TMEFF2 Recombinant Monoclonal Antibody

What is TMEFF2 and why is it an important research target?

TMEFF2 is a 374-residue long single polypeptide, type-I transmembrane proteoglycan containing an EGF-like domain and two follistatin-like domains. It has attracted considerable research interest due to its differential expression in various cancers and potential roles in diverse physiological processes .

TMEFF2 is expressed in several tissues including brain, lung, prostate, and breast, with reported functions spanning a wide range of physiological and pathological spectra . It has been identified as having roles in:

• Corticotropin release hormone stimulation in the anterior pituitary gland

• Neuroprotection in Alzheimer's disease by binding amyloid β protein derivatives

• JAK-STAT pathway signaling in colorectal cancer

• One-carbon metabolism modulation in prostate tissue

The protein's tumor-specific expression pattern makes it a particularly attractive target for therapeutic antibody development, especially given its high overexpression in prostate cancer with limited distribution in normal tissues .

What are the main applications of TMEFF2 monoclonal antibodies in research?

TMEFF2 monoclonal antibodies serve multiple critical research functions:

• Protein Detection: Used in western blotting, immunohistochemistry, and flow cytometry to detect TMEFF2 expression in cell lines and tissues

• Functional Studies: Help elucidate TMEFF2's role in cellular processes by blocking or activating its function

• Therapeutic Development: Serve as platforms for developing antibody-drug conjugates for targeted cancer therapy

• Diagnostic Research: Study methylated TMEFF2 as a potential diagnostic biomarker in various cancers

• Mechanistic Research: Investigate protein-protein interactions, such as the TMEFF2-SARDH interaction

Validated applications for commercially available antibodies typically include ELISA, flow cytometry, and functional assays .

How is TMEFF2 protein typically detected in experimental systems?

TMEFF2 detection methods vary depending on the experimental context. Western blot analysis reveals that TMEFF2 appears at different molecular weights depending on cell type, sample preparation conditions, and the specific antibody used:

This variability reflects the complex post-translational modifications of TMEFF2 including glycosylation and proteolytic processing . For consistent results, researchers should carefully select antibodies validated for their specific application and experimental system.

How can I reconcile contradictory findings regarding TMEFF2's role in cancer progression?

The literature presents seemingly contradictory data about TMEFF2's role in cancer, with evidence supporting both oncogenic and tumor-suppressive functions. To reconcile these findings, consider:

• Cellular Context Dependency: TMEFF2 function appears highly context-dependent. In prostate cancer studies, full-length TMEFF2 demonstrates tumor suppressor properties while the shed ectodomain exhibits oncogenic characteristics .

• Differential Protein Processing: The protein undergoes ectodomain shedding, which generates fragments with potentially opposing functions. Research indicates that:

  • Full-length TMEFF2 decreases cell proliferation by 20-30% compared to control cells

  • The TMEFF2 ectodomain fails to bind SARDH and modulate sarcosine levels, reversing the tumor suppressor phenotype

• Methodological Differences: Different detection methods, cell models, and functional assays contribute to variable findings.

When designing experiments, carefully consider which protein form you're studying (full-length vs. ectodomain), the cellular context, and the specific signaling pathways being examined. Conducting parallel experiments with both forms can help clarify these seemingly contradictory roles.

What are the considerations for using TMEFF2 antibodies for immunoprecipitation studies?

When conducting immunoprecipitation (IP) studies with TMEFF2 antibodies, researchers should consider:

• Epitope Accessibility: TMEFF2's complex structure with multiple domains may affect epitope accessibility. Select antibodies that target epitopes known to be accessible in native conditions.

• Protein-Protein Interactions: TMEFF2 interacts with proteins like SARDH . The antibody chosen should not interfere with the interaction being studied or deliberately block it depending on experimental goals.

• Cross-Reactivity: Verify specificity through proper controls. The high homology between TMEFF1 and TMEFF2 (35.8%) necessitates validation of antibody specificity.

• Post-Translational Modifications: Consider how glycosylation may affect antibody binding. TMEFF2 exhibits multiple bands on Western blots due to extensive glycosylation .

• Buffer Conditions: Optimize lysis and washing conditions to maintain protein interactions while minimizing non-specific binding.

For interaction studies with SARDH specifically, using antibodies targeting epitopes away from the SARDH-binding region would be ideal to avoid disrupting the interaction being studied .

How can TMEFF2 antibodies be utilized in studying the relationship between TMEFF2 expression and cancer progression?

TMEFF2 antibodies can be valuable tools for investigating the complex relationship between TMEFF2 expression and cancer progression through several methodological approaches:

• Tissue Microarray Analysis: Immunohistochemistry using validated TMEFF2 antibodies can quantify expression across cancer stages. Past studies have shown significant TMEFF2 protein expression in 74% of primary prostate cancers and 42% of metastatic lesions from lymph nodes and bone, representing both hormone-naïve and hormone-resistant disease .

• Correlation with Methylation Status: TMEFF2 methylation increases with breast, colon and gastric cancer progression . Combining antibody-based protein detection with methylation analysis can reveal epigenetic regulation mechanisms.

