Table 1.
Genetic aberrations of de novo neuroblastoma cell lines.
Figure 1.
Flow cytometric analysis of GD2 and CD56 expression.
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. (A) 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) Selected histograms of GD2 (upper panel, black filled curve) and CD56 (lower panel, black filled curve) expression on de novo NB cell lines HGW-1, HGW-2, HGW-3 and HGW-5. Respective isotype controls are shown as black dashed curves in both panels.
Figure 2.
RT-PCR analysis of human tyrosine hydroxylase and MDR1 mRNA expression and level of spontaneous release of calcein from NB cell lines.
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 (A) and MDR1 (B) gene expression (PCR-product sizes 405 bp and 202 bp, respectively). Expression of GAPDH mRNA served as internal control. (C) 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. (D) Levels of spontaneous release of calcein from NB cells examined after 4 h incubation of calcein-labeled cells with 12.5% heat inactivated serum.
Figure 3.
Assessment of variables for the complement-dependent cytotoxicity bioassay.
(A) 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) 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. (C) 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. t-test; *P < 0.05.
Figure 4.
Evaluation of donor-specific CDC.
Serum samples (12.5% final concentration) of four healthy donors D1 (A), D2 (B), D3 (C) and D4 (D) were analyzed using calcein-AM-based cytotoxicity assay as described in section “Materials and Methods”. Serial dilutions of 1.0 µg/ml ch14.18/CHO were used for CDC (closed circles). Rituximab served as a negative control (1 µg/ml; open triangles). To show GD2-specific target cell lysis, samples were pre-incubated with excess of GD2-mimicking anti-Id Ab ganglidiomab (5 µg/ml; closed squares). Data are shown as mean values ± SE of three independent experiments performed at least in triplicates.
Figure 5.
Evaluation of CDC-mediated by different anti-GD2 Ab.
Serial dilutions (final concentration of 1.0, 0.5, 0.25, 0.12, 0.06 and 0.03 µg/ml) of murine 14G2a (A, closed circles), chimeric human/murine ch14.18/CHO (B, closed circles), and IL-2 conjugated humanized hu14.18-IL-2 (D, closed circles) were used for CDC. Complement fixation deficient mutants chimeric ch14.18-delta-CH2 (C, closed circles) and humanized hu14.18 (E, 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.
Figure 6.
Evaluation of ADCC-mediated by different effector cell subsets.
Three populations of freshly isolated effector cells of a healthy donor were compared for ADCC: leukocytes (A), PBMCs (B) and granulocyte rich fraction (C). ADCC were performed using calcein-AM-based cytotoxicity assay as described in Materials and Methods. 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 (D), PBMCs (E) and effector cells of granulocytes rich fraction (F) 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 (G), IL-2 treated PBMCs (H) and IL-2 treated cells of granulocyte rich fraction (I) were calculated as described in Materials and Methods. 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. (J) 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).
Figure 7.
Determination of ADCC sensitivity.
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 Materials and Methods. Data are shown as mean values ± SE of three independent experiments performed at least in triplicates.
Figure 8.
Within-assay precision, inter-assay precision and sample stability.
For reliable and reproducible evaluation of cytotoxicity, within-assay and inter-assay precision analyses were performed (A and C). Both parameters were calculated according to the formula: SD/mean×100% and found to be under 20%. To determine within-assay precision (A), 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 (C) 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 and D), 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) or to five freeze-thaw cycles (D). Rituximab containing (closed triangles) and Ab-free controls (open triangles) were included as described in Materials and Methods. Data are shown as mean CDC values ± SE of two independent experiments performed at least in triplicates.
Table 2.
Effect of anticoagulants on leukocyte count and viability.
Figure 9.
Evaluation of CDC and ADCC in high-risk NB patients treated with a combination of IL-2 and ch14.18/CHO mAb.
Serum samples (12.5% final concentration) and effector cells (E:T ratio of 40∶1) collected from three selected NB patients (patient 1, patient 2, patient 3) treated with 100 mg/m2 ch14.18/CHO in combination with 6×106 IU/m2 s.c. IL-2 were analyzed for CDC (A) and ADCC (B) on two days (day 1, prior to Ab infusion, white column and day 15, eight days after the start of Ab application, black column) using both established bioassays. Patient-specific CDC and ADCC were calculated as described in Materials and Methods. Rituximab and anti-Id Ab ganglidiomab containing controls (grey columns) were included as described in Materials and Methods. Data are shown as mean values ± SE of independent experiments performed at least in triplicates. t-test; *P<0.05.