelet-monocyte conjugates. Our study reveals an increased CD63 expression and P-selectin expression/ secretion of circulating platelets in GBM patients. In parallel a down-modulated PSGL-1 expression in circulating monocytes and a trend towards a decreased formation of heterotypic platelet-monocyte conjugates in GBM patients was seen. Whether this and the observed decreased plasma level of the immunomodulatory S1P reflects a systemic anti-inflammatory status needs to be addressed in future studies.