2.1 Construction of the rhHER2-mAb expression vector
The eukaryotic expression vector pCDNA3.4 was designed and built in this study utilizing the TGE system with mammalian cell HEK293F as the host cell. The variable region sequence obtained via screening of the laboratory phage library was spliced and cloned into the pCDNA3.4 plasmid to construct the rhHER2-mAb expression vector (Fig1A,1B). The primers were designed according to the gene sequence shown in Table 1. NheI and Hind Ⅲ restriction sites that are homologous to the sequences of the expression vector pCDNA 3.4 were introduced at the 5'-end of the primer. Overlap PCR was used to splice the gene fragments of light and heavy chains. The light chain was obtained by overlap PCR using primers 1, 2, 3, and 4, while the heavy chain was obtained by primers 1, 5, 6, and 7. The pCDNA3.4 plasmid double-digested with NheⅠand HindⅢ endonuclease were cut and recovered and connected with the Overlap PCR product using a homologous recombination kit. The homologous recombination product was then transformed into Escherichia coli DH5a. The positive clones are selected for culture and sent for sequencing, and mutation-free clones are selected for mass sampling of non-endotoxic plasmid. The result of 0.8% agarose gel electrophoresis showed that light and heavy chain sizes were 708 bp and 1413 bp, respectively (Fig 1C). The final sequencing results confirmed that the expression vectors, namely pCDNA3.4-Anti-Her2 Light chain and pCDNA3.4-Anti-Her2 Heavy chain, were successfully constructed.
2.2 Transient gene expression of rhHer2-mAb
Conditions of TGE, such as DNA/PEI and heavy/light chain plasmid ratios, were optimized using HEK293F as the host cell and pCDNA3.4 as the eukaryotic expression plasmid.
2.2.1 Optimization of DNA/PEI ratio
The heavy and light chain plasmids were extracted using endotoxin-free plasmid extraction kit before filtration with the 0.22 mm filter; HEK293F cells were inoculated into Freestyle 293 medium and placed in a constant temperature shaker at 37°C, 5% CO2, and 125 rpm. The cell density and viability were maintained at between 1-2×106 cells/ml and above 95%, respectively. Twenty-four hours before transfection, cells were centrifuged and resuspended in fresh medium before the expansion of cells to a density of 0.6×106 cells/ml. On the day of transfection, the cell concentration was measured before identification of cell types. Cell density was then adjusted to 1-1.2×106 cells/ml and 0.6 ml of the cells was added to a 12-well plate. Based on the predetermined condition that 0.6 mg of plasmid is needed to transfect every 106 cells, the light/heavy chain plasmids were matched with a mass ratio of 1:1 and DNA and PEI were mixed with mass ratios of 1:1, 1:2, 1:3, 1:4 and 1:5, respectively and inoculated at room temperature for 25 min before being added to the cell suspension to continue culturing. The supernatant of the cell fermentation broth was collected by centrifugation 48 h later for subsequent cell identifications.
Meanwhile, the green fluorescent protein (GFP) and control plasmids were mixed with PEI using the same gradient ratio for transfection. After 48 h, 1 × 106 cells were collected from each group, centrifuged at 300 × g at 4°C for 5 min, washed twice with 1 mL of 2% FBS-PBS buffer before resuspension to 100 ml, and passed through a flow cytometer. The transfection efficiency was detected by the FITC channel. The results showed that the transfection efficiencies of DNA/PEI ratio from 1:1 to 1:5 were 5.02%, 12.38%, 35.99%, 90.22% and 92.34%(Fig 2A), respectively, suggesting that the transfection efficiency can exceed 90% when the DNA/PEI ratio is 1:4 and 1:5. In terms of cost, a DNA/PEI ratio of 1:4 was determined for subsequent transfection steps with a transfection efficiency of 90.22%.
2.2.2 Optimization of light/heavy chain to plasmid ratio
Based on the aforementioned transfection method using different co-transfection ratios of light/heavy chain plasmid (Table 2), DNA and PEI were mixed at a mass ratio of 1:4 and inoculated at room temperature for 25 minutes before being added to the cell suspension for further culture. After 48 h, the supernatant of the cell fluid was collected by centrifugation for subsequent analysis.
