The process of Velaglucerase alpha was design to modify the glycosylation profile of the protein toward oligomannose N-glycans to improve mannose-receptor mediated uptake of the drug into macrophages, the target cells. Here, we determined the optimal concentration of kifunensine and demonstrate that kifunensine addition at a concentration of 0.375 µM in the Agrobacterium infiltration solution of N. benthamiana plants during the vacuum infiltration process allows the production of exclusively high-mannose recombinant proteins. The anti-CD20 monoclonal antibody rituximab, approved for the treatment of non-Hodgkin’s lymphoma, was selected to evaluate the effectiveness of kifunensine for the production of an anti-cancer antibody with enhanced antibody-dependent cell mediated cytotoxicity . ADCC efficacy of rituximab is inversely correlated with the content of core fucose, suggesting that a rituximab variant with altered glycosylation would lower dosing requirements. More importantly, we demonstrate that afucosylated high-mannose decorated antibody, derived from the treated plants, exhibits increased ADCC effector function, as compared with rituximab derived from non-treated plants. The increased ADCC activity was verified using effector cells carrying both FcγRIIIa-V158 and FcγRIIIa-F158 allotypes. Several strategies could be implemented to modulate the plant-specific glycans: Protein containment in the Endoplasmic Reticulum using specific signal sequences, knockdown of fucosyltransferase and xylosyltransferase enzymesin N. benthamiana with RNA interference technology,hydroponic growing system knockout of fucosyltransferase and xylosyltransferase enzymes in N. benthamiana using gene editing, and replacement of plant glycans with human glycans through glyco-remodeling.
Gene editing using sequence-specific transcription-activator-like effector nucleases was only partially effective, while the use of transgenic knockdown lines at manufacturing require more exigent containment and cleaning procedures. Our approach eliminates the need for modification of the primary sequence or the use of transgenic, regulated material for manufacturing. Combined with the scalability and low manufacturing cost associated with the N. benthamiana transient expression system, this method represents an excellent alternative to the use of either glycoengineered or kifunensine-treated mammalian cell lines for the production of afucosylated anti-cancer antibody.We have hypothesized that a treatment with kifunensine would inhibit trimming of mannose residues in the endoplasmic reticulum , subsequently preventing the addition of α1,3-fucose and β1,2-xylose residues on the polysaccharide core . To do so, 60 plants per conditions were vacuum infiltrated in a solution of Agrobacteria with or without kifunensine, ranging from 0 to 5 µM . Visual observation of infiltrated plants from three to seven days post infiltration revealed no noticeable phenotypic or morphological differences between treated and untreated control plants . All leave and stems were collected from each infiltration and pooled for protein extraction and purification. Rituximab expression levels were quantified at 7 dpi and revealed a low to moderate increase in antibody expression between untreated and treated plants, with the average rituximab expression level ranging from 288 mg/kg to 385 mg/kg whole plant fresh weight in treated plants, compared to 287 mg/kg whole plant FW in untreated plants . These observations demonstrated that the kifunensine treatments were not detrimental to plant growth or protein expression. To evaluate the effect of kifunensine treatments on the integrity and assembly of rituximab, SDS-PAGE analysis was carried out under reduced and non-reduced conditions.
As illustrated in Figure 1D, rituximab derived from kifunensine treated and untreated conditions appeared intact and fully assembled. Non-reduced rituximab migrated at the expected molecular weight of ~145 kDa, while the reduced heavy and light chains migrated at the expected MW of ~50 kDa and ~25 kDa, respectively . Infiltration experiments were run in duplicates.The N-glycosylation profiles of purified rituximab expressed in N. benthamiana were evaluated by LC-MS/MS analysis . The major glycoforms were compared based on the relative intensity of the Asn297 glycopeptide masses identified by LC-MS/MS.As previously described in the literature, this plant-derived rituximab control exhibited primarily complex-type N-glycans, with the most abundant N-glycan structure being GlcNAc2Man3GlcNAc2 . On the other hand, there were significant differences in rituximab N-glycan profiles between untreated and treated samples. Complete conversion of plant complex glycans to oligomannose-type glycans was observed when N. benthamiana plants were infiltrated with ‘higher range’ and ‘medium range’ concentrations of kifunensine . The GlcNAc2Man9 and GlcNAc2Man8 were the major glycoforms observed with Man9 being the most abundant. In fact, the same oligomannose-type glycoform distribution was observed whether 0.375 µM or higher kifunensine concentration was used, indicating that 0.375 µM is sufficient to provide homogeneous rituximab with oligomannose-type glycans . When lower concentrations of kifunensine were used, a mixture of oligomannose, hybrid, and complex glycans was detected . For instance, the glycosylation profifile of rituximab from plants treated with 0.25 µM contained more than ~48% hybrid/complex glycan modififications and ~52% oligomannose glycosylation .
