ENPP3 Recombinant Monoclonal Antibody

What is ENPP3 and what biological functions does it serve?

ENPP3 is a transmembrane glycoprotein involved in multiple biological processes including cell growth regulation, differentiation, and mineralization. It functions primarily through hydrolysis of extracellular nucleotides to generate inorganic phosphate and nucleoside diphosphates, which are critical for purinergic signaling pathway regulation. ENPP3 is expressed in various tissues, including bone, kidney, and liver, with dysregulation linked to calcification disorders, cancer, and inflammatory conditions .

At the molecular level, ENPP3 acts as a hydrolase that metabolizes extracellular nucleotides including ATP, GTP, UTP, and CTP. It plays a crucial role in limiting mast cell and basophil responses during inflammation and chronic phases of allergic responses by eliminating extracellular ATP, which functions as a signaling molecule that activates these cells in an autocrine manner .

How are ENPP3 recombinant monoclonal antibodies generated and manufactured?

ENPP3 recombinant monoclonal antibodies are produced through a multi-step process that begins with immunization. For example, one approach uses recombinant human SLC39A6 protein as an immunogen to generate the initial antibody response. The cDNA of the resulting ENPP3 monoclonal antibody is then sequenced to obtain the antibody gene, which is subsequently cloned into a plasmid vector. This vector is transfected into host cells using appropriate transfection methods .

After expression, the ENPP3 recombinant monoclonal antibody undergoes purification through affinity chromatography and is tested for specificity through ELISA. Quality control typically includes binding assays where the antibody demonstrates specific binding capabilities to recombinant human ENPP3, with documented EC50 ranges (e.g., 3.313-4.724 ng/mL for some commercial antibodies) .

Alternative approaches include immunizing Balb/c mice with the ENPP3 extracellular domain (ECD) to generate mouse anti-ENPP3 monoclonal antibodies, as was done for the development of therapeutic antibodies like AGS16F .

What are the essential validation parameters for ENPP3 antibodies in research applications?

Validation of ENPP3 antibodies should include several key parameters:

• Binding specificity assessment: Typically performed through ELISA against recombinant ENPP3 proteins, with EC50 values documented. For example, one commercial ENPP3 recombinant antibody demonstrates binding to human ENPP3 with an EC50 of 2.151-2.492 ng/mL and to Macaca fascicularis ENPP3 with an EC50 of 3.313-4.724 ng/mL .

• Cross-reactivity testing: Determination of reactivity with ENPP3 from different species. Many commercial antibodies are validated for human and non-human primate (e.g., Macaca fascicularis) ENPP3 .

• Application-specific validation: Confirmation of functionality in specific applications such as flow cytometry, immunohistochemistry, or Western blotting. For example, anti-ENPP3 PE antibody [NP4D6] has been validated for flow cytometry and immunocytochemistry/immunofluorescence applications .

• Electrophoretic analysis: SDS-PAGE analysis under reducing conditions, typically using Tris-Glycine gels with 5% enrichment gel and 15% separation gel, to confirm antibody purity and integrity .

What are optimal conditions for using ENPP3 antibodies in flow cytometry?

For flow cytometry applications using ENPP3 antibodies, researchers should consider the following protocol elements:

• Sample preparation: Fresh isolation of target cells (e.g., basophils or mast cells) is recommended as ENPP3 expression can be affected by cell activation status. For basophil analysis, allergen-stimulated whole blood preparations have been successfully used .

• Antibody conjugation: PE-conjugated anti-ENPP3 antibodies are frequently used for flow cytometry. The clone NP4D6 (e.g., ab90751) has been validated for both surface and intracellular staining .

• Co-staining strategies: For identification of specific cell populations, co-staining with lineage-specific markers is recommended. For example, activated human basophils can be identified using FITC-conjugated anti-CD63 (clone MEM-259) combined with PE-conjugated anti-ENPP3 .

• Controls: Include appropriate isotype controls and unstained samples. For ENPP3 knockout or knockdown studies, cells with confirmed ENPP3 deficiency serve as excellent negative controls .

