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Trends in Pharmacological Sciences
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Engineering ACE2 decoy receptors to combat viral escapability

  • Takao Arimori
    Affiliations
    Laboratory for Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Osaka, Japan
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  • Nariko Ikemura
    Affiliations
    Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan
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  • Toru Okamoto
    Affiliations
    Institute for Advanced Co-Creation Studies, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

    Center for Infectious Disease Education and Research (CiDER), Osaka University, Osaka, Japan
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  • Junichi Takagi
    Affiliations
    Laboratory for Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Osaka, Japan

    Center for Infectious Disease Education and Research (CiDER), Osaka University, Osaka, Japan

    Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives (OTRI), Osaka University, Osaka, Japan
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  • Daron M. Standley
    Affiliations
    Center for Infectious Disease Education and Research (CiDER), Osaka University, Osaka, Japan

    Department of Genome Informatics, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
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  • Atsushi Hoshino
    Correspondence
    Correspondence:
    Affiliations
    Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan
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      Highlights

      • Recombinant angiotensin-converting enzyme 2 (ACE2) was developed as a biologic against acute respiratory distress syndrome and rapidly repositioned as a decoy receptor for SARS-CoV-2 after emergence of COVID-19.
      • High-affinity mutagenesis and fusion with IgG-Fc enables recombinant ACE2 to effectively neutralize SARS-CoV-2 in animal models of COVID-19.
      • ACE2 decoy receptors neutralize broadly SARS-CoV-2 variants as well as ACE2-utilizing sarbecoviruses.
      • Enhancement of Fc effector function has the potential to promote antiviral effects by the clearance of viral particles, cytotoxic elimination of virus-infected cells, and induction of antiviral T cell responses.
      • Direct drug delivery to infectious site by liquid aerosol inhalation potentially enhances therapeutic efficacy against COVID-19.
      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

      Engineered ACE2 decoy receptors as an alternative strategy to overcome SARS-CoV-2 evolution

      Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first identified in the Chinese city of Wuhan in late 2019. SARS-CoV-2 is a positive-stranded RNA virus that belongs to lineage B (sarbecovirus) of the betacoronavirus genus [
      • Zhou P.
      • et al.
      A pneumonia outbreak associated with a new coronavirus of probable bat origin.
      ,
      • Wu F.
      • et al.
      A new coronavirus associated with human respiratory disease in China.
      ,
      • Letko M.
      • et al.
      Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses.
      ]. ACE2 (see Glossary) is the major receptor for SARS-CoV-2 cell entry. The virus attaches to host cells through its trimeric spike glycoprotein in which the S1 subunit binds to the receptor and the S2 subunit mediates membrane fusion [
      • Wrapp D.
      • et al.
      Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.
      ]. Along with worldwide spread of the virus, viral RNA genome replication errors have continuously generated new mutants. Some of these have had advantages in viral transmissibility and have formed new waves of infections. To fight against COVID-19, intensive efforts have been made in the development of vaccines and drugs. The main drug targets are the site of initial interaction between the receptor binding domain (RBD) of the virus spike protein and host cell surface ACE2, and the main protease that cleaves virus nonstructural long polyproteins, as well as the cleaved product, RNA-dependent RNA polymerase (RdRp) [
      • Banerjee R.
      • et al.
      Potential SARS-CoV-2 main protease inhibitors.
      ,
      • Qiao J.
      • et al.
      SARS-CoV-2 M(pro) inhibitors with antiviral activity in a transgenic mouse model.
      ,
      • Drayman N.
      • et al.
      Masitinib is a broad coronavirus 3CL inhibitor that blocks replication of SARS-CoV-2.
      ,
      • Robson F.
      • et al.
      Coronavirus RNA proofreading: molecular basis and therapeutic targeting.
      ]. Among drugs developed for SARS-CoV-2, monoclonal antibodies (mAbs) have gained a leading role as therapeutics against COVID-19. However, mAbs have an Achilles’ heel in that mutations in the spike gene can lead to the SARS-CoV-2 adaptation to neutralizing antibodies, termed escape mutation [
      • Zhu Q.
      • et al.
      Analysis of respiratory syncytial virus preclinical and clinical variants resistant to neutralization by monoclonal antibodies palivizumab and/or motavizumab.
      ,
      • Jensen B.
      • et al.
      Emergence of the E484K mutation in SARS-COV-2-infected immunocompromised patients treated with bamlanivimab in Germany.
      ,
      • Starr T.N.
      • et al.
      Complete map of SARS-CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555 and its cocktail with LY-CoV016.
      ]. To prevent such viral evasion, some mAbs are administered in cocktail form and others are designed to target evolutionarily conserved regions [
      • Baum A.
      • et al.
      Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual antibodies.
      ,
      • Rappazzo C.G.
      • et al.
      Broad and potent activity against SARS-like viruses by an engineered human monoclonal antibody.
      ]. With these efforts, mAbs have effectively prevented infection and clinical progression for most SARS-CoV-2 strains [
      • Kreuzberger N.
      • et al.
      SARS-CoV-2-neutralising monoclonal antibodies for treatment of COVID-19.
      ].
      However, the striking evolution of omicron, which included numerous mutations in the spike, resulted in escape from most mAbs in clinical use, as well as from vaccinated sera [
      • VanBlargan L.A.
      • et al.
      An infectious SARS-CoV-2 B.1.1.529 Omicron virus escapes neutralization by therapeutic monoclonal antibodies.
      ,
      • Cameroni E.
      • et al.
      Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift.
      ,
      • Hoffmann M.
      • et al.
      The Omicron variant is highly resistant against antibody-mediated neutralization: Implications for control of the COVID-19 pandemic.
      ,
      • Dejnirattisai W.
      • et al.
      SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses.
      ].
      ACE2 decoy receptors are an alternative strategy to neutralize the virus in a manner that is robust against such escape [
      • Monteil V.
      • et al.
      Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2.
      ,
      • Lei C.
      • et al.
      Neutralization of SARS-CoV-2 spike pseudotyped virus by recombinant ACE2-Ig.
      ]. The concept of decoy receptors has already been clinically applied to modulate autoimmune diseases and angiogenesis, with a focus on tumor necrosis factor receptor (TNFR), cytotoxic T lymphocyte associated antigen‐4 (CTLA4) [
      • Smolen J.S.
      • et al.
      New therapies for treatment of rheumatoid arthritis.
      ], or vascular endothelial growth factor receptor (VEGFR) [
      • Kaiser P.K.
      Emerging therapies for neovascular age-related macular degeneration: drugs in the pipeline.
      ,
      • Gaya A.
      • Tse V.
      A preclinical and clinical review of aflibercept for the management of cancer.
      ]. In the case of viral infection, decoy receptors have a distinct advantage over mAbs in terms of resistance to escape mutations [
      • Higuchi Y.
      • et al.
      Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.
      ,
      • Chan K.K.
      • et al.
      An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants.
      ]. Viral mutations escaping from decoy receptors may appear, but in theory, they would have limited binding affinity toward the native receptors on host cells, leading to reduced or even absent infectivity [
      • Higuchi Y.
      • et al.
      Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.
      ]. In recent studies, virus culture treated with sparing concentrations of ACE2 decoy receptors has generated no escape mutants [
      • Higuchi Y.
      • et al.
      Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.
      ], and omicron and even other sarbecoviruses remain sensitive to ACE2 decoy receptors (Figure 1) [
      • Ikemura N.
      • et al.
      An engineered ACE2 decoy neutralizes the SARS-CoV-2 Omicron variant and confers protection against infection in vivo.
      ,
      • Zhang L.
      • et al.
      An engineered ACE2 decoy receptor can be administered by inhalation and potently targets the BA.1 and BA.2 omicron variants of SARS-CoV-2.
      ].
      Figure 1
      Figure 1Comparison 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 [
      • Ikemura N.
      • et al.
      An engineered ACE2 decoy neutralizes the SARS-CoV-2 Omicron variant and confers protection against infection in vivo.
      ].
      Historically, recombinant human soluble ACE2 (rhACE2) was developed as an anti-inflammatory drug for ARDS [
      • Khan A.
      • et al.
      A pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome.
      ]. Its therapeutic potential as a decoy receptor was investigated in the early phase of the COVID-19 pandemic [
      • Monteil V.
      • et al.
      Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2.
      ]. During the pandemic, several groups made efforts to engineer affinity-enhancing mutations or to fuse human Fc region of immunoglobulin with soluble ACE2 in order to improve its pharmacokinetics and virus neutralization efficacy. Emergence of the omicron variant reinforced the difficulty of drug development against mutational escape of viruses and increased interest in decoy receptors as potential universal effective drugs against current and future SARS-CoV-2 variants and coronavirus pandemics.

