TGFB3 Antibody

What is TGF-beta 3 and what is its biological function?

TGF-beta 3 (transforming growth factor beta 3) is one of three closely related mammalian members of the TGF-beta superfamily that share a characteristic cystine knot structure. TGF-beta 3 is a highly pleiotropic cytokine that regulates processes such as immune function, proliferation, and epithelial-mesenchymal transition. Unlike other TGF-beta isoforms, TGF-beta 3 has some non-redundant functions, particularly in palatogenesis and pulmonary development, as demonstrated in knockout mice studies . TGF-beta cytokines (TGFβ1, TGFβ2, and TGFβ3) play critical roles in tissue fibrosis, and increased expression of TGFB3 has been significantly associated with severity biomarkers in systemic sclerosis .

What are the molecular characteristics of TGF-beta 3?

Human TGF-beta 3 cDNA encodes a 412 amino acid precursor containing a 20 amino acid signal peptide and a 392 amino acid proprotein. This proprotein is processed by furin-like convertases to generate an N-terminal 220 amino acid latency-associated peptide (LAP) and a C-terminal 112 amino acid mature TGF-beta 3. After secretion, disulfide-linked homodimers of LAP and TGF-beta 3 remain non-covalently associated, forming the small latent TGF-beta 3 complex . The precursor/latent complex is typically observed at approximately 55 kDa in Western blot analyses, while the predicted band size of the mature protein is 47 kDa .

What are the most common applications for TGF-beta 3 antibodies?

TGF-beta 3 antibodies are used in multiple research applications including:

• Western Blot (WB) at dilutions typically ranging from 1:500-1:1000

• Immunohistochemistry (IHC) at dilutions ranging from 1:50-1:500

• Immunofluorescence (IF) at dilutions ranging from 1:50-1:500

• ELISA (Enzyme-Linked Immunosorbent Assay)

• Flow Cytometry (intracellular)

• Immunoprecipitation (IP)

These applications allow researchers to detect and quantify TGF-beta 3 expression in various cell and tissue types, study its localization, and investigate its functional role in different biological processes.

What are the optimal sample preparation protocols for detecting TGF-beta 3 in different applications?

For Western blot analysis, whole cell lysates from various cell lines have been successfully used for detecting TGF-beta 3, including HeLa, 293T, NIH/3T3, and PC-12 cells at approximately 20 μg of protein per lane . For immunofluorescence analysis, paraformaldehyde fixation (4%) followed by permeabilization with 0.1% TritonX-100 has been shown to be effective for cytoplasmic staining of TGF-beta 3 in cell lines such as A549 .

For immunohistochemistry, antigen retrieval methods may be necessary, with suggested protocols including TE buffer pH 9.0 or alternatively citrate buffer pH 6.0 . This optimization is particularly important as TGF-beta 3 detection in tissue sections often requires specific retrieval conditions to expose the epitope. When working with tissue samples, validation has been reported in human breast cancer tissue, mouse ovary tissue, and various fibrotic tissues .

How can researchers validate antibody specificity for TGF-beta 3 versus other TGF-beta isoforms?

Validating antibody specificity for TGF-beta 3 requires multiple approaches:

• Isoform selectivity testing: Compare antibody reactivity against recombinant TGF-beta 1, TGF-beta 2, and TGF-beta 3 proteins in direct ELISA or Western blot. High-quality TGF-beta 3 antibodies should show minimal cross-reactivity with other isoforms.

• Functional neutralization assays: Evaluate the antibody's ability to specifically neutralize TGF-beta 3 bioactivity. For example, TGF-beta 3 inhibits IL-4-dependent cell proliferation in the HT-2 mouse T cell line. A specific anti-TGF-beta 3 antibody should neutralize this inhibition in a dose-dependent manner without affecting TGF-beta 1 or TGF-beta 2 functions .

• Knockout/knockdown validation: Test antibody reactivity in samples where TGF-beta 3 expression has been genetically reduced or eliminated to confirm signal specificity.

