Proteinase inhibitor IIA Antibody

What is Proteinase Inhibitor IIA and how does it function in biological systems?

Proteinase Inhibitor IIA (PI-IIA) is a compact, globular protein that functions as a serine protease inhibitor. It forms a stable conformation in solution, characterized by conformation-dependent chemical shifts for aliphatic amino acid side-chains, numerous slowly exchanging amide protons, and unusual pH titrations of aromatic residues . The protein exhibits remarkable stability across a wide pH range (4-12) at 25°C and temperature range (5-50°C) at pH 4.9 .

PI-IIA functions by binding to target proteases through a specific recognition site, forming a tight complex that prevents the protease from carrying out its catalytic function. This inhibition is critical for regulating proteolytic activity in various biological processes, including inflammation, coagulation, and tissue remodeling. The inhibitor typically contains a reactive site that interacts with the active site of the target protease, forming either a reversible or irreversible complex depending on the specific inhibitor-protease pair.

How can researchers distinguish Proteinase Inhibitor IIA from other similar inhibitors?

Distinguishing Proteinase Inhibitor IIA from other similar inhibitors requires a multi-faceted approach:

• Structural characteristics: PI-IIA has a distinct conformation with three disulfide bridges and a single cis peptide bond . Nuclear magnetic resonance (NMR) studies have revealed its unique structural features.

• Specificity profile: Testing against a panel of proteases helps establish the inhibitory profile. For example, human PI9 specifically inhibits granzyme B but not other proteases .

• Molecular weight determination: Using techniques like SDS-PAGE or mass spectrometry can identify the characteristic molecular weight of PI-IIA.

• Immunological techniques: Using specific antibodies that recognize unique epitopes of PI-IIA can differentiate it from other inhibitors. Cross-reactivity tests, like those performed for PI9 (which does not cross-react with PI6, PI8, or PAI-2), are essential .

• Kinetic parameters: Determining inhibition constants (Ki) and mechanisms of inhibition provides distinctive characteristics for each inhibitor.

What techniques are most effective for determining the solution conformation of Proteinase Inhibitor IIA?

The solution conformation of Proteinase Inhibitor IIA can be most effectively determined through a combination of advanced biophysical techniques:

• Two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY): This NMR technique has been successfully used to obtain distance constraints between hydrogen atoms of the polypeptide chain, identify disulfide bridge positions, and locate cis peptide bonds in PI-IIA. In a key study, researchers obtained 202 distance constraints using this method .

• Vicinal spin-spin coupling analysis: This provides information about dihedral angles in the protein backbone, which helps determine the secondary structure elements.

• Hydrogen bond identification: NMR can detect slowly exchanging amide protons, indicating the presence of hydrogen bonds that stabilize the protein structure .

• Distance geometry calculations: Software programs like DISGEO can compute all-atom structures from NMR-derived distance constraints. For PI-IIA, researchers computed five conformers from NOE distance constraints alone and another five with supplementary constraints .

• Energy refinement: This computational approach validates that the constraints derived from NMR data are compatible with a low-energy spatial structure .

• Temperature-dependent studies: Investigating the protein's behavior at different temperatures provides insights into its stability and dynamics. For PI-IIA, equilibrium between different conformations has been observed at temperatures above 50°C .

The integration of these methods provides a comprehensive view of the inhibitor's three-dimensional structure in solution, critical for understanding its function and interactions.

How should researchers optimize protease inhibitor usage in experimental protocols?

Optimizing protease inhibitor usage requires careful consideration of multiple factors:

• Selection of appropriate inhibitors:

  • Determine the target molecule(s) requiring inhibition

  • Choose between broad-spectrum or specific inhibitors based on experimental needs

  • Consider the IC50 values of inhibitors for target and off-target proteases

  • Evaluate cell permeability if working with intact cells

• Timing of inhibitor addition:

  • Adding inhibitor at time zero with substrate and enzyme

  • Adding to an ongoing enzyme-substrate reaction

  • Preincubating enzyme with inhibitor before substrate addition

  Each approach will yield different results and must be interpreted accordingly. For valid comparisons, data should be collected under identical conditions .

