ECA2 Antibody

What is the mechanism of action for ACE2-targeting antibodies against coronaviruses?

ACE2-targeting antibodies work by binding to the human angiotensin-converting enzyme 2 (ACE2) receptor, which serves as the entry point for multiple coronaviruses. Unlike traditional approaches that target viral proteins, these antibodies block the interaction between the virus's spike protein and the host cell receptor. Research demonstrates that antibodies like 3E8 can effectively block S1-subunits and pseudo-typed virus constructs from multiple coronaviruses including SARS-CoV-2, several SARS-CoV-2 variants (D614G, B.1.1.7, B.1.351, B.1.617.1, and P.1), SARS-CoV, and HCoV-NL63 .

Importantly, these antibodies target the receptor-binding domain (RBD)-binding site on ACE2, which does not overlap with ACE2's catalytic site. This strategic targeting allows the antibodies to block viral entry while preserving ACE2's physiological activities . The non-interference with normal ACE2 function represents a significant advantage over approaches that might disrupt this enzyme's important role in blood pressure regulation and other physiological processes.

How are ACE2 antibodies generated for research purposes?

The generation of ACE2 antibodies typically follows established hybridoma technology protocols with specific modifications for targeting this receptor. In one documented approach, BALB/c mice were immunized with Fc-tagged human ACE2 protein, and their sera were screened for binding to ACE2 and blocking of SARS-CoV-2 S1-subunit and ACE2 interaction . Following successful immunization, hybridoma cells were constructed and their supernatants screened for neutralizing antibodies.

After identification of promising candidates like antibody 3E8, the variable regions of the heavy (VH) and light (VL) chains are cloned into a human IgG backbone (IgG4 in the case of 3E8), expressed in cells such as HEK293F, and purified . Alternative approaches include using AlivaMab mice, which produce chimeric antibodies consisting of human Fab domains and a murine Fc domain, to maximize diversity of the anti-hACE2 mAbs . The quality of purified antibodies is typically verified through reduced and non-reduced gel analysis, and binding affinity is assessed using methods like ELISA and biolayer interferometry (BLI) .

What experimental models are used to evaluate the efficacy of ACE2 antibodies?

Researchers employ a multi-tiered approach to evaluate ACE2 antibody efficacy, progressing from in vitro binding studies to cellular and animal models:

• Binding assays: ELISA and biolayer interferometry (BLI) are used to measure binding affinity of antibodies to ACE2 proteins. For example, antibody 3E8 demonstrated an EC50 value of 15.3 nM in ELISA and an apparent dissociation constant (KD) of 30.5 nM in BLI using dimeric ACE2 (residues 19-740) as the soluble analyte .

• Cell-based studies: Flow cytometry assays confirm antibody binding to cells expressing ACE2, including both cells engineered to overexpress human ACE2 (HEK293F) and those endogenously expressing the receptor (Vero E6) . Pseudovirus neutralization assays are conducted to assess blocking of viral entry.

• Animal models: ACE2 "knock-in" mice are frequently utilized to evaluate both efficacy and safety. These models help determine if the antibodies can block live SARS-CoV-2 infection in vivo in a prophylactic context while monitoring for potential toxicity related to interference with normal ACE2 function . Additionally, testing in macaque models is facilitated by antibodies that demonstrate cross-reactivity with macaque ACE2 .

• Live virus neutralization: The gold standard for evaluating protective efficacy involves testing against live SARS-CoV-2 infection both in vitro and in animal models under appropriate biocontainment conditions .

How can computational approaches optimize ACE2 antibodies against emerging viral variants?

Computational optimization represents a cutting-edge approach to enhance ACE2 antibody performance against multiple viral variants simultaneously. This "zero-shot" design strategy uses high-performance computing, simulation, and machine learning to improve binding affinity to multiple antigen targets without requiring iterative laboratory testing. One notable approach, the generative unconstrained intelligent drug engineering (GUIDE) platform, co-optimizes binding affinity to multiple targets (such as RBDs from several SARS-CoV-2 strains) while maintaining critical attributes like thermostability .

The process involves several sophisticated steps:

• Problem formulation by identifying a parental antibody, target antigens, and corresponding co-structures

• Sequence generation using predictions of multiple properties to propose multi-point mutant antibody candidates

• Selection of proposed sequences by a Bayesian optimization agent for simulation

• Experimental evaluation of computationally promising candidates using immunoassays

• Final validation through neutralization testing against multiple SARS-CoV-2 variants

This approach has demonstrated success in restoring potency to clinical antibodies. For example, researchers were able to repair the activity of COV2-2130 against Omicron variants by introducing just four amino acid substitutions, a process completed in only three weeks . This computational optimization strategy allows for rapid response to emerging variants while minimizing the experimental work required.

What are the key considerations in designing experiments to assess potential interference with ACE2's physiological functions?

