The tobacco-related species Nicotiana benthamiana has recently emerged as a promising host for the manufacturing of protein therapeutics. However, the production of recombinant proteins in N. benthamiana is frequently hampered by undesired proteolysis. Here, we show that the expression of the human anti-HIV antibodies 2F5, 2G12, and PG9 in N. benthamiana leaves leads to the accumulation of discrete heavy chain-derived degradation products of 30–40 kDa. Incubation of purified 2F5 with N. benthamiana intercellular fluid resulted in rapid conversion into the 40-kDa fragment, whereas 2G12 proved largely resistant to degradation. Such a differential susceptibility to proteolytic attack was also observed when these two antibodies were exposed to various types of proteinases in vitro. While serine and cysteine proteinases are both capable of generating the 40-kDa 2F5 fragment, the 30-kDa polypeptide is most readily obtained by treatment with the latter class of enzymes. The principal cleavage sites reside within the antigen-binding domain, the VH–CH1 linker segment and the hinge region of the antibodies. Collectively, these results indicate that down-regulation of endogenous serine and cysteine proteinase activities could be used to improve the performance of plant-based expression platforms destined for the production of biopharmaceuticals.

Plants are gaining increasingly acceptance as a production platform for recombinant proteins. One reason for this is their ability to carry out posttranslational protein modifications in a similar if not identical way as mammalian cells. The capability of plants to carry out human-like complex glycosylation is well known. Moreover, the targeted manipulation of the plant N-glycosylation pathway allows the production of proteins carrying largely homogeneous, human-type oligosaccharides. These outstanding results have placed plants in a favourable position compared to other eukaryotic expression systems. This review provides a comprehensive summary of the N-glycosylation of plant-produced recombinant proteins, the possible impact of plant-specific N-glycans on the human immune system, and recent advances in engineering the plant N-glycosylation pathway towards the synthesis of (complex) human-type glycan structures, highlighting challenges and achievements in the application of these powerful technologies.

Significance IgM antibodies are increasingly gaining interest as therapeutics; however, knowledge about this antibody class is scarce. Specifically the impact of N-glycans on the functional mechanism of this heavily glycosylated molecule is entirely unknown. To address this issue we produced different IgM glycoforms in plants and characterized them. Moreover, we present a computer model that explains the characteristic N-glycosylation pattern of IgMs. With the successful in planta generation of recombinant IgMs largely resembling the plasma-derived orthologue, we offer an efficient alternative to mammalian cell-based expression systems. IgMs with targeted glycoengineered N-glycans now enable detailed structure–function studies and will lead to the production of IgMs with optimized in vivo activities.

SummaryTo study how the P19 suppressor of gene-silencing protein can be used effectively for the production of therapeutic glycoproteins, the following factors were examined: the genetic elements used for expressing recombinant proteins; the effect of different P19 concentrations; compatibility of P19 with various Nicotiana tabacum cultivars for transgenic expression; the glycan profile of a recombinant therapeutic glycoprotein co-expressed with P19 in an RNAibased glycomodified Nicotiana benthamiana expression host. The coding sequences for the heavy and light chains of trastuzumab were cloned into five plant expression vectors (102-106) containing different 5′ and 3′ UTRs, designated as vector sets 102-106 mAb. The P19 protein of Tomato bushy stunt virus (TBSV) was also cloned into vector 103, which contained the Cauliflower mosaic virus (CaMV) 35S promoter and 5′UTR together with the terminator region of the nopaline synthase gene of Agrobacterium. Transient expression of the antibody vectors resulted in different levels of trastuzumab accumulation, the highest being 105 and 106 mAb at about 1% of TSP. P19 increased the concentration of trastuzumab approximately 15-fold (to about 2.3% of TSP) when co-expressed with 103 mAb but did not affect antibody levels with vectors 102 and 106 mAb. When 103 mAb was expressed together with P19 in different N. tabacum cultivars, all except Little Crittenden showed a marked discolouring of the infiltrated areas of the leaf and decreased antibody expression. Co-expression of P19 also abolished antibody accumulation in crosses between N. tabacum cv. I-64 and Little Crittenden, indicating a dominant mode of inheritance for the observed P19-induced responses.