• Concentration optimization:

  • Determine the appropriate inhibitor concentration based on IC50 values

  • Consider potential toxicity at higher concentrations

  • Account for the compatibility of reconstitution solvent with the assay

• Sample preparation considerations:

  • When working with tissue homogenates or cell lysates, add appropriate protease inhibitor cocktails to prevent nonspecific proteolysis

  • For mammalian samples, Protease Inhibitor Cocktail III is recommended

• Reaction termination methods:

  • Select appropriate methods based on downstream analysis requirements

  • Consider neutralization steps if acids are used to stop reactions

  • For radioisotope-labeled substrates, adding excess "cold" substrate can slow incorporation into the end product

• Data normalization:

  • Set aside sample aliquots for protein analysis

  • Calculate reaction rates as substrate converted or product formed per mg protein per unit time

Following these optimization strategies ensures reliable and reproducible results when using protease inhibitors in research protocols.

What are the critical parameters for evaluating antibody-based proteinase inhibitors?

Evaluating antibody-based proteinase inhibitors requires assessment of multiple critical parameters:

• Inhibitory potency:

  • Determine inhibition constants (Ki) through enzyme kinetics studies

  • Measure IC50 values under standardized conditions

  • For highly potent inhibitors, like the scFv antibody inhibitors of serine protease MT-SP1/matriptase, Ki values in the low picomolar range can be achieved

• Specificity profiling:

  • Test against a panel of related proteases to establish selectivity

  • Perform alanine scanning of loops surrounding the protease active site to understand the basis of specificity

  • Identify unique three-dimensional binding epitopes that contribute to selectivity

• Mechanism of inhibition:

  • Determine whether the inhibitor is competitive, non-competitive, or uncompetitive

  • Assess if it's a standard mechanism inhibitor (binding in a substrate-like manner)

  • Investigate if processing of the inhibitor occurs at specific conditions (e.g., low pH)

• Binding kinetics:

  • Measure association (kon) and dissociation (koff) rates

  • Determine binding affinity (KD)

  • Analyze the stability of the inhibitor-protease complex

• Structure-function relationships:

  • Map the binding epitope through mutagenesis studies

  • Identify critical residues for interaction using alanine scanning

  • Characterize the three-dimensional structure of the inhibitor-protease complex

For example, studies of scFv antibody inhibitors of MT-SP1/matriptase revealed that they gained specificity by making numerous critical interactions with surface loops on the protease while functioning as standard mechanism inhibitors by inserting a reactive loop into the active site .

A comprehensive evaluation of these parameters provides insights into the inhibitor's mechanism of action and potential utility in research and therapeutic applications.

How can researchers engineer protease-resistant antibodies with selective cell-killing functions?

Engineering protease-resistant antibodies with selective cell-killing functions involves a multi-step approach:

This approach has successfully yielded antibodies that resist proteolytic cleavage while maintaining or even enhancing their cell-killing functions, making them potentially valuable for targeting tumors or infectious microenvironments with high protease content.

What approaches can be used to design antibodies targeting specific epitopes in proteinase inhibitors?

Designing antibodies that target specific epitopes in proteinase inhibitors requires sophisticated approaches:

• Rational design method for disordered epitopes:

  • Sequence-based design of complementary peptides targeting a selected disordered epitope

  • Grafting of these peptides onto an antibody scaffold

  • This method has been successfully applied to design single-domain antibodies against disease-related intrinsically disordered proteins

• Alanine scanning of binding interfaces:

  • Systematically replace amino acids at the binding interface with alanine

  • Identify critical residues that contribute significantly to binding

  • Use this information to design antibodies that make optimal contacts with these residues

• Structure-guided epitope selection:

  • Analyze the three-dimensional structure of the target proteinase inhibitor

  • Identify surface-exposed regions that are unique to the specific inhibitor

  • Target these regions to achieve high specificity

  • For proteinase inhibitor IIA, NMR studies have provided detailed structural information that can guide epitope selection

• Computational modeling and docking:

  • Use computational approaches to predict antibody-antigen interactions

  • Optimize the binding interface through in silico mutagenesis

  • Validate predictions through experimental binding studies

• Phage display technology:

  • Generate diverse antibody libraries

  • Select for binders to specific epitopes of the proteinase inhibitor

  • Perform affinity maturation to improve binding characteristics

• Hybridoma technology with epitope-specific screening:

  • Immunize animals with the target proteinase inhibitor

  • Screen antibodies for binding to specific epitopes using competitive binding assays

  • Select clones that target the desired epitope

These approaches enable the development of highly specific antibodies that can distinguish between closely related proteinase inhibitors and target particular functional domains, providing valuable tools for research and potential therapeutic applications.

