Jurkat/NFAT-luc/FcγRⅢa as effector cells for the ADCC RGA assay

The ADCC effector cells Jurkat/NFAT-luc/FcγRⅢa were developed as described previously.¹⁴ Briefly, Jurkat T cells were first transfected by electroporation with plasmid pcDNA3.1, which expresses the human FcγRⅢa, and G418-resistant cell clones were screened by fluorescence-activated cell sorting for FcγRⅢa expression. The selected cell clone was then transfected with plasmid pcDNA3.1-puro that encodes the luciferase reporter under the control of the NFAT promoter, and puromycin-resistant cell clones were screened by phorbol 12-myristate 13-acetate (PMA) and ionomycin co-stimulation for high induction of relative luciferase units (RLU). The dual-resistant Jurkat/NFAT-luc/FcγRⅢa cell clone was confirmed by determining the ADCC activity of the anti-HER2 antibody against SK-BR-3 cells.

Generation of the CVS-N2c-293 cell line as target cells

The HEK293 cells were transfected with plasmid pcDNA3.1 that encodes the CVS-N2c G protein¹⁷ and cultured in selective medium (complete medium with 500 µg/mL G418). G418-resistant single cell clones were obtained by limited dilutions, then screened for clones that highly expressed luciferase and low background by co-culturing with Jurkat/NFAT-luc/FcγRⅢa effector cells and gradient dilutions of anti-RABV mAb-1. mAb-1 is a human IgG1 antibody that targets the antigenic site III of RABV G. The expression of the RABV G protein in the selected cell clone was determined by flow cytometry and named as the CVS-N2c-293 cell line.

ADCC RGA

CVS-N2c-293 cells were seeded into white-bottom 96-well plates at a density of 30,000 cells per well and incubated at 37°C and 5% CO₂ for 16–20 hr. After incubation, the supernatant in each well was removed, and 50 μL of three-fold serially diluted anti-RABV G mAb (20–0.019 µg/mL) in dilution buffer (RPMI-1640, 0.5% BSA) was added into the plate. Then, 240,000 cells per well of Jurkat/NFAT-luc/FcγRⅢa cells¹⁴ were added and incubated at 37°C in a humidified atmosphere with 5% CO₂. After 6 hr of incubation, 100 μL Bright-Glo luciferase assay reagent (Promega) was added into each well, and RLUs were recorded with a plate reader (Envision, PerkinElmer).

Anti-RABV G mAbs and vaccine-induced antisera

Seven anti-RABV neutralizing mAbs targeting different epitopes were included, and mAb-1 was used for method optimization. Both mAb-1 and mAb-2 target antigenic site III, mAb-3 binds to the discontinuous conserved residues, mAb-4 targets antigenic site I, whereas mAb-5 targets antigenic site II. Bispecific antibody (bsAb), bsAb-1, and -2 target antigenic sites I and II and their Fc regions were mutationally optimized to enhance binding affinity to FcγRⅢa. mAb-2 and -3 are components of an anti-RABV cocktail that are in a phase III clinical trial, and mAb-4 has been approved for clinical use in China.

Four anti-RABV sera, namely YRS, DN, TX, and PD, were obtained by immunization of vaccine comprising the Flury-LEP strain. A commercial HRIG was obtained from Tonrol Bio-Pharmaceutical Co., Ltd (Hefei, China). The 37.0 IU/mL national standard (NS) for anti-RIG was established and calibrated by the National Institutes for Food and Drug Control (NIFDC).

Pseudovirus-based neutralization assays

Pseudotyped RABV preparation, titration, and neutralization assays were performed as described previously.¹⁷ The pseudovirus-based neutralization assay was measured as a reduction in luciferase expression after a single-round infection of 293 T cells. Briefly, 100 μL serial diluted antisera or mAbs was added into 96-well plates, and 50 μL pseudoviruses (32,000 tissue culture infectious dose [TCID₅₀]/mL) were added, then incubated at 37°C for 1 hr. After incubation, 100 μL of 293 T cells (4 × 10⁵ cells/mL) was added in the presence of 10 μg/mL DEAE-dextran in a total volume of 250 μL, then incubated at 37°C in a humidified atmosphere with 5% CO₂. The luciferase activity was measured after 48 hr of incubation.

Method validation of established ADCC RGA assay

According to the ICH Q2 (R1) guideline, the specificity, accuracy, precision, and linearity of the ADCC assay were systematically validated. Anti-RABV G mAb-1 was diluted to seven concentrations of 5, 10, 15, 20, 25, 30, and 35 μg/mL, and another prepared sample (20 μg/mL) was used as the reference. The nominal relative bioactivity of anti-RABV mAb-1 dilutions should be 25%, 50%, 75%, 100%, 125%, 150%, and 175%, respectively. The ratio of measured relative bioactivity versus the nominal value indicated the accuracy of the RGA, and the linearity was demonstrated by plotting the measured versus the expected relative bioactivity. Repeatability and intermediate precision were assessed by testing the 100% level samples (20 μg/mL) thrice on three different days by three operators independently.

Data analysis

A four-parameter model was used to fit the dose–response curve between antibody concentrations (log₁₀) and RLU by Prism software 5.0. The relative ADCC potency was calculated by dividing the half-maximal effective concentration (EC₅₀) of the reference by EC₅₀ of the tested sample. The Reed–Muench method was used to calculate the half-maximal inhibitory dilution (ID₅₀) of the antisera.

