HPN Antibody

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

HPN (Hepsin) is a type II transmembrane serine protease, also known as TMPRSS1 (Transmembrane protease serine 1). It functions as a critical enzyme that cleaves extracellular substrates and contributes to the proteolytic processing of growth factors, such as HGF and MST1/HGFL . HPN plays essential roles in cell growth and maintenance of cell morphology .

The protein is particularly significant in cancer research because its expression is associated with the growth and progression of various cancers . In prostate cancer specifically, elevated levels of Hepsin appear to facilitate tumor progression by degrading extracellular matrix components, enabling cancer cells to invade new tissues . This makes HPN an important target for both basic research investigating cancer mechanisms and translational research developing potential therapeutic approaches.

What types of HPN antibodies are available for research applications?

Several types of HPN antibodies have been developed for research applications, each with specific characteristics and applications:

• Recombinant Monoclonal Antibodies: Examples include Rabbit Recombinant Monoclonal Hepsin/HPN antibody [EP7654] . These offer high specificity and batch-to-batch consistency.

• Monoclonal Antibodies: Such as Mouse monoclonal antibody (M02), clone 2D5 . These provide excellent specificity for particular epitopes.

• Polyclonal Antibodies: Including Anti-HPN antibody produced in rabbit (HPA006804) and HPN Polyclonal Antibody (E-AB-52191) . These typically recognize multiple epitopes on the target protein.

• Humanized Antibodies: Like the Human Anti-HPN Recombinant Antibody (clone mAb55), which can potentially be used in treating ovarian and prostate cancer .

The selection of an appropriate antibody depends on the specific research application, required specificity, and experimental conditions.

What applications are HPN antibodies validated for?

HPN antibodies have been validated for several experimental applications, including:

• Western Blot (WB): Most HPN antibodies are validated for WB, typically at dilutions between 1:500 and 1:5000 depending on the specific antibody . For example, the HPN Polyclonal Antibody E-AB-52191 is recommended at a dilution of 1:500-1:2000 for Western blot applications .

• Immunohistochemistry (IHC-P): Antibodies like the Rabbit Recombinant Monoclonal Hepsin/HPN antibody and HPA006804 are suitable for IHC-P, with recommended dilutions of 1:50-1:200 .

• ELISA: Several antibodies have been validated for ELISA applications, including Mouse monoclonal antibody (M02), clone 2D5, which has a detection limit of 3 ng/ml as a capture antibody for recombinant GST-tagged HPN .

• Flow Cytometry: Antibodies like Mab55 have demonstrated specific and saturable surface staining in flow cytometry experiments with cells expressing HPN .

• Confocal Microscopy: HPN antibodies such as Mab55 have been used successfully in confocal laser scanning microscopy to visualize cell surface HPN expression .

What species reactivity should I consider when selecting an HPN antibody?

When selecting an HPN antibody, species reactivity is a critical consideration. From the available information:

• Most HPN antibodies react with Human HPN, including all the antibodies mentioned in the search results .

• Some antibodies show cross-reactivity with rodent models:

  • The Rabbit Recombinant Monoclonal Hepsin/HPN antibody [EP7654] reacts with Human, Rat, and Mouse samples .

  • The HPN Polyclonal Antibody (E-AB-52191) shows reactivity to Human, Mouse, and Rat samples .

  • The Mouse monoclonal antibody (M02) shows interspecies antigen sequence similarity with Mouse (89%) and Rat (89%), suggesting potential cross-reactivity .

For research involving non-human primates, antibodies like Mab55 have been tested against cynomolgus monkey hepsin .

Always review the manufacturer's validation data for specific species reactivity before selecting an antibody for your research, especially if working with animal models.

How can I validate HPN antibody specificity for my particular experimental system?

Validating antibody specificity is critical for ensuring reliable experimental results. For HPN antibodies, consider the following validation approaches:

Positive and Negative Controls:

• Use cell lines known to express high levels of HPN, such as HepG2 cells, as positive controls .

• Include negative controls such as untransfected HEK293 cells (wild type) which show no detectable binding with HPN antibodies like Mab55 .

Multiple Detection Methods:

• Verify results using complementary techniques. For example, if using flow cytometry to detect HPN expression, confirm findings with confocal microscopy and Western blotting .

• For Western blot validation, compare observed molecular weight with the expected molecular weight of HPN (approximately 45 kDa) .

Epitope Mapping:

• Consider the immunogen sequence used to generate the antibody. For instance, HPA006804 was generated against the sequence "FDKTEGTWRLLCSSRSNARVAGLSCEEMGFLRALTHSELDVRTAGANGTSGFFCVDEGRLPHTQRLLEVISVCDCPRGRFLAAICQDCGRRKLPVDRIVGGRDTSLGRWPWQVSLRYDGAHL" .