How do environmental factors affect the stability and activity of Proteinase Inhibitor IIA?

Environmental factors significantly influence the stability and activity of Proteinase Inhibitor IIA:

• pH effects:

  • PI-IIA forms a stable, compact globular conformation between pH 4 and 12 at 25°C

  • Unusual pH titrations of two aromatic residues have been observed, indicating pH-dependent conformational changes

  • Researchers should consider these pH ranges when designing experiments to ensure optimal inhibitor function

• Temperature dependence:

  • The protein maintains stability between 5 and 50°C at pH 4.9

  • At temperatures above 50°C, evidence shows an equilibrium between several different conformations

  • The rate of exchange between these conformations is in the intermediate range on the NMR time scale

  • For Tyr32, a temperature-dependent transition from low-frequency to high-frequency flipping motions has been observed

• Aromatic ring dynamics:

  • Among the four aromatic rings in PI-IIA, Phe10, Phe38, and Tyr16 undergo rapid 180° flips over the entire temperature range tested

  • This dynamic behavior may play a role in the inhibitor's function and interactions

• Amide proton exchange rates:

  • Studies have presented preliminary data on the individual exchange rates of 18 backbone amide protons

  • These rates provide insights into the protein's dynamics and stability under different conditions

• Salt and solvent effects:

  • For mass spectrometry analysis, salt concentration significantly impacts signal detection

  • A protein sample in PBS requires 91-fold dilution to achieve 50% of the potential MS signal due to the 137 mM NaCl content

  • Researchers should consider sample cleanup methods like ultrafiltration using spin cartridges with MWCO-membrane when working with high salt concentrations

Understanding these environmental influences is crucial for designing experiments, interpreting results, and developing storage and handling protocols for Proteinase Inhibitor IIA.

What are the advantages and limitations of antibody-guided proteolytic enzymes compared to conventional proteinase inhibitors?

Antibody-guided proteolytic enzymes represent an innovative approach with distinct advantages and limitations compared to conventional proteinase inhibitors:

Advantages:

• Selective sub-stoichiometric turnover: Antibody-guided proteases enable selective targeting and catalytic turnover of therapeutic targets, requiring lower drug concentrations than conventional inhibitors .

• Enhanced target engagement: Increased target engagement through antibody-antigen recognition enhances the catalytic activity and specificity of genetically fused proteases .

• Applicability to challenging targets: This approach has shown promise for difficult-to-target proteins like amyloid-β (Aβ) and immunoglobulin G (IgG) .

• Potential for recycling: Properly designed antibody-protease fusions can facilitate rapid recycling of target antigen for cleavage by the fused protease .

• Tunability through antibody engineering: Altering antibody binding kinetics and affinity can optimize the performance of the fusion protein. For example, researchers have incorporated mutations like G33S(HC) and G33S(HC)/S56F(LC) to create slower off-rates and stronger affinities .

Limitations:

• Complex design requirements: Creating effective antibody-protease fusions requires sophisticated protein engineering and careful selection of both the antibody component and the proteolytic enzyme.

• Potential immunogenicity: The fusion proteins might elicit immune responses, particularly if the protease component is derived from non-human sources.

• Manufacturing challenges: Production of consistent antibody-protease fusion proteins may face technical hurdles in expression, folding, and purification.

• Regulatory considerations: As a novel therapeutic modality, regulatory pathways may be less well-defined compared to conventional antibodies or small molecule inhibitors.

• Target-specific optimization: Each target likely requires specific optimization of the antibody-protease construct, limiting generalizability of the platform.

This innovative approach may be particularly valuable for targets that are present at high abundance or within physiologic sites of low drug exposure, potentially addressing unmet medical needs that conventional inhibitors cannot adequately address .

How can researchers assess the in vivo efficacy of Proteinase Inhibitor IIA antibodies?

