Effect of plant produced Anti-hIL-6 receptor antibody blockade on pSTAT3 expression in human peripheral blood mononuclear cells

As a response to invasion by pathogens, the secretion of interleukin 6 (IL-6) which is a cytokine, activates IL-6/JAKs/STAT3 intracellular signaling via., phosphorylation. Over expression of pSTAT3 induces IL-6 positive feedback loop causing cytokine release syndrome or cytokine storm. Plants have gained momentum as an alternative expression system. Hence, this study aims to produce mAb targeting human IL-6 receptor (hIL-6R) in Nicotiana benthamiana for down regulating its cellular signaling thus, decreasing the expression of pSTAT3. The variable regions of heavy and light chains of anti-hIL-6R mAb were constructed in pBYK2e geminiviral plant expression vector and transiently co-expressed in N. benthamiana. The results demonstrate the proper protein assembly of anti-hIL-6R mAb with highest expression level of 2.24 mg/g FW at 5 dpi, with a yield of 21.4 µg/g FW after purification. The purity and N-glycosylation of plant produced antibody was analyzed, including its specificity to human IL-6 receptor by ELISA. Additionally, we investigated the effect to pSTAT3 expression in human PBMC’s by flow cytometry wherein, the results confirmed lower expression of pSTAT3 with increasing concentrations of plant produced anti-hIL-6R mAb. Although, further in vivo studies are key to unveil the absolute functionality of anti-hIL-6R, we hereby show the potential of the plant platform and its suitability for the production of this therapeutic antibody.

hIL-6R antibody in N. benthamiana, the V H and V L of anti-hIL-6R mAb were codon-optimized and fused to constant regions of IgG1 heavy chain and light chains respectively.pBY2eK geminiviral plant expression vector was used to clone the light and heavy chains, resulting in pBY2eK-Anti-hIL-6R HC and pBY2eK-Anti-hIL-6R LC (Figure 1).Each construct was transformed into Agrobacterium tumefaciens strain GV3101.Plant leaves were co-infiltrated with both anti-hIL-6R HC and LC.To confirm the expression of HC and LC, infiltrated leaf samples were analyzed by western blot with anti-human IgG gamma and anti-human kappa detection antibodies (data not shown).Non-infiltrated leaves were used as negative control.For day optimization experiment, leaf samples were harvested on 1-, 3-, 5-, 7-and 9-days post infiltration (dpi).The infiltrated leaves showed the onset of necrosis on 3 dpi with gradual increase until 9 dpi (Figure 2A).ELISA was used for quantifying the expression of plant-produced anti-hIL-6R antibody.The results showed that the maximum expression level of plant-produced anti-hIL-6R was observed at 5 dpi accumulating up to 2.24 mg/g fresh weight (FW) (Figure 2B).The purified protein was accumulated up to 21.4 µg/g of fresh leaf weight.
Purification of anti-hIL-6R antibody from infiltrated N. benthamiana leaves.Agroinfiltrated leaves were harvested, extracted in 1X PBS and centrifuged to obtain the crude extract.The crude extract was purified by Protein-A affinity column chromatography.Non-specific proteins were removed by washing the column with wash buffer and plant produced anti-hIL-6R was eluted by elution buffer.Plant produced anti-hIL-6R antibody was characterized by SDS-PAGE.The expected size under non-reducing condition for anti-hIL-6R HC and LC is approximately 150 kDa (Figure 3A,B,C).Under reducing conditions, the expected molecular weight of anti-hIL-6R-HC is 50 kDa and anti-hIL-6R-LC is 25 kDa.(Figure 3D,E,F) The purified anti-hIL-6R antibody was used for binding assay and in vitro studies.The results of size exclusion chromatography showed that most of the anti-hIL-6R produced by the plant was in the form of full and intact IgG molecules, which accounted for up to 90% of the sample and appeared as the major peak on the chromatogram.However, some smaller amounts of the anti-hIL-6 were found in dimeric (4.46%) and aggregated (4.08%) forms of the sample respectively (Figure 4).