When developing ACE2-targeting antibodies, carefully assessing potential interference with normal ACE2 physiological functions is crucial. This requires a multi-faceted experimental approach:

• Enzymatic activity assays: Researchers must measure ACE2 enzymatic activity in the presence and absence of the antibody. This ensures the antibody does not inhibit ACE2's catalytic function in converting angiotensin II to angiotensin 1-7, which is critical for blood pressure regulation. Studies have confirmed that properly designed antibodies like those reported can block viral entry without markedly affecting ACE2's physiological activities .

• Cell surface expression monitoring: Flow cytometry and immunofluorescence techniques should be employed to confirm that antibody binding does not induce cell-surface depletion of ACE2. Evidence shows that effective antibodies can bind ACE2 without causing significant internalization or downregulation .

• Animal model safety studies: ACE2 "knock-in" mice expressing human ACE2 serve as important models for toxicity assessment. These studies should monitor blood pressure, kidney function, and other physiological parameters dependent on ACE2 activity. Current research demonstrates that carefully designed ACE2 antibodies do not cause severe toxicity in these models .

• Cardiovascular assessment: Since ACE2 has important functions in the cardiovascular system, researchers should include comprehensive cardiac evaluation, including blood pressure measurements, cardiac tissue analysis, and functional assessments following antibody administration.

• Tissue distribution studies: Experiments should track antibody biodistribution to ensure it reaches appropriate target tissues while monitoring any unintended effects on ACE2 function across different organ systems.

What structural and functional analyses can determine the precise epitope of ACE2 antibodies?

Determining the precise epitope of ACE2 antibodies requires sophisticated structural and functional analyses:

• Cryo-electron microscopy (Cryo-EM): This technique provides high-resolution visualization of the antibody-ACE2 complex, revealing key interaction points. Researchers have used cryo-EM to identify binding residues on ACE2 that interact with antibody components, such as the CDR3 domain of the 3E8 heavy chain .

• "Alanine walk" studies: This mutagenesis approach systematically replaces amino acids in the potential binding region with alanine to identify critical residues for antibody interaction. When combined with binding assays, this technique precisely maps the epitope and determines which residues are essential for antibody recognition .

• Competitive binding assays: These experiments determine if the antibody competes with viral RBD for binding to ACE2. This confirms whether the antibody targets the virus-binding site on ACE2 and provides functional validation of the epitope.

• X-ray crystallography: When applicable, crystal structures of the antibody-ACE2 complex provide atomic-level resolution of the binding interface, offering detailed insights into the structural basis of recognition.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique identifies regions of altered solvent accessibility upon antibody binding, providing complementary data about the epitope and potential conformational changes.

Through these approaches, researchers have identified a broad-spectrum anti-coronavirus epitope on ACE2 that can be targeted without affecting the receptor's normal functions .

How does the neutralization breadth of ACE2 antibodies compare with spike protein-targeting antibodies?

ACE2-targeting antibodies demonstrate distinct advantages in neutralization breadth compared to spike protein-targeting approaches:

• Variant resistance: ACE2 antibodies show remarkable efficacy against multiple SARS-CoV-2 variants. Studies demonstrate that human mAbs binding the hACE2 receptor can block infection by all hACE2-binding sarbecoviruses tested, including SARS-CoV-2 ancestral, Delta, and Omicron variants at concentrations of approximately 7–100 ng/ml . This broad coverage occurs because these antibodies target the host receptor rather than the rapidly mutating viral proteins.

• Cross-coronavirus efficacy: ACE2 antibodies effectively neutralize multiple coronaviruses that use ACE2 as an entry receptor. For example, antibody 3E8 blocked S1-subunits and pseudo-typed virus constructs from SARS-CoV-2 and its variants, SARS-CoV, and HCoV-NL63 . This broad-spectrum activity provides protection against both current and potentially future ACE2-utilizing coronaviruses.

• Genetic barrier to resistance: ACE2 antibodies present a high genetic barrier to the acquisition of resistance since they target a conserved host protein rather than viral components . Viruses would need to evolve alternative entry mechanisms to escape neutralization, which represents a significant evolutionary challenge.

• Complementary approach: While spike-targeting antibodies can be highly potent against specific viral strains, they often lose efficacy as new variants emerge. In contrast, the research indicates that ACE2 antibodies maintain their effectiveness despite viral evolution, suggesting they could provide durable protection or serve as complementary therapeutics alongside optimized spike-targeting antibodies .

What are the optimal experimental protocols for evaluating ACE2 antibody binding affinity and specificity?