• Gating strategy: When analyzing tissue-resident cells, initial gating on viable single cells followed by lineage-specific markers before analyzing ENPP3 expression is recommended to minimize false positives.

How can ENPP3 function be assessed in cellular models?

Assessing ENPP3 function in cellular models can be approached through several methodologies:

• Knockdown/knockout studies: ENPP3 gene silencing through siRNA or CRISPR-Cas9 approaches can reveal its function. Previous studies have shown that ENPP3 knockdown cells exhibit significantly increased levels of intracellular nucleotide sugars and display changes in the total cellular glycosylation profile .

• Enzymatic activity assays: ENPP3 hydrolytic activity can be measured using specific substrates:

  • For phosphodiesterase activity: Measure the hydrolysis of nucleotide sugars like UDP-GlcNAc into UMP and GlcNAc-1-phosphate

  • For nucleotide metabolism: Quantify ATP, GTP, UTP, or CTP hydrolysis and resulting products

• Glycosylation profiling: Since ENPP3 influences cellular glycosylation through nucleotide sugar hydrolysis, techniques like lectin microarrays or mass spectrometry-based glycomics can reveal ENPP3-dependent changes in glycan profiles .

• Cloning approaches: For functional studies, mouse ENPP3 cDNA can be cloned by RT-PCR using total RNA from mouse brain tissue with specific primers (forward: 5′-ACGGGAACAATGGATTCCAG-3′; reverse: 5′-CCCCATTTTGTCAAATGGCT-3′) .

• Site-directed mutagenesis: Catalytically inactive ENPP3 can be generated by mutating the threonine 205 catalytic center to alanine using specific PCR primers designed for the mutation .

What are the recommended protocols for immunohistochemical detection of ENPP3?

For optimal immunohistochemical (IHC) detection of ENPP3:

• Tissue preparation: Formalin-fixed, paraffin-embedded (FFPE) tissue sections are commonly used. Fresh frozen sections may better preserve certain epitopes but require careful handling.

• Antigen retrieval: Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) is typically effective for ENPP3 detection.

• Antibody selection: Select antibodies validated specifically for IHC applications. Monoclonal antibodies generated against the ENPP3 extracellular domain (ECD) have shown good specificity in tissue staining .

• Detection systems: For clinical samples, polymer-based detection systems provide excellent sensitivity with minimal background. For research applications, brightfield or fluorescence-based detection can be selected based on experimental needs.

• Controls: Include positive controls (tissues with known ENPP3 expression, such as renal cell carcinoma samples) and negative controls (antibody diluent without primary antibody) .

• Expression evaluation: For quantitative assessment, use established scoring systems. For ENPP3 in cancer tissues, both staining intensity and percentage of positive cells should be recorded. In renal cell carcinoma studies, a high threshold for positivity is justified as 92.3% of samples show some ENPP3 expression, with 83.9% demonstrating high expression .

How does ENPP3 regulate nucleotide metabolism and influence cellular signaling?

ENPP3 plays a sophisticated role in nucleotide metabolism that extends beyond simple hydrolysis:

• Extracellular nucleotide regulation: ENPP3 metabolizes extracellular nucleotides including ATP, GTP, UTP, and CTP, directly influencing purinergic signaling pathways . This activity is particularly important in:

  • Limiting mast cell and basophil responses during inflammatory and allergic reactions

  • Preventing ATP-induced apoptosis of intestinal plasmacytoid dendritic cells

  • Modulating immune cell activation through control of extracellular ATP concentrations

• Dinucleoside polyphosphate hydrolysis: ENPP3 can hydrolyze extracellular dinucleoside polyphosphates, including vasoactive adenosine polyphosphates, affecting vascular tone regulation .

• Nucleotide sugar metabolism: ENPP3 hydrolyzes UDP-GlcNAc into UMP and GlcNAc-1-phosphate, as well as other nucleotide sugars including UDP-GalNAc, CMP-NeuAc, GDP-Fuc, and UDP-GlcA. This activity generates UMP, a potent competitive inhibitor of glycosyltransferases like GnT-IX .