Generation of the CVS-N2c-293 cell line as target cell for the ADCC assay

The CVS-N2c-293 cell line that stably expresses the G protein of the CVS-N2c strain was generated by transfecting HEK293 cells with plasmid pcDNA3.1 that encodes the CVS-N2c G protein, followed by selection with G418. Limited dilutions were applied to obtain monoclonal cell clones. Finally, 10 clones were obtained and isolated for effective induction of luciferase expression and a full dose–response curve using Jurkat/NFAT-luc/FcγRⅢa as the effector cell for the ADCC assay. Clone 197, which showed the highest induced luminescence intensity and signal-to-noise ratio (S/N) value (Figure 1b), was selected as the target cell clone for further method optimization. The expression of the G protein of the CVS-N2c strain on the cell surface of clone 197 was also confirmed by flow cytometry analysis (Figure 1c).

Optimization of the ADCC RGA

Key parameters of the ADCC assay, including the density of the target cells, the ratio of effector cells to target cells (E:T ratio), range of mAb working concentration, and induction time, were further optimized. The density of the target cells (CVS-N2c-293 cell) was adjusted to 0.5 × 10⁴, 1 × 10⁴, 3 × 10⁴, and 5 × 10⁴ cells/well (Figure 2a–d), and the density of effector cells (Jurkat/NFAT-luc/FcγRⅢa cell) was correspondingly adjusted at E:T ratios of 1:1, 2:1, 4:1, and 8:1. The dose–response curve was well fitted, and a high S/N was obtained when the density of CVS-N2c-293 cell was 3 × 10⁴ cells/well and the E:T ratio was 8:1 (Figure 2c and Table 1). Then, the anti-RABV mAb was pre-diluted to 20 μg/mL (working concentration: 10 μg/mL), and serial dilutions consisting of 10 concentration points with different dilution folds (2-, 3-, 4-, or 5-fold) were prepared. The optimal range of working concentration was the three fold serial dilution, and the concentration range was 0.152–10,000 ng/mL (Figure 2e). The induction time was optimized by co-culturing the target and effector cells for 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 22 hr, and 24 hr, and we observed the highest S/N at 6 hr (Figure 2f). Taken together, key parameters were optimized to obtain a well-fitted dose–response curve with a high S/N to determine the ADCC activity of anti-RABV G antibodies.

Validation of the ADCC RGA

According to ICH Q2 (R1) guideline, the specificity, accuracy, linearity, and precision of the ADCC assay were validated. We found mAbs that target non-RABV epitopes, including the trastuzumab (anti-HER2 mAb), pembrolizumab (anti-PD-1 mAb), bevacizumab (anti-VEGF mAb), infliximab (anti-TNFα mAb), rituximab (anti-CD20 mAb), and denosumab (anti-RANKL mAb) could not induce luciferase expression or generate a dose–response curve in our established ADCC assay, where only the anti-RABV G mAb showed a specific signal curve (Figure 2a). These data indicated that our established ADCC assay is highly specific (Figure 3).

mAb-1 was pre-diluted to seven samples at different concentrations with an expected potency of 25%, 50%, 75%, 100%, 125%, 150%, and 175%. The ADCC activity of all seven samples were measured, and the relative bioactivities were calculated using another independent 100% level sample as the reference. The recoveries of the seven samples with different bioactivity levels were within 100 ± 15% (Table 2), which indicated good accuracy. Linearity was demonstrated by plotting the measured versus the expected relative bioactivity with a fitted R² ＞ 0.99 (Figure 2b).

The repeatability and intermediate precision of the RGA assay were assessed by testing the 100% level samples thrice on three different days by three operators (O1, O2, and O3) independently. The RSD of relative bioactivities measured from plate to plate, day to day, and operator to operator was below 9%, which indicated good repeatability (plate-to-plate precision) and intermediate precision (day-to-day and operator-to-operator precision) (Table 3).

Further applications of the RGA

Using the established RGA, we measured the ADCC activity of anti-RABV Abs (five mAbs and two bsAb) and also the anti-RABV polyclonal antibodies, including one HRIG, one NS, and four vaccine-induced antisera. Seven anti-RABV Abs targeting different antigenic sites of the RABV glycoprotein showed dose–response curves but different ADCC strengths (Figure 4a), which indicated that this assay can be used in assessing the relative ADCC bioactivities of anti-RABV Abs. Polyclonal antibodies HRIG and NS were pre-diluted to 16 IU/mL (resulting in an initial working concentration of 8 IU/mL) and prepared in two-fold serial dilutions to generate 10 concentration points. As shown in Figure 4b, both HRIG and NS showed dose–response curves and comparable ADCC potencies (EC₅₀: 101.8 mIU/mL for NS, 127.5 mIU/mL for HRIG). It is of note that the HRIG induced much higher RLU compared with NS. The vaccine-induced antisera also showed ADCC activity (Figure 4c), and we found that there was no correlation between ADCC strength and neutralization potency (Figure 4d). Although sera DN had the lowest neutralization potency and also showed the lowest ADCC activity (lowest RLU value), the other three sera varied. The neutralizing Ab titer of sera TX was three-fold higher than sera PD; however, the ADCC strength of these two sera was similar (comparable RLU values). Sera YRS, which had an intermediate-level neutralizing Ab titer, showed the most potent ADCC activity. We also found that the ADCC activity of mAb-1, which was denatured by heating at 70°C for 30 min, had decreased compared with the untreated mAb-1 (Figure 4e), whereas a higher percentage of aggregates was also detected in heated mAb-1 (47.9% vs. 2.6%, Figure 4f), suggesting that the established ADCC assay may be utilized in assessing the stability of anti-RABV mAbs.