TMEFF2 Antibody

What is TMEFF2 and why is it considered a promising antibody target?

TMEFF2 is a 374-residue long single polypeptide, type-I transmembrane proteoglycan with two follistatin-like domains and one epidermal growth factor-like domain. Its value as an antibody target stems from its highly restricted expression pattern in normal tissues (primarily brain and prostate) coupled with significant overexpression in prostate cancer . Immunohistochemistry studies have confirmed significant TMEFF2 protein expression in 73-74% of primary prostate tumors and 42% of metastatic lesions from lymph nodes and bone that represented both hormone-naïve and hormone-resistant disease . More recent studies have detected TMEFF2 in 91% (41/45) of metastatic castration-resistant prostate cancer (mCRPC) samples .

What types of TMEFF2 antibodies are currently used in research?

Research into TMEFF2 antibodies has developed several distinct types:

• Conventional monoclonal antibodies: Used primarily for detection in techniques like immunohistochemistry and flow cytometry .

• Antibody-drug conjugates (ADCs): Exemplified by Pr1-vcMMAE, which combines anti-TMEFF2 antibodies with the cytotoxic agent auristatin E via a cathepsin B-sensitive valine-citrulline linker .

• Bispecific T-cell engagers: JNJ-70218902 (JNJ-902) represents this approach, engaging both TMEFF2 on tumor cells and CD3 on T cells to promote T cell-mediated killing of cancer cells .

• Humanized antibodies: Modified versions like huPr1-vcMMAE with reduced immunogenicity for potential clinical applications .

How is TMEFF2 expression validated in experimental models?

Validation of TMEFF2 expression typically involves multiple complementary approaches:

• Binding assays: JNJ-902 demonstrated concentration-dependent binding to TMEFF2-positive LNCaP-AR cells (EC50 = 9.6 nM) with no binding to TMEFF2-negative DU145 prostate cancer cells, confirming specificity .

• Immunohistochemistry (IHC): Using specific monoclonal antibodies to detect TMEFF2 protein in tissue samples, allowing for quantification of expression rates across tumor types .

• Gene expression profiling: Identifying TMEFF2 as highly expressed in prostate cancer with limited normal tissue distribution .

• Cross-reactivity testing: Evaluating antibody reactivity with murine TMEFF2 to assess potential off-target effects, which is particularly important given the 100% sequence homology between human and monkey TMEFF2 .

What are the recommended protocols for evaluating TMEFF2 antibody efficacy in vitro?

Based on current research, effective evaluation protocols include:

T cell-mediated cytotoxicity assays:

• Incubate antibodies with healthy human donor T cells and target tumor cells at specific effector-to-target ratios (typically 3:1)

• Measure caspase-3 activity as an indicator of cell death (JNJ-902 showed EC50 = 1.4 nM in such assays)

• Include appropriate negative controls (TMEFF2-negative cell lines and control antibodies)

T cell activation assessment:

• Measure concentration-dependent increases in cell surface markers (CD8+CD25+)

• Quantify proinflammatory cytokine production (GM-CSF, IFN-γ, IL-10, TNF-α)

• Assess granzyme B expression and proliferation markers (Ki-67)

How should researchers design in vivo experiments to evaluate TMEFF2 antibody efficacy?

Optimal experimental design should include:

Model selection:

• T cell humanized NSG mice bearing LNCaP xenografts or LuCaP 86.2 patient-derived xenografts have proven effective

• Models should represent clinical scenarios of interest, particularly treatment-resistant disease

Dosing considerations:

• Test multiple dose levels (3-10 mg/kg showed efficacy in preclinical models)

• Compare dosing schedules (once weekly vs. biweekly)

• Consider step-up dosing approaches, which showed enhanced CD8+ T cell infiltration in non-human primate studies

Efficacy measurements:

• Tumor growth inhibition (TGI) relative to control-treated mice (JNJ-902 demonstrated 72-122% TGI in preclinical models)

• Intratumoral T cell infiltration analysis by flow cytometry

• Assessment of T cell activation markers within the tumor microenvironment

What analytical methods are most informative when characterizing pharmacodynamic responses to TMEFF2 antibodies?

