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. 2021 Jun 28;18(1):17.
doi: 10.1186/s12977-021-00560-6.

Characterisation of a highly potent and near pan-neutralising anti-HIV monoclonal antibody expressed in tobacco plants

Catherine M Moore  1 , Melanie Grandits  1 , Clemens Grünwald-Gruber  2 , Friedrich Altmann  2 , Maria Kotouckova  1 , Audrey Y-H Teh  3 , Julian K-C Ma  4
Affiliations

Characterisation of a highly potent and near pan-neutralising anti-HIV monoclonal antibody expressed in tobacco plants

Catherine M Moore et al. Retrovirology. .

Abstract

Background: HIV remains one of the most important health issues worldwide, with almost 40 million people living with HIV. Although patients develop antibodies against the virus, its high mutation rate allows evasion of immune responses. Some patients, however, produce antibodies that are able to bind to, and neutralise different strains of HIV. One such 'broadly neutralising' antibody is 'N6'. Identified in 2016, N6 can neutralise 98% of HIV-1 isolates with a median IC50 of 0.066 µg/mL. This neutralisation breadth makes N6 a very promising therapeutic candidate.

Results: N6 was expressed in a glycoengineered line of N. benthamiana plants (pN6) and compared to the mammalian cell-expressed equivalent (mN6). Expression at 49 mg/kg (fresh leaf tissue) was achieved in plants, although extraction and purification are more challenging than for most plant-expressed antibodies. N-glycoanalysis demonstrated the absence of xylosylation and a reduction in α(1,3)-fucosylation that are typically found in plant glycoproteins. The N6 light chain contains a potential N-glycosylation site, which was modified and displayed more α(1,3)-fucose than the heavy chain. The binding kinetics of pN6 and mN6, measured by surface plasmon resonance, were similar for HIV gp120. pN6 had a tenfold higher affinity for FcγRIIIa, which was reflected in an antibody-dependent cellular cytotoxicity assay, where pN6 induced a more potent response from effector cells than that of mN6. pN6 demonstrated the same potency and breadth of neutralisation as mN6, against a panel of HIV strains.

Conclusions: The successful expression of N6 in tobacco supports the prospect of developing a low-cost, low-tech production platform for a monoclonal antibody cocktail to control HIV in low-to middle income countries.

Keywords: HIV; Immunotherapy; Molecular pharming; Monoclonal antibodies; Plants; bNAbs.

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Conflict of interest statement

The authors are aware of no potential conflicts of interest.

Figures

Fig. 1
Fig. 1
Expression of N6 antibody in N. benthamiana. A Human IgG kappa (HuIgGk) from human serum (Sigma), mock-infiltrated sample from leaf disk (mock), N6 extracted from leaf disk (pN6), and Precision Plus Protein™ All Blue Pre-stained Protein Standards (M) were run, either reduced (R) or not reduced (NR), on SDS-PAGE before blotting onto nitrocellulose. Heavy chain was detected with mouse anti-human IgG Fc domain and light chain was detected with goat anti-human kappa light chain. Secondary antibodies were donkey anti-mouse with green fluorescent tag, and donkey anti-goat with red fluorescent tag. Black arrows indicate, from top to bottom, fully assembled antibody (yellow), heavy chain (green) and light chain (red). B Sandwich ELISA detecting fully assembled antibody in plant crude extract. Leaf disks taken from N. benthamiana transiently expressing N6 (triangle), human IgG kappa positive control (circle) or plants mock-infiltrated with infiltration solution only (square) were extracted in PBS and introduced to an ELISA plate coated with goat anti-human IgG Fc domain antibody. Bound antibodies were detected using HRP-conjugated goat anti-human IgG kappa light chain antibody. Representative of 3 biological replicates (i.e. separate plants and infiltration experiments). Each ELISA was performed with 2 technical replicates. Means derived from 2 leaf disks per sample ± S.D. Yields were estimated using Graphpad Prism software, fitting to Michaelis Menton equation
Fig. 2
Fig. 2
N6 antigen binding assessment. A ELISA demonstrating specific binding of anti-HIV antibody to its cognate antigen—gp140. Purified pN6 antibody (triangle), commercially-sourced human IgG1 kappa (square) and VRC01 (circle) were incubated on an ELISA plate coated with gp140. VRC01 (circle) was previously purified from tobacco plants in-house. Bound antibodies were detected using HRP-conjugated goat anti-human IgG Fc domain antibody. Human IgG kappa (square) was included as a negative control at 100 ng/ml only. Representative of 3 biological replicates. ELISAs were performed with 2 technical replicates. Data shown are mean ± S.D. B Surface plasmon resonance measuring binding kinetics of pN6 antibody compared to mN6. Protein A was immobilised onto a CM5 chip and N6 antibody was captured to 5000 RU. C Calculated association constant (ka), dissociation constant (kd) and affinity (KD) from surface plasmon resonance were estimated using the Langmuir model of binding (1:1), with BIAcore™ Evaluation software. Both versions of N6 bind to gp140 with an equivalent affinity
Fig. 3
Fig. 3
Neutralisation of HIV-1 pseudoviruses by pN6. A Neutralisation by pN6 was assessed against a panel of 10 HIV-1 ENV pseudoviruses, and HIV-1 strain BaL.26 as an internal control, using TZM-bl cells. pN6 neutralisation assay was carried out in triplicate, and mN6 BaL.26 set was carried out in duplicate. B Correlation of published mN6 IC50s with pN6 IC50s from pseudovirus neutralisation assay. Open circle denotes assay-derived IC50 of pN6 compared with mN6 for BaL.26, rather than published value, as an internal control. Pearson correlation analysis calculated r value of 0.92 (p ≤ 0.0001)
Fig. 4
Fig. 4
Glycosylation analysis of pN6. A PNGaseF assay where 1 µg each of PNGaseF-digested antibody (+P) was compared to undigested antibody (−P), including the positive control HuIgGk (human IgG1 kappa antibody, Sigma). Marker (M) is Precision Plus Protein™ All Blue Pre-stained Protein Standards. Heavy and light chains are indicated by black arrows. PNGase F enzyme visible at 36 kDa. B Percent abundance, derived from mass spectrometry, of various glycoforms in the heavy (Fc) and light (K) chains. C Mass spectra of pN6 heavy and light chain glycoforms. Purified proteins were analysed by digestion with trypsin followed by LC–ESI–MS. Glycopeptides from the kappa-chain variable region occurred as doubly charged ions, partly with ammonium
Fig. 5
Fig. 5
N6 Fc effector function assessment. A Binding kinetics of FcγRIIIa to pN6 and mN6 measured by surface plasmon resonance. Protein A was immobilised onto a CM5 chip and N6 antibody was captured to 5000 RU. Association constant (ka), dissociation constant (kd) and affinity (KD) were estimated using the Langmuir model of binding (1:1), with BIAcore™ Evaluation software. B Surface plasmon resonance measuring binding kinetics of pN6 compared to mN6. C Antibody dependent cellular cytotoxicity (ADCC) assay comparing activation of ADCC by pN6 (black) with mN6 (white). ADCC is detected by reporter effector cells expressing luciferase when activated. Results are from 3 technical replicates ± S.D. Statistical analyses carried out using Graphpad Prism software Student’s T-test. * p ≤ 0.05. ** p ≤ 0.006
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