• Feedback regulation: ENPP3-generated UMP acts as a competitive inhibitor of glycosyltransferases, creating a feedback loop that regulates glycosylation processes. Kinetic analysis has demonstrated that ENPP3-induced inhibition of GnT-IX is mediated through this mechanism .

• Alkaline phosphodiesterase activity: Beyond its primary functions, ENPP3 displays alkaline phosphodiesterase activity in vitro, potentially contributing to additional signaling pathways .

What is the significance of ENPP3 in cancer biology and as a therapeutic target?

ENPP3 has emerged as an important molecule in cancer biology with promising therapeutic applications:

• Cancer-specific expression patterns: ENPP3 shows highly selective expression in certain cancer types:

  • In clear cell renal cell carcinoma (ccRCC), 92.3% of samples are positive for ENPP3 and 83.9% show high expression

  • Expression is less frequent in papillary renal cell carcinoma and hepatocellular carcinoma

  • Normal tissues show negligible expression, with the exception of kidney

• Antibody-drug conjugate development: The selective expression pattern makes ENPP3 an excellent target for antibody-drug conjugates:

  • AGS16F, an anti-ENPP3 antibody conjugated with maleimidocaproyl monomethyl auristatin F via a noncleavable linker (mcMMAF), has shown promising results in preclinical studies

  • AGS16F demonstrated tumor growth inhibition in three different renal cell carcinoma xenograft models

  • The drug localizes to tumors, forms the active metabolite Cys-mcMMAF, induces cell-cycle arrest and apoptosis

• Pharmacodynamic markers: ENPP3-targeted therapies can be monitored using specific biomarkers:

  • Increased blood levels of caspase-cleaved cytokeratin-18, a marker of epithelial cell death, correlate with AGS16F activity

  • Tumor biopsies show evidence of cell-cycle arrest and apoptosis following treatment

• Clinical development: AGS16F has progressed to clinical evaluation:

  • Phase I clinical trials have been initiated to evaluate safety and preliminary efficacy

  • The selective expression pattern suggests potential for therapeutic window with minimal off-target effects

How does ENPP3 influence glycosylation pathways and cellular glycophenotypes?

ENPP3 exerts profound effects on cellular glycosylation through multiple mechanisms:

• Nucleotide sugar donor regulation: ENPP3 hydrolyzes nucleotide sugar donors (UDP-GlcNAc, UDP-GalNAc, CMP-NeuAc, GDP-Fuc, UDP-GlcA), directly affecting substrate availability for glycosyltransferases .

• Competitive inhibition mechanism: The UMP produced by ENPP3-mediated hydrolysis of UDP-GlcNAc acts as a potent competitive inhibitor of glycosyltransferases like GnT-IX. Kinetic analysis has confirmed this as the mechanism for ENPP3-induced inhibition of glycosyltransferase activity .

• Global glycosylation impact: ENPP3 knockdown cells show significantly increased levels of intracellular nucleotide sugars and display widespread changes in the total cellular glycosylation profile. This indicates that ENPP3 acts as a broad regulator of cellular glycosylation .

• Novel regulatory system: ENPP3 represents a novel regulatory system for cellular glycosylation processes that operates alongside other known mechanisms such as chaperone-mediated regulation of glycosyltransferases .

• Cell-specific effects: The impact of ENPP3 on glycosylation appears to be cell-type specific. In Neuro2a cells, ENPP3 was identified as an inhibitory factor for N-acetylglucosaminyltransferase GnT-IX (GnT-Vb), suggesting potential tissue-specific roles in regulating glycosylation .

What is the current status of ENPP3-directed antibody-drug conjugates in cancer therapy?

ENPP3-directed antibody-drug conjugates (ADCs) represent a promising approach in targeted cancer therapy:

• Development rationale: ENPP3 was identified through suppression subtractive hybridization as a potential human cancer-specific antigen, particularly in renal cell carcinoma. Its limited expression in normal tissues (except kidney) provides a favorable therapeutic window .