• Peptide competition: Pre-incubate the antibody with the immunogen peptide to demonstrate specific blocking of the signal in subsequent detection assays.

What controls should be included when using TGF-beta 3 antibodies in experimental designs?

When designing experiments with TGF-beta 3 antibodies, several controls should be included:

• Positive control: Include samples known to express TGF-beta 3, such as HeLa cells, 293T cells, NIH/3T3 cells, or PC-12 cells for Western blot applications .

• Negative control: Include samples with low or no TGF-beta 3 expression, or use siRNA knockdown models.

• Isotype control: Include the corresponding isotype antibody (e.g., Rabbit IgG for rabbit monoclonal antibodies) at equivalent concentrations to assess non-specific binding.

• Secondary antibody control: Include samples treated only with secondary antibody to identify potential background signal, particularly important in immunofluorescence applications .

• Recombinant protein standard: Include purified recombinant TGF-beta 3 protein as a reference standard for molecular weight confirmation in Western blots or as a standard curve in quantitative applications.

How can TGF-beta 3 antibodies be effectively utilized in fibrosis research?

TGF-beta 3 has been implicated as a key mediator in fibrotic diseases, with increased expression observed in human lung and liver fibrotic tissue and in the skin of patients with systemic sclerosis (SSc) . Researchers can utilize TGF-beta 3 antibodies in fibrosis research through several approaches:

• Biomarker correlation studies: TGF-beta 3 antibodies can be used to quantify protein expression in fibrotic tissues and correlate with disease biomarkers. Increased TGFB3 expression has been significantly associated with higher levels of biomarkers of SSc disease severity and prognosis, including cartilage oligomeric matrix protein (COMP) and periostin .

• Therapeutic target validation: Specific neutralizing antibodies against TGF-beta 3, such as RO7303509, can be used in experimental models to assess the therapeutic potential of TGF-beta 3 inhibition. In preclinical mouse studies, TGF-beta 3-specific antibodies significantly inhibited endogenous activation of TGF-beta target genes in a model of fibrotic lung disease .

• Mechanistic studies: TGF-beta 3 antibodies can be employed in mechanistic studies to investigate the specific role of this isoform versus other TGF-beta family members in fibrogenic processes, potentially revealing unique therapeutic opportunities.

What are important considerations when developing neutralizing antibodies against TGF-beta 3?

Developing effective neutralizing antibodies against TGF-beta 3 requires careful consideration of several factors:

• Isoform specificity: While pan-TGF-beta inhibitors have demonstrated unacceptable toxicities in clinical settings, isoform-specific antibodies like RO7303509 may offer improved safety profiles by targeting only TGF-beta 3 .

• Neutralization potency: The neutralization dose (ND₅₀) should be determined, which is typically 0.1-0.3 μg/mL in the presence of 0.1 ng/mL Recombinant Human TGF-beta 3 for effective neutralization in functional assays .

• Epitope selection: Targeting specific epitopes that affect either activation or receptor binding is critical. The mature TGF-beta 3 domain (C-terminal 112 amino acid segment) is often the target for neutralizing antibodies .

• Formulation and delivery: For therapeutic applications, antibody formulation and route of administration (e.g., intravenous vs. subcutaneous) can significantly impact pharmacokinetics and bioavailability .

• Safety profile assessment: As demonstrated in the RO7303509 clinical study, careful evaluation of safety and tolerability is essential, particularly given the known toxicities associated with pan-TGF-beta inhibition .

How can researchers monitor TGF-beta 3 pathway inhibition in experimental and clinical settings?

Monitoring TGF-beta 3 pathway inhibition can be accomplished through several biomarker approaches:

• Downstream target gene expression: Measure the expression of TGF-beta responsive genes such as collagen, fibronectin, or α-smooth muscle actin using qPCR, Western blot, or immunostaining.

