ACE2 (18-740) Human, Fc

What is ACE2 (18-740) Human, Fc and what does the nomenclature indicate?

ACE2 (18-740) Human, Fc refers to a recombinant protein containing the extracellular domain of human ACE2 from amino acid positions 18 to 740, fused to an immunoglobulin Fc tag at the C-terminal end. The numbering is significant because it includes the zinc metallopeptidase domain (residues 18-615) that functions as the receptor for SARS-CoV-2, as well as the ACE2 dimerization domain (CLD) up to residue 740 . This construct excludes the signal peptide (residues 1-17), transmembrane domain, and intracellular C-terminal tail .

The Fc fusion provides several advantages for research applications:

• Increases the half-life of the protein in circulation

• Enables dimerization, potentially enhancing avidity for target binding

• Confers Fc-mediated effector functions

• Facilitates purification via Protein-G chromatography

• Improves stability and solubility of the recombinant protein

What is the structure and functional domains of ACE2 (18-740) Human, Fc?

Human ACE2 has several distinct functional domains, with the (18-740) construct incorporating key extracellular portions:

• Signal peptide (residues 1-17): Not included in the construct

• Zinc metallopeptidase domain (residues 18-615): Primary binding site for SARS-CoV-2 spike protein

• ACE2 dimerization domain (residues within 616-740): Contributes to stability

• TMPRSS2 protease cleavage site (residues 697-716): Included in the construct

The metallopeptidase domain contains an HEXXH zinc-binding motif critical for its enzymatic activity. In some engineered variants, mutations (H374A and H378A) are introduced to eliminate enzymatic activity while preserving binding to SARS-CoV-2 .

The Fc portion typically consists of the hinge, CH2, and CH3 domains of human IgG1, and may contain modifications like the GASDALIE mutations (G236A/S239D/A330L/I332E) to enhance Fcγ receptor binding and effector functions .

How is ACE2 (18-740) Human, Fc produced and purified?

The production process for ACE2 (18-740) Human, Fc involves:

Production system:

• Expression in human embryonic kidney (HEK293) cells

• This mammalian expression system ensures proper folding and post-translational modifications

Purification methods:

• Primary purification via Protein-G affinity chromatography, which binds specifically to the Fc portion

• Quality control typically includes:

  • SDS-PAGE to confirm purity (typically >95% pure)

  • HPLC-SEC-MALS (High-Performance Liquid Chromatography with Size-Exclusion Chromatography and Multi-Angle Light Scattering) for additional validation

Formulation:

• Typically supplied in buffers containing either:

  • 50mM Tris-HCl, pH 7.5, 150mM NaCl, and glycerol

  • Or PBS, pH 7.4 with 10% glycerol

The choice of HEK293 cells ensures human-compatible glycosylation patterns and proper folding, critical for both binding studies and therapeutic applications .

What are the storage and stability conditions for ACE2 (18-740) Human, Fc?

For optimal maintenance of ACE2 (18-740) Human, Fc activity:

Storage conditions:

• Shipping: Typically shipped on ice packs

• Long-term storage: -20°C

• Avoid repeated freeze-thaw cycles

Stability parameters:

• Shelf life: Generally stable for 6 months from receipt when stored properly

• The glycerol in the formulation buffer helps maintain protein stability during freeze-thaw cycles

Quality control specifications:

• Purity: >90-95% as determined by SDS-PAGE

• Endotoxin levels: High-quality preparations have <1 EU/μg

• Functional activity: Should be verified before use after extended storage

Following these storage guidelines ensures that the protein maintains both structural integrity and functional activity for research applications.

What is the biological activity of ACE2 (18-740) Human, Fc protein and how is it measured?

The biological activity of ACE2 (18-740) Human, Fc can be assessed through several functional assays:

Binding to SARS-CoV-2 Spike Protein:

• Primarily measured by functional ELISA where immobilized ACE2 binds to SARS-CoV-2 Spike RBD

• Typical binding protocols use 2μg/ml of immobilized ACE2-Fc protein

• Binding affinity is reported as ED50 (effective dose for 50% binding), with high-quality preparations showing ED50 ≤ 100ng/ml

Enzymatic Activity (if not inactivated):

• Measured as specific activity in pmol/min/μg

• Natural substrates include angiotensin I and II, converting them to angiotensin 1-9 and 1-7 respectively

• Some preparations report specific activity >250 pmol/min/μg

• Engineered variants with H374A and H378A mutations should show significantly reduced or absent enzymatic activity

Fc-mediated Effector Functions:

• When the construct contains Fc modifications like GASDALIE mutations, additional assays can measure:

  • Antibody-dependent cellular cytotoxicity (ADCC)

  • Antibody-dependent cellular phagocytosis (ADCP)

  • Antibody-dependent complement deposition (ADCD)

Researchers should select the appropriate activity assay based on their specific application and whether they are using wild-type or engineered ACE2-Fc variants.

