ACA2 Antibody

What are anti-ACE2 antibodies and how do they differ from autoantibodies against ACE2?

Anti-ACE2 antibodies represent a broad category of antibodies that bind to the angiotensin-converting enzyme 2 (ACE2) receptor. In research contexts, these can be divided into two major categories: therapeutic monoclonal antibodies deliberately designed to target ACE2, and autoantibodies that develop naturally against ACE2 during disease states. Therapeutic anti-ACE2 monoclonal antibodies are engineered immune proteins that bind to specific epitopes on ACE2, typically to block interactions with pathogens that use ACE2 as an entry receptor, such as SARS-CoV-2 . These antibodies are specifically designed to have high affinity and specificity for ACE2 without inhibiting its enzymatic function. Autoantibodies against ACE2, by contrast, are produced by the immune system against self-ACE2 molecules, typically following infections or in autoimmune conditions .

The key functional difference between these antibody types lies in their effect on ACE2 activity. Therapeutic anti-ACE2 antibodies are typically designed to block viral binding without affecting ACE2's physiological functions, maintaining its enzymatic activity and avoiding internalization of the receptor . Researchers have developed human monoclonal antibodies that bind the ACE2 receptor with affinities in the low nanomolar to picomolar range, effectively blocking infection by all human ACE2 binding sarbecoviruses tested, including various SARS-CoV-2 variants . Conversely, autoantibodies against ACE2 that develop during SARS-CoV-2 infection often target epitopes in the catalytic domain and can inhibit ACE2 enzymatic function, potentially contributing to dysregulated inflammation and more severe disease outcomes .

Research has demonstrated that individuals with severe COVID-19 develop higher levels of ACE2 autoantibodies across multiple immunoglobulin isotypes (IgG, IgA, and IgM) compared to healthy individuals or those with mild disease . Furthermore, plasma from individuals with severe COVID-19 significantly decreases ACE2 enzymatic activity in functional assays, indicating that these autoantibodies may interfere with the normal regulatory role of ACE2 in inflammation . Understanding these fundamental differences is critical for researchers designing studies involving anti-ACE2 antibodies, whether for therapeutic development or for investigating the role of autoantibodies in disease pathogenesis.

How do anti-ACE2 antibodies interact with viral infection processes?

Anti-ACE2 antibodies interact with viral infection processes primarily by disrupting the critical binding between the viral spike protein and the ACE2 receptor. In the case of SARS-CoV-2, the virus initiates infection by binding its spike protein's receptor binding domain (RBD) to the host cell ACE2 receptor. Anti-ACE2 antibodies can bind to epitopes on ACE2 that overlap with or are proximal to the RBD binding site, thereby preventing virus attachment through steric hindrance or conformational changes . This mechanism differs from spike-targeting antibodies, which bind directly to the virus rather than the host receptor.

The structural basis for this viral neutralization has been elucidated through high-resolution studies. Cryo-electron microscopy analysis of one potent anti-ACE2 monoclonal antibody (05B04) revealed that it binds to the N-terminal helices of human ACE2 at a 3.3 Å resolution . This binding orientation specifically sterically hinders and competes with the SARS-CoV-2 RBD for binding to ACE2 . The antibody's complementarity-determining regions (CDRs) - particularly CDRH2, CDRH3, CDRL1, and CDRL3 - contribute to binding an epitope that comprises residues from the α1 and α2 helices of ACE2 . This strategic binding explains the antibody's broad neutralization capabilities against multiple sarbecoviruses that use ACE2 for entry.

Importantly, well-designed therapeutic anti-ACE2 antibodies demonstrate neutralizing activity against all tested ACE2-binding sarbecoviruses, including diverse SARS-CoV-2 variants like ancestral, Delta, and Omicron . This broad-spectrum activity represents a significant advantage over spike-targeting antibodies, which have often been rendered obsolete by emerging viral variants with mutations in the spike protein. The IC50 values for these antibodies against pseudotyped virus infection ranged from approximately 7-100 ng/ml, which approaches the potency of spike-targeting therapeutic monoclonal antibodies . This capacity for pan-sarbecovirus neutralization suggests that ACE2-targeting antibodies could serve as effective prophylactic and treatment agents against both current SARS-CoV-2 variants and future sarbecovirus pandemic threats.

What are the current methods for detecting and quantifying anti-ACE2 antibodies?

Multiple methodological approaches exist for detecting and quantifying anti-ACE2 antibodies, each with distinct advantages depending on the research question. For detecting ACE2 autoantibodies in patient samples, isotype-specific enzyme-linked immunosorbent assays (ELISAs) represent the most common approach . These assays typically involve coating plates with recombinant ACE2 protein, incubating with patient plasma or serum, and then detecting bound antibodies using isotype-specific secondary antibodies (anti-IgG, anti-IgA, or anti-IgM) . This approach allows researchers to characterize the isotype distribution of the autoantibody response, which may have important implications for understanding disease pathogenesis.

