The B cell death function of obinutuzumab-HDEL produced in plant (<i>Nicotiana benthamiana</i> L.) is equivalent to obinutuzumab produced in CHO cells

<div><p>Plants have attracted attention as bio-drug production platforms because of their economical and safety benefits. The preliminary efficacy of ZMapp, a cocktail of antibodies produced in <i>N</i>. <i>benthamiana</i> (<i>Nicotiana benthamiana</i> L.), suggested plants may serve as a platform for antibody production. However, because the amino acid sequences of the Fab fragment are diverse and differences in post-transcriptional processes between animals and plants remain to be elucidated, it is necessary to confirm functional equivalence of plant-produced antibodies to the original antibody. In this study, Obinutuzumab, a third generation anti-CD20 antibody, was produced in <i>N</i>. <i>benthamiana</i> leaves (plant-obinutuzumab) and compared to the original antibody produced in glyco-engineered Chinese hamster ovary (CHO) cells (CHO-obinutuzumab). Two forms (with or without an HDEL tag) were generated and antibody yields were compared. The HDEL-tagged form was more highly expressed than the non-HDEL-tagged form which was cleaved in the N-terminus. To determine the equivalence in functions of the Fab region between the two forms, we compared the CD20 binding affinities and direct binding induced cell death of a CD20-positive B cells. Both forms showed similar CD20 binding affinities and direct cell death of B cell. The results suggested that plant-obinutuzumab was equivalent to CHO-obinutuzumab in CD20 binding, cell aggregation, and direct cell death via binding. Therefore, our findings suggest that Obinutuzumab is a promising biosimilar candidate that can be produced efficiently in plants.</p></div

Comparison of the integrity and expression of light and heavy chains with or without HDEL.

<p>(<b>A</b>) Total protein extracts were subjected to PAGE under non-reducing and reducing conditions. Immunoblot was performed using HRP-conjugated human Ig Fc-specific antibody and HRP-conjugated mouse Ig Fc-specific antibody to detect both chains. CHO-rituximab (50 ng, 100 ng, and 200 ng) was used as the standard. Coomassie-stained gel images were used to show equivalent loading of proteins. (<b>B</b>) Comparison of localization and expression between plant-obinutuzumab-HDEL and plant-obinutuzumab from <i>N</i>. <i>benthamiana</i> leaves. Immunohistochemistry was performed to detect the localisation of plant-obinutuzumab-HDEL and plant-obinutuzumab in <i>N</i>. <i>benthamiana</i> leaves. Formalin-fixed and paraffinised <i>N</i>. <i>benthamiana</i> leaves expressing plant-obinutuzumab-HDEL and plant-obinutuzumab were sectioned at 10 μm thickness and immunostained. Fluorescein isothiocyanate (FITC; green)-conjugated anti-human Ig Fc-specific 2^nd antibody was used for detection. BiP protein fused with Ds-RED (red) was used to indicate the localisation of ER in <i>N</i>. <i>benthamiana</i> leaves. DraQ was used to indicate the nucleus (blue). Bar: 20 μm.</p

Specificity and affinity of epitope binding by plant-obinutuzumab-HDEL compared to CHO-obinutuzumab.

<p>(<b>A</b>) Antibody protein concentrations were measured via BCA method and quantification of PAGE gel analysis with Coomassie blue staining. To validate the concentration of each antibody, 1 μg of antibodies was subjected to PAGE under non-reducing conditions with BSA (0.1, 0.2, 0.5, 1, 2 μg) as the standard. Gels were then stained with Coomassie blue. (<b>B</b>) Specific epitope recognition by plant-obinutuzumab-HDEL was tested by immunocytochemistry. mCherry-tagged CD20 was expressed in HEK cells. CHO-obinutuzumab and plant-obinutuzumab-HDEL were used for immunocytochemistry with FITC-conjugated human Fc-specific secondary antibodies. Bar 1 μm. C. Representative FACS images for affinity comparison with 10 μg/ml antibodies. (<b>C, D</b>) Dose-dependent binding capacity of CHO-rituximab, plant-obinutuzumab-HDEL and CHO-obinutuzumab (1 ng/ml, 10 ng/ml, 100 ng/ml, 1 μg/ml, and 10 μg/ml) using flow cytometry. Representative FACS images of binding affinity with 10 μg/ml antibodies are shown in <b>C</b>. FITC intensities of 10 μg/ml of each antibody bound to cells are depicted by normalized mean fluorescence intensity (MFI). Results of triplicate assays are summarised in <b>D.</b></p

