MUC16 Human
Description
Structure of MUC16
MUC16 is the largest membrane-associated mucin, spanning ~22,000 amino acids . Its structure comprises three domains:
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N-terminal domain: Extracellular, heavily O-glycosylated.
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Tandem repeat domain: Contains serine/threonine/proline-rich repeats, critical for glycosylation and antibody binding .
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C-terminal domain: Includes SEA modules, a transmembrane region, and a cytoplasmic tail that interacts with cytoskeletal proteins like ezrin .
Genetic and Clinical Correlations
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Mutations in MUC16 correlate with higher tumor mutational burden (TMB) and improved survival in gastric/lung cancers .
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Overexpression predicts poor prognosis in ovarian, pancreatic, and breast cancers .
Biomarker Utility
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CA125: FDA-approved serum marker for ovarian cancer monitoring, though limited by false positives (e.g., endometriosis) .
Emerging Therapies
Challenges: Proteolytic shedding of MUC16’s extracellular domain limits drug delivery .
Research Frontiers
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Structural insights: Crystal structures of SEA domains (e.g., PDB 7SA9) reveal antibody-binding surfaces, aiding immunotherapy design .
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Immune microenvironment: MUC16 mutations alter immune cell infiltration (e.g., dendritic cells, Tregs) in lung adenocarcinoma .
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Exosome-mediated signaling: MUC16-rich exosomes induce EMT in non-cancerous cells, amplifying invasiveness .
Product Specs
Human carcinoma cell line.
Q&A
How do MUC16-targeting antibodies differ in specificity between the cleaved CA125 domain and the retained carboxy-terminal (CT) fragment?
MUC16 antibodies targeting the cleaved CA125 domain (e.g., M11) primarily recognize tandem repeats in the N-terminal region, which are shed into circulation as CA125 . In contrast, carboxy-terminal (CT)-specific antibodies (e.g., 5E6) bind retained juxtamembrane regions post-cleavage, offering improved tumor specificity due to reduced off-target binding in normal tissues . This distinction is critical for therapeutic strategies: CT-targeting antibodies minimize interference with CA125 in serosal fluids, enhancing tumor-to-normal tissue contrast .
Table 1: Comparative Reactivity of MUC16 Antibodies in Ovarian Cancer
| Antibody Type | Target Domain | Tumor Reactivity (Serous/Papillary) | Normal Tissue Binding |
|---|---|---|---|
| CA125 (M11) | N-terminal | 87% | High (serosal sites) |
| CT-specific (5E6) | Juxtamembrane | 93.5% | Low |
What experimental approaches reconcile discrepancies in MUC16 tandem repeat counts between early studies (63 repeats) and recent revisions (19 repeats)?
Early estimates of 63 tandem repeats relied on short-read sequencing, which struggles with repetitive regions . Recent Nanopore long-read sequencing and in silico modeling (AlphaFold) revealed a consensus model of 19 tandem repeats, validated via proteomics . Researchers should prioritize long-read sequencing for structural analysis and use AlphaFold to predict epitope accessibility in therapeutic antibody design .
Key Methodological Takeaway:
“Nanopore sequencing resolves repetitive regions more accurately than Illumina, enabling precise tandem repeat count determination. AlphaFold modeling of the 19-repeat structure identifies unstructured linker regions rich in proline/serine/threonine, critical for epitope recognition.”
How do MUC16-targeting therapies compare in efficacy and safety across different formats (CAR T-cells, ADCs, bispecific antibodies)?
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CAR T-cells: Phase 1 trials demonstrated safety in ovarian cancer but faced challenges in persistence and tumor penetration .
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ADCs: Cytotoxic payloads raise toxicity concerns, particularly in tissues with low MUC16 expression .
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Bispecific Antibodies (e.g., IMV-M): IMV-M clusters DR5 on MUC16+ cells, inducing apoptosis without cytotoxic payloads. It showed superior safety in non-human primates and potent activity in xenograft models .
Therapeutic Advantages:
| Format | Mechanism | Safety Profile | Efficacy in Models |
|---|---|---|---|
| CAR T-cells | T-cell activation | Moderate (Phase 1) | Variable persistence |
| ADCs | Payload delivery | High toxicity risk | Target-dependent |
| Bispecific IMV-M | DR5 clustering via antibody | Low toxicity | High (tumor regression) |
What methodological challenges arise when validating MUC16 expression in normal vs. cancer tissues?
MUC16 is expressed in normal mesothelial, respiratory, and reproductive tissues, complicating IHC interpretation . To mitigate this:
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Use CT-specific antibodies (e.g., 5E6) that avoid cross-reactivity with shed CA125 .