      ACE2 as an enzymatic drug for ARDS and decoy receptor for COVID-19

      ACE2, a homolog of ACE, is a zinc metallopeptidase and predominantly hydrolyzes angiotensin (Ang) II to Ang 1–7 [
      • Vickers C.
      • et al.
      Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase.
      ]. Ang II is the main effector of the renin–angiotensin system (RAS) and plays a key role in maintaining fluid and salt balance. It also mediates proinflammatory actions including vasoconstriction, apoptosis, capillary leak, and fibroproliferation via the angiotensin type I receptor (AT1R), while Ang 1–7 induces anti-inflammatory responses through the Mas receptor pathway [
      • Khan A.
      • et al.
      A pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome.
      ]. Since increased Ang II and imbalance between AT1R and Mas receptor signals are involved in the pathogenesis of ARDS, ACE2 which converts Ang II to Ang 1–7, is expected to resolve this imbalance and improve lung inflammatory injury [
      • Vickers C.
      • et al.
      Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase.
      ,
      • Zhang H.
      • Baker A.
      Recombinant human ACE2: acing out angiotensin II in ARDS therapy.
      ]. Indeed, the development of rhACE2 was motivated by its therapeutic effect in animal models of ARDS [
      • Kuba K.
      • et al.
      Angiotensin-converting enzyme 2 (ACE2) in the pathogenesis of ARDS in COVID-19.
      ]. In clinical studies, although rhACE2 was well-tolerated in both healthy human volunteers and ARDS patients, and treatment resulted in rapid conversion of Ang II to Ang 1–7, no beneficial changes in acute physiology or clinical outcomes was detected [
      • Khan A.
      • et al.
      A pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome.
      ,
      • Haschke M.
      • et al.
      Pharmacokinetics and pharmacodynamics of recombinant human angiotensin-converting enzyme 2 in healthy human subjects.
      ].
      Following the emergence of SARS-CoV-2, rhACE2 was repositioned as a decoy receptor for COVID-19 therapy. Treatment of vessel and kidney organoids with rhACE2 in vitro effectively blocked SARS-CoV-2 infection [
      • Monteil V.
      • et al.
      Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2.
      ], but Phase 2 trials failed to demonstrate significantly improved clinical outcomes in the treatment of hospitalized patients upon intravenous administration, even though there were small improvements in pulmonary gas exchange efficiency (ClinicalTrials.gov identifier NCT04335136). Because rhACE2 monomers have a fast clearance rate in human blood with a half-life of ~3 h, one likely reason for the lack of efficacy is inadequate drug concentration at the infection site [
      • Haschke M.
      • et al.
      Pharmacokinetics and pharmacodynamics of recombinant human angiotensin-converting enzyme 2 in healthy human subjects.
      ,
      • Huentelman M.J.
      • et al.
      Structure-based discovery of a novel angiotensin-converting enzyme 2 inhibitor.
      ]. Currently, Phase 1 trials to evaluate the safety and tolerability of rhACE2 inhalation, which is a more direct administration to site of infection, are ongoing (NCT05065645). In parallel, ACE2 mutagenesis to increase binding affinity and fusion to Fc for extended half-life, avidity effects, and antiviral immune responses, are expected to improve the therapeutic potential of rhACE2 as a decoy receptor against COVID-19.

