Sweet Rules: Linking Glycosylation to Antibody Function

Nimmerjahn, Falk; Werner, Anja

doi:10.1007/978-3-030-76912-3_12

Cited by 4 publications

(5 citation statements)

References 179 publications

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“…Also, relatively little attention is paid to the characterization and engineering of posttranslational modifications (PTMs), like N-glycosylation. Control of N-glycosylation is not only important for product homogeneity but also for efficacy [reviewed ( Nimmerjahn and Werner, 2021 )]. Individual studies, mainly using magnICON vectors, indicate the capacity to generate mAbs with designed PTMs, thereby improving their activities [e.g., ( Zeitlin et al, 2011 ; Loos et al, 2015 ; Kallolimath et al, 2020 )].…”

Section: Introductionmentioning

confidence: 99%

Comparative analysis of plant transient expression vectors for targeted N-glycosylation

Eidenberger

Eminger

Castilho

et al. 2022

Front. Bioeng. Biotechnol.

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While plant-based transient expression systems have demonstrated their potency to rapidly express economically feasible quantities of complex human proteins, less is known about their compatibility with posttranslational modification control. Here we investigated three commonly used transient expression vectors, pEAQ, magnICON and pTra for their capability to express a multi-component protein with controlled and modified N-glycosylation. Cetuximab (Cx), a therapeutic IgG1 monoclonal antibody, which carries next to the conserved Fc an additional N-glycosylation site (GS) in the Fab-domain, was used as model. While pEAQ and pTra produce fully assembled Cx at similar levels in N. benthamiana, the yield of magnICON-Cx was twice as high. When expressed in wild type plants, both Cx-GSs exhibited typical plant N-glycans decorated with plant-specific xylose and fucose. Likewise, Cx generated in the glycoengineered ΔXTFT line carried mainly complex N-glycans lacking plant specific residues. Exposure to different engineering settings (encompassing stable lines and transient approaches) towards human galactosylation and sialylation resulted in Cx carrying targeted N-glycans at similar quantities using all three expression vectors. Collectively, our results exhibit the universal application of plant-based glycoengineering, thereby increasing the attractivity of the ambitious expression platform.

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Section: Introductionmentioning

confidence: 99%

Comparative analysis of plant transient expression vectors for targeted N-glycosylation

Eidenberger

Eminger

Castilho

et al. 2022

Front. Bioeng. Biotechnol.

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“…4D-E) unveils a layer of functional specialization that may define the fine-tuning of immune responses. For instance, B4GALT1 is instrumental in the addition of GlcNAc moieties to N-glycans, while FUT8 is responsible for adding fucose residues to the basal GlcNAc core, modifications known to increase and dampen ADCC activity, respectively (Nimmerjahn and Werner 2021; Wang and Ravetch 2019). Furthermore, in the thousands of MBCs profiled in the MBC gate, high MGAT3 and SLC35C1expression was not only restricted to a minority of clones but was also mutually exclusive.…”

Section: Discussionmentioning

confidence: 99%

De novo antibody discovery in human blood from full-length single B cell transcriptomics and matching haplotyped-resolved germline assemblies

Beaulaurier,

Ly,

Duty

et al. 2024

Preprint

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Immunoglobulin (IGH, IGK, IGL) loci in the human genome are highly polymorphic regions that encode the building blocks of the light and heavy chain IG proteins that dimerize to form antibodies. The processes of V(D)J recombination and somatic hypermutation in B cells are responsible for creating an enormous reservoir of highly specific antibodies capable of binding a vast array of possible antigens. However, the antibody repertoire is fundamentally limited by the set of variable (V), diversity (D), and joining (J) alleles present in the germline IG loci. To better understand how the germline IG haplotypes contribute to the expressed antibody repertoire, we combined genome sequencing of the germline IG loci with single-cell transcriptome sequencing of B cells from the same donor. Sequencing and assembly of the germline IG loci captured the IGH locus in a single fully-phased contig where the maternal and paternal contributions to the germline V, D, and J repertoire can be fully resolved. The B cells were collected following a measles, mumps, and rubella (MMR) vaccination, resulting in a population of cells that were activated in response to this specific immune challenge. Single-cell, full-length transcriptome sequencing of these B cells resulted in whole transcriptome characterization of each cell, as well as highly-accurate consensus sequences for the somatically rearranged and hypermutated light and heavy chain IG transcripts. A subset of antibodies synthesized based on their consensus heavy and light chain transcript sequences demonstrated binding to measles antigens and neutralization of measles live virus.

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“…Moreover, it was shown that IgG antibodies with eliminated fucose exhibit increased effector functions. [ 7 ]…”

Section: Introductionmentioning

confidence: 99%

“…[ 3–6 ] These proteins are usually heavily glycosylated with significant impacts of this central post‐translational modification on biochemical and functional features. [ 7 ] Therefore, controlling glycosylation is an important quality parameter in recombinant protein production. While plants synthesize complex N‐glycans similar to humans, they are simpler and less diverse.…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, it was shown that IgG antibodies with eliminated fucose exhibit increased effector functions. [7] Recent developments in genome editing (i.e., CRISPR/Cas9) have created new possibilities for genomic manipulations. While efficient protocols exist for plants with diploid genomes, targeted gene editing is more challenging in complex genomes, like N. benthamiana with an allotetraploid genome.…”

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confidence: 99%

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A genome‐edited N. benthamiana line for industrial‐scale production of recombinant glycoproteins with targeted N‐glycosylation

Kogelmann,

Melnik,

Bogner

et al. 2023

Biotechnology Journal

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Control over glycosylation is an important quality parameter in recombinant protein production. Here, we demonstrate the generation of a marker‐free genome edited Nicotiana benthamiana N‐glycosylation mutant (NbXF‐KO) carrying inactivated β1,2‐xylosyltransferase and α1,3‐fucosyltransferase genes. The knockout of seven genes and their stable inheritance was confirmed by DNA sequencing. Mass spectrometric analyses showed the synthesis of N‐glycans devoid of plant‐specific β1,2‐xylose and core α 1,3‐fucose on endogenous proteins and a series of recombinantly expressed glycoproteins with different complexities. Further transient glycan engineering towards more diverse human‐type N‐glycans resulted in the production of recombinant proteins decorated with β1,4‐galactosylated and α2,6‐sialylated structures, respectively. Notably, a monoclonal antibody expressed in the NbXF‐KO displayed glycosylation‐dependent activities. Collectively, the engineered plants grow normally and are well suited for upscaling, thereby meeting industrial and regulatory requirements for the production of high‐quality therapeutic proteins.

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Sweet Rules: Linking Glycosylation to Antibody Function

Cited by 4 publications

References 179 publications

Comparative analysis of plant transient expression vectors for targeted N-glycosylation

Comparative analysis of plant transient expression vectors for targeted N-glycosylation

De novo antibody discovery in human blood from full-length single B cell transcriptomics and matching haplotyped-resolved germline assemblies

A genome‐edited N. benthamiana line for industrial‐scale production of recombinant glycoproteins with targeted N‐glycosylation

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