For establishing positivity thresholds, researchers often use plasma samples from healthy individuals to determine a cut-off value, typically set at twice the background reading for each immunoglobulin isotype . This approach helps distinguish true autoantibody responses from background signals. When comparing autoantibody levels between groups (such as healthy individuals versus those with disease), statistical methods like the Wilcoxon-Mann-Whitney test are commonly employed to assess the significance of observed differences .

Beyond simple detection, functional assays are crucial for understanding the biological impact of anti-ACE2 antibodies. In vitro ACE2 functional enzymatic assays can measure whether antibodies inhibit ACE2 enzymatic activity . These assays typically involve incubating plasma or purified antibodies with enzymatically active recombinant ACE2 protein, then measuring the conversion of an ACE2-specific fluorogenic substrate . Comparing the resulting fluorescence to unblocked positive control ACE2 provides a quantitative measure of inhibitory activity. For therapeutic monoclonal antibodies, binding affinity is commonly assessed using surface plasmon resonance (SPR), which can determine association constants with high precision, revealing affinities ranging from 1.45 × 10^-9 M to 4.99 × 10^-12 M for potent anti-ACE2 monoclonal antibodies .

Flow cytometry provides another valuable approach for assessing antibody binding to cell-surface ACE2, allowing researchers to confirm specificity by comparing binding to ACE2-expressing cells versus parental cells lacking ACE2 expression . This method also enables evaluation of whether antibodies induce ACE2 internalization, which would be undesirable for therapeutic applications. For structural characterization of antibody-ACE2 interactions, cryo-electron microscopy has proven invaluable, allowing visualization of binding epitopes at resolutions as high as 3.3 Å and providing insights into mechanisms of virus neutralization .

How are therapeutic monoclonal antibodies against ACE2 developed?

The development of therapeutic monoclonal antibodies against ACE2 follows a systematic process that combines immunization strategies, hybridoma technology, and recombinant antibody engineering. The initial step involves immunizing specialized mice capable of producing antibodies with human variable domains. For example, AlivaMab mice, which produce chimeric antibodies consisting of human Fab domains and murine Fc domains, have been successfully used for this purpose . To maximize antibody diversity, researchers may employ different strains, such as the KP AlivaMab mouse strain that generates human Kappa light chain (κ) containing antibodies and the AV AlivaMab mouse strain that generates both human Kappa (κ) and Lambda (λ) light chains .

The immunization protocol involves administering recombinant human ACE2 extracellular domains, which may be either monomeric or rendered dimeric by fusion to the Fc portion of human IgG1 . Following immunization, researchers monitor the immune response by testing sera for inhibition of SARS-CoV-2 pseudotyped viruses. Once mice with neutralizing sera are identified, researchers harvest B cells and generate hybridomas through fusion with myeloma cells, creating immortalized cell lines that secrete monoclonal antibodies . Hybridoma supernatants are then screened for ACE2-binding antibodies using ELISA with ACE2-coated plates, allowing identification of hybridomas producing antibodies of interest .

To generate fully human monoclonal antibodies suitable for therapeutic use, researchers clone the variable domains from promising chimeric human-mouse antibodies into human immunoglobulin-γ1 (IgG1) expression vectors . These vectors may include specific mutations to optimize antibody properties, such as L234/L235 (LALA) mutations to reduce Fc receptor binding and M428/N434 (LS) mutations to extend serum half-life . The complete human monoclonal antibodies are then produced by co-expression of corresponding heavy and light chains in suitable expression systems.

Following production, candidate antibodies undergo extensive characterization to assess their binding properties, neutralization potency, and functional effects. Key assessments include pseudovirus neutralization assays to determine IC50 values, flow cytometry to confirm binding to ACE2-expressing cells, surface plasmon resonance to measure binding affinity, and functional assays to verify that antibodies do not inhibit ACE2 enzymatic activity or induce receptor internalization . Advanced structural studies, such as cryo-electron microscopy, may also be performed to understand the molecular basis of antibody binding and neutralization . This comprehensive development and characterization process ensures the identification of antibodies with optimal properties for therapeutic applications.

What experimental models are used to evaluate anti-ACE2 antibodies?

A diverse array of experimental models is employed to evaluate anti-ACE2 antibodies, spanning from in vitro cellular systems to animal models that recapitulate human disease. Cell-based assays represent the foundation of initial evaluations, with pseudotyped virus neutralization assays serving as a critical tool for assessing the ability of antibodies to prevent viral entry. These assays typically utilize SARS-CoV-2 (or other sarbecovirus) spike pseudotyped HIV-1 particles to infect target cells such as Huh-7.5 cells, allowing measurement of infection inhibition and determination of half-maximal inhibitory concentration (IC50) values . This approach provides a safe, quantitative method for comparing neutralization potency across multiple antibody candidates and viral variants.