SummarySeed-specific expression is an appealing alternative technology for the production of recombinant proteins in transgenic plants. Whereas attractive yields of recombinant proteins have been achieved by this method, little attention has been paid to the intracellular deposition and the quality of such products. Here, we demonstrate a comparative study of two antiviral monoclonal antibodies (mAbs) (HA78 against Hepatitis A virus; 2G12 against HIV) expressed in seeds of Arabidopsis wild-type (wt) plants and glycosylation mutants lacking plant specific N-glycan residues. We demonstrate that 2G12 is produced with complex N-glycans at great uniformity in the wt as well as in the glycosylation mutant, carrying a single dominant glycosylation species, GnGnXF and GnGn, respectively. HA78 in contrast, contains additionally to complex N-glycans significant amounts of oligo-mannosidic structures, which are typical for endoplasmic reticulum (ER)-retained proteins. A detailed subcellular localization study demonstrated the deposition of both antibodies virtually exclusively in the extracellular space, illustrating their efficient secretion. In addition, although a KDELtagged version of 2G12 exhibited an ER-typical N-glycosylation pattern, it was surprisingly detected in protein storage vacuoles. The different antibody variants showed different levels of degradation with hardly any degradation products detectable for HA78 carrying GnGnXF glycans. Finally, we demonstrate functional integrity of the HA78 and 2G12 glycoforms using viral inhibition assays. Our data therefore demonstrate the usability of transgenic seeds for the generation of mAbs with a controlled N-glycosylation pattern, thus expanding the possibilities for the production of optimally glycosylated proteins with enhanced biological activities for the use as human therapeutics.

SARS-CoV-2 infection results in viral burden in the respiratory tract, enabling transmission and leading to substantial lung pathology. The 1212C2 fully human monoclonal antibody was derived from an IgM memory B cell of a COVID-19 patient, has high affinity for the Spike protein Receptor Binding Domain, neutralizes SARS-CoV-2 and exhibits in vivo prophylactic and therapeutic activity in hamsters when delivered intraperitoneally, reducing upper and lower respiratory viral burden and lung pathology. Inhalation of nebulized 1212C2 at levels as low as 0.6mg/kg, corresponding to 0.03mg/kg of lung deposited dose, reduced viral burden below the detection limit, and mitigated lung pathology. The therapeutic efficacy of an exceedingly low-dose of inhaled 1212C2 supports the rationale for local lung delivery for dose-sparing benefits as compared to the conventional parenteral route of administration. These results suggest clinical development of 1212C2 formulated and delivered via inhalation for the treatment of SARS-CoV-2 infection should be considered.

