Specific location of galactosylation in an afucosylated antiviral monoclonal antibody affects its FcγRIIIA binding affinity
Bibliographic record
Abstract
Monoclonal antibodies (mAbs) comprise an essential type of biologic therapeutics and are used to treat diseases because of their anti-cancer and anti-inflammatory properties, and their ability to protect against respiratory infections. Its production involves post-translational glycosylation, a biosynthetic process that conjugates glycans to proteins, which plays crucial roles in mAb bioactivities including effector functions and pharmacokinetics. These glycans are heterogeneous and have diverse chemical structures whose composition is sensitive to manufacturing conditions, rendering the understanding of how specific glycan structures affect mAb bioactivity challenging. There is a need to delineate the effects of specific glycans on mAb bioactivity to determine whether changes in certain glycosylation profiles (that can occur during manufacturing) will significantly affect product quality. Using enzymatic transglycosylation with chemically-defined N-glycans, we show that galactosylation at a specific location of N-glycans in an afucosylated anti-viral mAb is responsible for FcγRIIIA binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity. We report a facile method to obtain purified asymmetric mono-galactosylated biantennary complex N-glycans, and their influence on bioactivity upon incorporation into an afucosylated mAb. Using ELISA, surface plasmon resonance and flow cytometry, we show that galactosylation of the α6 antenna, but not the α3 antenna, consistently increases FcγRIIIA binding affinity. We confirm its relevance in an anti-viral model of respiratory syncytial virus (RSV) using an adapted ADCC reporter assay. We further correlate this structure-function relationship to the interaction of the galactose residue of the α6 antenna with the protein backbone using 2D-1H-15N-NMR, which showed that galactosylation of at this location exhibited chemical shift perturbations compared to glycoforms lacking this galactose residue. Our results highlight the importance of identifying and quantifying specific glycan isomers to ensure adequate quality control in batch-to-batch and biosimilar comparisons.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".