Engineering ACE2 decoy receptors to combat viral escapability

Abstract

Decoy receptor proteins that trick viruses to bind to them should be resistant to viral escape because viruses that require entry receptors cannot help but bind decoy receptors. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for coronavirus cell entry. Recombinant soluble ACE2 was previously developed as a biologic against acute respiratory distress syndrome (ARDS) and verified to be safe in clinical studies. The emergence of COVID-19 reignited interest in soluble ACE2 as a potential broad-spectrum decoy receptor against coronaviruses. In this review, we summarize recent developments in preclinical studies using various high-affinity mutagenesis and Fc fusion approaches to achieve therapeutic efficacy of recombinant ACE2 decoy receptor against coronaviruses. We also highlight the relevance of stimulating effector immune cells through Fc-receptor engagement and the potential of using liquid aerosol delivery of ACE2 decoy receptors for defense against ACE2-utilizing coronaviruses.

Keywords: ACE2 decoy receptor; COVID-19; escape mutation; high-affinity mutagenesis.

Figure 1

Comparison of engineered angiotensin-converting enzyme 2 (ACE2) decoy receptor and monoclonal antibody against neutralization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). (A) The spike protein of SARS-CoV-2 binds to ACE2 on the surface of host cells through its trimeric spike glycoprotein and its interaction is essential for internalization of SARS-CoV-2 into host cells. The receptor binding domain (RBD) of spike protein is a direct binding site for ACE2 and a common target of neutralizing antibodies. (B) Evolutionary mutations in the spike gene can lead to the SARS-CoV-2 adaptation to neutralizing antibodies, termed escape mutation. In this case, existing monoclonal antibodies (mAbs) against a particular epitope of the original coronavirus spike RBD might inhibit infection by partially masking the viral spike RBD. Engineered high affinity ACE2 mutants decoy receptors, some fused with human Fc region of immunoglobulin, have been developed by directed evolution, deep mutational scanning, computer-assisted design, and a combination of these approaches to improve neutralization of viruses and escape mutants. (C) The effective recognition of monoclonal antibody against SARS-CoV-2 largely depends on the virus variants. Recently developed ACE2 decoy receptors have been shown to neutralize all variant of concerns including SARS-CoV-2 omicron variant in cultured cells, while existing mAbs therapy developed for Wuhan strain SARS-CoV-2, for example, imdevimab and casirivimab, show reduced affinity to SARS-CoV-2 omicron strain [26].

Figure 2

Directed evolution to enhance angiotensin-converting enzyme 2 (ACE2) affinity toward the spike receptor binding domain (RBD). An illustration depicting workflow for directed evolution of ACE2 as described in [24]. The protease domain (PD) of ACE2 is the interface to viral spike RBD. Random mutations are introduced to the N-terminal residues 18–102 of the PD by error-prone PCR at the rate of ~10 mutations per 1 kb (up to three mutations in residues 18–102). The PCR fragments are inserted into the backbone plasmid in the context of full-length ACE2 expression with an N-terminal HA tag. The resultant library are composed of ~10⁵ ACE2 mutants and packaged into lentiviruses. The mutant library are transduced to 293T cells in less than 0.3 multiplicity of infection to yield no more than one mutant ACE2 per cell, followed by incubation with recombinant RBD of SARS-CoV-2 spike protein fused to superfolder GFP (sfGFP). Using fluorescence-activated cell sorting, the top 0.05% of cells showing higher binding affinity (sfGFP signal) relative to protein expression level (HA signal) are harvested from ~5×10⁷ cells. To exclude mutants with impaired structural stability, only cells with preserved signal of surface ACE2 are gated. Genomic DNA are extracted from collected cells and used as the template for error-prone PCR to pursue the next round of mutagenesis and selection.

Figure 3

Location of mutation sites for affinity enhancement in angiotensin-converting enzyme (ACE)2. ACE2 and receptor-binding domain (RBD) in the complex structure (PDB: 6m0j [20,34]) are shown as a light orange surface model and a blue translucent cartoon model, respectively. Mutation sites for affinity enhancement introduced in ACE2 are indicated in red. Reported amino acid substitutions of each residue are shown in parentheses. Asterisks indicate mutation sites for deletion of the N90 and N322 glycans.