• Comparative Expression Studies: Using antibodies to compare TMEFF2 expression in:

  • Normal vs. tumor tissue

  • Primary vs. metastatic sites

  • Treatment-responsive vs. resistant samples

• Functional Blocking Studies: Antibodies that block specific domains of TMEFF2 can help determine which regions are critical for its tumor-suppressive or oncogenic functions.

• Circulating TMEFF2 Detection: Developing assays to detect shed TMEFF2 in blood samples as potential liquid biopsy markers.

For prostate cancer specifically, research indicates that the TMEFF2 downregulation signature equals and sometimes outperforms the Gleason and pathological scores, suggesting its potential value as a prognostic marker .

What validation steps should be performed when using TMEFF2 antibodies for research?

Thorough validation of TMEFF2 antibodies is essential for reliable experimental results. The following methodological validation steps are recommended:

• Specificity Testing:

  • Western blot analysis using positive controls (e.g., LNCaP cells with known endogenous TMEFF2 expression)

  • Comparison of signals between TMEFF2-expressing and non-expressing cell lines

  • Knockdown/knockout validation to confirm specificity

  • Testing for cross-reactivity with TMEFF1 due to the 35.8% homology

• Application-Specific Validation:

  • For immunohistochemistry: Include tissue samples with known TMEFF2 expression patterns

  • For flow cytometry: Compare staining patterns with isotype controls

  • For functional assays: Verify blocking or activating effects with appropriate biological readouts

• Technical Controls:

  • Include recombinant TMEFF2 as a positive control

  • Use appropriate negative controls (isotype control antibodies)

  • Evaluate binding under reducing and non-reducing conditions, as TMEFF2 detection varies significantly between these conditions

• Epitope Mapping:

  • Confirm which domain of TMEFF2 the antibody recognizes (EGF-like domain, follistatin-like domains, transmembrane region, or cytoplasmic domain)

  • This is critical as different domains may exhibit different functions

• Batch-to-Batch Consistency Testing:

  • Verify consistent performance across different antibody lots using standardized positive controls

How can I design experiments to investigate the dual role of TMEFF2 in oncogenesis and tumor suppression?

To effectively investigate TMEFF2's dual role, consider these methodological approaches:

• Compare Full-Length vs. Ectodomain:

  • Express full-length TMEFF2 and the shed ectodomain separately in the same cell line

  • Assess differential effects on proliferation, migration, and invasion

  • Examine downstream signaling pathways activated by each form

  Research has shown that full-length TMEFF2 decreases cell numbers by 20-30% compared to controls, while the ectodomain shows no tumor suppressor activity .

• Domain-Specific Function Analysis:

  • Generate domain deletion constructs to identify regions responsible for tumor suppression or oncogenic activity

  • Create chimeric proteins swapping domains between TMEFF1 and TMEFF2 to identify critical functional domains

• Context-Dependent Studies:

  • Test TMEFF2 function across multiple cell lines representing different cancer stages

  • Investigate effects under various conditions (hormone treatment, hypoxia, inflammation)

• Mechanistic Investigations:

  • Examine TMEFF2-SARDH interaction and sarcosine regulation

  • Investigate ectodomain shedding mechanisms and regulation

  • Study effects on one-carbon metabolism pathways

• In vivo Models:

  • Compare xenograft growth with full-length vs. ectodomain TMEFF2 expression

  • Evaluate metastatic potential and response to therapy

When designing these experiments, include appropriate controls and consider using inducible expression systems to study the temporal effects of TMEFF2 expression.

What are the key considerations when developing TMEFF2 antibody-drug conjugates for therapeutic applications?

Developing TMEFF2 antibody-drug conjugates (ADCs) requires careful consideration of several methodological factors:

• Target Expression Profile:

  • Confirm target specificity in disease tissues vs. normal tissues

  • TMEFF2 shows significant expression in 74% of primary prostate cancers and 42% of metastatic lesions but limited normal tissue distribution

  • Quantify expression levels to predict potential efficacy and toxicity

• Antibody Characteristics:

  • Select antibodies with high affinity and specificity for TMEFF2

  • Evaluate internalization efficiency, as ADCs require internalization for payload delivery

  • Consider humanization to reduce immunogenicity (as demonstrated with huPr1-vcMMAE)

• Linker-Drug Chemistry:

  • Select appropriate linker chemistry (e.g., cathepsin B-sensitive valine-citrulline linker used in Pr1-vcMMAE)

  • Choose payload based on potency requirements and mechanism of action

  • Optimize drug-antibody ratio for efficacy/toxicity balance

• Preclinical Testing Strategy:

  • Test in models expressing varying levels of TMEFF2

  • Include TMEFF2-negative models as specificity controls

  • Evaluate potential on-target, off-tumor toxicity

  In preclinical studies, doses of 3-10 mg/kg of Pr1-vcMMAE resulted in significant and sustained tumor growth inhibition in xenografted prostate cancer models, while an isotype control ADC had no significant effect .

• Cross-Species Reactivity Assessment:

  • Determine cross-reactivity with murine TMEFF2 to better predict toxicity

  • Past research noted no overt in vivo toxicity with either murine or human ADC despite significant cross-reactivity with murine TMEFF2

• Resistance Mechanisms:

  • Investigate potential resistance mechanisms such as decreased target expression or increased efflux

These considerations should guide the methodological approach to developing effective TMEFF2-targeted therapeutic ADCs.