Assessing the in vivo efficacy of Proteinase Inhibitor IIA antibodies requires a comprehensive evaluation strategy:

• Animal model selection:

  • Choose disease models where the target protease is implicated

  • For alpha1-proteinase inhibitor (related to PI-IIA), studies have assessed efficacy in lung damage models, as demonstrated with Respreeza

  • Consider humanized mouse models if the inhibitor is specific to human proteases

• Dosing optimization:

  • Determine appropriate dosing regimens based on pharmacokinetic studies

  • For reference, alpha1-proteinase inhibitor therapy (Respreeza) uses 60 mg per kilogram body weight given once weekly

  • Consider route of administration (intravenous, subcutaneous, etc.) based on the therapeutic target

• Efficacy endpoints:

  • Define clear, measurable endpoints related to the disease process

  • For lung applications, changes in lung density measured by imaging can be an effective indicator, as demonstrated in studies where Respreeza showed a decrease of around 2.6 g/l compared to 4.2 g/l in placebo groups

  • Include functional endpoints relevant to the specific disease mechanism

• Safety monitoring:

  • Assess potential side effects, including allergic reactions

  • Monitor for development of antibodies against the therapeutic

  • Be particularly vigilant in subjects lacking IgA who may develop antibodies against it, increasing the risk of allergic reactions

• Pharmacokinetic/pharmacodynamic (PK/PD) correlation:

  • Measure antibody levels in plasma and relevant tissues

  • Correlate these levels with observed biological effects

  • Determine the relationship between exposure and efficacy

• Biomarker development:

  • Identify biomarkers that reflect target engagement

  • For protease inhibitors, measure levels of protease-specific cleavage products in accessible biological fluids

  • Develop assays to detect cleaved IgGs in target tissues, similar to methods used to detect cleaved IgGs in tumor microenvironments

• Comparative studies:

  • Compare efficacy to existing standards of care

  • Include appropriate control groups (placebo, active comparator)

  • Consider dose-ranging studies to establish optimal therapeutic dosing

This multifaceted approach provides a robust assessment of in vivo efficacy, essential for advancing Proteinase Inhibitor IIA antibodies toward clinical applications.

What considerations are important when designing clinical trials for proteinase inhibitor antibody therapeutics?

Designing clinical trials for proteinase inhibitor antibody therapeutics involves several important considerations:

• Patient selection criteria:

  • Identify patients with confirmed deficiency or dysfunction of the relevant proteinase inhibitor

  • For alpha1-proteinase inhibitor deficiency therapies like Respreeza, patients with severe disease are selected

  • Consider genetic testing to identify specific mutations or variants that may affect treatment response

• Endpoint selection:

  • Choose clinically meaningful primary endpoints

  • Include surrogate markers that can be measured objectively

  • For lung-related applications, endpoints might include:

    • Changes in lung density measured by imaging

    • Pulmonary function tests

    • Frequency of exacerbations

    • Quality of life measures

• Trial duration and design:

  • Plan for sufficient follow-up to observe clinically significant changes

  • Consider adaptive trial designs for dose optimization

  • Include crossover designs when appropriate to maximize data from limited patient populations

• Safety monitoring:

  • Implement robust monitoring for allergic reactions, which are a primary safety concern for protein therapeutics

  • Develop risk mitigation strategies, such as excluding patients at higher risk for severe reactions (e.g., those lacking IgA who have developed antibodies against it)

  • Monitor for development of neutralizing antibodies against the therapeutic

• Dosing considerations:

  • Establish optimal dosing regimens based on preclinical and early clinical data

  • Consider the need for loading doses versus maintenance therapy

  • Evaluate different administration routes and frequencies

  • For reference, Respreeza uses weekly infusions of 60 mg/kg, with first infusions supervised by healthcare professionals experienced in treating the relevant deficiency

• Biomarker integration:

  • Include pharmacodynamic biomarkers to confirm target engagement

  • Collect samples for exploratory biomarker analysis to identify potential predictors of response

  • Consider patient stratification based on biomarker profiles

• Post-approval studies:

  • Plan for long-term safety and efficacy monitoring

  • Consider studies of extended dosing regimens or alternative doses

  • For instance, plans for studying whether an increased dose of 120 mg/kg may provide improved effects compared to the standard 60 mg/kg dose for alpha1-proteinase inhibitor therapy

These considerations help ensure that clinical trials for proteinase inhibitor antibody therapeutics are scientifically rigorous, ethically sound, and optimized to demonstrate clinical benefit in the intended patient population.

How can researchers overcome the challenges in determining the binding kinetics of antibodies to proteinase inhibitors?

Determining binding kinetics of antibodies to proteinase inhibitors presents several challenges, which can be overcome with specialized approaches:

• Surface Plasmon Resonance (SPR) optimization:

  • Challenge: Immobilization may alter the conformation of the proteinase inhibitor

  • Solution: Compare multiple immobilization strategies (amine coupling, streptavidin-biotin, etc.)