Cell binding assays using flow cytometry complement neutralization studies by confirming antibody binding to cell-surface ACE2. A549 cells expressing human ACE2 are commonly used, with parental A549 cells serving as negative controls to ensure binding specificity . For therapeutic development, it's important to verify cross-reactivity with ACE2 from animal species used in preclinical studies. For instance, researchers have confirmed that potent anti-ACE2 monoclonal antibodies bind equivalently to cells expressing human ACE2 and macaque ACE2, facilitating translation of findings from macaque models to humans .

Functional cellular assays assess whether antibodies affect normal ACE2 biology. Fluorescence-based internalization assays using ACE2 tagged with epitopes such as influenza hemagglutinin (HA) at its intracellular C-terminus allow researchers to monitor whether antibody binding induces receptor downregulation from the cell surface . This is crucial information for therapeutic antibodies, as inducing ACE2 internalization could potentially disrupt normal physiological functions. Similarly, enzymatic activity assays using fluorogenic substrates help determine whether antibodies interfere with ACE2's catalytic function .

For in vivo evaluation, human ACE2 knock-in mice represent a valuable model system. These mice express human rather than murine ACE2, allowing assessment of antibody efficacy and pharmacological properties in the context of SARS-CoV-2 infection . The protection against lung infection in this model provides important proof-of-concept for therapeutic development. Researchers may also evaluate antibody pharmacokinetics, tissue distribution, and safety parameters in both rodent and non-human primate models prior to clinical translation.

How do ACE2 autoantibody levels correlate with COVID-19 severity?

ACE2 autoantibody levels demonstrate a significant correlation with COVID-19 disease severity, with research revealing distinct patterns across different patient populations. Individuals who recover from severe COVID-19 requiring hospitalization exhibit significantly higher levels of ACE2 autoantibodies compared to both healthy individuals and those recovering from mild COVID-19 (defined as not requiring hospitalization) . This pattern holds true across all three major immunoglobulin isotypes - IgG, IgA, and IgM - suggesting a robust and multi-faceted autoimmune response against ACE2 in severe disease . The most pronounced differences are typically observed for IgG autoantibodies, which represent the mature, affinity-matured antibody response.

The functional consequences of these elevated autoantibody levels are particularly noteworthy. Plasma from individuals with severe COVID-19 demonstrates significantly greater inhibition of ACE2 enzymatic activity compared to plasma from healthy individuals not previously infected with SARS-CoV-2 (P=0.0035; Wilcoxon-Mann-Whitney) . This inhibition of ACE2 function may contribute to dysregulated inflammation, as ACE2 plays a key role in the renin-angiotensin system that regulates both systemic and local inflammatory processes . ACE2 levels have been inversely correlated with markers of inflammation, and genetic knockout of ACE2 in mice results in a hyperinflammation phenotype . The development of function-inhibiting autoantibodies during severe COVID-19 may therefore represent a mechanism by which initial inflammation becomes amplified and sustained.

The timeline of autoantibody development and persistence may also influence disease outcomes. The studies referenced examined samples obtained 30-60 days after COVID-19, indicating that these autoantibody responses persist well into the recovery period . This persistence may have implications for long-term complications and sequelae following COVID-19. Interestingly, preexisting ACE2 autoantibodies are detectable in some healthy individuals (12 IgG, 16 IgA, and 17 IgM ACE2 autoantibody positive individuals among 35 healthy individuals tested), suggesting that baseline autoimmunity to ACE2 exists in the general population . Whether these preexisting autoantibodies influence susceptibility to severe COVID-19 upon SARS-CoV-2 infection remains an important area for investigation.

What is the significance of different isotypes of ACE2 autoantibodies?

The IgA predominance is particularly noteworthy given that ACE2 is highly expressed in mucosal tissues, including the respiratory and gastrointestinal tracts. IgA represents the primary antibody isotype in mucosal secretions and plays a critical role in maintaining homeostasis at mucosal surfaces. The elevated baseline levels of IgA autoantibodies against ACE2 may reflect ongoing interactions between the immune system and ACE2-expressing mucosal tissues. Following severe COVID-19, significant increases are observed across all isotypes, but the relative proportions shift, suggesting maturation and diversification of the autoimmune response .

The functional properties of different antibody isotypes likely contribute to their distinct biological effects. IgG antibodies, with their longer half-life and ability to activate complement and Fc receptor-bearing cells, may mediate more sustained inhibition of ACE2 function. IgA antibodies, particularly in their secretory form, may primarily affect ACE2 in mucosal tissues, potentially influencing local inflammatory responses. IgM antibodies, while typically short-lived, have high avidity due to their pentameric structure and may be particularly effective at neutralizing ACE2 function during early immune responses.

