Genetic aberrations of <i>de novo</i> neuroblastoma cell lines.

<p>Genetic aberrations of <i>de novo</i> neuroblastoma cell lines were evaluated using high resolution SNP array analysis. N.D; not done.</p><p>Genetic aberrations of <i>de novo</i> neuroblastoma cell lines.</p

Effect of anticoagulants on leukocyte count and viability.

<p>Leukocytes were isolated from sodium-heparin- and EDTA whole blood samples followed by analysis of cell viability and count. Values are represented as mean ± SE of two independent experiments.</p><p>Effect of anticoagulants on leukocyte count and viability.</p

Evaluation of CDC-mediated by different anti-GD₂ Ab.

<p>Serial dilutions (final concentration of 1.0, 0.5, 0.25, 0.12, 0.06 and 0.03 µg/ml) of murine 14G2a (<b>A</b>, closed circles), chimeric human/murine ch14.18/CHO (<b>B</b>, closed circles), and IL-2 conjugated humanized hu14.18-IL-2 (<b>D</b>, closed circles) were used for CDC. Complement fixation deficient mutants chimeric ch14.18-delta-CH2 (<b>C</b>, closed circles) and humanized hu14.18 (<b>E</b>, closed circles) as well as rituximab (closed triangles) served as negative controls. To show GD₂-dependent specificity of CDC induced, additional controls containing respective anti-GD₂ Ab were pre-incubated with excess of GD₂-mimicking anti-Id Ab ganglidiomab (5 µg/ml; open circles). Data are shown as mean values ± SE of three independent experiments performed at least in triplicates.</p

Flow cytometric analysis of GD₂ and CD56 expression.

<p>NB cells were stained with 1.0 µg ch14.18/CHO and 0.05 µg anti-CD56-APC, followed by incubation with 0.017 µg of PE-labeled anti-human IgG secondary antibody. Chimeric anti-CD20 antibody and APC-labeled mouse IgG1 were utilized as isotype controls, respectively. (<b>A</b>) Expression of GD₂ (upper panel, black filled curve) and CD56 (lower panel, black filled curve) on NB cell lines LA-N-1, CHLA-20, SK-N-SH and the melanoma cell line M21. Respective isotype controls are indicated in both panels as black dashed curves. Results are presented as representative histograms. (<b>B</b>) Selected histograms of GD₂ (upper panel, black filled curve) and CD56 (lower panel, black filled curve) expression on <i>de novo</i> NB cell lines HGW-1, HGW-2, HGW-3 and HGW-5. Respective isotype controls are shown as black dashed curves in both panels.</p

Determination of ADCC sensitivity.

<p>Different concentrations of anti-GD₂ mAb ch14.18/CHO (10,000, 1,000, 100, 10, 1, 0.1 and 0.01 ng/ml, closed circles) were used to investigate a concentration range of Ab inducing a detectable ADCC. Activated leukocytes of a healthy donor were incubated with calcein-labeled LA-N-1 cells at E:T ratio of 40∶1. Rituximab (closed triangles) and anti-Id Ab ganglidiomab (open circles) containing controls were included as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107692#s2" target="_blank">Materials and Methods</a>. Data are shown as mean values ± SE of three independent experiments performed at least in triplicates.</p

Evaluation of ADCC-mediated by different effector cell subsets.

<p>Three populations of freshly isolated effector cells of a healthy donor were compared for ADCC: leukocytes (<b>A</b>), PBMCs (<b>B</b>) and granulocyte rich fraction (<b>C</b>). ADCC were performed using calcein-AM-based cytotoxicity assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107692#s2" target="_blank">Materials and Methods</a>. Effector cells were incubated with 10 µg/ml ch14.18/CHO (closed circles) and calcein-labeled target cells LA-N-1 at different E:T ratios (80∶1, 40∶1, 20∶1, 10∶1, 5∶1, 2.5∶1 and 1.25∶1) and ADCC mediated by leukocytes (<b>D</b>), PBMCs (<b>E</b>) and effector cells of granulocytes rich fraction (<b>F</b>) were examined. In order to simulate the use of IL-2 in combination with ch14.18/CHO in clinical trials, effector cells were incubated for 64 h in cell culture medium supplemented with 1,000 IU/ml IL-2. ADCC mediated by IL-2 treated leukocytes (<b>G</b>), IL-2 treated PBMCs (<b>H</b>) and IL-2 treated cells of granulocyte rich fraction (<b>I</b>) were calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107692#s2" target="_blank">Materials and Methods</a>. Rituximab served as a negative control (closed triangles). GD₂-specific ADCC of NB cells was demonstrated by pre-incubation of ch14.18/CHO with excess of anti-Id Ab ganglidiomab (open circles). Data are shown as mean values ± SE of three independent experiments performed at least in triplicates. (<b>J</b>) Comparison of ADCC mediated by leukocytes at E:T ratio of 40∶1 and PBMCs at E:T ratio 20∶1 isolated from the same blood sample of five selected NB patients treated with a combination of IL-2 and ch14.18/CHO (black columns). Rituximab served as a negative control (white columns).</p

Assessment of variables for the complement-dependent cytotoxicity bioassay.

