DHS Antibody

What is the DHS mutation in antibody engineering?

The DHS mutation represents an engineered IgG1 Fc domain that functions as an hFcRn binding pH toggle switch, specifically designed to eliminate residual binding to FcRn at physiological pH while maintaining strong binding at endosomal pH . This engineered Fc domain confers significantly improved pharmacokinetic properties compared to wild-type antibodies and other half-life extension variants, making it valuable for therapeutic antibody development . The DHS mutation works by optimizing the pH-dependent binding characteristics that govern antibody recycling through the neonatal Fc receptor pathway, which is critical for extending antibody circulation time in vivo.

How does the DHS mutation compare to other half-life extension technologies (YTE, LS)?

The DHS mutation demonstrates superior pharmacokinetic properties compared to clinical-stage half-life extension variants like YTE and LS. In humanized mouse models, antibodies with the DHS mutation exhibited a β-phase half-life that was 2.0-fold and 3.1-fold higher than YTE and LS variants, respectively (290.9 ± 25.6 h for DHS compared to 148.4 ± 36.8 h for YTE and 92.9 ± 6.1 h for LS) . Furthermore, the total drug exposure over time (AUC inf) for DHS-IgG1 was 1.6-, 1.9-, and 5.3-fold greater than YTE-, LS-, and wild-type IgG1 variants, respectively . The clearance rate of DHS-IgG1 (0.11 ± 0.01 mL/day/kg) was approximately 6-fold lower than wild-type IgG1 and 1.9- and 2.2-fold lower than YTE- and LS-IgG1, indicating superior persistence in circulation .

What experimental methods are used to engineer and screen for DHS mutations?

Engineering and screening for DHS mutations involves a multi-step process using advanced molecular biology techniques:

• Spheroplast display system: Spheroplasts expressing mutated human IgG1 (e.g., Trastuzumab) anchored on the external leaflet of the inner membrane are screened using FACS for binding to fluorescently labeled hFcRn:hβ2m .

• pH-dependent selection: Multiple rounds of FACS with hFcRn:hβ2m at pH 5.8 are performed to enrich antibodies with Fc domains exhibiting higher binding affinity at endosomal pH .

• Negative selection: A competitive, two-step labeling process is employed to eliminate variants with detectable binding at pH 7.4 to high-avidity, dimeric GST-hFcRn:hβ2m. This involves labeling spheroplasted cells with Alexa647-labeled GST-hFcRn:hβ2m at pH 7.4, followed by washing with pH 7.4 PBS .

• Validation in animal models: Promising candidates are then validated in humanized mouse models that recapitulate key processes for antibody persistence in circulation .

Can DHS mutations be applied across different IgG subclasses?

Yes, the DHS mutations can be successfully applied across all IgG subclasses. Research has demonstrated that introducing DHS mutations into the Fc domain of IgG2, IgG3 (G3m16 allotype with H435 residue), or IgG4 subclasses results in no detectable interaction with FcRn at pH 7.4 while conferring significantly improved pharmacokinetic properties for all four IgG subclasses . This versatility makes DHS mutations applicable to various therapeutic antibody formats, allowing researchers to select the most appropriate IgG subclass for their specific therapeutic target while still benefiting from the enhanced half-life properties.

What are the potential limitations or challenges when implementing DHS mutations?

While DHS mutations show promise, researchers should be aware of potential limitations:

• Effect on other antibody functions: Although studies indicate that antibodies with the DHS Fc domain maintain intact effector functions important for clearing target pathogenic cells , researchers should still verify that specific effector functions relevant to their therapeutic target are preserved.

• Need for optimization: Despite improved pharmacokinetics, some studies suggest that further optimization may be needed to maximize therapeutic efficacy . The performance of DHS mutations may vary depending on the antibody context and target.

• Species differences: While DHS mutations have been validated in humanized mouse models, the translation to human pharmacokinetics should be carefully evaluated, as species differences in FcRn binding and antibody recycling exist.

• Manufacturing considerations: Researchers should assess whether DHS mutations affect antibody expression, stability, or other developability characteristics before large-scale production.

What is the function of DHPS/DHS enzyme in cellular biology?

Deoxyhypusine synthase (DHPS/DHS) is an essential enzyme that catalyzes the NAD-dependent oxidative cleavage of spermidine and subsequently transfers the butylamine moiety of spermidine to the epsilon-amino group of a critical lysine residue in the eIF-5A precursor protein . This reaction forms an intermediate deoxyhypusine residue, which represents the first step in the post-translational modification pathway that produces hypusine, an unusual amino acid residue unique to mature eIF-5A factor . This hypusination process is essential for eIF-5A function, which plays critical roles in cell proliferation, translation elongation, and other cellular processes. Understanding DHPS function is therefore crucial for research into fundamental cellular processes and potential therapeutic interventions.