• Serum biomarkers: Evaluate levels of TGF-beta pathway-associated biomarkers such as periostin and COMP, which have been used as exploratory biomarkers in clinical studies of TGF-beta 3 inhibition. These markers were assessed at multiple timepoints (baseline, day 5, 8, 15, 29, and 85) after treatment with RO7303509 to provide evidence of pathway inhibition .

• Phosphorylation of SMAD proteins: Detect changes in the phosphorylation status of SMAD2/3 proteins, which are direct downstream mediators of TGF-beta signaling.

• Cell-based functional assays: Utilize the HT-2 mouse T cell line, where TGF-beta 3 inhibits IL-4-induced proliferation, as a functional readout of TGF-beta 3 activity and neutralization .

What are common technical challenges when working with TGF-beta 3 antibodies and how can they be addressed?

Several technical challenges may arise when working with TGF-beta 3 antibodies:

• Variable molecular weight detection: The TGF-beta 3 precursor/latent complex is typically observed around 55 kDa, while the predicted mature protein is 47 kDa . This variability can be addressed by:

  • Including positive control samples with known TGF-beta 3 expression

  • Using reducing vs. non-reducing conditions appropriately to account for disulfide-linked complexes

  • Understanding post-translational modifications that may affect protein migration

• Antigen retrieval optimization: For immunohistochemistry applications, optimal antigen retrieval conditions may vary. Researchers should test both TE buffer pH 9.0 and citrate buffer pH 6.0 to determine which provides optimal staining .

• Antibody dilution optimization: Optimal dilutions should be determined for each application and sample type. Recommended ranges are 1:500-1:1000 for WB, 1:50-1:500 for IHC, and 1:50-1:500 for IF-P .

• Distinguishing latent versus active TGF-beta 3: Since TGF-beta 3 exists in both latent and active forms, researchers should carefully consider which form they are targeting and whether their detection method can distinguish between them.

How should researchers interpret differences in results obtained with different TGF-beta 3 antibody clones?

When different antibody clones produce varying results, researchers should consider:

• Epitope differences: Different antibody clones recognize distinct epitopes on the TGF-beta 3 protein. For example, some antibodies target the mature domain (C-terminal) while others may recognize the LAP region (N-terminal) . This can affect detection of different forms of TGF-beta 3.

• Clone validation status: Review the validation data for each clone across different applications and sample types. For instance, clone 20724 has been validated for neutralization assays , while clone EPR27093-72 has been validated for Western blot, immunofluorescence, and flow cytometry .

• Specificity profiles: Compare cross-reactivity profiles between antibodies. Some may have higher specificity for TGF-beta 3 over other TGF-beta isoforms.

• Species reactivity differences: Verify species reactivity for each clone. Some antibodies may work well in human samples but not in mouse or rat samples, or vice versa .

• Application suitability: An antibody that works well for Western blot may not be optimal for immunohistochemistry or other applications. Review application-specific validation data.

What approaches can resolve contradictory findings when studying TGF-beta 3 expression in different disease models?

Contradictory findings regarding TGF-beta 3 expression or function in different disease models may be resolved through:

• Multi-antibody approach: Use multiple validated antibodies targeting different epitopes of TGF-beta 3 to confirm expression patterns.

• Multi-technique validation: Combine protein detection (Western blot, IHC, IF) with mRNA analysis (qPCR, RNA-seq) to establish concordance between transcript and protein levels.

• Context-dependent expression: Consider that TGF-beta 3 expression and function may be highly context-dependent. For example, in cancer contexts, cellular localization in breast cancer tissue shows specific cytoplasmic staining in cancer cells .

• Temporal dynamics: Assess TGF-beta 3 expression at multiple timepoints in disease progression, as expression patterns may change over time.

• Active vs. latent forms: Distinguish between latent and active forms of TGF-beta 3, as the activation status may vary between disease models and affect detection and functional outcomes.

• Pathway crosstalk: Consider potential crosstalk with other signaling pathways that may modulate TGF-beta 3 expression or activity in a disease-specific manner.

