Construction of scFv-IgG Fc plasmid vectors

Nucleotide sequence of mouse scFv against GD2 clone 14G2a (a kind gift from Prof. Dr. Suradej Hongeng, Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand) was designed to be inserted into mammalian expression plasmid fusion-hIgG1-Fc2 tag vector (pFUSE-hIgG1-Fc2) using SnapGene software (version 1.1.3, GSL Biotech LLC, San Diego, CA, USA). The gene encoding scFv against GD2 was synthesized and amplified by polymerase chain reaction (PCR) using forward primer with EcoRI site; 5’-GAGGAGGAATTCggatattttgctgacccaaact-3’ and reverse primer with NcoI site; 5’-GAGGAGCCATGGctgaggagacggtgact-3’. The PCR products of gene encoding scFv against GD2 were cloned into vector pFUSE-hIgG1-Fc2 (InvivoGen, Toulouse, France). The genes were inserted between IL-2 signal sequence (IL2ss) and the hinge region of the hIgG1 Fc part (CH2-CH3 domain). The constructed vector was named pFuse-scFv-GD2-hIgG1-Fc2 vector. The constructed vectors were verified by DNA sequencing. The synthesis of nucleotide sequence of mouse scFv against GD2, amplification, cloning, and DNA sequencing processes were carried out by Bio Basic Inc. (Singapore).

To increase the vector yield for expression of GD2 specific scFv-IgG Fc antibody, the pFUSE-scFv-GD2-hIgG1-Fc2 vector was transformed into competent Escherichia coli (E. coli) DH5-α. E. coli carrying vectors were selected by spreading on Luria-Bertani (LB) agar containing 25 µg/mL of zeocin (cat# ant-zn-1p, InvivoGen). The single colonies were picked up and the GD2 gene was verified by PCR. A bacterial clone containing constructed plasmid vectors was cultured in LB broth containing 25 µg/mL of zeocin at large scale. The plasmid vectors were extracted using plasmid midi preparation kit (cat# K0481, Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania).

Production of scFv-IgG Fc antibody against GD2 in mammalian expression system

The generated pFuse-scFv-GD2-hIgG1-Fc2 vectors were transfected into human embryonic kidney 293T (HEK293T) cell line. In brief, cells (2 × 10⁵ cells) were plated into 6-well plate (cat# 3516, Corning Inc., NY, USA) and incubated at 37^°C, 5% CO₂ for 2 days. The plasmid vector (500 ng) was incubated with 3 µL of Lipofectamine® 2000 reagent (cat# 11668-027, Invitrogen, Carlsbad, CA, USA) at room temperature for 20 min and added into HEK293T cells. After transfection, the cells were incubated at 37^°C, 5% CO₂ for three days. The transfected cells were harvested and intracellular immunofluorescence was stained to determining protein expression.

For staining intracellular protein expression, cells were fixed with 4% paraformaldehyde (cat# A11313, Alfa Aesar, Ward Hill, MA, USA) and permeabilized with 0.1% saponin (cat# 97061-114, VWR Life Science, Radnor, PA, USA). Later, cells were blocked at the Fc receptor using 10% fetal bovine serum (FBS, cat# A5256701, Gibco, GrandIsland, NY, USA) and stained with 50 µL of Alexa Fluor 488 conjugated goat anti-hIgG antibody (1:500, cat# 109-545-098, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) or Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (1:250, cat# A11029 Life technology, Eugene, OR, USA) as a control. The expression of scFv-Fc antibody was determined by flow cytometry (Accuri^TM C6 flow cytometer, BD Biosciences, San Jose, CA, USA).

For selection of stable expression of HEK293T cells, the successfully transfected HEK293T cells were plated at 100 cells in 10% FBS-Dulbecco’s Modified Eagle Medium (DMEM, cat# 12800-017, Gibco) containing 100 µg/mL of zeocin drug in a 96-well plate (cat# 3599, Corning Inc). Cells that received vector harboring zeocin drug resistant gene were grown. Cells were screened for the expression of scFv-IgG Fc antibody against GD2 by intracellular immunofluorescence staining as described above.