N-linked glycosylation pattern of anti-hIL-6R antibody.
To determine the N-glycosylation of anti-hIL-6R antibody produced in wild-type N. benthamiana, the purified antibody was subjected to trypsin digestion and analyzed by LC-ESI-MS.The plant produced anti-hIL-6R heavy chain which carries the conserved N-glycosylation site in the Fc region showed the glycosylated peptide EEQYNSTYR with oligomannosidic N-glycans due to the ER retention tag (KDEL) at C-terminal of amino acid sequence (Figure 5).The graphic representation (data shown in supplementary information) of plant produced glycosylated and aglycosylated anti-PD-L1 mAb was used as control.

Binding activity of plant-produced anti-hIL-6R antibody to human recombinant IL-6R.
To investigate the specific binding activity of plant-produced anti-hIL-6R, the purified antibody was used in binding ELISA with its target human recombinant IL-6 receptor protein.Plant-produced H4 monoclonal antibody (mAb) 35 was used as negative control.Each antibody was serially diluted before incubation with hIL-6R.The binding activity was detected by anti-human kappa HRP conjugate (dilution 1:2,500) at OD 450 .The plant-produced anti-hIL-6R antibody elicit specific binding to human recombinant IL-6 receptor protein with a dissociation constant K D value of 1.748 µg/mL (Figure 6), whereas the negative control did not bind as anticipated.Positive control, commercial TCZ was not used in the binding assay due to its limited accessibility in the region 36 .Altogether, the anti-hIL-6R produced in the plant is functional, as indicated by the specific binding to its target.
In vitro activity of anti-hIL-6R antibody in human PBMCs.PBMCs were isolated from whole blood cells and were investigated for the efficacy of plant produced anti-hIL-6R mAb in suppression of the IL-6/IL-6R/ STAT3 pathway by flow cytometry.The pSTAT3 expression and mean flow intensity (MFI) data showed that pSTAT3 expression level was decreased significantly at 10 µg/mL of plant produced anti-hIL-6R mAb in CD3 + T cells (Figure 7A,D).Interestingly, 0.1, 1.0 and 10 µg/mL concentrations of plant produced anti-hIL-6R mAb were able to diminish the pSTAT3 expression by almost 2-fold in IL-6 activated CD14 + monocytes without much difference (Figure 7B,E).However, there was no significant change in pSTAT3 expression level in CD19 + B cells (Figure 7C,F) at all concentrations of plant produced anti-hIL-6R antibody.