Importantly, no α1,3-fucose or β1,2-xylose residues were detected in rituximab derived from plants treated with ≥0.375 µM kifunensine.In this study, we have investigated the use of kifunensine in a plant expression platform that is established for large-scale manufacturing for recombinant protein. More specifically, we evaluated the concentration of kifunensine sufficient for the production of an afucosylated rituximab with enhanced biological activity. The vacuum infiltration of plants in a solution containing Agrobacterium culture supplemented with kifunensine at a concentration varying from 0.0625 µM to 5 µM did not affect the seven-day post-infiltration growth of the plants or the expression of rituximab. In fact, the expression of rituximab was slightly higher from kifunensine-treated plants. Similar tolerance to kifunensine treatment has also been described for the expression of antibodies in Chinese Hamster Ovary cells. However, it has been reported in one instance that kifunensine treatment of N. benthamiana plants via the growth medium led to a decrease in expression of a recombinant protein]. When applied during plant vacuum infiltration in the Agroinfiltration solution, kifunensine enters the interstitial spaces of the leaf tissue in contact with the host cells where recombinant protein expression occurs, rather than through uptake via the root system. The positive impact of infiltrated kifunensine on host cell tolerance and protein production may be due to its suppressing effects on the ER-associated degradation pathway where proteins with trimmed oligomannose glycans may be degraded if the polysaccharide chain is not further processed or proteins transported to the Golgi apparatus.Kifunensine treatment during the agroinfiltration ultimately results in protein afucosylation as it stops mannose trimming in the endoplasmic reticulum, yielding Man5-Man9 N-glycan structures. When delivered in this fashion, a minimum concentration of 0.375 µM kifunensine was sufficient to generate rituximab harboring only oligomannose glycan structures lacking fucose residues. In agreement with reports using mammalian cell cultures, the minimum required kifunensine concentration to generate antibody devoid of fucose residues in N. benthamiana falls somewhere between 0.25 and 0.375 µM . Kifunensine has the practical advantage of being active at 2-to-4-fold-lower concentrations than other inhibitors of the glycosylation pathway making it more cost-effective. Moreover, as described here and in mammalian cell cultures, treatments with kifunensine leads to a highly homogeneous product, with ultimately no formation of core-fucosylated hybrid structures.Proteins produced with glycan-engineering technologies not only lack potentially immunogenic plant-specific glycoforms,hydroponic growing but also provide enhanced effector function. The in vitro bioassay described in this study demonstrated enhanced ADCC activity from rituximab containing high-mannose glycoforms. It is expected that the reason for this increased ADCC activity lies in the absence of fucose residues on the glycosylation core rather than the high content of mannose residues, as many studies have reported the effect of afucosylation on the ADCC activity of anti-cancer antibodies including rituximab. In fact, similar ADCC results were obtained with antibodies derived from CHO cell cultures treated with kifunensine. It is important to note that ADCC activity was linearly proportional to the relative abundance of oligomannose glycoforms. This was particularly evident with ‘low-range’ concentrations of kifunensine applied, which generated a mixture of complex, hybrid, and oligomannose structures. With rituximab derived from a treatment of 0.25 µM kifunensine, generating a relatively small increase of oligomannose glycoforms, there was a significant but lower increase in ADCC activity . Importantly, plant-derived high-mannose rituximab glycoforms exhibited the same affinity for CD20 as Rituxan®, the commercial standard. Thus, the kifunensine treatment does not affect the paratope conformation of the plant-derived antibody.