      ACE2 mutagenesis approaches to achieve increased affinity

      In comparison to native ACE2, the higher affinity of ACE2 decoy receptors for SARS-CoV-2 spike protein is important for competitive neutralization of infectious virus at the host cell surface at standard biologic doses. The equilibrium dissociation constant (KD) between ACE2 monomer and SARS-CoV-2 spike RBD ranges from ~10 to ~60 nM, depending on the virus variant [
      • Wrapp D.
      • et al.
      Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.
      ,
      • Cameroni E.
      • et al.
      Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift.
      ,
      • Higuchi Y.
      • et al.
      Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.
      ,
      • Lan J.
      • et al.
      Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor.
      ,
      • Shang J.
      • et al.
      Structural basis of receptor recognition by SARS-CoV-2.
      ]. By contrast, mAbs in clinical use for COVID-19 treatment have KD <10 nM [
      • Cao Y.
      • et al.
      Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies.
      ]. Multifaceted approaches have been recently used to enhance binding affinity of ACE2 decoy receptors to the level of therapeutic antibodies, including directed evolution, deep mutational scanning (DMS) and computational design.

      Directed evolution as an established protein engineering method

      Directed evolution is a powerful method to design proteins with desirable functions by repeated random mutagenesis and selection (Box 1) [
      • Packer M.S.
      • Liu D.R.
      Methods for the directed evolution of proteins.
      ]. Recent studies demonstrate the utility of this method to achieve high-affinity virus neutralizers. For example, Hoshino and coworkers conducted directed evolution of ACE2 in an assay using human cell line. They focused on the protease domain of ACE2 which is the interface for SARS-CoV-2 spike RBD binding and performed mutagenesis by error-prone PCR of the N-terminal residues 18–102 of the protease domain (Figure 2). Three cycles of such mutagenesis and selection experiments identified three major high-affinity ACE2 mutants, termed 3N39, 3J113, and 3J320. Each high-affinity ACE2 mutant contained six or seven mutations. Substitution with wild-type residues resulted in a second version (v2) that contained four essential mutations for affinity enhancement. Among these, the 3N39v2 monomer (amino acids 18–615) harboring A25V/K31N/E35K/L79F substitutions exhibited the best KD value (0.64 nM) for the SARS-CoV-2 spike RBD, which indicates a 100-fold increased affinity compared to wild-type ACE2 (Table 1) [
      • Higuchi Y.
      • et al.
      Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.
      ]. In another study, Wilson and coworkers performed directed evolution with error-prone PCR and staggered extension process (StEP) in yeast surface display and identified several high affinity ACE2 mutants. Among these, a dimer form with IgG4-Fc fusion version of CDY14HL that contains K31M/E35K/S47A/L79F/L91P/N330Y; and H345L substitution, which confers enzymatic inactivation was shown to have 1000-fold improved affinity (31 pM) [
      • Sims J.J.
      • et al.
      Intranasal gene therapy to prevent infection by SARS-CoV-2 variants.
      ] (Table 1). This CDY14HL decoy was then applied as an adeno-associated virus (AAV)-mediated intranasal gene therapy in mice and nonhuman primates. The result showed significant prophylactic effect against SARS-CoV-2 challenge. Although directed evolution is a classical approach to protein engineering, these studies indicate that it is still one of the efficient methods to achieve the saturated binding affinity between virus glycoprotein and its receptor. (See Figure 3.)
      Directed evolution
      Directed evolution is an experimental method to improve protein function for desired purpose by mutagenesis and selection, imitating evolution in nature. Sol Spiegelman pioneered this technology in 1967 and Frances H. Arnold contributed to the development of artificial enzymes and received the Nobel Prize in Chemistry in 2018. Evolution in nature consists of repeated random mutations to the genome and selection in terms of adaptation to the surrounding environment. Directed evolution is, in other words, a method of evolving a protein in vitro with an accelerated time course. Here, we review the general process of directed evolution. First, a random mutation is introduced into the gene of the protein of interest to prepare a mutant library. One of the common methods for mutagenesis is error-prone PCR using low-fidelity DNA polymerase. DNA shuffling and StEP are other methods to obtain a chimeric gene through realignment of mutated DNA fragments by a PCR reaction [
      • Packer M.S.
      • Liu D.R.
      Methods for the directed evolution of proteins.
      ]. The mutant library is then introduced into a host, such as yeast, Escherichia coli, phage, or mammalian cells, to express mutant proteins. Yeast has advantages in the library size, resistance to incorporation of multiple mutants due to the exclusive nature of different plasmids, and established systems for displaying the proteins on the cell surface. By contrast, screening with mammalian cells allows analysis of a wide variety of cellular phenotypes that include virus infection. More recently, a method for autonomously introducing random mutations in the host cell was developed. This method optimized base editor and protospacer adjacent motif (PAM)-free nuclease in CRISPR genome editing [
      • Richter M.F.
      • et al.
      Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity.
      ]. Here, after obtaining library cells, screening is performed under the desired selective pressure. In case of binding affinity analysis, it is useful to pool the library and collect the target cell population by fluorescence-activated cell sorting. The target phenotypic cells are collected and random mutations are reintroduced in the extracted DNA for the next round of mutagenesis and selection. The desired function becomes stronger upon repetition of this cycle.
      Figure 2
      Figure 2Directed 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 [
      • Higuchi Y.
      • et al.
      Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.
      ]. 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.
      Table 1Summary of ACE2 mutants to neutralize SARS-CoV-2
      NameMutations for affinity