The most informative analytical approaches include:

Tissue analysis:

• Flow cytometry to assess T cell infiltrates and myeloid cells in target tissues and blood

• Quantification of surface markers of T cell function (activation, proliferation, and suppression)

• Measurement of CD4+CD25hi Foxp3+ regulatory T cells to monitor potential immunosuppression

Inflammatory response monitoring:

• Assessment of pro-inflammatory cell influx (dendritic cells, myeloid cells, macrophages, and B cells) into target tissues

• Cytokine profiling in serum and tissue

Exposure-response relationship analysis:

• Correlation of pharmacokinetic parameters with biological responses

• Consideration that clinical activity may not always be dose-related, as observed in the phase 1 study of JNJ-902

How can researchers address the dual role of TMEFF2 as both oncogenic and tumor-suppressive?

The multifaceted nature of TMEFF2 presents a significant challenge to researchers . A methodological approach to resolving these contradictions includes:

• Context-dependent analyses: Design experiments that investigate TMEFF2 function across different cancer stages, microenvironments, and genetic backgrounds

• Signaling pathway dissection: Determine how TMEFF2 interacts with various signaling networks that might lead to opposing outcomes in different cellular contexts

• Proteolytic processing studies: Examine whether cleaved forms of TMEFF2 have different functions than the membrane-bound protein

• Expression level effects: Investigate whether different expression levels trigger different cellular responses

• Interaction partner identification: Characterize proteins that interact with TMEFF2 in different contexts to elucidate mechanistic differences

What strategies can enhance efficacy of TMEFF2 x CD3 bispecific antibodies in treatment-resistant prostate cancer?

Based on current research, promising strategies include:

Dosing optimization:

• Step-up dosing approaches (starting with 0.075 mg/kg followed by 0.3 mg/kg one week later) demonstrated greater CD8+ T cell infiltration compared to fixed dosing in non-human primates

• Exploration of different administration schedules (once weekly versus biweekly) as evaluated in clinical studies

Combination approaches:

• Consider rational combinations with other immunomodulatory agents

• Target the tumor microenvironment to overcome immunosuppression

Patient selection biomarkers:

• Develop predictive biomarkers based on TMEFF2 expression levels

• Consider tumor immune microenvironment characteristics that may influence response

How should researchers interpret the disparities between preclinical efficacy and clinical outcomes with TMEFF2 antibodies?

Addressing translational gaps requires systematic analysis:

• Pharmacokinetic/pharmacodynamic (PK/PD) assessment:

  • In the phase 1 study of JNJ-902, clinical activity was not dose-related and no clear exposure-response relationship was observed

  • Compare tissue penetration and target engagement between preclinical models and clinical samples

• Response heterogeneity analysis:

  • In clinical studies, only 15.2% of patients with measurable disease had confirmed partial responses to JNJ-902

  • Investigate molecular features that distinguish responders from non-responders

• Resistance mechanism characterization:

  • Serial biopsies to study adaptive changes during treatment

  • Development of patient-derived models from non-responders

• Translational biomarker development:

  • Correlation of PSA response (12.2% of patients showed ≥50% PSA reduction with JNJ-902 ) with imaging and survival outcomes

  • Identification of early pharmacodynamic markers that predict therapeutic benefit

What approaches can mitigate potential toxicity concerns when targeting TMEFF2?

Despite TMEFF2's expression in normal brain and prostate, research suggests several strategies to manage toxicity:

Preclinical safety assessment:

• Leverage the cross-reactivity of anti-TMEFF2 antibodies with murine TMEFF2 to evaluate potential off-target effects

• Studies with both Pr1-vcMMAE and huPr1-vcMMAE demonstrated no overt in vivo toxicity despite this cross-reactivity, suggesting a high safety profile

Dose optimization:

• In the JNJ-902 clinical study, dose-limiting toxicities were observed in only 2.4% of patients

• Step-up dosing approaches may improve tolerability while maintaining efficacy

Molecular design considerations:

• Engineering antibodies with optimized binding properties

• Exploring conditional activation mechanisms that restrict activity to the tumor microenvironment

How can researchers effectively troubleshoot inconsistent results in TMEFF2 antibody experiments?