• Competitive binding assays with the immunizing peptide can confirm specificity.

Cross-Reactivity Testing:

• Test for cross-reactivity with related proteases. For example, Mab55 was shown to be protease-specific, with negligible activity against other serine proteases like Matriptase, HAT, Enteropeptidase, and Trypsin under the same test conditions .

What are the optimal experimental conditions for detecting HPN in cancer tissues using immunohistochemistry?

For optimal detection of HPN in cancer tissues using immunohistochemistry (IHC), researchers should consider these protocol optimization steps:

Antibody Selection and Dilution:

• Anti-HPN antibody HPA006804 is recommended at a dilution range of 1:50-1:200 for IHC applications .

• The Rabbit Recombinant Monoclonal Hepsin/HPN antibody [EP7654] has been extensively validated for IHC-P applications .

Antigen Retrieval:

• Heat-induced epitope retrieval in citrate buffer (pH 6.0) is typically effective for HPN detection in formalin-fixed, paraffin-embedded tissues.

• The specific protocol may need optimization based on tissue type and fixation conditions.

Blocking and Incubation Conditions:

• For Western blot applications (which can inform IHC optimization), blocking with 5% NFDM/TBST has been reported effective .

• Primary antibody incubation should typically be performed overnight at 4°C to enhance specific binding while minimizing background.

Detection Systems:

• HRP-conjugated secondary antibodies with DAB substrate provide good visualization for brightfield microscopy.

• For fluorescent detection, select secondary antibodies compatible with your primary antibody species (rabbit or mouse, depending on which HPN antibody is used).

Tissue Considerations:

• HPN expression varies across tissue types, with liver tissues often showing strong expression that can serve as positive controls .

• In cancer studies, include both tumor and adjacent normal tissue for comparative analysis, as HPN is often overexpressed in certain cancers .

How do different HPN antibodies compare in terms of inhibitory potential against HPN enzymatic activity?

Different HPN antibodies exhibit varying inhibitory potentials against HPN enzymatic activity, which is an important consideration for functional studies:

Inhibitory Potency:

• Antibody Mab55 (humanized HPN antibody) shows potent inhibition of human hepsin activity in FRET activity assays .

• The inhibition appears to be dose-dependent, with IC50 values that can be calculated after fitting the inhibition data to a four-parameter equation .

Species Specificity in Inhibition:

• Some antibodies like Mab55 demonstrate species-specific inhibition, showing different activity profiles against human hepsin compared to cynomolgus monkey hepsin .

• This species specificity is an important consideration for translational studies moving between animal models and human applications.

Inhibition Mechanism:

• Some HPN antibodies exhibit non-linear inhibition types, suggesting complex binding interactions with the enzyme .

• The inhibitory effect typically involves binding to the enzyme and preventing substrate access or catalytic activity.

Comparative Analysis:

• When comparing different antibodies such as mouse Mab 2.7.35 (mH35), chimeric Mab 2.7.35 (chH35), and Mab55 (hH35), variations in their inhibition profiles and binding affinities have been observed .

• These differences are characterized by surface plasmon resonance measurements to determine binding and dissociation constants .

What considerations are important when using HPN antibodies to study cancer progression mechanisms?

When studying cancer progression mechanisms using HPN antibodies, researchers should consider:

Expression Level Analysis:

• HPN is overexpressed in various cancer types, including prostate, ovarian, and pancreatic cancers .

• Quantitative analysis of HPN expression levels relative to normal tissues is essential for understanding its role in cancer progression.

Functional Role Investigation:

• In prostate cancer, elevated HPN levels appear to facilitate tumor progression by degrading extracellular matrix components .

• Design experiments to assess whether HPN inhibition (using inhibitory antibodies) affects cancer cell invasion and metastatic potential.

Pathway Analysis:

• HPN contributes to the proteolytic processing of growth factors such as HGF and MST1/HGFL .

• Consider using HPN antibodies in combination with analysis of downstream signaling pathways to elucidate mechanistic connections.

Model System Selection:

• HepG2 cells (human hepatocellular carcinoma) abundantly express HPN and can serve as useful model systems .

• For prostate cancer studies, consider established cell lines with varying levels of HPN expression to study concentration-dependent effects.

Therapeutic Potential Assessment:

• Some humanized HPN antibodies like clone mAb55 have potential therapeutic applications in ovarian and prostate cancer .

• Design experiments to evaluate antibody effects on tumor growth, invasion, and response to standard therapies.

What controls should be included when using HPN antibodies for Western blot analysis?

