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

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

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

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

Engineering the N-glycosylation pathway of Nicotiana tabacum for molecular pharming using CRISPR/Cas9.

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

Investigation of allergen proteins in five tomato cultivars

Investigation of putative allergens from tomato berries is challenging as differences between human serum IgE specificity and reactivity as well as the non-specific binding of the primary and secondary antibodies often cause difficulties. In this study five tomato cultivars were investigated to evaluate their potential allergenicity in Hungarian tomato sensitive patients. The major allergens proved to be low molecular weight proteins, but several previously described allergens could be identified as well using IgE-Western blotting. IgE binding to cross-reactive carbohydrate determinants (CCDs) was ruled out through the use of CCD inhibitor, but non-specific binding of the secondary antibody remained an issue. IgE binding activity of a purified, immunoblot positive protein (band at 40 kDa), and non-specific binding of the secondary antibody to the same protein, was demonstrated with an Optical Waveguide Lightmode Spectroscopy (OWLS) based immunosensor. LC-ESI-MS/MS analysis showed this protein is an as-yet undescribed vicilin-type putative allergen

Improving the efficacy of plant-made anti-HIV monoclonal antibodies for clinical use

IntroductionBroadly neutralising antibodies are promising candidates for preventing and treating Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS), as an alternative to or in combination with antiretroviral therapy (ART). These mAbs bind to sites on the virus essential for virus attachment and entry, thereby inhibiting entry into the host cell. However, the cost and availability of monoclonal antibodies, especially combinations of antibodies, hampers implementation of anti-HIV bNAb therapies in low- to middle- income countries (LMICs) where HIV-1 prevalence is highest.MethodsWe have produced three HIV broadly neutralizing antibodies (bNAbs), 10-1074, VRC01 and 3BNC117 in the Nicotiana benthamiana transient expression system. The impact of specific modifications to enhance potency and efficacy were assessed. To prolong half-life and increase bioavailability, a M252Y/S254T/T256E (YTE) or M428L/N434S (LS) mutation was introduced. To increase antibody dependent cellular cytotoxicity (ADCC), we expressed an afucosylated version of each antibody using a glycoengineered plant line.ResultsThe majority of bNAbs and their variants could be expressed at yields of up to 47 mg/kg. Neither the expression system nor the modifications impacted the neutralization potential of the bNAbs. Afucosylated bNAbs exhibit enhanced ability to bind to FcγRIIIa and trigger ADCC, regardless of the presence of Fc amino acid mutations. Lastly, we demonstrated that Fc-modified variants expressed in plants show enhanced binding to FcRn, which results in a favourable in vivo pharmacokinetic profile compared to their unmodified counterparts. ConclusionTobacco plants are suitable expression hosts for anti-HIV bNAbs with increased efficacy and an improved pharmacokinetic profile

Impact of mutations on the plant-based production of recombinant SARS-CoV-2 RBDs

Subunit vaccines based on recombinant viral antigens are valuable interventions to fight existing and evolving viruses and can be produced at large-scale in plant-based expression systems. The recombinant viral antigens are often derived from glycosylated envelope proteins of the virus and glycosylation plays an important role for the immunogenicity by shielding protein epitopes. The receptor-binding domain (RBD) of the SARS-CoV-2 spike is a principal target for vaccine development and has been produced in plants, but the yields of recombinant RBD variants were low and the role of the N-glycosylation in RBD from different SARS-CoV-2 variants of concern is less studied. Here, we investigated the expression and glycosylation of six different RBD variants transiently expressed in leaves of Nicotiana benthamiana. All of the purified RBD variants were functional in terms of receptor binding and displayed almost full N-glycan occupancy at both glycosylation sites with predominately complex N-glycans. Despite the high structural sequence conservation of the RBD variants, we detected a variation in yield which can be attributed to lower expression and differences in unintentional proteolytic processing of the C-terminal polyhistidine tag used for purification. Glycoengineering towards a human-type complex N-glycan profile with core α1,6-fucose, showed that the reactivity of the neutralizing antibody S309 differs depending on the N-glycan profile and the RBD variant

Implications of O-glycan modifications in the hinge region of a plant-produced SARS-CoV-2-IgA antibody on functionality.