Discussion
Interleukin -6 is an indication of severe cytokine storm in many cases such as chimeric antigen receptor-T (CAR-T) cell therapy, malignant tumor progression, autoimmune disorders, inflammatory diseases and COVID-19, which could be life-threatening 37,38 .There are many inhibitors that are efficient in the interruption of IL-6 signaling and its pathway 11 .TCZ, a monoclonal antibody targeting human IL-6R, that is approved for treatment of rheumatoid arthritis, Castleman's disease, juvenile idiopathic arthritis 39 , and newly used for treatment of cytokine release syndrome caused by CAR-T cell therapy 40 or COVID-19 infection 41 has gained momentum in the recent few years.Although the drug is effective, the key downside is high treatment expenses due to the involvement of pricey upstream-downstream production technology.The findings by Sinha and Linas, 2021 specifies a total cost of $83,130 in the combination of TCZ and dexamethasone for COVID-19 treatment and were estimated to achieve a gain of 9.36 QALYs (Quality-of-Life-Adjust-Year).Although it was determined that the combination of TCZ and dexamethasone is a cost-effective strategy for persons with COVID-19 who are extremely unwell, it would be important to have a less expensive option that is equally effective as TCZ 42 .Another study in Thailand, reported the arbitration of the price of TCZ currently available on the market to reduce the financial burden of treating refractory systemic juvenile idiopathic arthritis 36 .Hence, the transient expression of recombinant proteins in plants is an alternative, that can overcome the limitations of mammalian cell culture systems which require high manufacturing and scale-up costs, with the risk of contaminations 29,[43][44][45][46] .Lately, there have been various examples of successful biopharming in plants 47,48 including virus-like particle (VLP) production as vaccines against influenza H1N1 49 , rotavirus-like particle vaccines 50 , growth factors such as aFGF (acidic fibroblast growth factor) 51 , and antibodies such as mAbs 6D8 (87.2 ± 25.2 μg/g), which is a protective mAb against EBV GP1 52 , anti-CD20-human (2B8) antibody (28 mg/kg); 2B8-Fc-hIL2 immunocytokine (16 mg/kg) 53 , and anti-PD-1 mAb (pembrolizumab) 54 and diagnostic reagents 55 .Consequently, we produced monoclonal antibody targeting human IL-6R using transient expression in N. benthamiana.Geminiviral vector pBYK2e utilizes the CaMV (cauliflower mosaic virus) 35S promoter for optimal gene expression.The current study showed that anti-hIL-6R mAb was expressed with maximum expression level, accumulating to 2239.92 µg/g fresh weight on 5 dpi (Fig. 2).Previous studies reported that the highest yield of various transiently expressed proteins in N. benthamiana at 6 dpi for anti-PD-L1 (atezolizumab) (86.76 µg/g) 56 , anti-IgE (omalizumab) (41.2 µg/g) 57 , B38 (4 µg/g), and H4 mAb (35 µg/g) 35 .Other plant produced proteins expressed showed yields of 18.49 mg/kg of human interleukin-6 58 , 3 mg/kg of cocaine-hydrolase Fc protein 59 , 730 mg/kg of CMG2-Fc protein 60 .These short time periods provide essential advantages of protein expression in terms of speed over other expression approaches including mammalian cells [61][62][63][64] and stable plant expression [65][66][67] systems.The significant reduction in antibody expression after 5 dpi might be due to the progressive development of necrosis in the infiltrated leaves.Overexpression of recombinant proteins can result in misfolding of the recombinant proteins, trigger necrosis and subsequent degradation of recombinant proteins 68 .Fine tuning of the purification strategies might mitigate the loss of target protein, thus improving its yield.The N-glycan profile of   www.nature.com/scientificreports/heavy chain peptide sequence in anti-hIL-6R mAb expression in N. benthamiana 69,70 , to avoid the addition of plant specific glycan sugar moieties during the series of glycosylation reactions in the secretory pathway.Previous studies showed that antibodies with mannosidic N-glycans have increased rates of antibody clearance from circulation 71 , but it is expected that the mannosidic N-glycans have no adverse impact on allergic reactions or hypersensitivity responses 72,73 .The use of glycoengineered plants 70 and methods to increase the glycosylation efficiency 74 are alternatives to overcome these bottlenecks for use in potential therapeutic applications.
The specific binding activity of plant-produced anti-hIL-6R mAb to recombinant human IL-6R protein was also investigated by immunoassay.The ELISA results (Fig. 6) of the plant produced anti-hIL-6R mAb was found to have effective binding with human IL-6R, indicating specificity to the target protein whereas plant-produced H4 mAb 35 showed no binding activity.The K D value reported for mammalian cell-produced tocilizumab is 2.5 nM 75 and our plant-produced anti-hIL-6R mAb was found to be 1.748 µg/mL.The reason for the difference in binding affinity is currently unclear.The K D value for the mammalian cell-produced antibody was determined using a radioactive cell-based assay employing human myeloma cell line 76 , while this study employed ELISA to assess the affinity of plant produced anti-hIL-6R to the target protein.Due to the limited access for commercial TCZ (control), we could not directly compare the mammalian TCZ with the plant produced variant in our binding and in vitro assays.Occurrence of protein aggregates as evident from size exclusion chromatography, might  Cell blockade assay of anti-hIL-6R antibody.Whole blood was treated with anti-hIL-6R at a concentration of 0.1, 1, and 10 ug/mL for 20 min, followed by hIL-6 at 30 ng/mL for 15 min.After staining the cells with CD14 (APC-H7) and CD19 (APC), the cells were evaluated using flow cytometry for the pSTAT3 expression.Each value is expressed as the mean ± SEM (n = 3).Dunnett's test, *p < 0.05, **p < 0.01, and ***p < 0.001 versus group with hIL-6 at 30 ng/mL.also have interference in the binding activity.However, the study by Heinrich et al., 2010 shows that kinetic constants characterized for antibody interaction to its respective receptors by different methods are discrete, as the principle varies in each biophysical technique adopted. 77.
To determine the biological function of plant-produced anti-hIL-6 mAb, we performed the inhibitory effect of the mAb effect on STAT3 phosphorylation.Our results demonstrated that pSTAT3 expression in CD14 + cells (monocytes and macrophages) and CD3 + T cells was decreased, whereas almost no change was found in CD19 + (B cells).A study by Zhang et al., 2020b showed that the decrease in pSTAT3 expression in CD3 + T cells and CD14 + monocytes from human PBMCs depends on the concentration of anti-hIL-6R mAb or tocilizumab, which is consistent with our findings using plant produced anti-hIL-6R mAb in these two cell populations.The data showed that tocilizumab completely repressed the pSTAT3 expression in CD3 + T cells and CD14 + monocytes at 12.5 µg/mL 78 which is similar to our plant produced anti-hIL-6R mAb at 10 µg/mL.Activation of IL-6R/JAK/ STAT3 signaling has been shown to induce systemic hyperinflammation during cytokine storm 79 .In addition, IL-6/JAK/STAT3 signaling has been demonstrated to play important roles in tumor progression and aggressiveness in several cancers 11,26,[80][81][82][83][84] .Hence, STAT3/pSTAT3 was chosen as the biomarker in this preliminary study.In addition, our study results are analogous with sarilumab, an anti-IL-6R mAb expressed in N. benthamiana in certain characteristics that was evident from the study by Jugler et al., 2021 85 .However, further research including in vivo studies in animal models must be conducted to compare the efficacy of our plant produced mAb with commercial anti-hIL-6R mAb.