Large-scale production and purification of scFv-IgG Fc antibody

The HEK293T clone expressing scFv-IgG Fc antibody against GD2 was seeded in T 75 cm² flask (cat# 156499, Thermo Scientific, Rockford, IL, USA) and grown in 10% FBS-DMEM containing 100 µg/mL zeocin. After cells reached 70% confluence, culture media was removed and gently washed with DMEM for 3 times. The media were then replaced with Chinese Hamster Ovary-Suspension-Serum Free Media II (CHO-S-SFM II, cat# 12052-098, Gibco). Cells were cultured in serum-free media containing 100 µg/mL zeocin at 37^°C in 5% CO₂ for 72 h. Culture supernatant was harvested. Two hundred milliliters of culture supernatant was subjected to purification of the scFv-IgG Fc antibody by affinity chromatography using HiTrap Protein G column (cat# 17-0404-03, GE Healthcare, Uppsala, Sweden). The concentration of purified antibody was measured by bicinchoninic acid assay (BCA) protein assay kit (cat# 23227, Thermo scientific). The purity and structure of purified antibodies were validated by SDS-PAGE and western blotting (WB) using horseradish peroxidase (HRP)-conjugated rabbit anti-human Ig antibodies (cat# P0212, Dako, Glostrup, Denmark). The binding activity of purified antibodies was also determined by immunofluorescence staining using GD2 expressing cells.

Validation of the binding activity of purified scFv-IgG Fc antibody against GD2

Human NB cell line, SH-SY5Y, which expressed GD2 on cell surface, was stained with purified scFv-Fc antibodies. The bound scFv-Fc antibodies were detected by Alexa Fluor 488-conjugated anti-hIgG antibody. The binding activity of antibodies was analyzed by flow cytometry.

ADCC assay

Peripheral blood was collected from healthy donors using sodium heparin as an anti-coagulant or buffy coat of healthy individuals obtained from The Reginal Blood Center X, Thai Red Cross Society, Chiang Mai, Thailand. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation.

For ADCC assay, SH-SY5Y was labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE, cat# 21888, Sigma-Aldrich, Saint Louis, MO, USA) and used as a target cell. CFSE labeled SH-SY5Y was incubated with or without purified GD2-scFv-IgG Fc antibodies and co-cultured with PBMCs (effector cells) at various effector cell: target cell (E:T) ratios or without effector cells. After being incubated for 4 h at 37^°C in a 5% CO₂ incubator, cells were harvested, washed twice with 200 µL of phosphate buffered saline (PBS, cat# 524650, EMD Millipore, Burlington, MA, USA), and then stained with 200 µL of propidium iodide (PI, cat# P4170, Sigma-Aldrich) solution at final concentration of 2 µg/mL. The percentage of dead target cells (CFSE⁺PI⁺) was determined using an Accuri C6 flow cytometer (BD Biosciences).

ADCP assay

Human monocyte-enriched PBMCs were prepared from PBMCs by Percoll gradient centrifugation. Briefly, PBMCs were diluted with PBS at a concentration of 1 × 10⁷ cells/mL. Then, diluted PBMCs were overlaid on Percoll solution (cat# 17-0891-02, GE Healthcare) and centrifugated at 25^°C, 895 g, for 40 min. After centrifugation, the monocyte-enriched PBMCs were collected and washed. Thereafter, the monocyte-enriched PBMCs were determined for a percentage of monocyte using flow cytometry. The obtained monocyte-enriched PBMCs were plated in 24-well plates (cat# 3524, Corning Inc) at the number of monocytes of 5 × 10⁵ cells per well and incubated in a 5% CO₂ incubator for 2 h. Then, non-adherent cells were removed by washing with PBS and macrophage colony-stimulating factor (M-CSF, cat# 11343113 Immuno Tools, Friesoythe, Germany), 50 ng/mL, was added and incubated for six days to induce macrophage differentiation. Upon culturing, 50 ng/mL M-CSF was added every two days.