Researchers investigating ACE2 autoantibodies should consider measuring all three major isotypes to gain a comprehensive understanding of the autoimmune response. Methodologically, this requires isotype-specific secondary antibodies in ELISA or other detection systems . Furthermore, the presence of different isotypes may have distinct implications for disease pathogenesis, progression, and prognosis, making isotype determination a valuable component of clinical research protocols examining ACE2 autoimmunity in COVID-19 and other conditions.

How do therapeutic anti-ACE2 antibodies differ from antibodies targeting viral proteins?

Therapeutic anti-ACE2 antibodies represent a fundamentally different approach to viral neutralization compared to antibodies targeting viral proteins, with distinct advantages and considerations for research and development. The primary mechanistic difference lies in the target: anti-ACE2 antibodies bind to the host receptor rather than the virus itself . This strategy blocks the critical interaction between the virus and its cellular entry point, effectively preventing infection regardless of variations in viral surface proteins. In contrast, antibodies targeting viral proteins, such as the SARS-CoV-2 spike, bind directly to the virus to prevent receptor engagement or disrupt other aspects of the viral entry process.

The most significant advantage of the anti-ACE2 approach is its broad-spectrum activity against multiple viral variants and even different viruses that use the same receptor. Human monoclonal antibodies targeting ACE2 have demonstrated the ability to block infection by all human ACE2-binding sarbecoviruses tested, including ancestral SARS-CoV-2 and its Delta and Omicron variants, with IC50 values ranging from approximately 7-100 ng/ml . This pan-sarbecovirus neutralization capacity represents a crucial advantage over spike-targeting antibodies, which have frequently been rendered ineffective by the emergence of new viral variants with mutations in the spike protein . For researchers, this suggests that anti-ACE2 antibodies may provide more durable protection against evolving viral threats.

The design considerations for these two antibody types also differ substantially. For anti-ACE2 antibodies, the critical requirement is to block viral binding without disrupting the normal physiological functions of ACE2. Cryo-electron microscopy studies of antibody-ACE2 complexes have revealed that effective antibodies, such as 05B04, bind to epitopes on the α1 and α2 helices of ACE2 that sterically hinder SARS-CoV-2 RBD binding . Importantly, these antibodies must be carefully engineered to avoid inhibiting ACE2 enzymatic activity or inducing receptor internalization, which could potentially disrupt normal physiological processes . For spike-targeting antibodies, the challenge is different – they must recognize conserved epitopes that are functionally constrained and less prone to mutation-driven escape.

What epitopes on ACE2 are targeted by therapeutic antibodies versus autoantibodies?

The epitope specificity of antibodies targeting ACE2 differs substantially between engineered therapeutic antibodies and naturally occurring autoantibodies, with important implications for their functional effects and potential applications. Therapeutic anti-ACE2 monoclonal antibodies are strategically designed to target regions of ACE2 that overlap with the SARS-CoV-2 binding site without affecting the receptor's enzymatic function. Structural studies using cryo-electron microscopy have revealed that effective therapeutic antibodies, such as 05B04, bind primarily to epitopes on the N-terminal helices of ACE2, particularly the α1 and α2 helices . This binding orientation places the antibody in direct competition with the SARS-CoV-2 receptor binding domain (RBD) through steric hindrance . The complementarity-determining regions of the antibody - specifically CDRH2, CDRH3, CDRL1, and CDRL3 - form contacts with these helical structures, creating a binding interface with substantial buried surface area .

In contrast, autoantibodies that develop during SARS-CoV-2 infection often target epitopes in the catalytic domain of ACE2 . This targeting pattern likely explains why these autoantibodies inhibit ACE2 enzymatic function, as demonstrated by in vitro functional enzymatic assays . The inhibition of ACE2 catalytic activity may contribute to dysregulated inflammation and more severe disease outcomes, as ACE2 plays a key role in regulating inflammatory processes through the renin-angiotensin system . The development of autoantibodies targeting the catalytic domain may represent an unfortunate consequence of immune responses to viral infection, potentially driven by molecular mimicry or exposure of normally sequestered ACE2 epitopes during tissue damage.

The distinct epitope preferences of therapeutic antibodies versus autoantibodies highlight the precision required in antibody engineering for therapeutic applications. Effective therapeutic antibodies must thread a narrow path - binding with high affinity to regions that block viral attachment while avoiding interference with ACE2's enzymatic pocket or inducing conformational changes that might alter its function . Understanding these epitope differences provides researchers with valuable guidance for antibody selection and optimization processes. Furthermore, epitope mapping of naturally occurring autoantibodies in diverse patient populations may yield insights into the mechanisms driving autoimmunity against ACE2 and potential strategies for intervention in cases where these autoantibodies contribute to disease pathology.