Potential of an anti-bevacizumab idiotype scFv DNA-based immunization to elicit VEGF-binding antibody response

Cell culture

HEK 293 T cells (ATCC, Manassas, VA, USA) were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA), and B16-F10 cells (ATCC) in RPMI-1640 (Thermo Fisher Scientific). Both media were supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific). 10.D7 hybridoma cells were maintained in RPMI-1640 containing 10% FBS and 50 μM 2-mercaptoethanol. All cells were cultured at 37 °C in a humidified atmosphere with 5% CO2 and were regularly checked for mycoplasma contamination.

Construction of 10.D7 scFv gene

Total RNA was isolated from 106 10.D7 hybridoma cells with TRIzol (Thermo Fisher Scientific). The cDNA of VH and VL were synthesized with oligos A and C, respectively, using High-capacity cDNA reverse transcription kit (Applied Biosystems, Waltham, MA, USA) (Table 1), as previously described [14]. Briefly, a solution containing 1 μg RNA in 5 μl of sterile water, 1 μl of 10 ×  buffer, 0.4 μl dNTP at 100 mM, 1 μl 10x random primer, 0.5 μl MultiScribe reverse transcriptase (Thermo Fisher Scientific) at 50 U/µl, and 2.1 µl of sterile water, totaling a 10-µl final volume, was prepared. The reaction was conducted in a thermocycler at 25 °C for 10 min, 37 °C for two hours, and 85 °C for 5 min.

Table 1 Sequence of primers used for the isolation of VH and VL from 10.D7 mAb.

The DNA fragments encoding for the VH and VL domains of 10.D7 mAb were then amplified by PCR using A + B and C + D oligo pairs (1 μM each), respectively (Table 1). PCR work solution was prepared according to the manufacturer’s instructions: 5 µl of cDNA diluted 1:100 in sterile water, 0.25 µl of sense primer and 0.25 µl of antisense, 12.5 µl of Master Mix (Thermo Fisher Scientific), and 7 µl of sterile water. The antibody heavy chain amplification conditions were: 94 °C for 3 min; 40 cycles at 94 °C for 1 min, 62 °C for 30 s, and 72 °C for 1 min; and a final incubation of 5 min at 72 °C. The light chain amplification conditions were: 94 °C for 3 min; 40 cycles at 94 °C for 1 min, 60 °C for 30 s and 72 °C for 1 min; 5 min at 72 °C. The products were analyzed by electrophoresis on a 1% agarose gel in TAE (40 mM Tris-acetate, 1 mM EDTA). The DNA bands were detected with SYBR Safe DNA stain (Thermo Fisher Scientific) on a Gel Doc EZ Imager system (Bio-Rad, Hercules, CA, USA). DNA fragments corresponding to VH and VL were purified from the agarose gel with Geneclean (MP Biomedicals, Solon, OH, USA) and further analyzed using BigDye Terminator Ready (Thermo Fisher Scientific) on a genetic analyzer sequencer. The 10.D7 scFv was designed in the VH-linker-VL orientation, using (Gly4Ser)3 as a linker [15], and was synthesized by a gene service (GenScript, Piscataway, NJ, USA) into a pcDNA3.1 expression vector containing the EcoRI and HindIII restriction sites. To confirm the final product, the plasmid vector was digested with EcoRI and HindIII, and the insert englobing the scFv gene was sequenced.

Animals

6–8-week-old male C57Bl/6 mice were obtained from “Centro de Desenvolvimento de Modelos Experimentais para Medicina e Biologia” (CEDEME/UNIFESP, Brazil) animal facility and maintained with a 12:12 h light:dark cycle and water ad libitum. Experiments were performed after anesthetic induction with ketamine (100 mg/kg; Syntec, Brazil) and xylazine (10 mg/kg; Syntec). Mice were euthanized by anesthetic overdose. All procedures were in accordance with the guidelines of the US National Research Council for care and use of laboratory animals [16].

Enzyme immunoassay (ELISA) studies

Cell-bound ELISA was performed to assess whether 10.D7 scFv gene-transfected cells are recognized by bevacizumab. For that, HEK 293 T cells were transfected with pcDNA3.1(+) containing or not the scFv gene, using the Superfect Transfection Reagent (Qiagen, Germany) following the manufacturer’s instructions. Transfected cells were plated on a 96-well plate (2 × 104 cells/well) and fixed with 0.05% glutaraldehyde (Sigma). Endogenous peroxidase was neutralized with 1% H2O2 (Sigma). After blocking with 1% bovine serum albumin (BSA; Sigma) in phosphate-buffered saline (PBS), wells were incubated for 1 h at 37 °C in the presence or absence of 1 µg/ml bevacizumab (Avastin; Roche, Switzerland). After washes with PBS containing 0.1% BSA and 0.05% Tween 20 (Sigma), wells were incubated with biotin-conjugated anti-human IgG (Sigma).

Indirect ELISA was carried out to detect VEGF-binding antibodies in serum samples. 96-well plates were coated with 50 ng/ml recombinant VEGF (Thermo Fisher Scientific), blocked with 1% BSA in PBS, and incubated overnight at 4 °C with bevacizumab (1 μg/ml) or serum samples from animals immunized with pcDNA3.1 or pcDNA3.1-scFv10.D7. Sera were obtained by bleeding the retro-orbital plexus of ketamine/xylazine-anesthetized mice 15 days after the last immunization dose. After washes, wells were incubated with anti-mouse or anti-human IgG secondary antibody (Thermo Fisher Scientific), both conjugated with biotin.

In both assays, after incubation with peroxidase-streptavidin (Sigma) for 30 min at room temperature, the wells were developed with o-phenylenediamine substrate (Sigma). The reaction was stopped with 4 N H2SO4 (Merck, Germany) and absorbance values were read at 490 nm.

Mouse immunization

For gene immunization studies, animals were intramuscularly injected with DNA plasmid (50 µg in each quadriceps) four times at 15-day intervals. Immediately after each administration, six electric pulses (100 V; 40 milliseconds per pulse; 1-second interval) were applied through 10-mm tweezer electrodes (T820-BTX; Genetronics, San Diego, CA, USA) positioned close to the injection sites [17, 18].

In vivo tumor growth

The potential antitumor effect of immunization with 10.D7 scFv gene was assessed in a subcutaneous tumor model. For that, on the 15th day after the last immunization dose, mice were subcutaneously challenged with 5 × 105 B16-F10 cells in the left flank. Tumor growth was monitored daily with a caliper. Two groups: pcDNA3.1-scFv10.D7-immunized, and pcDNA3.1-immunized (empty vector control) mice. Tumor volume was calculated considering the equation: Volume = (large diameter) × (small diameter)2 × 0.52 [19].

Statistical analyses

Statistical analyses were performed with GraphPad Prism, version 7.0 (GraphPad Software, La Jolla, CA, USA). Data were analyzed by Student’s t-test, when two groups were compared, or by one-way ANOVA followed by Bonferroni’s post-test, in the case of multiple comparisons, as indicated in the figure legends. The differences were considered significant when p < 0.05.

留言 (0)

沒有登入
gif