• AGS16F development: A lead ADC candidate, AGS16F, was developed by conjugating an anti-ENPP3 antibody with maleimidocaproyl monomethyl auristatin F via a noncleavable linker (mcMMAF) .

• Preclinical efficacy: AGS16F demonstrated:

  • Effective tumor growth inhibition in three different renal cell carcinoma (RCC) xenograft models

  • Successful localization to tumors and formation of the active metabolite Cys-mcMMAF

  • Induction of cell-cycle arrest and apoptosis in target tissues

  • Increased blood levels of caspase-cleaved cytokeratin-18, indicating epithelial cell death

• Clinical evaluation: AGS16F has progressed to clinical trials:

  • Phase I clinical trial was initiated to evaluate safety, pharmacokinetics, and preliminary efficacy

  • Results will determine optimal dosing and potential for further development in RCC and potentially other ENPP3-expressing tumors

How can researchers evaluate antibody specificity and cross-reactivity for ENPP3?

Thorough evaluation of antibody specificity and cross-reactivity is essential for reliable ENPP3 research:

• ELISA-based binding assays: Functional ELISA using immobilized recombinant ENPP3 proteins can quantify binding affinity and specificity:

  • Human ENPP3 at 2 μg/ml can bind anti-ENPP3 recombinant antibody with an EC50 of 2.151-2.492 ng/mL

  • Macaca fascicularis ENPP3 at 2 μg/ml can bind the same antibody with an EC50 of 3.313-4.724 ng/mL

• Species cross-reactivity: Systematic testing against ENPP3 from multiple species determines the range of research applications:

  • Commercial antibodies are often validated for human and non-human primate ENPP3

  • Western blot analysis using tissue lysates from different species can confirm cross-reactivity

• Cellular validation: Flow cytometry on cells with known ENPP3 expression:

  • Human basophils are excellent positive controls for surface ENPP3 expression

  • Comparison of resting versus activated states can confirm detection of physiologically relevant expression changes

• Knockout/knockdown controls: Cells with ENPP3 knockout or knockdown provide the gold standard negative control:

  • CRISPR-Cas9 engineered cell lines

  • siRNA-treated cells with confirmed knockdown efficiency

• Immunohistochemical validation: Testing on tissues with known ENPP3 expression patterns:

  • Clear cell renal cell carcinoma samples as positive controls (92.3% positivity rate)

  • Normal tissues (except kidney) as negative controls

What methodological approaches are most effective for studying ENPP3 enzymatic activity?

Several complementary approaches can be employed to characterize ENPP3 enzymatic activity:

• Site-directed mutagenesis: Generation of catalytically inactive ENPP3 through mutation of the threonine 205 catalytic center to alanine provides an excellent control for enzymatic studies. This can be accomplished using specific PCR primers designed for the mutation:

  • Forward primer: 5′-ATCCCACCAAAGCCTTCCCAAATC-3′

  • Reverse primer: 5′-GATTTGGGAAGGCTTTGGTGGGAT-3′

• Nucleotide sugar hydrolysis assay: Measurement of ENPP3-catalyzed hydrolysis of UDP-GlcNAc to UMP and GlcNAc-1-phosphate:

  • Incubate purified ENPP3 with UDP-GlcNAc substrate

  • Quantify reaction products (UMP and GlcNAc-1-phosphate) using HPLC or mass spectrometry

  • Calculate kinetic parameters (Km, Vmax) to characterize enzyme efficiency

• Cell-based nucleotide sugar quantification: Comparative analysis of intracellular nucleotide sugar levels in ENPP3 knockout/knockdown versus wild-type cells can reveal the physiological impact of ENPP3 activity .

• Glycosyltransferase inhibition assay: Assessment of UMP-mediated competitive inhibition of glycosyltransferases like GnT-IX:

  • Measure glycosyltransferase activity in the presence of varying concentrations of UMP

  • Determine IC50 values and inhibition constants (Ki)

  • Compare with ENPP3-containing reaction mixtures to confirm the mechanism

• Extracellular nucleotide hydrolysis: Quantification of ATP, GTP, UTP, or CTP hydrolysis using luciferase-based assays or HPLC-based methods to assess ENPP3 activity toward these physiological substrates .

