Codon optimization regulates IgG3 and IgM expression and glycosylation in N. benthamiana

Plants are being increasingly recognized for the production of complex human proteins, including monoclonal antibodies (mAbs). Various methods have been applied to boost recombinant expression, with DNA codon usage being an important approach. Here, we transiently expressed three complex human mAbs in Nicotiana benthamiana, namely one IgG3 and two IgM directed against SARS-CoV-2 as codon optimized(CO) and non-codon optimized (NCO) variants. qRT-PCR exhibited significantly increased mRNA levels of all CO variants compared to the non-codon optimized orthologues, in line with increased protein expression. Purified CO and NCO mAbs did not exhibit obvious biochemical differences, as determined by SDS-PAGE and antigen binding activities. By contrast, enhanced production selectively impacts on glycosite occupancy and N-glycan processing, with increased mannosidic structures. The results point to a careful monitoring of recombinant proteins upon enhancing expression. Especially if it comes to therapeutic application even subtle modifications might alter product efficacy or increase immunogenicity

Glyco engineered pentameric SARS-CoV-2 IgMs show superior activities compared to IgG1 orthologues

Immunoglobulin M (IgM) is the largest antibody isotype with unique features like extensive glycosylation and oligomerization. Major hurdles in characterizing its properties are difficulties in the production of well-defined multimers. Here we report the expression of two SARS-CoV-2 neutralizing monoclonal antibodies in glycoengineered plants. Isotype switch from IgG1 to IgM resulted in the production of IgMs, composed of 21 human protein subunits correctly assembled into pentamers. All four recombinant monoclonal antibodies carried a highly reproducible human-type N-glycosylation profile, with a single dominant N-glycan species at each glycosite. Both pentameric IgMs exhibited increased antigen binding and virus neutralization potency, up to 390-fold, compared to the parental IgG1. Collectively, the results may impact on the future design of vaccines, diagnostics and antibody-based therapies and emphasize the versatile use of plants for the expression of highly complex human proteins with targeted posttranslational modifications

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

\u3ci\u3eIn Vitro and In Vivo\u3c/i\u3e Efficacy of Anti-Chikungunya Virus Monoclonal Antibodies Produced in Wild-Type and Glycoengineered \u3ci\u3eNicotiana benthamiana\u3c/i\u3e Plants

Chikungunya virus (CHIKV) is a mosquito‐transmitted alphavirus, and its infection can cause long‐term debilitating arthritis in humans. Currently, there are no licensed vaccines or therapeutics for human use to combat CHIKV infections. In this study, we explored the feasibility of using an anti‐CHIKV monoclonal antibody (mAb) produced in wild‐type (WT) and glycoengineered (∆XFT) Nicotiana benthamiana plants in treating CHIKV infection in a mouse model. CHIKV mAb was efficiently expressed and assembled in plant leaves and enriched to homogeneity by a simple purification scheme. While mAb produced in ∆XFT carried a single N‐glycan species at the Fc domain, namely GnGn structures, WT produced mAb exhibited a mixture of N‐glycans including the typical plant GnGnXF3 glycans, accompanied by incompletely processed and oligomannosidic structures. Both WT and ∆XFT plant‐produced mAbs demonstrated potent in vitro neutralization activity against CHIKV. Notably, both mAb glycoforms showed in vivo efficacy in a mouse model, with a slight increased efficacy by the ∆XFT‐produced mAbs. This is the first report of the efficacy of plant‐produced mAbs against CHIKV, which demonstrates the ability of using plants as an effective platform for production of functionally active CHIKV mAbs and implies optimization of in vivo activity by controlling Fc glycosylation

DataSheet_1_Impact of mutations on the plant-based production of recombinant SARS-CoV-2 RBDs.pdf

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.</p

Engineering of complex protein sialylation in plants

Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP &gt;40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions