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

Abstract

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.

Fig 1. Schematic overview of generation of human/mouse chimeric anti-Id Ab ganglidiximab.

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).

Fig 2. Amplification of DNA fragments encoding for GD₂-mimicking paratopes of ganglidiximab.

(A) 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) 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).

Fig 3. Establishment of a cell line stably producing ganglidiximab.

CHO cells were stably co-transfected with two generated expression plasmids (p3-ganglidiximab-HC/ p3-ganglidiximab-LC) and a cell clone “VH-VL-1” stably producing high levels of ganglidiximab was selected for further Ab production. Permanent ganglidiximab expression was confirmed after 15 passages and two freeze-thaw cycles by RT-PCR (A) and standard ELISA (B). (A) To amplify coding sequences of ganglidiximab VH (440 bp) and -VL (420 bp), RNA of “VH-VL-1” was used for RT-PCR followed by agarose gel electrophoresis. RNA of ganglidiomab-producing hybridoma cells served as a positive control (+) and RNA of non-transfected CHO cells as a negative control. One representative image is shown. NTC—no-template-control. M—Marker (100-bp). (B) Ganglidiximab production by “VH-VL-1” was analyzed in supernatants collected during 15 passages and after two freeze-thaw cycles (P2, 5, 10 and 15). Supernatants of non-transfected CHO cells or cells incubated with control plasmids (mock) or transfection reagent only were utilized as negative controls (white columns). Human/mouse chimeric mAb rituximab and cell culture medium were included as additional negative controls (white-striped columns). Data are shown as mean values ± SEM of three independent experiments performed at least in triplicates. One-way ANOVA on ranks followed by appropriate post hoc comparison test; *P 0.05 vs. negative controls.

Fig 4. Binding of anti-GD₂ antibodies to ganglidiximab and competitive inhibition of binding to nominal antigen GD₂.

(A) 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; ***P 0.001 vs. rituximab. (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; ***P 0.001 vs. rituximab.

Fig 5. Binding of ganglidiximab to NK-92tr and ganglidiximab-dependent inhibition of NK-92tr-mediated GD₂-specific cytotoxicity against NB.

(A) 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) 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; *P 0.05 vs. w/o Ab.

Fig 6. Ganglidiximab-dependent inhibition of GD₂-specific ch14.18/CHO-mediated ADCC and CDC.

GD₂-specific ch14.18/CHO-mediated ADCC (A) and CDC (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; *P 0.05 vs. ch14.18/CHO.

Fig 7. Induction of GD₂-specific humoral immunity after vaccination with ganglidiximab.

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; ***P 0.001 vs. baseline (prior to the first immunization); ^§P < 0.05 vs. NaCl control group after six immunizations.