plant-obinutuzumab-HDEL binding to Ramos cells showed typical type II CD20 antibody characteristics, similar to CHO-obinutuzumab.

<p>(<b>A</b>) Two-panel photomicrograph showing binding of the antibodies to CD20 localized on the surface of Ramos cells. Binding was visualised with FITC-conjugated human Fc-specific secondary antibody. Bar, 2 μm. (<b>B</b>) In addition to the same antibodies used in A, caveolin was stained with anti-caveolin antibody and Alexa 568-conjugated secondary antibody. Merged DIC image shows CD20 and caveolin co-localised on the surface of the Ramos cell. Bar, 1 μm. (<b>C</b>) Photomicrograph showing cell aggregation (homotypic adhesion: HA) 30 minutes after treatment with each antibody. (<b>D</b>) Direct binding-induced cell death caused by CHO-obinutuzumab and plant-obinutuzumab-HDEL were compared to IgG and CHO-rituximab. Each antibody (at 1 μg/ml, 10 μg/ml, and 30 μg/ml) was incubated with Ramos cells for 14 hours and cell death was measured by lose of calcein-AM dye via FACS analysis. Three independent experiments are shown as means ± s.e.m. **P < 0.01; ***P < 0.001.</p

Light and heavy chains obinutuzumab constructed with or without an HDEL tag were successfully incorporated into the <i>N</i>. <i>benthamiana</i> genome.

<p>(A) Schematic representation of plant-obinutuzumab Heavy Chain and Light Chain to produce in <i>N</i>. <i>benthamiana</i> leaves by using transient infiltration method. Each construct (BiP ss-light chain, BiP ss-heavy chain-HDEL, and BiP ss-heavy chain) was used 35S, cauliflower mosaic virus 35S promoter; BiP ss, ER targeting signal sequence; HDEL, ER retention signal; NOS, nos terminator; Hyg, hygromycin-resistance cassette; LB, left border; RB, right border. Binding regions of each set of primers used in Fig. 1A are indicated by pairs of red arrows. (B) Validation of constructs incorporated into the <i>N</i>. <i>benthamiana</i> genome. Genomic DNA was extracted from <i>N</i>. <i>benthamiana</i> leaves engineered to express each construct via agrobacterium-mediated infiltration and was used as template for PCR with primer pairs indicated in Fig. 1A (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191075#pone.0191075.s004" target="_blank">S4 Fig</a> for primer sequences). (PCR product sizes: light chain, 1350 bp; heavy chain, 2050 bp).</p

Quantification of plant-obinutuzumab-HDEL production yield in <i>N</i>. <i>benthamiana</i> leaves.

<p>(A) Various amounts (1, 12.5, 25, 50, 75, 100, and 200 ng) of CHO-obinutuzumab and each 20 μg of total soluble protein extract from the leaves expressed plant-obinutuzumab-HDEL and plant-obinutuzumab-no HDEL were subjected to immunoblot with human and mouse IgG specific-HRP conjugated antibody. The heavy chain bands of CHO-obinutuzumab were quantified to obtain a standard curve then expression amounts of each antibody was estimated by the heavy chain intensities of total soluble lysate. (B). The immunoblot band of 20 μg plant-obinutuzumab-HDEL of total soluble protein extracted in <i>N</i>. <i>benthamiana</i> leaves was used to calculate the production yield.</p