Western blotting was used to verify whether rhHER2-mAb was correctly assembled. Samples were isolated by 10% SDS-PAGE gel electrophoresis and transferred to PVDF membrane before incubation overnight at 4°C with TBST containing 5% skimmed milk powder. The next day, the PVDF membrane was incubated with anti-human IgG (H+L) secondary antibody before rinsing with TBST for 3 times, each lasting for 5 min each time. Western blotting films were then developed. The impacts of DNA/PEI and light/heavy chain plasmid ratios on the transient expression of rhHER2-mAb were compared. Results showed that the protein yield is higher when DNA/PEI ratio was 1:4 and the light/heavy chain ratio was 2:1. Thus, the optimal ratios were adopted for subsequent large-scale expression of rhHer2-mAb.(Fig 2B,2C)
Therefore, based on the aforementioned transfection method, 250 ml HEK293F cells were transfected with a light-to-heavy chain ratio of 2:1 and a DNA/PEI ratio of 1:4. When the cell viability is lower than 60%, cell fermentation supernatant was collected by centrifugation at 800×g for 20 min at 4°C before undergoing a second centrifugation at 8000 rpm for 30 min and passing through a 0.22 mm filter for protein purification.
2.3 Purification and identification of rhHER2-mAb r
rProteinA has high specificity and affinity against the Fc segment at the stable region of the heavy chain, as well as has good versatility for antibody purification. All purification reagents are filtered with a 0.22 mm membrane, and cell fermentation broth supernatant was subsequently filtered through a 0.45 mm membrane. Specific processes of rProteinA affinity chromatography was as follows: The AKTA system was rinsed with 20% ethanol and ultrapure water, and the column was connected to Position 1 of the AKTA purifier. Binding buffer (pH 7.2, 20 mM phosphate buffer, 150 mM NaCl) was used to equilibrate the column and the sample was loaded at a speed of 1 ml/min. After loading, the column was equilibrated again using binding buffer before non-specific impurities were removed by using the Wash buffer (pH 5.0, 100 mM citrate buffer). The protein was eluted with elution buffer (100 mmol/L citric acid buffer, pH 3.0). The eluted sample was then adjusted to neutral pH with neutralization buffer (1 mol/L Tris-HCl buffer, pH 8.5), and the column was eventually washed with 0.5M NaOH and 20% ethanol. The obtained product antibody was dialyzed into PBS (pH 7.2), filtered and sterilized, and stored in a -80°C refrigerator.
Results of rProtein A affinity chromatography was shown in( Fig 3A)The upper column was cleaned with citric acid washing solution (pH 5.0) and replacement solution (pH 3.0) for purified products with no impurities eluted. rhHER2-mAb can be eluted in large scale using the replacement solution, presenting with a unified peak pattern. Finally, unintended products were eluted using NaOH. SDS-PAGE gel electrophoresis results showed that the molecular weight of rhHER2 antibody was greater than 180 kDa, and the bands of interest for the reduced sample was clear. The heavy and light chains’ molecular weights were measured to be 55 and 25 kDa, respectively (Fig 3B). The purified sample was dialyzed, concentrated and underwent protein quantification. The protein concentration was determined to be 7.3824 mg/ml, with a yield of about 102.45 mg/l.
2.4 Affinity detection of rhHER2-mAb
The affinity of rhHER2-mAb is determined by bio-layer interferometry (BLI) using OctetRed96 system with the following protocol: The streptomycin affinity (SA) sensor was pre-soaked in PBS buffer for 15 minutes and the program was set for antibody affinity detection. The SA sensor was then infiltrated into the equilibrium solution (0.02% PBST containing 0.05% BSA) for 180 s as benchmark. The sensor was infiltrated into the solidification solution (5 mg/ml biotin-labeled Her2 protein) for 600 s. After the signal was stable, the sensor was soaked in the fresh equilibrium solution for 180 s, before being soaked to solutions with rhHER2-mAb concentrations of 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.125 nM, as well as PBS control, for 450 s. After the signal was stable, the sensor was immersed in PBS and dissociated for 1800 s. The above experiments were carried out at 30°C with a rotating speed of 1000 r/min, and the PBS (pH 7.4) used in all steps was of the same batch. Data analysis was performed with the OctetRed96 analysis software. After subtracting the data of PBS control group from the that of the experimental group, the curve of binding and dissociation was depicted (Fig 4). The binding rate constant (kon) was calculated as 2.69×105 1/Ms and the dissociation rate constant (koff) was 9.43× 10-7 1/s. The equilibrium dissociation constant, i.e. the antibody affinity (KD) was 2.35×10-13 M with an R2 of 0.9538.