As the glycosylation profile of an anti-cancer antibody is correlated to its biological activity, it is therefore considered as a critical quality attribute that needs to be maintained during the manufacturing process. To that end, the glycosylation profile of kifunensine-treated antibodies is homogeneous, consistent, and easy to control at scale, which represent a significant advantage for this technology. There are reports that antibodies carrying high mannose glycans have a shorter serum half-life, as compared with other glycoforms. However, other pharmacokinetic studies with afucosylated high mannose antibodies indicated no impact on clearance. Thus, the pharmacokinetics property of any antibody will have to be evaluated based on its biological activity, the target indication, and dosage regimen. In conclusion, the application of kifunensine during transient agroinfiltration of the N. benthamiana host leverages the scalability and cost-effectiveness of the plant expression platform for the production of biobetter anti-cancer antibodies. First, the scale-up of rituximab expression was also demonstrated at the iBio CDMO facility using manufacturing procedures without affecting the expression or product quality. Second, using the process model described by Holtz et al. 2015, and the findings from this study , the cost of cGMP -grade kifunensine to produce high-mannose, afucosylated antibodies at manufacturing scale was estimated to be less than $0.80/g of antibody produced. Thus, kifunensine can be incorporated into already established manufacturing protocols without affecting production cost significantly. Further studies will focus on determining how long the inhibition effect of kifunensine lasts during and after the plant infiltration process. This attempt to increase anti-cancer efficacy of recombinant antibodies through in-process glycan engineering represents a promising alternative to meet unmet medical needs.The ADCC reporter assay was performed using Wil2-S cells as targets along with Jurkat-CD16 reporter cell lines. Two reporter cell lines stably expressing the FcγRIIIa receptor, V158 or F158 variants were used. Wil2-S cells were plated in a 96-well white bottom assay plate at 5000 cells per well. Serial dilutions of test antibodies were added to the plates containing the target cells and incubated at 4 ◦C for 15 min to allow opsonization. Jurkat-CD16 reporter cells were then added to assay plates already containing Wil2-S cells and antibodies. The fifinal concentration of antibodies ranged from 2 to 0.0003 µg/mL following several 3-fold dilutions. The effector:target cell ratio was 10:1. After a 6 h. incubation at 37 ◦C, One-Glo™ Luciferase Assay Reagent was added and luminescence was determined using a Gen5 microplate reader. Samples and controls were tested in triplicate, and the mean reporter signals of sample dilutions in Relative Luminescence Units were plotted against the antibody concentration. Antibody independent cellular cytotoxicity was measured in wells containing target and effector cells without antibodies. GraphPad prism software was used to plot normalized RLU versus Log10. The half maximal effective concentration values of plant-made rituximab and Fc variants were derived as dose responses obtained from non-linear regression curves. Fold of induction was calculated by taking the ratio of background subtracted induced RLU and background subtracted untreated control. Nitrogen is a crucial plant nutrient; to encourage large yields, farmers tend to apply excess nitrogen fertilizers to their crops. However, crop plants are generally inefficient at nitrogen uptake from the soil, with as much as 50 to 75% of applied N being unused by the plants . Crop plants compete for soil N against soil microbes involved with denitrification and nitrification, volatization to the atmosphere, as well as loss of N by leaching into waterways . It is important to breed and/or design nitrogen use efficient crop plants that can produce the same, or higher yields with less applied N fertilizer. Growth of NUE crop plants, coupled with implementation of best fertilizer management practices, would allow for a reduction of applied N fertilizer per hectare. This would both greatly reduce the N fertilizer expense for the farmer and greatly reduce the environmental pollution from excess N fertilizers. We have recently developed genetically engineered rice by introducing a barley alanine aminotransferase cDNA driven by a rice tissue specific promoter, OsAnt1 . This modification significantly increased biomass and grain yield in the transgenic plants compared to control plants when the plants were grown at a fixed, high amount of ammonium as the N source. As well, we analysed the transcriptomic profile of these transgenic plants grown at the fixed N concentration using Affymetrix Rice GeneChip microarrays to provide further insights into the nature of increased NUE of these transgenics . In this study, we compared various physiological and genetic data from alanine aminotransferase over-expressing transgenic plants to control plants grown at three different nitrogen levels and demonstrated significant changes between them.