What are common challenges in ENPP3 detection and quantification?

Researchers frequently encounter several challenges when working with ENPP3:

• Variable expression levels: ENPP3 expression can vary significantly between cell types and activation states. For example, basophil activation can alter surface ENPP3 levels, potentially leading to inconsistent detection .

• Glycosylation heterogeneity: As a transmembrane glycoprotein, ENPP3 exhibits heterogeneous glycosylation patterns that can affect antibody recognition. Different glycoforms may show varying molecular weights on SDS-PAGE (around 130 kDa), complicating quantification .

• Enzymatic activity preservation: ENPP3's enzymatic function can be lost during sample preparation. Cell lysis buffers containing certain detergents or incorrect pH can diminish enzymatic activity, necessitating careful buffer optimization .

• Cross-reactivity with other ENPP family members: ENPP3 shares sequence similarity with other ENPP family members, potentially leading to cross-reactivity. Validation using specific knockout controls is essential to confirm antibody specificity .

• Technical variability in functional assays: When measuring ENPP3's impact on glycosylation or nucleotide metabolism, technical variability can arise from multiple sources in the experimental workflow. Standardized protocols with appropriate controls are necessary for reproducible results .

How can researchers optimize ENPP3 expression systems for functional studies?

Optimizing ENPP3 expression systems requires attention to several key parameters:

• Expression vector selection: For mammalian expression, vectors like pcDNA3.1/Myc-His version C have been successfully used for ENPP3 expression:

  • C-terminally Myc-His-tagged mouse ENPP3 constructs allow for easy purification and detection

  • The addition of epitope tags should be validated to ensure they don't interfere with enzymatic activity

• Mutation strategies: For structure-function studies, site-directed mutagenesis of the threonine 205 catalytic center to alanine produces catalytically inactive ENPP3 that serves as an excellent control:

  • Upstream and downstream halves of the ENPP3 coding sequence can be separately amplified using specific primers designed to introduce the mutation

  • The two PCR products are then combined and used as templates for final PCR amplification

• Cell line selection: Different host cells may provide varying post-translational modifications and expression levels:

  • Neuro2a cells have been successfully used for ENPP3 functional studies

  • HEK293 cells are often preferred for high-yield recombinant protein production

  • Cell-type specific effects should be considered, as ENPP3 may have different impacts on glycosylation depending on the cellular context

• Purification strategies: For recombinant ENPP3 purification:

  • Affinity chromatography using tags like His or Myc provides good initial purification

  • Additional purification steps may be needed for highest purity

  • Validation through SDS-PAGE (Tris-Glycine gel with 5% enrichment gel and 15% separation gel) confirms purity

What quality control parameters should be evaluated for ENPP3 antibodies?

Rigorous quality control for ENPP3 antibodies should include:

• Binding affinity determination: EC50 values should be established through ELISA using recombinant ENPP3 proteins:

  • Human ENPP3: EC50 of 2.151-2.492 ng/mL

  • Macaca fascicularis ENPP3: EC50 of 3.313-4.724 ng/mL

• Specificity validation: Multiple approaches confirm specificity:

  • Western blot showing single band at expected molecular weight (~130 kDa)

  • Flow cytometry on positive and negative control cells

  • Immunoprecipitation followed by mass spectrometry identification

  • Testing on ENPP3 knockout/knockdown systems

• Functional impact assessment: Some applications require antibodies that don't interfere with ENPP3 function, while others (therapeutic applications) may benefit from function-blocking antibodies:

  • Enzymatic activity assays in the presence of antibody

  • Cell-based functional assays with antibody treatment

• Batch-to-batch consistency: Verification that different antibody lots maintain consistent:

  • Binding affinity (EC50)

  • Specificity profile

  • Application performance in flow cytometry, IHC, etc.

• Application-specific validation: Performance in specific applications using standardized protocols:

  • Flow cytometry on human basophils or other ENPP3-expressing cells

  • IHC on renal cell carcinoma samples (known high expressors)

  • Western blotting under reducing conditions