How does ACE2 (18-740) Human, Fc interact with SARS-CoV-2 RBD compared to membrane-bound ACE2?

The interaction between ACE2 (18-740) Human, Fc and SARS-CoV-2 RBD differs from membrane-bound ACE2 in several important aspects:

Structural considerations:

• Soluble ACE2-Fc lacks membrane anchoring constraints, allowing greater conformational freedom

• Dimerization through the Fc domain creates a bivalent molecule that may enhance avidity through simultaneous binding of two RBDs

• Crystal structure analysis using structures like 6M0J and 6VW1 identifies key interface contacts that can be optimized in engineered variants

Neutralization mechanism:

• Unlike membrane-bound ACE2 which facilitates viral entry, soluble ACE2-Fc acts as a decoy receptor

• ACE2-Fc competitively inhibits viral binding to cell-surface ACE2

• The Fc component provides additional neutralization mechanisms not present in membrane-bound ACE2:

  • Enhanced steric hindrance due to the large Fc domain

  • Recruitment of immune effector cells through Fc receptor interactions

  • Potential complement activation

  • Extended half-life in circulation

These differences make ACE2 (18-740) Human, Fc a promising therapeutic agent that leverages the natural viral entry mechanism while avoiding the facilitating effect of membrane-bound ACE2 .

What structural modifications have been engineered in ACE2-Fc variants to enhance binding affinity and reduce enzymatic activity?

Several strategic modifications have been developed to optimize ACE2-Fc for therapeutic applications:

Modifications to enhance RBD binding affinity:

• Structure-based design utilizing high-resolution ACE2-RBD structures (6M0J and 6VW1)

• Systematic analysis of interface contacts to identify positions for optimization

• Introduction of mutations to strengthen hydrogen bonds at the binding interface

• Addition of mutations that enhance electrostatic and hydrophobic interactions

Modifications to eliminate enzymatic activity:

• Mutation of zinc-binding histidines: H374A and H378A

• These residues are part of the HEXXH zinc-binding domain critical for catalytic function

• Inactivation prevents the enzymatic conversion of angiotensin substrates

• This modification avoids potential interference with the renin-angiotensin system when used therapeutically

Fc region modifications:

• GASDALIE mutations (G236A/S239D/A330L/I332E) to enhance binding to Fcγ receptors

• These mutations significantly increase Fc-mediated effector functions including ADCC, ADCP, and ADCD

• Some variants use IgG3 Fc instead of IgG1 to potentially enhance effector activity

• C220S mutation to eliminate unpaired cysteine that could cause instability

Domain optimization:

• Variants include either just the peptidase domain (PD) (residues 18-615, "ACE2 615 variant")

• Or both the PD and CLD (ACE2 dimerization domain) (residues 18-740, "ACE2 740 variant")

These engineered modifications create ACE2-Fc variants with optimized therapeutic properties compared to the wild-type protein.

How do Fc-effector functions contribute to the therapeutic potential of ACE2 (18-740) Human, Fc?

The Fc portion of ACE2 (18-740) Human, Fc contributes multiple mechanisms beyond extending half-life:

ADCC (Antibody-Dependent Cellular Cytotoxicity):

• When ACE2-Fc binds to viral spike proteins on infected cells, the Fc region can engage Fcγ receptors on NK cells

• This interaction triggers NK cells to release cytotoxic granules containing perforin and granzymes

• The resulting cytolysis can eliminate infected cells before they produce additional virions

• Enhanced by GASDALIE mutations in the Fc region

ADCP (Antibody-Dependent Cellular Phagocytosis):

• ACE2-Fc bound to viral particles can be recognized by Fcγ receptors on macrophages and neutrophils

