Ganglidiximab-dependent inhibition of GD₂-specific ch14.18/CHO-mediated ADCC and CDC.

<p>GD₂-specific ch14.18/CHO-mediated ADCC (<b>A</b>) and CDC (<b>B</b>). Ch14.18 induced ADCC and CDC of NB cells LA-N-1 (black column) was compared to rituximab used as negative control (white-striped column). Pre-incubation of ch14.18 with excess of ganglidiximab (grey column) or ganglidiomab (white column) resulted in inhibition of both ADCC and CDC. Results are expressed as percentage of cytotoxicity (mean values ± SEM) of three independent experiments performed at least in triplicates. One-way ANOVA on ranks followed by appropriate post hoc comparison test; *<i>P</i> 0.05 <i>vs</i>. ch14.18/CHO.</p

Binding of ganglidiximab to NK-92tr and ganglidiximab-dependent inhibition of NK-92tr-mediated GD₂-specific cytotoxicity against NB.

<p>(<b>A</b>) Binding of ganglidiximab to NK-92tr expressing a GD₂-specific chimeric antigen receptor was analyzed by flow cytometry. Cells were stained with chimeric ganglidiximab (black solid line), murine anti-Id ganglidiomab (positive control; grey solid line), chimeric rituximab (isotype control; black dashed line) or murine IgG1 (isotype control; grey dashed line) followed by incubation with biotinylated ch14.18/CHO and PE-labeled streptavidin. Staining of the parental NK-92 cell line lacking GD₂-specific CAR expression was further included as negative control. Results from one representative experiment are shown. (<b>B</b>) Inhibition of GD₂-specific NK-92tr-mediated NB cell lysis (w/o Ab; white-striped column) was analyzed after pre-incubation with excess of ganglidiximab (black column) using a calcein-AM-based cytotoxicity assay. Murine anti-Id ganglidiomab served as a positive control (grey column). Rituximab (white column) and murine IgG1 (white column) were utilized as negative controls. Results are expressed as percentage of lysis inhibition (mean values ± SEM) of two independent experiments performed using six replicates. One-way ANOVA on ranks followed by appropriate post hoc comparison test; *<i>P</i> 0.05 <i>vs</i>. w/o Ab.</p

Dissociation constants (K_D) of ganglidiximab and ganglidiomab from anti-GD₂ antibodies of the 14.18 family.

<p>Dissociation constants (K_D) of ganglidiximab and ganglidiomab from anti-GD₂ antibodies of the 14.18 family.</p

Amplification of DNA fragments encoding for GD₂-mimicking paratopes of ganglidiximab.

<p>(<b>A</b>) Visualization of coding sequences of GD₂-mimicking variable heavy (VH; 440 bp) and light chain (VL; 420 bp) amplified by RT-PCR. RNA was isolated from hybridoma cells producing murine anti-Id ganglidiomab. PCR products were analyzed by agarose gel electrophoresis. Representative image is shown. NTC—no-template-control. M—Marker (100-bp). (<b>B</b>) PCR products were cloned into pCR^®2.1-TOPO^® plasmids and analyzed by restriction enzyme digest to excise DNA sequences encoding for VH and VL (product sizes 427 bp and 407 bp, respectively). Resulting DNA fragments were analyzed by agarose gel electrophoresis. Representative image is shown. M—Marker (2-log, 0.1–10.0 kbp).</p

Binding of anti-GD₂ antibodies to ganglidiximab and competitive inhibition of binding to nominal antigen GD₂.

<p>(<b>A</b>) Concentration-dependent binding of ganglidiximab to anti-GD₂ Abs of the 14.18 family was analyzed by ELISA. Chimeric mAb rituximab and murine IgG2a were utilized as isotype controls. Data are expressed relative to binding of 1 μg/ml of ch14.18/CHO to ganglidiximab (100%) and are presented as mean values ± SEM of four independent experiments performed in duplicates. One-way ANOVA followed by appropriate post hoc comparison test; ***<i>P</i> 0.001 <i>vs</i>. rituximab. (<b>B</b>) Inhibition of anti-GD₂ Ab binding to GD₂ by ganglidiximab was analyzed using competitive ELISA. Chimeric mAb rituximab and murine IgG2a served as isotype controls. Data are expressed as percentage of binding inhibition and presented as mean values ± SEM of four independent experiments performed in duplicates. One-way ANOVA followed by appropriate post hoc comparison test; ***<i>P</i> 0.001 <i>vs</i>. rituximab.</p

Induction of GD₂-specific humoral immunity after vaccination with ganglidiximab.

<p>Female A/J mice (n = 8) were immunized six times with ganglidiximab combined with the adjuvant Al(OH)₃ every two weeks. Control groups received Al(OH)₃ or 0.9% NaCl. Serum samples were taken before (baseline; white column) and after the last immunization step (black column) and analyzed for Abs directed against GD₂-mimicking paratopes of ganglidiximab using ELISA. Mann-Whitney Rank Sum test or One-way ANOVA followed by appropriate post hoc comparison test; ***<i>P</i> 0.001 <i>vs</i>. baseline (prior to the first immunization); ^§<i>P</i> < 0.05 vs. NaCl control group after six immunizations.</p

Schematic overview of generation of human/mouse chimeric anti-Id Ab ganglidiximab.

<p>The human/mouse chimeric anti-Id Ab ganglidiximab is composed of GD₂ mimicking variable regions (VH, VL) of murine anti-Id ganglidiomab and human IgG1 constant regions. Coding sequences of VH and VL were synthesized and inserted into mammalian expression plasmids containing DNA sequences for human IgG1 heavy (p3-IgG1-HC) and light chain (p3-IgG1-LC), respectively. For ganglidiximab production, CHO cells were stably co-transfected with the two generated expression plasmids (p3-ganglidiximab-HC and p3-ganglidiximab-LC).</p