  • Validate that the immobilized protein retains its functional activity

  • Example application: SPR has been used successfully to measure binding responses of antibodies to immobilized proteins like PF4, showing differences between patient groups with mean binding responses ranging from 0.29 ± 0.18 nm to 1.18 ± 0.73 nm

• Addressing sample heterogeneity:

  • Challenge: Polyclonal antibodies exhibit complex binding patterns

  • Solution: Purify specific antibody fractions or use monoclonal antibodies

  • When sample constraints prevent further purification (a common limitation), test total IgG binding and compare with serum samples to confirm antibody-specific binding

• Assessing concentration-independent parameters:

  • Challenge: Variable antibody concentrations can complicate kinetic analysis

  • Solution: Focus on dissociation rates, which are concentration-independent

  • This approach has been successfully applied to compare antibody-antigen interactions across different samples

• Ensuring reproducibility:

  • Challenge: Binding kinetics measurements can show variation between experiments

  • Solution: Include control samples in separate experiments to demonstrate reproducibility

  • Example: Studies have shown reproducibility when retesting samples (two VITT, one HIT, and two healthy control samples) in separate experiments

• Distinguishing specific from non-specific binding:

  • Challenge: Non-specific binding can mask true kinetic parameters

  • Solution: Include proper reference surfaces and blocking agents

  • Use negative controls (non-binding antibodies) to establish baseline responses

• Managing avidity effects:

  • Challenge: Bivalent antibodies show apparent higher affinity due to avidity

  • Solution: Use Fab fragments for true monovalent affinity measurements

  • Compare with intact antibodies to quantify the avidity contribution

By implementing these specialized approaches, researchers can obtain accurate binding kinetics data for antibodies to proteinase inhibitors, providing crucial information for both basic research and therapeutic development.

What strategies can be employed to improve the specificity of antibodies targeting Proteinase Inhibitor IIA?

Improving the specificity of antibodies targeting Proteinase Inhibitor IIA requires strategic approaches:

These strategies can yield highly specific antibodies against Proteinase Inhibitor IIA, providing valuable tools for research and potential therapeutic applications.

What are the best practices for long-term storage and handling of Proteinase Inhibitor IIA antibodies to maintain activity?

Maintaining the activity of Proteinase Inhibitor IIA antibodies during long-term storage and handling requires attention to several critical factors:

• Formulation considerations:

  • Liquid formulations typically include stabilizers like BSA (e.g., 0.7% BSA) and preservatives like sodium azide (e.g., 0.1%)

  • For lyophilized antibodies, reconstitute in appropriate buffers according to manufacturer recommendations

  • Consider the compatibility of reconstitution solvents with intended applications

• Storage temperature:

  • Most antibodies should be stored at -20°C (freezer) or -80°C (ultra-low freezer) for long-term stability

  • For short-term storage (weeks to months), 4°C is generally suitable

  • Avoid repeated freeze-thaw cycles by preparing small working aliquots

• Protection from environmental factors:

  • Protect from light if light sensitivity is indicated on the label

  • Minimize exposure to moisture, which may damage the molecule

  • Avoid contamination by using sterile technique when handling

• Stability testing protocol:

  • Periodically verify antibody activity using consistent assay conditions

  • For PI-IIA antibodies, test binding to the target via ELISA or functional inhibition assays

  • Monitor for changes in specificity by testing against related proteins

• Proper thawing procedures:

  • Thaw frozen antibodies slowly at 4°C or room temperature

  • Avoid rapid temperature changes that can cause protein denaturation

  • Mix gently by inversion rather than vortexing to prevent aggregation

• Centrifugation before use:

  • Briefly centrifuge antibody vials before opening to collect liquid at the bottom

  • This helps prevent sample loss and potential contamination

• Carrier protein consideration:

  • For dilute antibody solutions, consider adding carrier proteins (e.g., BSA, gelatin) to prevent adsorption to container surfaces

  • Typical concentrations range from 0.1-1% BSA

• Documentation practices:

  • Maintain detailed records of antibody source, lot number, aliquoting dates, and freeze-thaw cycles

  • Document any observed changes in activity or appearance

  • Record successful experimental conditions for reproducibility

By following these best practices, researchers can maintain the activity and specificity of Proteinase Inhibitor IIA antibodies throughout long-term storage and handling, ensuring reliable and reproducible experimental results.