When facing experimental variability, consider these methodological approaches:

• Antibody validation:

  • Confirm antibody specificity through positive and negative control cell lines (LNCaP-AR vs. DU145)

  • Validate binding properties before and after any modification (conjugation to toxins, fluorophores)

• T cell functionality assessment:

  • Ensure consistent T cell activation status across experiments

  • Consider donor variability in T cell-based assays

  • Standardize effector-to-target ratios (typically 3:1)

• Target expression verification:

  • Quantify TMEFF2 expression levels in experimental models

  • Consider heterogeneity within cell populations

• Experimental controls:

  • Include isotype control antibodies

  • Use TMEFF2-negative cell lines as specificity controls

What methodological approaches best address heterogeneous TMEFF2 expression in patient samples?

Given the variable expression of TMEFF2 across patient samples (73-74% of primary tumors, 42% of metastatic lesions) , researchers should consider:

Comprehensive expression profiling:

• Multi-region sampling to account for intratumoral heterogeneity

• Correlation of expression with clinical parameters and outcomes

Threshold determination:

• Establish clinically relevant expression thresholds for patient selection

• Consider both percentage of positive cells and intensity of staining

Complementary biomarkers:

• Identify additional markers that may enhance patient selection

• Develop algorithms that incorporate multiple parameters

Serial monitoring:

• Assess changes in TMEFF2 expression during disease progression and treatment

• Develop liquid biopsy approaches for longitudinal assessment

What novel antibody formats beyond current bispecific and ADC approaches show promise for targeting TMEFF2?

While bispecific antibodies (JNJ-902) and ADCs (Pr1-vcMMAE) dominate current TMEFF2 research, several innovative approaches warrant investigation:

• Trispecific antibodies: Targeting TMEFF2, CD3, and an additional immune checkpoint molecule

• CAR-T cell therapy: Engineering T cells with TMEFF2-specific chimeric antigen receptors

• Antibody-cytokine fusions: Combining TMEFF2 targeting with localized cytokine delivery

• Proteolytically-activated antibodies: Engineered to become fully active only in the tumor microenvironment

• Radioimmunotherapy conjugates: TMEFF2 antibodies coupled with therapeutic radioisotopes

How might combinatorial approaches enhance the efficacy of TMEFF2-targeted therapies?

Strategic combinations to explore include:

Immune checkpoint inhibition:

• Combining TMEFF2 bispecific antibodies with PD-1/PD-L1 blockade

• Addressing T cell exhaustion mechanisms observed in persistent disease

Tumor microenvironment modulation:

• Targeting immunosuppressive cell populations

• Enhancing T cell trafficking and infiltration

Conventional therapies:

• Investigating synergy with androgen receptor-targeted therapies

• Exploring radiation therapy combinations to enhance immunogenic cell death

Sequencing strategies:

• Determining optimal treatment sequences

• Developing rational switching protocols based on response assessment

What research approaches will advance our understanding of resistance mechanisms to TMEFF2-targeted therapies?

To address inevitable treatment resistance, researchers should prioritize:

Longitudinal monitoring studies:

• Serial biopsies during treatment and at progression

• Liquid biopsy approaches for noninvasive monitoring

Resistance model development:

• Generation of resistant cell lines through prolonged exposure

• Patient-derived xenografts from non-responding patients

Mechanism characterization:

• TMEFF2 expression changes (downregulation, mutation, alternative splicing)

• Altered signaling pathway activation

• Immune escape mechanisms (T cell exhaustion, immunosuppressive cell recruitment)

Computational modeling:

• Integrative analysis of multi-omics data from resistant samples

• AI-assisted prediction of resistance mechanisms and rational combination strategies