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Exclude non-specific binding via blocking peptides or isotype controls.
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Quantify expression using automated image analysis (e.g., HALO software) to distinguish tumor vs. normal epithelia .
Case Study:
In ovarian cancer, CT-specific antibodies (5E6) stained 93.5% of serous/papillary tumors, while CA125 antibodies (M11) showed 87% reactivity but higher false positives in normal serosal tissues .
How can researchers address conflicting data on MUC16’s role in ovarian cancer progression?
Conflicts often stem from:
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Antibody specificity: CA125-targeting antibodies may detect shed fragments in circulation, not cell-bound MUC16 .
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Tumor heterogeneity: MUC16 expression varies across subtypes (e.g., serous vs. mucinous) .
Resolution Strategies:
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Combine IHC and mass spectrometry to confirm cell-surface MUC16 retention .
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Use orthogonal models: Validate findings in patient-derived xenografts (PDX) and syngeneic mouse models .
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Focus on CT-specific epitopes to avoid confounding with CA125 .
What novel epitopes could improve CA125 assays for ovarian cancer monitoring?
Current CA125 assays detect shed tandem repeats but lack sensitivity for detecting cell-bound MUC16. Ongoing efforts focus on:
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Glycoproteomic profiling to identify underglycosylated epitopes in cancer-specific MUC16 isoforms .
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CT-specific antibodies that avoid competition with CA125 in serum .
Future Directions:
“Deep learning models like AlphaFold could predict epitopes in the revised 19-repeat structure, enabling next-gen biomarkers with higher tumor specificity.”
How do MUC16-targeting therapies address minimal residual disease (MRD) in ovarian cancer?
MRD eradication requires targeting small tumor deposits. Strategies include:
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Bispecific antibodies: IMV-M clusters DR5 on MUC16+ cells, inducing apoptosis even in low-density tumors .
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CAR T-cells: Engineered to persist longer, though challenges remain in penetrating peritoneal tumors .
Experimental Design Tip:
“Use Incucyte live-cell imaging to monitor real-time apoptosis in MRD models, combining Caspase-3/7 dyes with nuclear tracking agents.”
What are the key considerations for developing MUC16-targeting bispecific antibodies?
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Antigen Density: Ensure MUC16 expression is sufficient for antibody clustering (e.g., >10,000 copies/cell) .
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DR5 Crosslinking: Optimize arm lengths and hinge regions to enable effective DR5 trimerization .
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Tumor Microenvironment: Test in models with peritoneal metastases to mimic clinical scenarios .
Example:
IMV-M’s dual anti-MUC16/DR5 arms enable clustering on a single MUC16 molecule, bypassing the need for secondary crosslinkers and reducing off-target effects .
Product Science Overview
Mucin-16, also known as CA125, is a high-molecular-weight glycoprotein encoded by the MUC16 gene. It is a member of the mucin family, which comprises glycoproteins produced by epithelial cells to protect and lubricate the surfaces of various organs. MUC16 is particularly significant due to its role as a biomarker for ovarian cancer .
MUC16 is the largest membrane-associated mucin, consisting of more than 22,000 amino acids . It is composed of three distinct domains:
- N-terminal domain: This domain is entirely extracellular and highly O-glycosylated.
- Tandem repeat domain: This domain contains repeating amino acid sequences rich in serine, threonine, and proline.
- C-terminal domain: This domain includes multiple extracellular SEA (sea urchin sperm protein, enterokinase, and agrin) modules, a transmembrane domain, and a cytoplasmic tail .
The extracellular region of MUC16 can be released from the cell surface through proteolytic cleavage, which is thought to occur at a site within the SEA modules .
MUC16 is expressed on the ocular surface, respiratory tract, and female reproductive tract epithelia. Its high glycosylation creates a hydrophilic environment that acts as a lubricating barrier against foreign particles and infectious agents on the apical membrane of epithelial cells . Additionally, the cytoplasmic tail of MUC16 interacts with the cytoskeleton by binding to members of the ERM protein family .
MUC16 is best known for its application as a tumor marker, particularly in ovarian cancer. Elevated levels of CA125 in the blood can indicate the presence of ovarian cancer or other conditions, both malignant and benign . The identification of CA125 as MUC16 has led to various studies investigating its expression, functional, and mechanistic involvement in multiple cancer types .
Efforts have been made to develop MUC16-targeted therapies, primarily using antibodies against the tandem repeat domains of MUC16. However, these approaches have met with limited success . Recent studies have focused on disrupting the functional cooperation of MUC16 and its interacting partners, such as using a novel immunoadhesin HN125 to interfere with MUC16 binding to mesothelin .