How might advanced computational approaches enhance the design of next-generation proteinase inhibitor antibodies?

Advanced computational approaches offer transformative potential for designing next-generation proteinase inhibitor antibodies:

• Distance geometry algorithms:

  • New distance geometry programs like DISGEO have enabled computing all-atom structures for proteins the size of BUSI IIA

  • These algorithms can process hundreds of distance constraints from NMR data to generate accurate protein conformers

  • Future improvements may integrate machine learning to predict constraints not directly measured experimentally

• Energy refinement methods:

  • Preliminary energy refinement has shown that constraints derived from NMR data are compatible with low-energy spatial structures

  • Advanced energy functions that better account for solvent effects and entropy could improve structure prediction accuracy

  • Molecular dynamics simulations with specialized force fields can model the flexibility of antibody-inhibitor complexes

• De novo design platforms:

  • Computational approaches that generate small (<75 amino acids) hyperstable de novo binding proteins with high specificity

  • These platforms can engineer precisely controlled receptor binding interfaces optimal for treating disease

  • Similar approaches could be adapted for designing antibodies against proteinase inhibitors

• Epitope mapping and targeting:

  • Computational methods can predict antigenic determinants on proteinase inhibitors

  • Algorithms can identify highly restricted binding sites, similar to approaches used to map antibody epitopes in vaccine-induced immune thrombotic thrombocytopenia

  • Machine learning models trained on existing antibody-antigen complexes can predict optimal binding configurations

• Affinity and specificity optimization:

  • In silico affinity maturation through computational mutagenesis and energy calculations

  • Virtual screening of antibody variants against panels of related proteases to identify mutations that enhance specificity

  • Integration with experimental data from high-throughput methods to train more accurate prediction algorithms

• Structure-based antibody engineering:

  • Leveraging detailed structural information about proteinase inhibitors like PI-IIA to design complementary binding surfaces

  • Modeling antibody-antigen complexes to optimize interactions at atomic resolution

  • Predicting the effects of mutations on binding kinetics and thermodynamics

These computational approaches promise to accelerate the development of proteinase inhibitor antibodies with enhanced properties, potentially reducing the time and resources required for experimental screening while improving outcomes in terms of affinity, specificity, and stability.

What role might proteinase inhibitor antibodies play in emerging therapeutic areas beyond traditional applications?

Proteinase inhibitor antibodies are poised to play transformative roles in several emerging therapeutic areas:

• Neurodegenerative disease therapies:

  • Antibodies targeting specific epitopes within disordered proteins associated with neurodegeneration

  • Demonstrated potential for inhibiting aggregation of α-synuclein at substoichiometric concentrations, with binding occurring at selected epitopes

  • Similar approaches could target other disease-related proteins like Aβ42 and IAPP

• Targeted protein degradation strategies:

  • Antibody-guided proteolytic enzymes enabling selective sub-stoichiometric turnover of therapeutic targets

  • Enhanced enzyme activity and specificity through antibody-mediated substrate targeting

  • Proof of concept exists for challenging targets like amyloid-β and immunoglobulin G

  • Potential applications in diseases characterized by protein accumulation or dysregulation

• Microbiome-related disorders:

  • Targeting proteases produced by pathological microorganisms that cleave human IgG1 as an immune evasion mechanism

  • Engineered protease-resistant antibodies with enhanced cell-killing functions could provide novel approaches to treating microbiome dysbiosis

• Tumor microenvironment modulation:

  • Addressing proteases secreted by invasive tumors that cleave human IgG1 in the lower hinge

  • Detection of cleaved IgGs within tumor microenvironments highlights the need for protease-resistant platforms

  • Potential for combining protease inhibition with immune checkpoint blockade for synergistic anti-tumor effects

• Cell-selective targeting in inflammatory diseases:

  • Engineered antibodies with selective cell-killing functions tailored to specific inflammatory cell populations

  • Protease-resistant variants that maintain function in inflamed tissues with high protease content

• Biomarker development and theranostics:

  • Using proteinase inhibitor antibodies as imaging agents to detect elevated protease activity in disease states

  • Dual-function antibodies that both detect and inhibit pathological protease activity

  • Integration with emerging liquid biopsy technologies for minimally invasive disease monitoring