What are the key experimental applications for anti-DHPS/DHS antibodies?

Anti-DHPS/DHS antibodies serve multiple experimental purposes in biomedical research:

• Western Blotting (WB): Anti-DHPS/DHS antibodies such as Boster's A30637 and Abcam's ab190266 have been validated for western blot applications, allowing researchers to detect and quantify DHPS expression in cell and tissue lysates .

• Immunoprecipitation (IP): Some antibodies like ab190266 are suitable for immunoprecipitation, enabling researchers to isolate DHPS and its interacting partners for further analysis .

• ELISA: Antibodies such as A30637 can be used in ELISA assays for quantitative detection of DHPS in various sample types .

• Studying post-translational modifications: These antibodies are valuable tools for investigating the hypusination pathway and its role in protein synthesis and cellular function.

• Cancer research: Given the role of eIF-5A and hypusination in cell proliferation, DHPS antibodies are useful for studying cancer biology and potential therapeutic targets.

What is the molecular weight discrepancy observed with DHPS/DHS in western blotting?

A notable technical challenge when working with DHPS/DHS antibodies is the discrepancy between observed and calculated molecular weights. While the calculated molecular weight of human DHPS is approximately 41 kDa (40,971 Da to be precise) , western blot analysis often reveals a band at approximately 72 kDa . This discrepancy could be attributed to several factors:

• Post-translational modifications: The native protein may undergo extensive modifications that significantly increase its apparent molecular weight.

• Protein dimerization: DHPS may exist as dimers that are not fully denatured during SDS-PAGE.

• Isoforms: Alternative splicing or proteolytic processing may produce higher molecular weight variants.

• Antibody specificity: Some antibodies may detect cross-reactive proteins with similar epitopes.

Researchers should be aware of this discrepancy when interpreting western blot results and might need to confirm DHPS identity through additional techniques such as mass spectrometry or knockdown/knockout validation studies.

What are the optimal experimental conditions for using anti-DHPS/DHS antibodies?

For optimal results when using anti-DHPS/DHS antibodies, researchers should:

• Store antibodies at -20°C for long-term storage and at 4°C for up to one month for frequent use .

• Avoid repeated freeze-thaw cycles as they can degrade antibody quality and affect experimental outcomes .

• When performing western blot, use appropriate loading controls and optimize transfer conditions for proteins in the 40-80 kDa range to account for the molecular weight discrepancy.

• For immunoprecipitation experiments, use 0.5-1 mg of cell lysate per IP reaction for optimal results .

• Always include appropriate positive controls (such as K562 cells for western blot) and negative controls (such as control IgG for immunoprecipitation) to validate experimental results .

How can researchers validate the specificity of anti-DHPS/DHS antibodies?

Validating antibody specificity is critical for reliable research outcomes. For anti-DHPS/DHS antibodies, researchers should consider:

• Positive and negative control samples: Use cell lines known to express DHPS (such as K562) as positive controls and samples with DHPS knockdown or knockout as negative controls .

• Blocking peptide experiments: Competition assays with the immunizing peptide can confirm specificity. Some manufacturers offer blocking peptides corresponding to the immunogen used to generate the antibody .

• Multiple antibody validation: Use multiple antibodies targeting different epitopes of DHPS to confirm consistent detection patterns.

• Mass spectrometry validation: Following immunoprecipitation, mass spectrometry analysis can confirm the identity of the detected protein.

• siRNA knockdown: Reduced signal following DHPS knockdown provides strong evidence of antibody specificity.

• Cross-reactivity testing: When working with samples from multiple species, confirm specificity across species boundaries, especially if the antibody is being used in non-validated species.

How can DHS-mutated antibodies be integrated into therapeutic development pipelines?

Integrating DHS-mutated antibodies into therapeutic development requires a systematic approach:

• Candidate selection: Begin with a high-affinity antibody against your therapeutic target and incorporate DHS mutations into the Fc region .

• Functional assessment: Verify that DHS mutations preserve critical effector functions relevant to the therapeutic mechanism, as DHS-IgG has been shown to retain intact effector functions important for target cell clearance .

• Comparative pharmacokinetic (PK) studies: Conduct head-to-head comparisons with wild-type and other half-life extension variants (YTE, LS) in relevant animal models. The expected improvement includes β-phase half-life extension (2.0- to 3.1-fold higher than YTE and LS variants) and increased drug exposure (AUC) .

• Developability assessment: Evaluate biophysical properties including thermal stability, aggregation propensity, and expression yields. DHS-IgG has demonstrated favorable developability properties for clinical development .