      (mutations for enzyme)
      LengthFcKD (nM)IC50 (μg/ml)Efficacy for omicronAnimal model, virus strain, delivery time, route and doseRefs
      Directed evolution
      3N39v2A25V/K31N/E35K/L79F

      (WT)
      615WT0.64

      17.6 (WT)
      0.082

      24.8 (WT)
      Hamster, Wuhan, post-infection, 20mg/kg i.p.[
      • Higuchi Y.
      • et al.
      Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.
      ]
      3J113v2K31M/E35K/Q60R/L79F

      (WT)
      1.140.33
      3J320v2T20I/H34A/T92Q/Q101H

      (WT)
      3.980.068Validated in in vitro
      3N39v4A25V/K31N/E35K/T92Q

      (S138C/V343C)
      740WTN.D.0.036

      0.45 (WT)
      Validated in miceHamster and CAG-hACE2 mice, Omicron, post-infection, 20mg/kg i.p.[
      • Ikemura N.
      • et al.
      An engineered ACE2 decoy neutralizes the SARS-CoV-2 Omicron variant and confers protection against infection in vivo.
      ]
      CDY14HLK31M/E35K/S47A/L79F/L91P/N330Y

      (H345L)
      615IgG40.031
      Avidity value of ACE2 dimer with collectrin domain and/or Fc.


      29
      Avidity value of ACE2 dimer with collectrin domain and/or Fc.
      (WT)
      0.037

      11(WT)
      Validated in vitroK18-hACE2 mice, rhesus/cynomolgus macaques, AAV, pre-infection[
      • Sims J.J.
      • et al.
      Intranasal gene therapy to prevent infection by SARS-CoV-2 variants.
      ]
      Deep mutational scanning
      sACE2v2.4T27Y/L79T/N330Y

      (WT)
      740WT0.6
      Avidity value of ACE2 dimer with collectrin domain and/or Fc.


