Intracellular reprogramming of expression, glycosylation, and function of a plant-derived antiviral therapeutic monoclonal antibody

Abstract

Plant genetic engineering, which has led to the production of plant-derived monoclonal antibodies (mAb(P)s), provides a safe and economically effective alternative to conventional antibody expression methods. In this study, the expression levels and biological properties of the anti-rabies virus mAb(P) SO57 with or without an endoplasmic reticulum (ER)-retention peptide signal (Lys-Asp-Glu-Leu; KDEL) in transgenic tobacco plants (Nicotiana tabacum) were analyzed. The expression levels of mAb(P) SO57 with KDEL (mAb(P)K) were significantly higher than those of mAb(P) SO57 without KDEL (mAb(P)) regardless of the transcription level. The Fc domains of both purified mAb(P) and mAb(P)K and hybridoma-derived mAb (mAb(H)) had similar levels of binding activity to the FcγRI receptor (CD64). The mAb(P)K had glycan profiles of both oligomannose (OM) type (91.7%) and Golgi type (8.3%), whereas the mAb(P) had mainly Golgi type glycans (96.8%) similar to those seen with mAb(H). Confocal analysis showed that the mAb(P)K was co-localized to ER-tracker signal and cellular areas surrounding the nucleus indicating accumulation of the mAb(P) with KDEL in the ER. Both mAb(P) and mAb(P)K disappeared with similar trends to mAb(H) in BALB/c mice. In addition, mAb(P)K was as effective as mAb(H) at neutralizing the activity of the rabies virus CVS-11. These results suggest that the ER localization of the recombinant mAb(P) by KDEL reprograms OM glycosylation and enhances the production of the functional antivirus therapeutic antibody in the plant.

Figure 1. Expression of mAb SO57 in transgenic plants.

(A) A schematic diagram of the mAb SO57 HC and LC DNA constructs . (B) PCR analysis of LC (729 bp), HC (1431 bp) and HC fused to KDEL (HCK, 1443 bp) in the genomic DNA. NT, non-transgenic plant; mAb^P, non-KDEL-tagged mAb^P; mAb^P K, KDEL- tagged mAb^P.

Figure 2. The RT-qPCR and immunoblot analysis of mAb SO57 HC and LC in transgenic tobacco plants.

(A) Relative expression levels (Y axis) of the HC and LC gene as determined by RT-qPCR. Amplicons generated with the RT-qPCR were confirmed by 1% agarose gel electrophoresis (upper panel). The results of RT-qPCR are expressed as the average of three independent experiments after normalization with N. tabacum actin bands (lower panel). Error bars represents mean relative expression values (mean ± SD) of HC and LC to actin from the mAb^P and mAb^PK samples. Transcript levels of HC and LC were not significantly different between the mAb^P and mAb^PK (p>0.05). mAb^P, non-KDEL-tagged mAb^P; mAb^PK, KDEL-tagged mAb^P. (B) Western blot analysis of proteins extracted from leaves of randomly selected 4 transgenic plants without KDEL and 3 transgenic plants with KDEL, respectively. The bands for HC (50 kDa) and LC (25 kDa) were detected with HRP-conjugated goat anti-human Fcγ- or F(ab′)₂-specific antibodies, respectively. *p<0.05 compared to mAb^P samples (Student's t-test analysis). Error bars represent the mean ± SD.

Figure 3. Confocal analysis of the subcellular localization of mAb SO57 and mAb SO57 K in plant leaves.

Immunofluorescent confocal microscopic photomicrographs displayed localization of mAb SO57 in subcellular organelles of transgenic tobacco plant leaf cells. The red signal for ER-Tracker Blue-White DPX, a photo-stable probe selective for the ER in live cells, shows specific ER localization of mAb. The mAb SO57-immunoreactive green fluorescence was detected by FITC-conjugated anti-human IgG (green). The nuclei (blue) were labeled with TO-PRO-3. Each image was merged to analyze the subcellular localization of mAb SO57 in transgenic plants. The green fluorescent signal of mAb SO57 K closely overlapped with the blue fluorescent signal of ER-Tracker. Scale bars, 20 µm.

Figure 4. SDS/PAGE of the mAb SO57 purified from transgenic plants.

Purified mAbs were loaded onto the gels and stained with Coomassie brilliant blue R250. TPS, total protein extract from plant leaves; mAb^H, hybridoma-derived mAb SO57; mAb^P, non-KDEL-tagged mAb^P; mAb^PK, KDEL-tagged mAb^P; HC, heavy chains of mAb^P; LC, light chain of mAb^P.

Figure 5. N-glycan profiles of anti-rabies mAb^PK and mAb^P.

mAbs were expressed from transgenic tobacco plants as dual forms with mAb^PK or mAb^P. Glycan profiles of mAb^P (A) and mAb^PK (B) were obtained by DNA sequencer-based method for the plant N-glycan analysis. Golgi-type and OM-type glycans were classified and confirmed by hexosamindase (H) and α(1,2)-mannosidase (α1,2-Man) digestions. The symbols of the glycan structures are as follow: GlcNAc, black square; Man, white circle; Xyl, white triangle; Fuc, diamond with a dot inside. Non, non-digested sample.

Figure 6. Stability profiles of mAb^H, mAb^PK and mAb^P in mice.

The % value was calculated with the formula [100×(concentration of each day/concentration of the first day (24 hr) after injection)], followed up to day 10. The concentration of mAb in the serum from BALB/C mice injected i.p. with mAb^H, mAb^PK or mAb^P was determined by ELISA. Data are presented as means ± SD.

Figure 7. Flow cytometric analysis of binding activity of mAb^H, mAb^P, and mAb^P K SO57 to the FcγRI (CD64).

U937 cells with IFN-γ to stimulate Fc receptor expression were incubated with mAb^H, mAb^P or mAb^P K SO57, respectively. Binding activity of mAb^H (upper), mAb^P (center), and mAb^P K (bottom) to the activated cells expressing CD64 were analyzed by flow cytometry. Non-bold and bold lines indicate IFN-γ stimulated cells without any mAb, and with mAb^H, mAb^P and mAb^PK, respectively. The binding signals were detected with treatment of RPE-conjugated goat anti-human IgG.