Researchers should employ multiple complementary techniques to comprehensively characterize ACE2 antibody binding properties:

• Enzyme-Linked Immunosorbent Assay (ELISA):

  • Coat plates with recombinant hACE2 extracellular domain

  • Incubate with serial dilutions of test antibodies

  • Detect binding with appropriate secondary antibodies

  • Calculate EC50 values to quantify binding affinity (e.g., 15.3 nM for antibody 3E8)

  • Include controls such as irrelevant antibodies and known ACE2 binders

• Biolayer Interferometry (BLI):

  • Immobilize antibodies on biosensor tips

  • Expose to varying concentrations of soluble ACE2

  • Measure association and dissociation rates

  • Calculate apparent dissociation constants (KD, e.g., 30.5 nM for 3E8 with dimeric ACE2)

  • Analyze binding kinetics to understand interaction dynamics

• Flow Cytometry:

  • Test binding to cells expressing hACE2 (both transfected and endogenous)

  • Include proper controls (parental cells without ACE2 expression)

  • Evaluate cross-reactivity with ACE2 from other species (e.g., macaque) when relevant

  • Quantify binding strength and percentage of positive cells

• Surface Plasmon Resonance (SPR):

  • Precisely measure association constants between antibodies and hACE2

  • Determine on/off rates for binding kinetics

  • Compare affinity across different antibody candidates

  • Assess binding under various buffer conditions to determine stability

• Competitive Binding Assays:

  • Evaluate ability to block interaction between ACE2 and viral S1 subunits

  • Compare blocking efficiency with other antibodies or ACE2-Fc constructs

  • Determine IC50 values for competitive inhibition

What are the challenges in scaling up production of research-grade ACE2 antibodies?

Producing research-grade ACE2 antibodies at scale presents several challenges that researchers must address:

• Expression system selection: Different expression systems (hybridoma, CHO cells, HEK293 cells) yield antibodies with varying glycosylation patterns and post-translational modifications that may affect function. Researchers must carefully select and optimize the expression system based on intended applications.

• Construct design considerations: When cloning antibody variable regions into expression vectors, researchers must ensure proper signal sequence inclusion, codon optimization, and vector compatibility. For ACE2 antibodies derived from mice, the variable regions may need to be cloned into human IgG backbones (like IgG4) to reduce immunogenicity in human applications .

• Purification challenges: Maintaining antibody functionality requires gentle purification processes. Multi-step chromatography (typically Protein A followed by size exclusion) must be optimized to achieve high purity while preserving binding activity. Quality should be verified through reduced and non-reduced gel analysis .

• Stability optimization: ACE2 antibodies must maintain stability during storage and use. Buffer optimization (pH, ionic strength, excipients) is essential to prevent aggregation, denaturation, or loss of binding capacity over time.

• Functional validation: Each production batch requires comprehensive functional validation, including:

  • Binding affinity confirmation via ELISA, BLI, or SPR

  • Virus neutralization assays with pseudotyped or live virus

  • Consistency checks between batches to ensure reproducible results

  • Endotoxin testing for applications in animal models

• Specificity verification: Cross-reactivity testing should confirm that antibodies maintain their specificity for ACE2 without off-target binding to related proteins, which could complicate research interpretations or cause unexpected effects in vivo.

How can researchers establish appropriate controls when testing ACE2 antibodies in neutralization assays?

Robust experimental design for ACE2 antibody neutralization assays requires comprehensive controls:

• Positive control antibodies:

  • Include well-characterized ACE2 antibodies with known neutralization activity

  • Use broadly cross-reactive, non-ACE2-competitive control antibodies (e.g., S309) as reference standards

  • Include the parental antibody when testing optimized variants to directly assess improvements

• Negative controls:

  • Non-binding isotype-matched antibodies to control for non-specific effects

  • Irrelevant target antibodies (antibodies targeting proteins unrelated to ACE2)

  • Buffer-only conditions to establish baseline infection levels

• Comparative controls:

  • ACE2-Fc fusion proteins as alternative ACE2-blocking agents

  • RBD-targeting antibodies to compare host versus viral targeting approaches

  • Previously characterized antibodies like B38 (an RBD-targeting antibody) for comparison of potency and efficacy

• Cell controls:

  • ACE2-knockout cells to confirm specificity of neutralization mechanism

  • Cells expressing ACE2 from different species to assess cross-reactivity

  • ACE2-overexpressing cells to evaluate efficacy under varying receptor density conditions

• Virus controls:

  • Multiple coronavirus types to assess breadth (SARS-CoV-2, SARS-CoV, HCoV-NL63)

  • Various SARS-CoV-2 variants (ancestral, D614G, B.1.1.7, B.1.351, B.1.617.1, P.1) to evaluate variant resistance

  • Pseudotyped viruses expressing different spike proteins for initial screening

  • Live virus confirmation under appropriate containment for definitive validation

• Dose-response assessments:

  • Test multiple antibody concentrations to generate complete neutralization curves

  • Calculate IC50/IC90 values for quantitative comparisons between antibodies

  • Assess maximum neutralization plateaus to determine efficacy

What are the prospects for developing antibody cocktails combining ACE2 and spike protein targeting approaches?