      22
      Avidity value of ACE2 dimer with collectrin domain and/or Fc.
      (WT)
      N.D.Validated

      in in vitro
      K18-hACE2 mice, Wuhan & Gamma, pre-/post-infection, 10 mg/kg i.v., Inhalation 3×[
      • Zhang L.
      • et al.
      An engineered ACE2 decoy receptor can be administered by inhalation and potently targets the BA.1 and BA.2 omicron variants of SARS-CoV-2.
      ,
      • Chan K.K.
      • et al.
      Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2.
      ,
      • Zhang L.
      • et al.
      Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants.
      ]
      Computational design
      CVD293K31F/H34I/E35Q

      (WT)
      740WT0.23
      Avidity value of ACE2 dimer with collectrin domain and/or Fc.


      2.8
      Avidity value of ACE2 dimer with collectrin domain and/or Fc.
      (WT)
      0.036

      0.7(WT)
      [
      • Glasgow A.
      • et al.
      Engineered ACE2 receptor traps potently neutralize SARS-CoV-2.
      ]
      CVD313K31F/N33D/H34S/E35Q

      (H345L)
      N.D.0.028
      M81L79F/M82Y/Q325Y

      (H374A/H378A)
      740GASDALIE
      GASDALIE: G236A/S239D/A330L/I332E, LALA: L234A/L235A, LALA-PG: L234A/L235A/P329G.
      2.74
      Avidity value of ACE2 dimer with collectrin domain and/or Fc.


      26.0
      Avidity value of ACE2 dimer with collectrin domain and/or Fc.
      (WT)
      N.D.K18-hACE2 mice, Wuhan, pre-/post-infection, 12.5 mg/kg i.n., 6.25 mg/kg i.v.[
      • Chen Y.
      • et al.
      Engineered ACE2-Fc counters murine lethal SARS-CoV-2 infection through direct neutralization and Fc-effector activities.
      ]
      FFWFS19F/T27F/K31W/N330F

      (WT)
      740WT1.8
      Avidity value of ACE2 dimer with collectrin domain and/or Fc.


      16
      Avidity value of ACE2 dimer with collectrin domain and/or Fc.
      (WT)
      N.D.[
      • Havranek B.
      • et al.
      Computationally designed ACE2 decoy receptor binds SARS-CoV-2 spike (S) protein with tight nanomolar affinity.
      ]
      S19W/N330Y

      (H374A/H378A/E402A)
      615WT10.1

      81.8 (WT)
      0.09

      15.5 (WT)
      [
      • Ye F.
      • et al.
      S19W, T27W, and N330Y mutations in ACE2 enhance SARS-CoV-2 S-RBD binding toward both wild-type and antibody-resistant viruses and its molecular basis.
      ]
      S19W/T27W/N330Y

      (H374A/H378A/E402A)
      19.30.53
      Q24E/T27K/H34S/N49E/N90D

      (H374N/H378N)
      615WT5.45
      Avidity value of ACE2 dimer with collectrin domain and/or Fc.


      11.6
      Avidity value of ACE2 dimer with collectrin domain and/or Fc.
      (WT)
      0.39

      0.82 (WT)
      [
      • Mou H.
      • et al.
      Mutations derived from horseshoe bat ACE2 orthologs enhance ACE2-Fc neutralization of SARS-CoV-2.
      ]
      Deglycosylation

      (WT)
      N.AWT7.6
      Avidity value of ACE2 dimer with collectrin domain and/or Fc.


      16.2
      Avidity value of ACE2 dimer with collectrin domain and/or Fc.
      (WT)
      N.D.[
      • Capraz T.
      • et al.
      Structure-guided glyco-engineering of ACE2 for improved potency as soluble SARS-CoV-2 decoy receptor.
      ]
      Multimer ACE2 and others
      WT

      (H374N/H378N)
      740LALA-PG
      GASDALIE: G236A/S239D/A330L/I332E, LALA: L234A/L235A, LALA-PG: L234A/L235A/P329G.
      11.5
      Avidity value of ACE2 dimer with collectrin domain and/or Fc.


