Audrey Yi‐Hui Teh

Thirty‐five years after the first recombinant protein was produced in potato and 30 years after clinical trials of edible vaccines from its tubers, the crop is being reconsidered as a molecular farming chassis. Potatoes can accumulate recombinant proteins in tubers, enabling long‐term storage and simplified logistics. Clonal propagation, access to minitubers and microtubers, and an established production infrastructure further distinguish the platform. Limited pollen dispersal and reliance on vegetative propagation also provide biosafety advantages. Despite these features, potato lost ground to other hosts due to low expression levels, high downstream processing (DSP) costs in water‐ and starch‐rich tissues and limited scalability relative to seed‐propagated crops. We review the technical and economic factors behind this decline and assess new opportunities to overcome them. Recent advances include refined expression cassettes, ER and secretory pathway engineering (including targeted glycoengineering), multigene stacking, genome editing and enzyme‐assisted DSP. Seed‐propagated diploid potatoes and alternative Solanum germplasm offer additional chassis options. Together with the emergence of start‐ups revisiting potato as a production platform, these advances point to practical routes for re‐evaluating its role in plant molecular farming. We argue that a rationally engineered, diploid‐based ‘bioreactor potato’ could complement existing hosts and re‐establish relevance in specific niches of next‐generation biomanufacturing.

The dawn of antibody therapy was heralded by the rise of IgG therapeutics. However, other antibody classes are at our disposal—one of the most exciting is IgA and is the most abundant antibody class within humans. Unlike IgG, it is uniquely specialized for mucosal applications due to its ability to form complex Secretory IgA (SIgA) molecules. Since the mucosa is constantly exposed to potential infectious agents, SIgA is pivotal to disease prevention as an important component of the mucosal barrier. Compared to IgG, SIgA has proven superior effectiveness in mucosal surfaces, such as the airway epithelium or the harsh gut environment. Despite this, hurdles associated with low yield and challenging purification have blocked SIgA therapeutic advancement. However, as a result of new antibody engineering strategies, we are approaching the next generation of (IgA-based) antibody therapies. Strategies include fine-tuning SIgA assembly, exploring different production platforms, genetic engineering to improve purification, and glycoengineering of different components. Due to its stability in mucosal environments, SIgA therapeutics would revolutionize passive mucosal immunotherapy—an avenue still underexploited by current therapeutics. This chapter will focus on the current perspectives of SIgA engineering and explore different approaches to unlocking the full therapeutic potential of SIgAs.

Antibody-dependent enhancement of infection (ADE) is clinically relevant to Dengue virus (DENV) infection and poses a major risk to the application of monoclonal antibody (mAb)-based therapeutics against related flaviviruses such as the Zika virus (ZIKV). Here, we tested a two-tier approach for selecting non-cross-reactive mAbs combined with modulating Fc glycosylation as a strategy to doubly secure the elimination of ADE while preserving Fc effector functions. To this end, we selected a ZIKV-specific mAb (ZV54) and generated three ZV54 variants using Chinese hamster ovary cells and wild-type (WT) and glycoengineered ΔXF Nicotiana benthamiana plants as production hosts (ZV54^CHO, ZV54^WT, and ZV54^ΔXF). The three ZV54 variants shared an identical polypeptide backbone, but each exhibited a distinct Fc N-glycosylation profile. All three ZV54 variants showed similar neutralization potency against ZIKV but no ADE activity for DENV infection, validating the importance of selecting the virus/serotype-specific mAbs for avoiding ADE by related flaviviruses. For ZIKV infection, however, ZV54^CHO and ZV54^ΔXF showed significant ADE activity while ZV54^WT completely forwent ADE, suggesting that Fc glycan modulation may yield mAb glycoforms that abrogate ADE even for homologous viruses. In contrast to the current strategies for Fc mutations that abrogate all effector functions along with ADE, our approach allowed the preservation of effector functions as all ZV54 glycovariants retained antibody-dependent cellular cytotoxicity (ADCC) against the ZIKV-infected cells. Furthermore, the ADE-free ZV54^WT demonstrated in vivo efficacy in a ZIKV-infection mouse model. Collectively, our study provides further support for the hypothesis that antibody–viral surface antigen and Fc-mediated host cell interactions are both prerequisites for ADE, and that a dual-approach strategy, as shown herein, contributes to the development of highly safe and efficacious anti-ZIKV mAb therapeutics. Our findings may be impactful to other ADE-prone viruses, including SARS-CoV-2.

Summary: Immune checkpoint inhibitors (ICIs) have achieved huge clinical success. However, many still have limited response rates, and are prohibitively costly. There is a need for effective and affordable ICIs, as well as local manufacturing capacity to improve accessibility, especially to low‐to‐middle income countries (LMICs). Here, we have successfully expressed three key ICIs (anti‐PD‐1 Nivolumab, anti‐NKG2A Monalizumab, and anti‐LAG‐3 Relatimab) transiently in Nicotiana benthamiana and Nicotiana tabacum plants. The ICIs were expressed with a combination of different Fc regions and glycosylation profiles. They were characterized in terms of protein accumulation levels, target cell binding, binding to human neonatal Fc receptors (hFcRn), human complement component C1q (hC1q) and various Fcγ receptors, as well as protein recovery during purification at 100 mg‐ and kg‐scale. It was found that all ICIs bound to the expected target cells. Furthermore, the recovery during purification, as well as Fcγ receptor binding, can be altered depending on the Fc region used and the glycosylation profiles. This opens the possibility of using these two parameters to fine‐tune the ICIs for desired effector functions. A scenario‐based production cost model was also generated based on two production scenarios in hypothetical high‐ and low‐income countries. We have shown that the product accumulation and recovery of plant production platforms were as competitive as mammalian cell‐based platforms. This highlights the potential of plants to deliver ICIs that are more affordable and accessible to a widespread market, including LMICs.