Introduction: Prolyl-4-hydroxylases (P4H) catalyse the irreversible conversion of proline to hydroxyproline, constituting a common posttranslational modification of proteins found in humans, plants, and microbes. Hydroxyproline residues can be further modified in plants to yield glycoproteins containing characteristic O-glycans. It is currently unknown how these plant endogenous modifications impact protein functionality and they cause considerable concerns for the recombinant production of therapeutic proteins in plants. In this study, we carried out host engineering to generate a therapeutic glycoprotein largely devoid of plant-endogenous O-glycans for functional characterization. Methods: Genome editing was used to inactivate two genes coding for enzymes of the P4H10 subfamily in the widely used expression host Nicotiana benthamiana. Using glycoengineering in plants and expression in human HEK293 cells we generated four variants of a potent, SARS-CoV-2 neutralizing antibody, COVA2-15 IgA1. The variants that differed in the number of modified proline residues and O-glycan compositions of their hinge region were assessed regarding their physicochemical properties and functionality. Results: We found that plant endogenous O-glycan formation was strongly reduced on IgA1 when transiently expressed in the P4H10 double mutant N. benthamiana plant line. The IgA1 glycoforms displayed differences in proteolytic stability and minor differences in receptor binding thus highlighting the importance of O-glycosylation in the hinge region of human IgA1. Discussion: This work reports the successful protein O-glycan engineering of an important plant host for recombinant protein expression. While the complete removal of endogenous hydroxyproline residues from the hinge region of plant-produced IgA1 is yet to be achieved, our engineered line is suitable for structure-function studies of O-glycosylated recombinant glycoproteins produced in plants

Investigation of a monoclonal antibody against enterotoxigenic Escherichia coli, expressed as secretory IgA1 and IgA2 in plants.

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

A comprehensive overview of radioguided surgery using gamma detection probe technology

The concept of radioguided surgery, which was first developed some 60 years ago, involves the use of a radiation detection probe system for the intraoperative detection of radionuclides. The use of gamma detection probe technology in radioguided surgery has tremendously expanded and has evolved into what is now considered an established discipline within the practice of surgery, revolutionizing the surgical management of many malignancies, including breast cancer, melanoma, and colorectal cancer, as well as the surgical management of parathyroid disease. The impact of radioguided surgery on the surgical management of cancer patients includes providing vital and real-time information to the surgeon regarding the location and extent of disease, as well as regarding the assessment of surgical resection margins. Additionally, it has allowed the surgeon to minimize the surgical invasiveness of many diagnostic and therapeutic procedures, while still maintaining maximum benefit to the cancer patient. In the current review, we have attempted to comprehensively evaluate the history, technical aspects, and clinical applications of radioguided surgery using gamma detection probe technology

Improving the efficacy of plant-made anti-HIV monoclonal antibodies for clinical use

Introduction: Broadly neutralising antibodies are promising candidates for preventing and treating Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS), as an alternative to or in combination with antiretroviral therapy (ART). These mAbs bind to sites on the virus essential for virus attachment and entry, thereby inhibiting entry into the host cell. However, the cost and availability of monoclonal antibodies, especially combinations of antibodies, hampers implementation of anti-HIV bNAb therapies in low- to middle- income countries (LMICs) where HIV-1 prevalence is highest. Methods: We have produced three HIV broadly neutralizing antibodies (bNAbs), 10-1074, VRC01 and 3BNC117 in the Nicotiana benthamiana transient expression system. The impact of specific modifications to enhance potency and efficacy were assessed. To prolong half-life and increase bioavailability, a M252Y/S254T/T256E (YTE) or M428L/N434S (LS) mutation was introduced. To increase antibody dependent cellular cytotoxicity (ADCC), we expressed an afucosylated version of each antibody using a glycoengineered plant line. Results: The majority of bNAbs and their variants could be expressed at yields of up to 47 mg/kg. Neither the expression system nor the modifications impacted the neutralization potential of the bNAbs. Afucosylated bNAbs exhibit enhanced ability to bind to FcγRIIIa and trigger ADCC, regardless of the presence of Fc amino acid mutations. Lastly, we demonstrated that Fc-modified variants expressed in plants show enhanced binding to FcRn, which results in a favourable in vivo pharmacokinetic profile compared to their unmodified counterparts. Conclusion: Tobacco plants are suitable expression hosts for anti-HIV bNAbs with increased efficacy and an improved pharmacokinetic profile