Conclusion
IL-6 is a hallmark cytokine of CRS that can impose life-threatening response events.Although there are various IL-6 signaling inhibitors approved in the market, the production and treatment costs are high-priced.Altogether, we produced a recombinant anti-hIL-6R mAb that can recognize human IL-6 receptor using plant as an expression platform.The results of plant produced anti-hIL-6R mAb demonstrate specific binding to human IL-6 receptor, in vitro inhibition of IL-6:IL-6R complex formation and decreased STAT3 expression.Further, to reveal the effectiveness of anti-hIL-6R mAb produced in plants, animal studies need to be performed for better insights on its functioning in the IL-6 signaling pathway and disease progressions.

Methods
Study details.The study was permitted to be carried out by the internal CU-IBC (Chulalongkorn University-Institutional Biosafety Committee) following all the Biosafety guidelines for Modern biotechnology.All methods were performed in accordance with the relevant guidelines/regulations/legislation.The seeds of N. benthamiana used in the present study were kindly gifted by Dr. Supaart Sirikantaramas, Faculty of Science, Chulalongkorn University.

Construction of plant expression vector. The variable region of heavy chain (V H ) and light chain (V L )
from previous study report 86 were codon-optimized to N. benthamiana using Invitrogen GeneArt® Gene Synthesis (Thermo Fisher Scientific) and commercially synthesized (Genewiz, Suzhou, China).The synthesized V H and V L were fused to human IgG1 constant regions of heavy chain (C H and light chain (C L ) and were flanked with signal peptide on the N-terminus respectively and a SEKDEL (Ser-Glu-Lys-Asp-Glu-Leu) sequence on C-terminus of heavy chain.Further, they were ligated to pBYR2eK2Md (pBYK2e) geminiviral expression vector to make pBYK2e-Anti-hIL-6R heavy chain (HC) and pBYK2e-Anti-hIL-6R light chain (LC).Each ligation product was transformed into Escherichia coli DH10B strain via., heat shock and confirmed by colony PCR (polymerase chain reaction).Subsequently, the extracted plasmid was transformed into Agrobacterium tumefaciens (GV3101) via., electroporation.