For rigorous Western blot analysis using HPN antibodies, researchers should include these key controls:

Positive Controls:

• HepG2 cells (human hepatocellular carcinoma) serve as excellent positive controls due to their abundant HPN expression .

• Human liver tissue lysate has also been validated as a positive control for HPN Western blot .

Negative Controls:

• Include cell lines or tissues known not to express HPN or with HPN knocked down through siRNA or CRISPR.

• For transfection studies, untransfected cells (e.g., HEK293 wild type) can serve as negative controls .

Loading Controls:

• Include housekeeping proteins (β-actin, GAPDH, or α-tubulin) to normalize protein loading across samples.

• This is particularly important when comparing HPN expression levels between different tissues or treatment conditions.

Antibody Controls:

• Include no-primary-antibody controls to assess background from secondary antibodies.

• When available, use blocking peptides (the immunogen used to generate the antibody) to confirm specificity.

Molecular Weight Validation:

• The expected molecular weight of HPN is approximately 45 kDa .

• For recombinant GST-tagged HPN, the molecular weight is approximately 67.43 kDa (including the 26 kDa GST tag) .

Dilution Optimization:

• Test different antibody dilutions to determine optimal signal-to-noise ratio.

• For example, HPN Polyclonal Antibody (E-AB-52191) is recommended at dilutions of 1:500-1:2000 for Western blot , while Anti-Hepsin/HPN antibody [EP7654] can be used at higher dilutions of 1:5000 .

How can HPN antibodies be effectively used to study protein-protein interactions involving HPN?

To effectively study protein-protein interactions involving HPN using antibodies:

Co-Immunoprecipitation (Co-IP):

• Use HPN antibodies to immunoprecipitate HPN from cell lysates, then probe for interacting partners.

• Alternatively, immunoprecipitate suspected binding partners and probe for HPN.

• Consider using recombinant monoclonal antibodies for higher specificity in pull-down experiments.

Proximity Ligation Assay (PLA):

• This technique can detect protein interactions in situ with high sensitivity.

• Requires antibodies raised in different host species against HPN and its potential interacting partners.

• Can visualize interactions at subcellular resolution.

FRET/BRET Assays:

• For investigating dynamic interactions, consider Fluorescence or Bioluminescence Resonance Energy Transfer.

• May require genetically tagged versions of HPN and interaction partners.

Cross-Linking Studies:

• Chemical cross-linking followed by immunoprecipitation with HPN antibodies can capture transient interactions.

• Mass spectrometry analysis of cross-linked complexes can identify novel binding partners.

Considerations for Known Interactions:

• HPN is known to interact with and process growth factors such as HGF and MST1/HGFL .

• HPN plays a role in the proteolytic processing of ACE2 .

• Design experiments to probe these specific interactions using appropriate antibodies against both HPN and these known partners.

What methodological approaches can be used to study HPN inhibition in functional assays?

Several methodological approaches can be employed to study HPN inhibition in functional assays:

FRET-Based Activity Assays:

• Fluorescence Resonance Energy Transfer (FRET) assays using peptide substrates like Ac-KQLR-AMC have been successfully employed to measure HPN inhibition by antibodies .

• The assay involves preincubating antibodies with hepsin for 30 minutes, then initiating the hydrolysis reaction by adding the peptide substrate.

• Fluorescence increase is measured after a defined incubation period (e.g., 40 minutes), and IC50 values can be calculated by fitting the percentage inhibition data to a four-parameter equation .

Surface Plasmon Resonance:

• This technique allows real-time analysis of antibody binding to HPN.

• By injecting HPN at different concentrations (e.g., 0-200 nM) over immobilized antibodies, binding and dissociation kinetics can be determined using models such as 1:1 Langmuir binding .

• This approach has been used to compare binding and dissociation constants for different HPN antibodies, including mouse, chimeric, and humanized variants .

Cell-Based Assays:

• Flow cytometry can be used to detect binding of HPN antibodies to cells expressing HPN on their surface.

• Experiments with HEK293 cells stably overexpressing full-length hepsin with a C-terminal GFP fusion tag have demonstrated specific and saturable surface staining with increasing amounts of HPN antibodies .

• Confocal laser scanning microscopy can confirm surface staining and localization patterns .

Matrix Degradation Assays:

• Since HPN in cancer facilitates tumor progression by degrading extracellular matrix components , matrix degradation assays can be used to assess the functional impact of HPN inhibition.

• These assays typically involve culturing cells on fluorescently labeled matrix proteins and quantifying degraded areas.

What are the key considerations for developing multiplex assays using HPN antibodies alongside other cancer biomarkers?