• Species-specific optimization: Consider that DHS mutation performance may vary across species due to differences in FcRn binding. Validation in humanized mouse models recapitulating human FcRn interaction is crucial .

• Combination with affinity-enhancing strategies: For maximal therapeutic efficacy, consider combining DHS mutations with affinity-enhancing mutations in the variable regions, as demonstrated in recent studies integrating machine learning approaches for antibody affinity enhancement .

What are the challenges in studying DHPS/DHS in relation to protein translation and cell proliferation?

Studying DHPS/DHS presents several methodological challenges:

• Redundancy and compensation: The hypusination pathway may have redundant mechanisms or compensatory responses when DHPS is inhibited, complicating interpretation of knockdown/knockout studies.

• Temporal dynamics: The rapid turnover of eIF-5A and dynamic nature of hypusination require careful experimental timing and synchronization of cell populations.

• Specificity of inhibitors: Small molecule inhibitors of DHPS may have off-target effects, necessitating careful validation through complementary genetic approaches.

• Tissue-specific expression: DHPS expression and importance may vary across tissues and cell types, requiring context-specific experimental designs.

• Technical limitations in detecting hypusinated proteins: The unusual nature of hypusine modifications presents analytical challenges for standard proteomic techniques, often requiring specialized methods for detection and quantification.

• Distinguishing primary from secondary effects: Given the fundamental role of eIF-5A in translation, distinguishing direct effects of DHPS inhibition from downstream consequences requires sophisticated experimental designs with appropriate controls.

How can researchers address discrepancies in experimental results when using anti-DHPS/DHS antibodies?

When facing discrepancies in experimental results with anti-DHPS/DHS antibodies, researchers should consider:

• Antibody batch variation: Different lots of the same antibody may show variation in specificity and sensitivity. Maintain consistent lot numbers for critical experiments or validate each new lot against previous ones.

• Cross-reactivity assessment: Determine if the antibody cross-reacts with related proteins or isoforms that might be differentially expressed in your experimental system.

• Sample preparation effects: Variations in lysis buffers, protein extraction methods, and handling can affect epitope accessibility and detection efficiency.

• Experimental conditions optimization: Systematically optimize antibody concentration, incubation time, temperature, and blocking conditions for each application and cell/tissue type.

• Alternative detection methods: Complement antibody-based detection with independent methods such as mass spectrometry or mRNA quantification.

• Biological variability: Consider that differences in cell cycle stage, confluency, passage number, or physiological state can affect DHPS expression levels and post-translational modifications.

• Data normalization approach: Evaluate whether different normalization strategies (e.g., different housekeeping proteins) could explain discrepancies in quantitative analyses.

What emerging technologies might enhance DHS antibody development and application?

The field of DHS antibody research is poised for advancement through several emerging technologies:

• Machine learning integration: Computational approaches integrating machine learning models can predict affinity-enhancing mutations to optimize DHS-containing antibodies against emerging targets, as demonstrated in recent studies enhancing antibodies against SARS-CoV-2 Omicron variants .

• Single-cell analysis: Advanced single-cell technologies could reveal heterogeneity in antibody uptake, recycling, and tissue distribution at unprecedented resolution.

• Cryo-EM and structural biology: High-resolution structural studies of DHS-modified Fc domains in complex with FcRn at different pH values could provide deeper mechanistic insights and guide further engineering efforts.

• In silico modeling: Improved computational models of antibody pharmacokinetics incorporating detailed molecular interactions could better predict in vivo behavior of novel DHS variants.

• Combination with other antibody engineering approaches: Integration of DHS mutations with bispecific formats, antibody-drug conjugates, or novel effector function engineering could create next-generation therapeutic modalities.

• Tissue-specific targeting: Combining DHS mutations with modifications that enhance tissue-specific distribution could improve therapeutic index for various disease applications.

What are the potential implications of DHPS/DHS research for cancer and infectious disease therapeutics?

DHPS/DHS research has significant implications for therapeutic development:

• Cancer therapeutics: Given the role of hypusinated eIF-5A in cancer cell proliferation, inhibiting DHPS represents a potential therapeutic strategy. Anti-DHPS antibodies are valuable tools for validating this target and understanding mechanism of action for DHPS inhibitors.

• Infectious disease applications: Hypusination is essential for various pathogens, including certain viruses and parasites. DHPS inhibition could represent a host-directed therapeutic approach with potentially broad-spectrum activity.

• Biomarker development: DHPS expression or activity levels might serve as biomarkers for certain disease states or treatment response prediction.

• Combinatorial therapy approaches: Understanding how DHPS inhibition interacts with established cancer therapies or antimicrobials could lead to synergistic therapeutic combinations.

• Selective delivery strategies: Knowledge of DHPS biology could inform the design of cell-type specific delivery systems for improved therapeutic targeting.