Structure of a Therapeutic Antibody in Complex with MUC16 Reveals a Conformational Epitope Influenced by Antigen Glycosylation
Bibliographic record
Abstract
The integral membrane glycoprotein Mucin‐16 (MUC16) has emerged as an important cancer antigen that displays a high degree of tumor selectivity. MUC16 is often overexpressed and involved in tumorigenesis in several malignancies, including pancreatic and ovarian cancer. The role of MUC16 in cancer progression is complex and provides multiple points for therapeutic intervention. However, despite clinical interest in MUC16 as an immunotherapy target, surprisingly little is known regarding how antibodies bind the protein. Here we report the humanization, epitope mapping, and structure determination of a MUC16 specific therapeutic antibody. The antibody was humanized using a germline complementary determining region (CDR) loop grafting approach and produced by transient transfection in Chinese hamster ovary (CHO) cells. The humanized and murine antibodies displayed nearly identical binding affinity to a recombinant MUC16 SEA ( S perm protein, E nterokinase, and Agrin) domain as measured by enzyme linked‐immunosorbent assay (ELISA) and surface plasmon resonance (SPR). High‐resolution x‐ray structures of the antibodies indicated no significant changes associated with humanization. Initial epitope mapping using an ELISA and overlapping MUC16 constructs suggested that the antibody epitope was localized to a SEA domain and was non‐linear and conformational in nature. A more detailed epitope mapping study carried out using hydrogen‐deuterium exchange mass‐spectrometry revealed five regions on the SEA domain that resulted in reduced deuteration when the antibody was bound to the antigen. These results were confirmed by x‐ray structures of the murine and humanized antibodies complex with the SEA domain. The structures revealed a complex, non‐linear structural epitope with a long b‐hairpin forming the center of the interaction. Finally, a fully glycosylated recombinant SEA domain was produced by transient transfection in CHO cells and used to assess the role of MUC16 glycosylation on antibody binding. Surprisingly, the densely glycosylated SEA domain bound the antibody with approximately 2‐fold higher affinity compared to the unglycosylated domain, suggesting a role for antigen glycosylation in mediating antibody binding. The results presented here represent the first structural characterization of an antibody in complex with MUC16 and reveal a complex, non‐linear epitope influenced by glycosylation. These findings will help to accelerate the clinical development of this promising therapeutic agent.
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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) | 0.001 | 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".