For ADCP assay, SH-SY5Y cells were labeled with CFSE (molecular probes). The CFSE labeled SH-SY5Y cells were incubated with or without purified GD2-scFv-IgG Fc antibodies. Monocyte-derived macrophages (effector cells) were added into CFSE labeled SH-SY5Y cells (target cells) and cultured in a 96-well flat plate at various E:T ratios. The co-cultured cells were incubated in a humidified atmosphere of 5% CO₂ at 37^°C for 30 min. After cultivation, cells were harvested and stained with PerCP-conjugated anti-CD14 mAb (cat# 325632, BioLegend, San Diego, CA, USA). Then, the cells were analyzed by flow cytometry for assessing the percentage of phagocytosed cells (CFSE⁺CD14⁺ cells).

Statistical analysis

All statistical analyses were performed using GraphPad Prism version 9.2.0 (GraphPad Software, CA, USA). One-way analysis of variance (ANOVA) was used as indicated in the figure legends. Statistical significance was accepted at a P value < 0.05.

Construction of scFv-IgG Fc plasmid vectors

In this study, we aimed to generate a chimeric antibody, which consists of mouse scFv against GD2 and hIgG1 Fc domain. This chimeric antibody was designed to lack heavy chain CH1 domain; thus, having a smaller size than the natural intact antibody. In order to obtain the chimeric antibody, the vector containing scFv-GD2 fused with hIgG1 Fc part was constructed. The graphical map of the constructed plasmid vector is shown in Figure 1A. The inserted genes were verified by DNA sequencing and showed 100% identity match to the original sequence. After transformation and dug selection, five single bacterial colonies were picked up and verified gene encoding scFv GD2. The PCR products at 741 base pairs (bp), as expected, were observed in all selected bacterial clones (Figure 1B). The plasmid vectors were used to express further.

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Figure 1. 

Graphical map of the constructed plasmid vector and PCR analysis of GD2 scFv gene. (A) Graphical map of pFUSE-hIgG1-Fc2 vector created by SnapGene software is shown. Nucleotide sequence of mouse scFv against GD2 was cloned into the vector at EcoRI and NcoI sites before the hinge region of the hIgG1 Fc part. The thin orange arrow indicates an open reading frame, which starts at the codon inside IL2ss and stops at the end of the Fc portion. Zeocin drug resistance gene (Zeo) was used for selection; (B) after cloning step, the constructed vector was transformed into DH5-α E. coli and selected by zeocin drug. Five bacterial colonies (as indicated) were picked up and grown. PCR was performed to detect the inserted GD2 scFv gene inside the constructed plasmid vectors. Bacteria clone number 1 was chosen for plasmid extraction. Standard DNA markers (M) in base pairs are shown on the left. Positive and negative controls are also demonstrated. Neg: negative; Pos: positive

Production of scFv-IgG Fc antibody against GD2 in mammalian cells

In order to establish stable-expressing mammalian cell line mouse scFv-hIgG1 Fc antibody against GD2, human HEK293T cell line was used as a host cell. Three days after transfection, transfected cells were intracellular immunofluorescence stained. The flow cytometric analysis showed that approximately 50% of transfected cells were positive with anti-hIgG antibody, but negative in anti-mouse IgG antibody control (Figure 2A).

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Figure 2. 

Determination of scFv-IgG Fc expression in transfected HEK293T cells. (A) Human HEK293T cell line was transfected with pFUSE-scFv-GD2-hIgG1-Fc2 vector and intracellular stained with Alexa Fluor 488-conjugated goat anti-hIgG antibody (GαhIgG-A488), Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (GαmIgG-A488), or without antibodies (no Abs); (B) transfected HEK293T cells were cultured in the presence of zeocin. The transfected cells and un-transfected cells were intracellularly stained with Alexa Fluor 488-conjugated goat anti-hIgG antibody (Blue color) or Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (red color). Overlay histograms are shown

Then, six transfected HEK293T containing wells were harvested to determine the expression of the scFv-IgG Fc antibody. All selected cells showed positive reactivity with Alexa Fluor 488-conjugated anti-hIgG antibody, but at different expression levels (Figure 2B). Reactivity with Alexa Fluor 488-conjugated anti-mouse IgG antibody was not detected. Un-transfected cells were also included in the experiment and showed negative reactivity with all Alexa Fluor 488-conjugated antibodies. These results indicated that stable human HEK293T cells expressing scFv-hIgG Fc antibodies were established. The transfected HEK293T well named C4, which had the highest expression level (100% positive), was selected to produce scFv-IgG Fc antibodies.