Combining ACE2-targeting antibodies with spike protein-targeting antibodies represents a promising strategy with several potential advantages:

• Complementary mechanisms: ACE2 antibodies block the host receptor, while spike antibodies neutralize the virus directly. This dual-targeting approach could provide synergistic protection and higher barriers to resistance development. The different mechanisms would require the virus to simultaneously evolve both a new receptor usage pathway and mutations in the spike protein to escape neutralization .

• Coverage optimization: Spike-targeting antibodies often show potent neutralization against specific variants but may lose efficacy as new mutations emerge. ACE2 antibodies demonstrate broader coverage against multiple variants and coronaviruses. Combining both approaches could provide both high potency and exceptional breadth .

• Experimental design considerations: To develop effective cocktails, researchers should:

  • Screen combinations for synergistic, additive, or antagonistic effects

  • Optimize antibody ratios for maximum neutralization

  • Evaluate cocktails against diverse viral variants and in multiple model systems

  • Assess potential competition between antibodies for binding sites

• Manufacturing and regulatory challenges: While theoretically promising, cocktail approaches face practical challenges:

  • Increased complexity in manufacturing and quality control

  • Higher regulatory hurdles for combination products

  • Potential for unexpected antibody interactions affecting stability or function

  • Increased development costs compared to monotherapy approaches

How might ACE2 antibodies be utilized in studying pathogenesis mechanisms of emerging coronaviruses?

ACE2 antibodies offer unique tools for investigating coronavirus pathogenesis mechanisms:

• Receptor biology research: ACE2-specific antibodies can help elucidate the normal physiological roles of ACE2 and how virus binding affects these functions. By blocking specific epitopes, researchers can determine which ACE2 domains are critical for various cellular processes versus viral entry .

• Virus-receptor interaction studies: These antibodies allow detailed examination of how different coronaviruses engage ACE2, potentially revealing:

  • Common binding mechanisms across coronavirus families

  • Unique features of highly pathogenic versus mild coronavirus-ACE2 interactions

  • Structural requirements for efficient viral entry

• Tissue tropism investigation: By using ACE2 antibodies in combination with viral challenge, researchers can:

  • Map ACE2 expression patterns in different tissues

  • Correlate ACE2 binding capacity with tissue susceptibility to infection

  • Identify additional factors beyond ACE2 that determine cell permissiveness

• Downstream signaling research: ACE2 antibodies that bind different epitopes can help determine:

  • Whether virus binding triggers specific signaling cascades through ACE2

  • How ACE2 internalization or shedding following virus binding affects cellular physiology

  • The role of ACE2-mediated signaling in disease pathogenesis

• Cross-species barrier studies: ACE2 antibodies with defined species cross-reactivity can help investigate:

  • Determinants of coronavirus zoonotic transmission potential

  • Species-specific ACE2 features that facilitate or restrict viral entry

  • Mechanisms underlying adaptation of animal coronaviruses to human hosts

What adaptive immune responses might be elicited against therapeutic ACE2 antibodies, and how can these be mitigated?

The potential for adaptive immune responses against therapeutic ACE2 antibodies requires careful consideration:

• Immunogenicity risk factors:

  • Source of antibody (murine, chimeric, humanized, or fully human)

  • Presence of non-human glycosylation patterns or post-translational modifications

  • Aggregation or degradation products in formulated antibody

  • Patient-specific factors (genetic background, immune status, concurrent medications)

• Assessment strategies:

  • In silico prediction of T-cell epitopes within the antibody sequence

  • In vitro T-cell assays using peripheral blood mononuclear cells from diverse donors

  • Animal model testing in immunocompetent species

  • Clinical monitoring for anti-drug antibodies during trials

• Mitigation approaches:

  • Engineering antibodies on human IgG4 backbones, which have reduced effector functions and potentially lower immunogenicity

  • Introduction of deimmunizing mutations in potential T-cell epitopes

  • Optimization of formulation to minimize aggregation

  • Consideration of combination with immunomodulatory agents for long-term use

• Special considerations for ACE2 antibodies:

  • Since ACE2 is a self-protein, antibodies binding to it might break immunological tolerance

  • T-cell help for anti-drug antibody responses might come from foreign sequences in the antibody

  • Careful epitope selection is crucial to avoid triggering autoimmune responses against endogenous ACE2

• Long-term monitoring requirements:

  • Testing for binding and neutralizing anti-drug antibodies

  • Evaluation of pharmacokinetic changes indicating accelerated clearance

  • Assessment of breakthrough infections suggesting neutralization of therapeutic effect

  • Monitoring for potential autoimmune phenomena against endogenous ACE2