<p>(<b>A</b>) Analysis of CDC mediated by different serum concentrations (100%, 50%, 25%, 12.5%, 6.3%, 3.1%, 1.6%, 0.8%, 0.4%) using NB cell line LA-N-1 as a target cell line and two defined concentrations of anti-GD₂ Ab ch14.18/CHO (1.0 µg/ml (black column) and 0.1 µg/ml (white column). (<b>B</b>) Concentration-dependent inhibition of CDC mediated by 1.0 µg/ml ch14.18/CHO (closed circle) by complement inhibitor eculizumab (closed triangles). Pre-incubation with excess of anti-Id Ab ganglidiomab (5.0 µg/ml; open circle) was performed to show GD₂-specific target cell lysis. Rituximab (1.0 µg/ml; closed square) served as a negative control. (<b>C</b>) Evaluation of CDC mediated by different NB cell lines, 12.5% serum and anti-GD₂ Ab ch14.18/CHO (1 µg/ml; black column). Ganglidiomab (5.0 µg/ml; grey columns) and rituximab (1.0 µg/ml; white columns) served as controls. Data are shown as mean values ± SE of three independent experiments performed at least in triplicates. <i>t</i>-test; *P < 0.05.</p

Within-assay precision, inter-assay precision and sample stability.

<p>For reliable and reproducible evaluation of cytotoxicity, within-assay and inter-assay precision analyses were performed (<b>A</b> and <b>C</b>). Both parameters were calculated according to the formula: SD/mean×100% and found to be under 20%. To determine within-assay precision (<b>A</b>), triplicated serum samples of a healthy donor (12.5% final concentration) with defined ch14.18/CHO concentrations (1.0 µg/ml) and calcein-labeled LA-N-1 target cells were analyzed. The cytotoxicity analysis was repeated ten times on the same plate. Results are presented as mean CDC of triplicates ± SD for ten data sets. Inter-assay precision (<b>C</b>) was determined on different days by different operators. Ten independent measurements of serum samples containing 1.0 µg/ml ch14.18/CHO and calcein-labeled LA-N-1 target cells were performed. The analyzed cytotoxicity levels are given as mean values ± SD for ten independent assays. To determine stability of CDC (<b>B</b> and <b>D</b>), two ch14.18/CHO concentrations 1.0 µg/ml (closed circles) and 0.1 µg/ml (open circles) prepared in 12.5% serum of a healthy donor were analyzed with established cytotoxicity assay. Samples were subjected to either storage at room temperature for up to 96 h (<b>B</b>) or to five freeze-thaw cycles (<b>D</b>). Rituximab containing (closed triangles) and Ab-free controls (open triangles) were included as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107692#s2" target="_blank">Materials and Methods</a>. Data are shown as mean CDC values ± SE of two independent experiments performed at least in triplicates.</p

RT-PCR analysis of human tyrosine hydroxylase and MDR1 mRNA expression and level of spontaneous release of calcein from NB cell lines.

<p>RNA of human NB cell lines LA-N-1, CHLA-20, HGW-1, HGW-2, HGW-3 and HGW-5 was used for RT-PCR analysis of hTH (<b>A</b>) and MDR1 (<b>B</b>) gene expression (PCR-product sizes 405 bp and 202 bp, respectively). Expression of GAPDH mRNA served as internal control. (<b>C</b>) Densitometric analysis of MDR1 mRNA expression relative to GAPDH. Values are given as means ± SE of three independent experiments. HTH-negative NB cell line SK-N-SH and melanoma cell line M21 served as negative controls for hTH mRNA RT-PCR analysis. NTC, no template control. (<b>D</b>) Levels of spontaneous release of calcein from NB cells examined after 4 h incubation of calcein-labeled cells with 12.5% heat inactivated serum.</p

Generation and Characterization of a Human/Mouse Chimeric GD2-Mimicking Anti-Idiotype Antibody Ganglidiximab for Active Immunotherapy against Neuroblastoma.

Vaccination with proteins mimicking GD2 that is highly expressed on neuroblastoma (NB) cells is a promising strategy in treatment of NB, a pediatric malignancy with poor prognosis. We previously showed efficacy of ganglidiomab in vivo, a murine anti-idiotype (anti-Id) IgG1. In order to tailor immune responses to variable regions, we generated a new human/mouse chimeric anti-Id antibody (Ab) ganglidiximab by replacing murine constant fragments with corresponding human IgG1 regions. DNA sequences encoding for variable regions of heavy (VH) and light chains (VL) were synthesized by RT-PCR from total RNA of ganglidiomab-producing hybridoma cells and further ligated into mammalian expression plasmids with coding sequences for constant regions of human IgG1 heavy and light chains, respectively. We established a stable production cell line using Chinese hamster ovarian (CHO) cells co-transfected with two expression plasmids driving the expression of either ganglidiximab heavy or light chain. After purification from supernatants, anti-idiotypic characteristics of ganglidiximab were demonstrated. Binding of ganglidiximab to anti-GD2 Abs of the 14.18 family as well as to NK-92tr cells expressing a GD2-specific chimeric antigen receptor (scFv(ch14.18)-zeta) was shown using standard ELISA and flow cytometry analysis, respectively. Ganglidiximab binding affinities to anti-GD2 Abs were further determined by surface plasmon resonance technique. Moreover, binding of anti-GD2 Abs to the nominal antigen GD2 as well as GD2-specific Ab-mediated cytotoxicity (ADCC, CDC) was competitively inhibited by ganglidiximab. Finally, ganglidiximab was successfully used as a protein vaccine in vivo to induce a GD2-specific humoral immune response. In summary, we report generation and characterization of a new human/mouse chimeric anti-Id Ab ganglidiximab for active immunotherapy against NB. This Ab may be useful to tailor immune responses to the paratope regions mimicking GD2 overexpressed in NB