Expression Profiling and Glycan Engineering of IgG Subclass 1–4 in Nicotiana benthamiana

Abstract

IgG, the main serum immunoglobulin isotype, exists in four subclasses which selectively appear with distinctive glycosylation profiles. However, very little is known about the biological consequences mainly due to the difficulties in the generation of distinct IgG subtypes with targeted glycosylation. Here, we show a comprehensive expression and glycan modulation profiling of IgG variants in planta that are identical in their antigen binding domain but differ in their subclass appearance. While IgG1, 2, and 4 exhibit similar expression levels and purification yields, IgG3 is generated only at low levels due to the in planta degradation of the heavy chain. All IgG subtypes are produced with four distinct N-glycosylation profiles, differing in sugar residues previously shown to impact IgG activities, i.e., galactosylation, sialylation and core fucosylation. Affinity purified IgG variants are shown to be fully assembled to heterodimers but display different biochemical/physical features. All subtypes are equally well amenable to targeted glycosylation, except sialylated IgG4 which frequently accumulates substantial fractions of unusual oligo-mannosidic structures. IgG variants show significant differences in aggregate formation and endotoxin contamination which are eliminated by additional polishing steps (size exclusion chromatography, endotoxin removal treatments). Collectively we demonstrate the generation of 16 IgG variants at high purity and large glycan homogeneity which constitute an excellent toolbox to further study the biological impact of the two main Fc features, subclass and glycosylation.

Nicotiana spp. for the Expression and Purification of Functional IgG3 Antibodies Directed Against the Staphylococcus aureus Alpha Toxin

“…This highlights the importance of subcellular targeting, because protease activity and glycosylation are compartment-specific (Hehle et al, 2011). The lower stability of IgG3 compared to IgG1 agreed with previous studies reporting >10-fold differences in the accumulation of these subclasses (Kallolimath et al, 2020). Interestingly, BY-2 cells cultivated in a stirred-tank reactor accumulated less of the product (16.02 ± 1.40 mg kg −1 , n 6) than the cells cultivated in shake flasks (20.69 ± 3.46 mg kg −1 , n 6).…”

“…We next assessed the functionality of the IgG3 and IgG1 constructs (Supplementary Figures S1A, B) by transient expression in PCPs, testing different 5′ UTRs and BY-2 cell cultivation conditions. We found that the IgG3 construct produced bands of ∼65 and ∼25 kDa whereas the IgG1 construct produced bands of ∼55 and ∼25 kDa (Supplementary Figure S1C), as anticipated based on previous reports (Kallolimath et al, 2020). The apparent mass of the IgG1 produced from a single expression cassette using the IntF2A linker matched the apparent mass of an IgG1 produced using separate expression cassettes for the heavy and light chains (Ma et al, 2015), confirming that the polypeptides were correctly processed.…”

“…We detected distinct degradation products for each mAb, including a ∼45 kDa product for IgG3 that matched earlier reports (Navarre et al, 2012). In plants, IgG3 heavy chain degradation was previously attributed to the activity of serine proteases (Navarre et al, 2012) as well as the lack of protective O-linked glycans on the IgG3 hinge region (Kallolimath et al, 2020). This highlights the importance of subcellular targeting, because protease activity and glycosylation are compartment-specific (Hehle et al, 2011).…”

“…We found that the quantity of IgG3 directed to the secretory pathway was about half that of the corresponding IgG1. Others have observed a >10-fold difference between the secretion of IgG3 (using the barley α-amylase signal peptide) and IgG1 ( Kallolimath et al, 2020). We predicted that the self-cleaving linker would favor mAb assembly with a theoretically optimal 1:1 ratio of heavy and light chain, thus helping to limit the degradation of monomeric IgG3, which is more severe than the degradation of IgG1 due to the elongated, proteasesensitive hinge region (Kallolimath et al, 2020).…”

“…In addition to the purification/detection challenges described above, IgG3 is more susceptible than IgG1 to proteolysis and aggregation (Saito et al, 2019), which limits the accumulation of recombinant IgG3 in higher eukaryotes (Mandal et al, 2016;Saito et al, 2019;Kallolimath et al, 2020). Therefore, we systematically investigated the suitability of plants (Nicotiana spp.)…”