Developing multiplex assays using HPN antibodies alongside other cancer biomarkers requires careful consideration of several technical aspects:

Antibody Compatibility:

• When selecting antibodies for multiplexing, ensure they are raised in different host species to avoid cross-reactivity of secondary antibodies.

• If using multiple rabbit antibodies, consider directly conjugated primary antibodies with different fluorophores.

Spectral Overlap Management:

• Choose fluorophores with minimal spectral overlap to reduce bleed-through in immunofluorescence or flow cytometry.

• Include appropriate single-stained controls for compensation in flow cytometry or spectral unmixing in confocal microscopy.

Biomarker Selection Strategy:

• Combine HPN with functionally related biomarkers for comprehensive pathway analysis. Since HPN cleaves extracellular substrates and contributes to the proteolytic processing of growth factors such as HGF and MST1/HGFL , consider including these as additional biomarkers.

• For cancer studies, include tissue-specific markers alongside HPN to improve diagnostic or prognostic value.

Sequential Staining Protocols:

• For challenging combinations, consider sequential staining protocols with blocking steps between each antibody application.

• This can be particularly useful for tissue sections where epitope accessibility may be an issue.

Validation of Multiplex Assays:

• Always validate multiplex results against single-marker controls to ensure antibody performance is not compromised in the multiplex setting.

• Use quantitative image analysis tools to assess colocalization and expression levels objectively.

Platform Selection:

• Choose appropriate technology platforms based on research needs:

  • Flow cytometry for single-cell quantification

  • Tissue microarrays for high-throughput screening across multiple samples

  • Mass cytometry (CyTOF) for highly multiplexed protein detection without fluorescence spectral limitations

How can HPN antibodies be used to evaluate potential therapeutic targets in cancer?

HPN antibodies can be valuable tools for evaluating potential therapeutic targets in cancer through several approaches:

Target Validation Studies:

• Use HPN antibodies to confirm expression levels in patient-derived samples compared to normal tissues, establishing HPN as a valid therapeutic target .

• Correlate HPN expression with clinical outcomes to determine prognostic significance.

Functional Blocking Studies:

• Inhibitory antibodies like Mab55 can be used to block HPN activity in vitro and assess effects on cancer cell phenotypes .

• Measure changes in proliferation, invasion, migration, and survival following HPN inhibition.

Mechanism-of-Action Studies:

• Combine HPN antibodies with pathway inhibitors to identify synergistic therapeutic combinations.

• Use phospho-specific antibodies against downstream signaling molecules to map how HPN inhibition affects cancer-relevant pathways.

Therapeutic Antibody Development:

• Humanized antibodies like clone mAb55 represent potential therapeutic agents for ovarian and prostate cancer .

• These can be further developed into various antibody formats, including antibody-drug conjugates or bispecific antibodies.

Trispecific T-Cell Engagers:

• While not directly using HPN antibodies, the development of constructs like HPN536 demonstrates how targeting approaches can be translated to clinical applications .

• HPN536 is a 53-kDa trispecific T-cell-activating protein that binds to MSLN-expressing tumor cells, CD3ε on T cells, and serum albumin .

• Similar approaches could potentially be developed for HPN-expressing tumors.

What are the challenges in developing therapeutic HPN antibodies for cancer treatment?

Developing therapeutic HPN antibodies for cancer treatment presents several challenges:

Target Specificity:

• Ensuring sufficient specificity for HPN over related serine proteases is critical to minimize off-target effects .

• Antibodies must be thoroughly tested against other serine proteases like Matriptase, HAT, Enteropeptidase, and Trypsin to confirm specificity .

Species Cross-Reactivity:

• Differences in inhibitory potential against human versus non-human primate HPN can complicate preclinical development .

• These differences may necessitate the development of surrogate antibodies for animal studies or careful interpretation of preclinical data.

Penetration into Solid Tumors:

• Full-sized antibodies face challenges penetrating solid tumors due to their large size.

• Alternative formats like antibody fragments or smaller bispecific constructs might offer improved tumor penetration.

Expression Heterogeneity:

• HPN expression may vary across tumor types and even within the same tumor .

• This heterogeneity necessitates careful patient selection strategies for clinical trials.

Potential Resistance Mechanisms:

• Cancer cells may develop resistance to HPN-targeting therapies through compensatory proteolytic pathways.

• Combination strategies may be needed to address potential resistance mechanisms.

Translational Considerations:

• The transition from preclinical models to human studies requires careful consideration of pharmacokinetics, immunogenicity, and safety profiles.

• The extended half-life observed with constructs like HPN536 in non-human primates demonstrates the importance of designing antibodies with favorable pharmacokinetic properties .