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

Abstract

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.

Figure 1

Schematic representation of Monoclonal antibody and geminiviral vector: pBYR2eK2Md (pBYK2e) used in this study. (A) Components of monoclonal antibody. The schematic and structural elements assembled in plant produced anti-hIL-6R mAb, T-DNA region of the pBYK2e vector; (B) Anti-hIL-6R HC: Heavy chain of anti-hIL-6R antibody; (C) Anti-hIL-6R LC: Light chain of anti-hIL-6R antibody.LB and RB: The left and right borders of the T-DNA region transferred by Agrobacterium into plant cells; Pin II 3′: The terminator from potato proteinase inhibitor II gene; P19: The RNA silencing suppressor from tomato bushy stunt virus; TMVΩ 5′-UTR: 5′ untranslated region of tobacco mosaic virus Ω; P35S: Cauliflower Mosaic Virus (CaMV) 35S promoter; LIR: Long intergenic region of BeYDV; P35Sx2e: CaMV 35S promoter with duplicated enhancer; NbPsalK2T1-63 5′UTR: 5′ untranslated region; XbaI: XbaI restriction enzyme site; Signal Peptide (SP): Barley alpha-amylase signal peptide; SEKDEL: Endoplasmic reticulum (ER) retention signal peptide; SacI: SacI-HF restriction enzyme site; Ext3′FL: 3′ region of tobacco (Nicotiana tabacum) extension gene; Rb7 5’ del: Tobacco RB7 promoter; SIR: Short intergenic region of BeYDV; C2/C1: Bean Yellow Dwarf Virus (BeYDV) ORFs C1 and C2 encoding for replication initiation protein (Rep) and RepA.

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

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 4

Size exclusion chromatography of plant produced anti-hIL-6R mAb. The chromatogram shows one major and two minor peaks corresponding to monomeric IgG, protein aggregates, dimeric IgG forms respectively.

Figure 5

Liquid chromatography-electrospray ionization-mass spectrometry (LC–ESI–MS) of plant produced anti-hIL-6R antibody. The N-glycosylation profiles of the heavy chain peptide EEQYNSTYR (mass: 1189.5120) is shown and the peaks assigned to oligomannosidic N-glycans (Man4-8GlcNAc2) are marked.

Figure 6

Plant produced anti-hIL-6R binding efficiency to human recombinant interleukin 6 receptor (hIL-6R) by ELISA. Anti-H4 mAb was used as negative control. K_D: Dissociation constant.

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.