Transient expression and day optimization of Anti-hIL-6R antibody in N. benthamiana. N.
benthamiana plants are grown approximately for 6-8 weeks before using for agroinfiltration.Agrobacterium cells containing either pBYK2e-Anti-hIL-6R HC or pBYK2e-Anti-hIL-6R LC were cultured with continuous shaking at 200 rpm at 28 °C for 20 h.Overnight, Agrobacterium cultures (HC, LC) were centrifuged at 5,000 × g for 8 min, and the bacterial pellets were resuspended in 1X infiltration buffer to get a final OD 600 of 0.2.The same amount of HC and LC harboring Agrobacterial cultures were mixed in the ratio 1:1 and syringe infiltrated into N. benthamiana leaves.The infiltrated plants were incubated in a controlled environment.To determine small scale protein expression level, the leaf discs from infiltrated leaves were collected on 1-, 3-, 4-, 5-, 6-, and 7 days post infiltration (dpi) in 1.5 mL centrifuge tubes.
Protein extraction and purification.Infiltrated leaves were harvested and extracted in 1X PBS (phosphate-buffered saline: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.47 mM KH 2 PO 4 , pH 7.4) in the presence of stainless-steel beads using a tissue homogenizer.The leaf extracts were centrifuged at 13,000 rpm for 15 min and the supernatant/crude extract collected was used for protein quantification and expression analysis.After the maximum expression of anti-hIL-6R antibody was assessed in the day optimization experiment, large scale vacuum infiltration was performed and harvested leaves were used for extraction in a blender with 1X PBS.The similar steps were followed as mentioned above before the large-scale purification of the anti-hIL-6R protein.
For purification, the plant leaves infiltrated on large-scale using vacuum infiltration were harvested on the day before expression yield analysis.The harvested leaves were blended in 1X PBS extraction buffer in the ratio of 1 g of leaf:3 mL of buffer.Then, the crude extract was filtered using a gauze cloth, centrifuged at 15,000 × g for 30 min at 4 °C and the supernatant was again filtered through 0.22 µm membrane filter.The protein-A affinity chromatography was employed for protein purification.Crude extract was loaded onto the Protein-A resin www.nature.com/scientificreports/column.The column was washed with 5X column volumes of 1X PBS.0.1 M glycine buffer at pH 2.7 was used to elute the target anti-hIL-6R antibody and further neutralized with 1.5 M Tris-HCl at pH 8.8.