• This recognition promotes phagocytosis and clearance of virus-ACE2-Fc complexes

• May be particularly important for clearing circulating virus

ADCD (Antibody-Dependent Complement Deposition):

• The Fc region can activate the classical complement pathway

• Complement deposition leads to formation of membrane attack complexes and virus neutralization

• Additionally recruits immune cells to sites of infection

Comparison of IgG isotypes:

• Some research has explored using IgG3 Fc instead of IgG1

• IgG3 may display greater Fc-effector activity for certain applications, similar to observations with some HIV neutralizing antibodies

These Fc-mediated mechanisms provide ACE2 (18-740) Human, Fc with multiple modes of action against SARS-CoV-2, potentially making it more difficult for the virus to develop resistance compared to single-epitope neutralizing antibodies .

What experimental approaches can validate ACE2 (18-740) Human, Fc activity against SARS-CoV-2 variants?

Comprehensive validation of ACE2 (18-740) Human, Fc against emerging variants requires a multi-faceted approach:

In vitro binding assays:

• Surface Plasmon Resonance (SPR) to determine binding kinetics (kon, koff, KD) with RBD from different variants

• Enzyme-Linked Immunosorbent Assay (ELISA) to measure binding at equilibrium

• Comparison of EC50 values across variants to identify potential resistance mutations

Neutralization assays:

• Pseudovirus neutralization assays using lentiviral particles bearing spike proteins from different variants

• Live virus neutralization using BSL-3 facilities to measure IC50/IC90 values

• Plaque reduction neutralization tests (PRNT) to confirm activity against infectious virus

Fc-effector function assays:

• ADCC reporter assays using engineered cells expressing Fcγ receptors

• Phagocytosis assays using fluorescent virus-like particles and primary monocytes or macrophages

• Complement deposition assays to measure C3b/C4b fixation on viral particles

• These are particularly important for variants with GASDALIE Fc mutations

In vivo studies:

• Transgenic mice expressing human ACE2 challenged with different SARS-CoV-2 variants

• Pharmacokinetic studies to ensure adequate biodistribution

• Dose-ranging studies to determine optimal therapeutic dosing

• Comparative studies against established therapeutic antibodies

Escape mutant selection:

• Serial passage of virus in the presence of sub-neutralizing concentrations of ACE2-Fc

• Deep sequencing to identify emerging resistance mutations

• Characterization of escape mutants for fitness and transmissibility

This comprehensive approach provides robust data on whether ACE2 (18-740) Human, Fc maintains efficacy across variants and is less susceptible to viral escape than epitope-specific antibodies.

How does ACE2 (18-740) Human, Fc compare to neutralizing antibodies as a therapeutic strategy?

ACE2 (18-740) Human, Fc offers distinct advantages and disadvantages compared to neutralizing antibodies (nAbs):

Resistance to viral escape:

• ACE2-Fc targets the functional receptor-binding interface that must be conserved for viral entry

• Mutations that reduce binding to ACE2-Fc would likely also reduce viral fitness

• Data suggest ACE2-Fc may retain efficacy against continuously developing variants

• In contrast, nAbs target specific epitopes that can more readily mutate without compromising viral function

Breadth of coverage:

• ACE2-Fc is potentially effective against all SARS-CoV-2 variants that use ACE2 for entry

• May also be effective against other coronaviruses that utilize ACE2 (e.g., SARS-CoV)

• nAbs typically have narrower specificity and may lose efficacy with new variants

Mechanism of action:

• ACE2-Fc functions as a decoy receptor, directly competing with cellular ACE2

• Additionally provides Fc-effector functions similar to nAbs when engineered with GASDALIE mutations

• nAbs can target various epitopes on the spike protein, not limited to the RBD

Production considerations:

• ACE2-Fc is a defined recombinant protein with potentially more consistent manufacturing

• nAbs require identification, isolation, and characterization from convalescent patients or immunized animals

• Both require mammalian cell expression systems for proper glycosylation

Safety profile:

• Enzymatically active ACE2 could potentially affect the renin-angiotensin system (addressed by introducing H374A/H378A mutations)

• nAbs are generally well-tolerated but could potentially trigger anti-idiotypic antibody responses

The data suggest that ACE2-Fc represents a promising therapeutic agent for COVID-19 treatment that may retain efficacy against continuously developing variants better than single-epitope antibodies .