How do TGF-beta 3-specific antibodies differ from pan-TGF-beta inhibitors in therapeutic applications?

TGF-beta 3-specific antibodies offer several advantages over pan-TGF-beta inhibitors in therapeutic contexts:

• Improved safety profile: Pan-TGF-beta inhibitors have demonstrated unacceptable toxicities in clinical settings due to the broad and essential functions of TGF-beta signaling in normal physiology. In contrast, isoform-specific inhibition of TGF-beta 3 may offer a more favorable safety profile by preserving essential functions of TGF-beta 1 and TGF-beta 2 .

• Targeted therapeutic approach: TGF-beta 3-specific antibodies like RO7303509 provide a more targeted approach by selectively inhibiting one isoform that may be particularly relevant in specific disease contexts, such as fibrotic diseases where TGFB3 expression is upregulated .

• Disease-specific relevance: Increased expression of TGFB3, but not TGFB1 or TGFB2, has been significantly associated with higher levels of biomarkers of systemic sclerosis disease severity and prognosis, suggesting that TGF-beta 3-specific inhibition may be particularly relevant in this context .

• Clinical development progress: High-affinity, TGF-beta 3-specific antibodies such as RO7303509 have advanced to clinical trials and demonstrated favorable safety and pharmacokinetic profiles in phase 1a studies in healthy volunteers, with doses up to 1200 mg being well-tolerated .

What role does TGF-beta 3 play in cancer research and how can antibodies facilitate its study?

TGF-beta 3 has emerging significance in cancer research, and antibodies can facilitate its study in several ways:

• Expression profiling: TGF-beta 3 antibodies can be used to characterize expression patterns in different cancer types. For example, TGF-beta 3 has been detected in human breast cancer tissue, with specific staining localized to the cytoplasm in cancer cells .

• Prognostic biomarker evaluation: Antibody-based detection methods can help evaluate TGF-beta 3 as a potential prognostic biomarker in various cancers, correlating expression levels with clinical outcomes.

• Therapeutic target validation: Neutralizing antibodies against TGF-beta 3 can be used to assess the functional consequences of TGF-beta 3 inhibition in cancer models, potentially revealing therapeutic opportunities.

• Tumor microenvironment analysis: TGF-beta 3 antibodies can help elucidate the role of this cytokine in shaping the tumor microenvironment, particularly in relation to immune cell function and fibrotic responses within tumors.

• Combination therapy studies: TGF-beta 3 antibodies can be used in combination therapy studies to evaluate potential synergies with other cancer therapies, particularly immunotherapies that may be affected by TGF-beta-mediated immunosuppression.

What recent technological advances have improved the development and application of TGF-beta 3 antibodies?

Recent technological advances that have enhanced TGF-beta 3 antibody development and application include:

• Humanized antibody engineering: The development of highly specific humanized monoclonal antibodies like RO7303509 (MTBT1466A) represents a significant advance for potential therapeutic applications, reducing immunogenicity concerns .

• High-throughput screening platforms: Advanced screening technologies have enabled the identification of antibodies with superior specificity and affinity for TGF-beta 3 over other TGF-beta isoforms.

• Improved detection systems: Enhanced detection systems for applications like immunohistochemistry, including the Anti-Mouse HRP-DAB Cell & Tissue Staining Kit, have improved sensitivity and specificity of TGF-beta 3 detection in complex tissue samples .

• Recombinant antibody technology: Recombinant monoclonal antibodies like EPR27093-72 offer advantages in terms of batch-to-batch consistency and reproducibility compared to conventional antibodies .

• Advanced biomarker analysis platforms: Technologies like the COBAS Elecsys system and ProteinSimple Ella platform have facilitated more sensitive and reproducible quantification of TGF-beta pathway-associated biomarkers such as periostin and COMP in clinical samples .

• PK/PD modeling approaches: Advanced pharmacokinetic/pharmacodynamic modeling approaches have improved dose selection and translation from preclinical models to clinical applications for TGF-beta 3-targeting therapeutics .