Purification and large-scale production of scFv-IgG Fc against GD2

The scFv-IgG Fc in the serum-free culture supernatant was purified by Protein G column. The purity and structure of purified scFv-IgG Fc were investigated. As shown in Figure 3A, by SDS-PAGE, the size of scFv-IgG Fc antibody in reducing conditions was monomeric at approximately 60 kilodalton (kDa). In non-reducing conditions, the majority band at 120 kDa, which was expected to be dimeric form, was observed. Unpredictable bands at 180 kDa and higher were also found. These bands were likely trimeric and tetrameric scFv-IgG Fc antibodies. The bands in SDS-PAGE were closely related to those observed in WB using anti-hIgG antibody.

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Figure 3. 

Validation of the purity, structure, and activity of purified GD2 scFv-hIgG Fc antibody. (A) Purified GD2 scFv-IgG Fc antibody was resolved in 10% SDS-PAGE under reducing (R) conditions and non-reducing (NR) conditions. Protein bands were stained with Coomassie blue. WB analysis was performed using HRP conjugated anti-hIgG antibody. The molecular weight (kDa) of protein markers is shown on the left; (B) human NB cell line (SH-SY5Y cell) was stained with purified GD2 scFv-IgG Fc antibody (blue color) or irrelevant IgG Fc fusion control protein (red color) followed by Alexa Fluor 488-conjugated goat anti-hIgG antibody and analyzed by flow cytometry. The overlay histogram is shown

Furthermore, the binding activity of the purified proteins was determined by staining with GD2 expressing SH-SY5Y cells [21]. The purified GD2 scFv-IgG Fc antibody showed positive reactivity to SH-SY5Y cells (Figure 3B). These results indicated that the scFv-hIgG Fc fusion antibody against GD2 could be produced and secreted by HEK293T cells. The purified scFv-IgG Fc antibody could be prepared from the HEK293T culture supernatant.

Anti-tumor activities of scFv-IgG Fc antibody against GD2

The GD2 expression cell line SH-SY5Y was used as target cell for determining antitumor activities. In ADCC assay, scFv-IgG Fc antibody at 10 µg/mL and 20 µg/mL significantly killed GD2 expressing SH-SY5Y cells [22]. This effect was observed at all tested E:T ratios (10:1, 20:1, 40:1) and compared with zero scFv-IgG Fc antibodies (0 µg/mL) as demonstrated in Figure 4A.

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Figure 4. 

ADCC and ADCP activities mediated by the generated scFv-IgG Fc antibody against GD2. (A) CFSE labeled SH-SY5Y cells were incubated with or without the generated scFv-IgG Fc antibody at 10 µg/mL and 20 µg/mL and PBMCs (n = 6) were added at the indicated E:T ratio. The dead target cells (CFSE⁺PI⁺) were analyzed by flow cytometry. Percent cytotoxicity was calculated as [dead target cells (%) − spontaneous death (%)]/(100 − spontaneous death); (B) for ADCP, macrophages (n = 4) were used instead of PBMCs and the percentage of phagocytosed target cells (CFSE⁺ CD14⁺ cells) is shown. Data normality was assessed using the Shapiro–Wilk test. All data sets were normally distributed except ADCP at E:T ratio of 10:1 in condition with GD2 scFv-IgG Fc 20 μg/mL. The values are shown as mean ± SD. One-way ANOVA followed by Tukey’s multiple comparison test was used for comparison

In addition, the activity of the generated scFv-IgG Fc antibody in ADCP was determined. As shown in Figure 4B, macrophages could phagocytose SH-SY5Y upon being sensitized with the generated scFv-IgG Fc antibody at 10 µg/mL and 20 µg/mL. This was statistically significant at an E:T ratio of 10:1. At lower E:T ratios (1:1 and 5:1), an increasing trend of ADCP could be observed but it was not statistically significant.

Taken together, the results demonstrated that the generated scFv-IgG Fc antibody against GD2 has anti-tumor activities in the induction of both ADCC and ADCP.