Analysis of anti-hIL-6R antibody by SDS-PAGE.
The expression and purity of anti-hIL-6R antibody was analyzed using SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and western blotting.In brief, approximately 20 µg of protein samples were mixed separately in 10X non-reducing (125 mM Tris-HCl pH 6.8, 12% Sodium Dodecyl Sulphate 10% Glycerol, 0.001% Bromophenol blue) and reducing (125 mM Tris-HCl pH 6.8, 12% Sodium Dodecyl Sulphate 10% Glycerol, 22% β-mercaptoethanol, 0.001% Bromophenol blue) dyes, heated at 95 °C for 5 min.Samples were then loaded on 4-15% gradient SDS polyacrylamide gel along with pre-stained SDS-PAGE standard (Bio-Rad, California, USA).Electrophoresis was performed at room temperature for 80 min using a constant voltage of 120 V in 1X running buffer (25 mM Tris, 192 mM Glycine, 1% SDS) until the dye front reached the end of the gel.
Protein staining and western blot.SDS-PAGE gels were stained using Instantblue Coomassie stain (Abcam, UK).Gels were washed three times in deionized water for 5 min, incubated in 10 mL of Coomassie blue stain for 4 h, de-stained 2-3 times in 10 mL of deionized water for 1 h, until clear protein bands were visualized.For western blotting, the electrophoretic separated proteins from the gel were subsequently transferred to a nitrocellulose membrane (Bio-Rad, California, USA) using 1X transfer buffer (25 mM Tris, 192 mM Glycine, 15% Methanol) at 100 V for 2 h.The nitrocellulose membrane after washing with deionized water, was blocked with 5% skim milk in 1X PBS for 30 min followed by 1X PBST (0.05% Tween 20 in 1X PBS) washes.The membrane was incubated either with 1:10,000 conjugated anti-human IgG gamma or with 1:2,500 conjugated antihuman IgG kappa antibody diluted in 1X PBS and 3% skim milk.After further washes, enhanced chemiluminescence (ECL) plus detection reagent (Abcam, UK) was used to record and capture the plant-expressed protein signals on nitrocellulose membrane to a light-sensitive X-ray film (Carestream, New York, USA).
Size exclusion chromatography of anti-hIL-6R antibody.5 µg of monoclonal antibody was injected into a UHPLC system (Waters, MA, USA) equipped with a BEH SEC 200 column (4.6 mm × 300 mm, Waters, MA, USA).The protein was eluted from the column using 1X PBS buffer (pH 7.4) at a flow rate of 0.3 mL/min, while the column was maintained at 25 °C, and the UV detector was set at 280 nm.The peak areas were automatically integrated using Empower3 software (Waters, MA, USA).
N-glycan analysis.The N-linked glycosylation of anti-hIL-6 antibody was analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS).The purified anti-hIL-6 antibody was run on SDS-PAGE under reducing conditions and then stained with Instant Blue Coomassie protein-dye (Abcam, UK) to view the separated antibody chains.Plant-produced anti-hIL-6 heavy chain was excised from the gel, S-alkylated and then digested with trypsin.The tryptic peptides were analyzed on a Vanquish™ Neo UHPLC (ThermoFisher Scientific, USA) system coupled to an Orbitrap Exploris 480 mass spectrometer (ThermoFisher Scientific, USA).The positive glycopeptides of m/z = 350-3200 were monitored and captured on the chromatogram.The expected glycopeptides on EEQYNSTYR site were manually detected and analyzed with FreeStyle 1.8 program (Thermo Scientific, USA).
Anti-hIL-6R antibody quantification by ELISA.The expression yield of crude and purified plant-produced anti-hIL-6R was determined by enzyme-linked immunosorbent assay (ELISA).A 96-half well microplate (Costa® Assay Plate, Corning, USA) was coated with 25 µL/well of anti-human IgG Fc fragment (Abcam, Cambridge, UK) diluted at a ratio of 1:1,000 in 1X PBS and incubated at 4 °C overnight.After incubation, the plate was washed with 170 µL/well of 1X PBS-T (1X PBS with 0.05% Tween-20) for 3 times and blocked with 5%(w/v) skim milk in 1X PBS at 37 °C for 2 h.The commercially available human IgG1 isotype control (Abcam®, Cambridge, UK), diluted twofold in 1X PBS starting at a concentration of 1 µg/mL was used as standard.Plant crude extracts and purified samples were used at a dilution of 1:1,000 and 1:2,000; respectively in 1X PBS.After washing, each dilution of the standard and samples were added into the microplate in duplicates.Then the microplate was incubated at 37 °C for 2 h followed by 3X washes with 1X PBS-T.The plate was then incubated with peroxidase-conjugated goat anti-human kappa antibody (Southern Biotech, Birmingham, USA) at 1:2,500 dilution in 1X PBS.After washing, the plate was developed by adding 25 µL/well of TMB (3,3′,5,5′-tetramethylbenzidine) (Promega®, USA) and incubated for 5 min.The reaction was stopped with 25 µL/well of 1 M H 2 SO 4 .Ultimately, the absorbance was measured at 450 nm in a 96-well plate reader (BMG Labtech, Germany).The standard curve was generated, and the concentration of samples were calculated from linear equation (y = mx + c).The experiments were performed in triplicates and data are presented as mean ± standard deviation (SD).