Molecular pharming in plants offers exciting possibilities to address global access to modern biologics. However, differences in the N-glycosylation pathway including the presence of β(1,2)-xylose and core α(1,3)-fucose can affect activity, potency and immunogenicity of plant-derived proteins. Successful glycoengineering approaches toward human-like structures with no changes in plant phenotype, growth, or recombinant protein expression levels have been reported for Arabidopsis thaliana and Nicotiana benthamiana. Such engineering of N-glycosylation would also be desirable for Nicotiana tabacum, which remains the crop of choice for recombinant protein pharmaceuticals required at massive scale and for manufacturing technology transfer to less developed countries. Here, we generated N. tabacum cv. SR-1 β(1,2)-xylosyltransferase (XylT) and α(1,3)-fucosyltransferase (FucT) knockout lines using CRISPR/Cas9 multiplex genome editing, targeting three conserved regions of the four FucT and two XylT genes. These two enzymes are responsible for generating non-human N-glycan structures. We confirmed full functional knockout of transformants by immunoblotting of total soluble protein by antibodies recognizing β(1,2)-xylose and core α(1,3)-fucose, mass spectrometry analysis of recombinantly produced VRC01, a broadly neutralizing anti-HIV-1 hIgG1 antibody, and Sanger sequencing of targeted regions of the putative transformants. These data represent an important step toward establishing Nicotiana tabacum as a biologics platform for Global Health.

MIDAS‐P is a plant expression vector with blue/white screening for iterative cloning of multiple, tandemly arranged transcription units (TUs). We have used the MIDAS‐P system to investigate the expression of up to five genes encoding three anti‐HIV proteins and the reporter gene DsRed in Nicotiana benthamiana plants. The anti‐HIV cocktail was made up of a broadly neutralizing monoclonal antibody (VRC01), a lectin (Griffithsin), and a single‐chain camelid nanobody (J3‐VHH). Constructs containing different combinations of 3, 4, or 5 TUs encoding different components of the anti‐HIV cocktail were assembled. Messenger RNA (mRNA) levels of the genes of interest decreased beyond two TUs. Coexpression of the RNA silencing suppressor P19 dramatically increased the overall mRNA and protein expression levels of each component. The position of individual TUs in 3 TU constructs did not affect mRNA or protein expression levels. However, their expression dropped to non‐detectable levels in constructs with four or more TUs each containing the same promoter and terminator elements, with the exception of DsRed at the first or last position in 5 TU constructs. This drop was alleviated by co‐expression of P19. In short, the MIDAS‐P system is suitable for the simultaneous expression of multiple proteins in one construct.

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. Graphic

Passive immunization with antibodies is a promising approach against enterotoxigenic Escherichia coli diarrhea, a prevalent disease in LMICs. The objective of this study was to investigate expression of a monoclonal anti-ETEC CfaE secretory IgA antibody in N. benthamiana plants, with a view to facilitating access to ETEC passive immunotherapy. SIgA1 and SIgA2 forms of mAb 68–81 were produced by co-expressing the light and engineered heavy chains with J chain and secretory component in N. benthamiana. Antibody expression and assembly were compared with CHO-derived antibodies by SDS-PAGE, western blotting, size-exclusion chromatography and LC-MS peptide mapping. N-linked glycosylation was assessed by rapid fluorescence/mass spectrometry and LC-ESI-MS. Susceptibility to gastric digestion was assessed in an in vitro model. Antibody function was compared for antigen binding, a Caco-2 cell-based ETEC adhesion assay, an ETEC hemagglutination inhibition assay and a murine in vivo challenge study. SIgA1 assembly appeared superior to SIgA2 in plants. Both sub-classes exhibited resistance to degradation by simulated gastric fluid, comparable to CHO-produced 68–61 SIgA1. The plant expressed SIgAs had more homogeneous N-glycosylation than CHO-derived SIgAs, but no alteration of in vitro functional activity was observed, including antibodies expressed in a plant line engineered for mammalian-like N glycosylation. The plant-derived SIgA2 mAb demonstrated protection against diarrhea in a murine infection model. Although antibody yield and purification need to be optimized, anti-ETEC SIgA antibodies produced in a low-cost plant platform are functionally equivalent to CHO antibodies, and provide promise for passive immunotherapy in LMICs.

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 49mg/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 10-fold 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.

Summary: Plants can provide a cost‐effective and scalable technology for production of therapeutic monoclonal antibodies, with the potential for precise engineering of glycosylation. Glycan structures in the antibody Fc region influence binding properties to Fc receptors, which opens opportunities for modulation of antibody effector functions. To test the impact of glycosylation in detail, on binding to human Fc receptors, different glycovariants of VRC01, a broadly neutralizing HIV monoclonal antibody, were generated in Nicotiana benthamiana and characterized. These include glycovariants lacking plant characteristic α1,3‐fucose and β1,2‐xylose residues and glycans extended with terminal β1,4‐galactose. Surface plasmon resonance‐based assays were established for kinetic/affinity evaluation of antibody–FcγR interactions, and revealed that antibodies with typical plant glycosylation have a limited capacity to engage FcγRI, FcγRIIa, FcγRIIb and FcγRIIIa; however, the binding characteristics can be restored and even improved with targeted glycoengineering. All plant‐made glycovariants had a slightly reduced affinity to the neonatal Fc receptor (FcRn) compared with HEK cell‐derived antibody. However, this was independent of plant glycosylation, but related to the oxidation status of two methionine residues in the Fc region. This points towards a need for process optimization to control oxidation levels and improve the quality of plant‐produced antibodies.