Broadly neutralizing anti-HIV-1 monoclonal antibodies, such as PG9, and its derivative RSH hold great promise in AIDS therapy and prevention. An important feature related to the exceptional efficacy of PG9 and RSH is the presence of sulfated tyrosine residues in their antigen-binding regions. To maximize antibody functionalities, we have now produced glycan-optimized, fucose-free versions of PG9 and RSH in Nicotiana benthamiana. Both antibodies were efficiently sulfated in planta on coexpression of an engineered human tyrosylprotein sulfotransferase, resulting in antigen-binding and virus neutralization activities equivalent to PG9 synthesized by mammalian cells ( CHO PG9). Based on the controlled production of both sulfated and nonsulfated variants in plants, we could unequivocally prove that tyrosine sulfation is critical for the potency of PG9 and RSH. Moreover, the fucose-free antibodies generated in N. benthamiana are capable of inducing antibody-dependent cellular cytotoxicity, an activity not observed for CHO PG9. Thus, tailoring of the antigen-binding site combined with glycan modulation and sulfoengineering yielded plant-produced anti-HIV-1 antibodies with effector functions superior to PG9 made in CHO cells.onoclonal antibodies (mAbs) offer great promise for AIDS treatment (1). In particular, the recent discovery of broadly neutralizing anti-HIV-1 mAbs (bNAbs) with extraordinary potency as exemplified by the antibodies PG9, PG16 (2), or those of the PGT series (3) creates hope for effective therapy by passive antibody transfer. PG9 and its close relative PG16 neutralize ∼80% of HIV-1 isolates across all clades (2, 4). The recognized epitopes are within the hypervariable and heavily glycosylated V1/V2 loops of the viral envelope glycoprotein gp120 and preferentially displayed in its trimeric state (2). Both mAbs use their unusually long complementarity-determining region (CDR) H3 domains (4-6) to penetrate the glycan shield of the virus and make contact with the underlying protein backbone (7). In addition, PG9 and PG16 recognize two highly conserved gp120 N-glycans attached to Asn 160 and Asn 156/173 , which flank the peptide epitope (7-9). Remarkably, the glycan-binding properties of the two antibodies could be combined by modification of the PG9 light chain with R L94

Intracellular trafficking and subcellular deposition are critical factors influencing the accumulation and posttranslational modifications of proteins. In seeds, these processes are not yet fully understood. In this study, we set out to investigate the intracellular transport, final destination, N-glycosylation status, and stability of the fusion of recombinant single-chain variable fragments to the crystallizing fragment of an antibody (scFv-Fc) of two antiviral monoclonal antibodies (2G12 and HA78). The scFv-Fcs were expressed in Arabidopsis (Arabidopsis thaliana) seeds and leaves both as secretory molecules and tagged with an endoplasmic reticulum (ER) retention signal. We demonstrate differential proteolytic degradation of scFv-Fcs in leaves versus seeds, with higher degradation in the latter organ. In seeds, we show that secretory versions of HA78 scFv-Fcs are targeted to the extracellular space but are deposited in newly formed ER-derived vesicles upon KDEL tagging. These results are in accordance with the obtained N-glycosylation profiles: complex-type and ER-typical oligomannosidic N-glycans, respectively. HA78 scFv-Fcs, expressed in seeds of an Arabidopsis glycosylation mutant lacking plant-specific N-glycans, exhibit custommade human-type N-glycosylation. In contrast, 2G12 scFv-Fcs carry exclusively ER-typical oligomannosidic N-glycans and were deposited in newly formed ER-derived vesicles irrespective of the targeting signals. HA78 scFv-Fcs exhibited efficient virus neutralization activity, while 2G12 scFv-Fcs were inactive. We demonstrate the efficient generation of scFv-Fcs with a controlled N-glycosylation pattern. However, our results also reveal aberrant subcellular deposition and, as a consequence, unexpected N-glycosylation profiles. Our attempts to elucidate intracellular protein transport in seeds contributes to a better understanding of this basic cell biological mechanism and is a step toward the versatile use of Arabidopsis seeds as an alternative expression platform for pharmaceutically relevant proteins.Recombinant monoclonal antibodies (mAbs) are of high therapeutic potential and have thus become a major product of the pharmaceutical industry (Aggarwal, 2009). In addition to full-length mAbs, antibodies are being engineered to alter their size, pharmacokinetics, specificity, valency, effector functions, etc. in order to better suit the intended applications (for review, see Filpula, 2007;Harmsen and De Haard, 2007). Of particular interest among these engineered fragments are single-chain variable fragments (scFvs), fusions of variable heavy and variable light domains that retain an antigen-binding function. Due to their smaller size as compared with their full-length counterparts, these molecules penetrate target tissues better and can even bind to intracellular targets. Furthermore, multimerization via disulfide bonds and/or multimerization domains allows for the production of divalent or higher order antibody-like molecules, where increased avidity