Specificity of anti-hIL-6R.
Plant-produced anti-hIL-6R antibody binding activity was investigated by ELISA using specific human IL-6 receptor (hIL-6R) recombinant protein.Three biological replicates were performed.Each 96 half well in the microplate was coated with 25 µL of 2 µg/mL human IL-6R (Sino Biological, USA) and incubated at 4 °C overnight.The incubated plate was washed with 1X PBS-T (3 times) and blocked with 5%(w/v) non-fat milk in 1X PBS at 37 °C for 2 h.The plate was further incubated with plant-produced anti-hIL-6R samples at 37 °C for 2 h.After washing, the plate was treated with goat anti-human kappa-HRP (1:2,500) (Southern Biotech, Birmingham, USA) for 1 h at 37 °C.Finally, the plate was washed and incubated with 25 µL/ well of 3,3′,5,5′-tetramethylbenzidine (TMB) (Promega®, USA).The reactions were stopped by addition of 25 µL/well of 1 M H 2 SO 4 and the absorbance was read at 450 nm by microplate reader (Hercuvan system, UK).
Plant-produced H4 mAb 35 was used as negative control.The data were analyzed by GraphPad Prism 9.3 software (San Diego, CA, USA).The dissociation constant (K D ) was determined by non-linear regression analysis using a one-site binding model.

Effect of plant produced anti-hIL-6R mAb in vitro.
Healthy individuals (n = 3) were recruited following written informed consent.The study was approved by the institutional review boards of the participating centers (Faculty of Medicine, Chulalongkorn University, COA No.1371/2022, IRB.Approval No. 633/64) and the experimental methods comply with the Helsinki Declaration.Briefly, healthy whole blood cells were incubated with plant produced anti-IL-6R antibody and was stimulated with human IL-6 (Sino Biological, USA).To lyse the cells, 1X Lysis buffer (BD Pharm Lyse Solution, BD Bioscience, USA) was added and incubated and the reaction was stopped by addition of 1X PBS.Then, the samples were incubated with APC Mouse Anti-Human CD19 (BD Pharmingen USA), and APC-H7 Mouse Anti-Human CD14 (BD Pharmingen USA).The samples were centrifuged, and the pellet was resuspended in fixation buffer (BD Pharmingen, USA) and followed by 2 washes with of staining buffer.

Figure 2 .
Figure 2. Expression level of plant produced anti-hIL-6R mAb on day-1, 3, 5, 7 and 9 post infiltration in Nicotiana benthamiana leaves were quantified by ELISA.The leaf necrosis (A) and comparison of anti-hIL-6R mAb yield (B) were shown.dpi: days post-infiltration; FW: Fresh Weight; Data are represented as means ± SD of triplicates.

Figure 3 .Figure 4 .
Figure 3. SDS-PAGE and Western blot analysis of plant produced anti-hIL-6R mAb.Non-infiltrated leaves were used as negative control (lane WT).Panels (A & D), (B & E) and (C & F) shows the antibody stained with coomassie, probed with anti-gamma and anti-kappa under non-reducing and reducing condition respectively.C: crude extract 20 µg/lane; P: Purified antibody 5 µg/lane; WT: Wild type.

Figure 7 .
Figure 7. Cell blockade assay of anti-hIL-6R antibody.Whole blood was treated with anti-hIL-6R at a concentration of 0.1, 1, and 10 ug/mL for 20 min, followed by hIL-6 at 30 ng/mL for 15 min.After staining the cells with CD14 (APC-H7) and CD19 (APC), the cells were evaluated using flow cytometry for the pSTAT3 expression.Each value is expressed as the mean ± SEM (n = 3).Dunnett's test, *p < 0.05, **p < 0.01, and ***p < 0.001 versus group with hIL-6 at 30 ng/mL. https://doi.org/10.1038/s41598-023-39106-5 Each sample was incubated with Perm Buffer III (BD Phosflow, BD Bioscience, USA), washed twice with staining buffer.Anti-STAT3 Phospho (Tyr705) antibody (PE, Biolegend, USA) or Mouse IgG1κ isotype antibody (PE, Biolegend, USA) was added and incubated in dark at room temperature for 30 min.Each sample was washed with staining buffer twice.Flow cytometry was performed to analyze the results.Post hoc Dunnett's test was used for statistical analysis in GraphPad Prism 9.3 software (San Diego, CA, USA).All the values were expressed as mean ± SD for duplicate samples in two biological replicates.Post hoc Dunnett's test was used for statistical analysis in flow cytometry.