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. 2023 Jan 8;24(2):1239.
doi: 10.3390/ijms24021239.

Minibody-Based and scFv-Based Antibody Fragment-Drug Conjugates Selectively Eliminate GD2-Positive Tumor Cells

Daniel V Kalinovsky  1 , Irina V Kholodenko  2 , Alexey V Kibardin  3 , Igor I Doronin  1   4 , Elena V Svirshchevskaya  1 , Dmitriy Y Ryazantsev  1 , Maria V Konovalova  1 , Fedor N Rozov  5 , Sergey S Larin  3 , Sergey M Deyev  1   6 , Roman V Kholodenko  1   4
Affiliations

Minibody-Based and scFv-Based Antibody Fragment-Drug Conjugates Selectively Eliminate GD2-Positive Tumor Cells

Daniel V Kalinovsky et al. Int J Mol Sci. .

Abstract

Ganglioside GD2 is a well-established target expressed on multiple solid tumors, many of which are characterized by low treatment efficiency. Antibody-drug conjugates (ADCs) have demonstrated marked success in a number of solid tumors, and GD2-directed drug conjugates may also hold strong therapeutic potential. In a recent study, we showed that ADCs based on the approved antibody dinutuximab and the drugs monomethyl auristatin E (MMAE) or F (MMAF) manifested potent and selective cytotoxicity in a panel of tumor cell lines and strongly inhibited solid tumor growth in GD2-positive mouse cancer models. Here, we employed two different GD2-binding moieties-minibodies and scFv fragments that carry variable antibody domains identical to those of dinutuximab, and site-directly conjugated them to MMAE or MMAF by thiol-maleimide chemistry with drug-to-antibody ratios (DAR) of 2 and 1, respectively. Specific binding of the antibody fragment-drug conjugates (FDCs) to GD2 was confirmed in direct ELISA, flow cytometry, and confocal microscopy. Selective cytotoxic and cytostatic effects of the conjugates were observed in GD2-positive but not GD2-negative neuroblastoma and melanoma cell lines. Minibody-based FDCs demonstrated more pronounced cytotoxic effects and stronger antigen binding compared to scFv-based FDCs. The developed molecules may offer considerable practical benefit, since antibody fragment-drug conjugates are capable of enhancing therapeutic efficacy of ADCs by improving their pharmacokinetic characteristics and reducing side effects.

Keywords: GD2-positive tumors; antibody fragments; antibody-drug conjugates; cancer; ganglioside GD2; immunotherapy; melanoma; minibodies; neuroblastoma; scFv fragments.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Design and expression of the GD2-specific antibody fragments. (A) Structures of the minibody 14.18 and the scFv fragment 14.18. (B) Size-exclusion chromatographic analysis of the minibody. (C) Polyacrylamide gel electrophoresis of the minibody; 1, molecular weight protein markers; 2, the minibody under reducing conditions, 10 min incubation at 95 °C; 3, the minibody under non-reducing conditions. (D) Size-exclusion chromatographic analysis of the scFv. (E) Polyacrylamide gel electrophoresis of the scFv; 1, molecular weight protein markers; 2, the scFv under reducing conditions, 10 min incubation at 95 °C; 3, the scFv under non-reducing conditions.
Figure 2
Figure 2
Antigen-binding properties of antibody fragments. (A) Confocal images of GD2-positive B78-D14 and GD2-negative B16 melanoma cell lines stained with FAM-labeled minibodies, scFv fragments, or control ch14.18 antibodies (1 µg per sample). Staining with fluorescently labeled anti-GD2 antibodies or antibody fragments (green) and counterstaining the nuclei with Hoechst 33342 (blue). Bar scale: 10 μm. (B) Flow cytometry analysis. Staining of B78-D14 and B16 cell lines with FAM-labeled minibodies and scFv fragments (1 µg per sample). Control–fluorescence intensity of intact cells. (C) Evaluation of the binding of minibodies and scFv fragments to gangliosides GD2, GD3, GM2, and GD1b in direct ELISA. Gangliosides were adsorbed on the plate; antibody fragments were added to the wells in 5 nM concentration. After incubating scFv fragments with anti-FLAG antibodies and minibodies with anti-human Fc-specific antibodies (both at 1:6000), the reaction was developed by Ultra TMB-ELISA Substrate Solution. Data are represented as OD mean ± SEM. scFv–scFv fragments 14.18, Mb–minibodies 14.18, Ab–antibodies 14.18.
Figure 3
Figure 3
Production of the FDCs and analysis of their antigen-binding properties. (A) Reaction scheme of the generation of scFv and minibody conjugates with MMAE and MMAF. (B) Absorption spectra of the scFv-based FDCs normalized at 280 nm. (C) Absorption spectra of the minibody-based FDCs normalized at 280 nm. (D) Absorption spectra of the auristatin drugs used for conjugation. (E) Polyacrylamide gel electrophoresis of the FDCs in reducing conditions, 5 min incubation at 95 °C. 1, molecular weight protein markers; 2, scFv; 3, scFv-MMAE; 4, scFv-MMAF; 5, minibody; 6, minibody-MMAE; 7, minibody-MMAF. (F) Direct ELISA of minibodies and minibody-based FDCs; ganglioside GD2 was adsorbed on the plate, serial dilutions of scFv fragments were added to the wells; after incubation with HRP-labeled anti-human antibodies (1:6000), the reaction was developed by Ultra TMB-ELISA Substrate Solution. Data are represented as mean ± SEM. (G) Direct ELISA of scFv fragments and scFv-based FDCs; ganglioside GD2 was adsorbed on the plate, serial dilutions of scFv fragments were added to the wells; after incubation with HRP-labeled anti-FLAG antibodies (1:6000), the reaction was developed by Ultra TMB-ELISA Substrate Solution. Data are represented as mean ± SEM. scFv–scFv fragments 14.18, Mb–minibodies 14.18.
Figure 4
Figure 4
Cytotoxic activity of anti-GD2 FDCs in GD2-positive (B78-D14 and IMR-32) and GD2-negative (B16 and NGP-127) cell lines. Viability of cell lines analyzed by MTT assay following 72 h incubation with scFv-MMAE and minibody-MMAE (A,C), scFv-MMAF and minibody-MMAF (B,D), MMAE (E), or MMAF (F). scFv–scFv fragments 14.18, Mb–minibodies 14.18.
Figure 5
Figure 5
PI assay. (A) Analysis of DNA fragmentation (propidium iodide staining) of GD2-positive (B78-D14 and IMR-32) and GD2-negative (B16 and NGP-127) cell lines treated with FDCs (100 nM) for 48 h. Control—untreated control cells. The percentage of cells with fragmented DNA in hypodiploid peaks is shown for each histogram. (B) Cell cycle analysis (propidium iodide staining) of GD2-positive human neuroblastoma IMR-32 treated with FDC-MMAE (100 nM) for 48 h. Control—untreated control cells. scFv–scFv fragments 14.18, Mb–minibodies 14.18.
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