2.5 Biological activity of rhHER2-mAb
2.5.1 Selection of HER2-high-expression cell lines
In terms of HER2 antigen over-expression on the cell surface, the SK-OV-3, OVCAR-3 and A2780 cell lines were selected as the cells of interest for the investigation of rhHER2-mAb bioactivity; Macoy'5A+10% FBS, RPMI 1640+10%FBS, and DMEM+10%FBS complete medium were prepared respectively to culture SK-OV-3, OVCAR-3 and A2780 cells in a 5% CO2 incubator at 37°C. Cell surface expression level of Her2 on our panel of cancer cells was tested using flow cytometry. （Fig 5A)
2.5.2 Detection of rhHer2-mAb bioactivity
In this study, the LDH cytotoxicity assay was used to detect the ADCC level mediated by rhHer2-mAb, in which lactate dehydrogenase (LDH) was quantitatively measured by CytoTox 96® Cytotoxicity Detection Kit. According to manufacturer’s instructions, the experimental and control groups, including effector cell spontaneous LDH release, target cell spontaneous LDH release, target cell maximum LDH release, volume correction control, and culture medium-only control groups, were set, with 3 replicates per group. Three strains of SK-OV-3, OVCAR-3 and A2780 cells respectively in growth phase were collected, centrifuged at 1000 rpm, resuspended in RPMI 1640 + 5% inactivated serum, sampled and counted, before subsequently plated in 96 well cell culture plates at a density of 8000 cells/well at 100 ml per well. A490 was detected by a multifunctional microplate reader after sequentially adding LDH matrix liquid with stop solution according to manufacturer’s instructions. Cytotoxicity ratios were calculated according to the formula, data were processed by GraphPad Prism 5, and the four-parameter analysis was adopted to fit a sigmoidal dose-dependent curve. The results showed that rhHER2-mAb exerted ADCC against all three cell lines at a PBMC ratio of 1:50, and the IC50 values of rhHER2-mAb against SK-OV-3, OVCAR-3, and A2780 cell lines were 6.06, 96.49, and 21.60 ng/ml, respectively.（Fig 5B)
2.6 rhHER2-mAb pharmacokinetics and anti-tumor animal experiments
The experimental protocol was approved by the Institutional Animal Use Committee of Changhai Hospital. Blood samples were collected separately and diluted 2000 folds for quantitative determination by enzyme-linked immunosorbent assay (ELISA) standard procedures. Specifically, goat anti-human IgG-kappa specific antibodies were incubated overnight on a 96-well high-affinity protein binding plate. rhHER2-mAB was captured using anti-human IgG-kappa antibody, and the signal was then amplified with goat anti-human FcHRP antibody. TMB solution was subsequently added to measure OD450 absorption. Finally, PK parameters were analyzed using a non-compartmental analysis model, and the half-life of rhHER2-mAb in vivo was found to be approximately 14 days (Fig 6A).
Antitumor efficacy evaluation of rhHER2-mAb mice were performed as follows: Fifteen female Balb/c mice (6-8 weeks old) were randomly divided into 3 groups, namely experimental rhHER2 group, Herceptin group and PBS group. Each mouse had subcutaneous implantation (on the right flank area) of 100ul of PBMC and SK-OV-3 cells, respectively, with an E/T ratio of 1:4. For each mouse, 5´106 SK-OV-3 cells cells were mixed with 2´106 inactivated PBMCs in a volume of 100 μL. On the second day, mice in each group were injected in situ with 10 mg/kg rhHER2 , Herceptin and PBS as blank controls, followed by weekly treatment via in situ injection of rhHER2-mAb at 10 mg/kg doses. The tumor size was measured with a vernier caliper every four days.
The anti-tumor bioactivity of rhHER2-mAb was evaluated on a xenograft NOD/SCID mouse model. Comparing the rhHER2-mAb, Herceptin and PBS groups after 60 days, results showed that rhHER2-mAb and Herceptin inhibited the growth of SK-OV-3 tumor. Further comparison between rhHER2-mAb and Herceptin was conducted and demonstrated that although both of them protected the mice from death at all, rhHER2-mAB was significantly more effective in inhibiting the growth of SK-OV-3 tumors after 60 days. Meanwhile, observations of subcutaneously denuded tumors found that mice in the PBS group had a more irregular tumor shape in addition to a larger size. (Fig 6B.6C.6D).