      18.3
      Avidity value of ACE2 dimer with collectrin domain and/or Fc.
      (WT)
      4.1 nM

      5.1 nM (WT)
      Hamster, Wuhan, post-infection, 5/50 mg/kg i.p.[
      • Ferrari M.
      • et al.
      Characterization of a novel ACE2-based therapeutic with enhanced rather than reduced activity against SARS-CoV-2 variants.
      ]
      MDR504WT

      (H345A)
      740LALA
      GASDALIE: G236A/S239D/A330L/I332E, LALA: L234A/L235A, LALA-PG: L234A/L235A/P329G.
      N.D.3.5nM
      IC50 value against authentic virus.


      7.5nM
      IC50 value against authentic virus.
      (WT)
      Ad5-hACE2-transduced/K18-ACE2 mice, Wuhan, pre-/post-infection, 15/30 mg/kg i.v.[
      • Iwanaga N.
      • et al.
      ACE2-IgG1 fusions with improved in vitro and in vivo activity against SARS-CoV-2.
      ]
      WT

      (H374N/H378N)
      N.AWT8.23
      Avidity value of ACE2 dimer with collectrin domain and/or Fc.


      1.59
      Avidity value of ACE2 dimer with collectrin domain and/or Fc.
      (WT)
      4.17

      1.02 (WT)
      Ad5-hACE2-transduced mice, Wuhan, pre-/post-infection, 50mg/kg i.p.[
      • Zhang Z.
      • et al.
      Potent prophylactic and therapeutic efficacy of recombinant human ACE2-Fc against SARS-CoV-2 infection in vivo.
      ]
      T27W-foldon

      (WT)
      615WT0.06

      76.8 (WT)
      0.21

      24.1 (WT)
      [
      • Xiao T.
      • et al.
      A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent.
      ]
      D30E-tetramer/hexamer

      (WT)
      740WTN.D.0.07/0.06 nM

      0.68 nM (WT)
      [
      • Li Y.
      • et al.
      SARS-CoV-2 and three related coronaviruses utilize multiple ACE2 orthologs and are potently blocked by an improved ACE2-Ig.
      ]
      a Avidity value of ACE2 dimer with collectrin domain and/or Fc.
      b GASDALIE: G236A/S239D/A330L/I332E, LALA: L234A/L235A, LALA-PG: L234A/L235A/P329G.
      c IC50 value against authentic virus.
      Figure 3
      Figure 3Location of mutation sites for affinity enhancement in angiotensin-converting enzyme (ACE)2.
      ACE2 and receptor-binding domain (RBD) in the complex structure (PDB: 6m0j [
      • Lei C.
      • et al.
      Neutralization of SARS-CoV-2 spike pseudotyped virus by recombinant ACE2-Ig.
      ,
      • Lan J.
      • et al.
      Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor.
      ]) 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.

      DMS for comprehensive mutagenesis analysis

      DMS is an approach to comprehensively evaluate the effect of all possible single amino acid substitutions on a protein property parameter such as binding affinity. Procko and coworkers developed a single mutant ACE2 library encompassing all 20 amino acid substitutions at 117 sites, that includes the interface with the SARS-CoV-2 spike RBD as well as those involved in ACE2 enzymatic activity. The results of this study revealed the advantage of N-glycosylation removal at N90 and amino acid substitutions at several other important sites that can increase affinity. Although the original paper mentioned no detailed methods for synergetic combination of affinity-enhancing single amino acid substitutions, several candidates made up of three or four mutations were further analyzed. Soluble ACE2 (sACE2)v2.4, containing mutations T27Y/L79T/N330Y and fused to the collectrin domain (amino acids 18–740), a homolog of collectrin (Tmem27) which induces ACE2 dimer formation [
      • Zhang H.
      • et al.
      Collectrin, a collecting duct-specific transmembrane glycoprotein, is a novel homolog of ACE2 and is developmentally regulated in embryonic kidneys.
      ], gained >10-fold higher virus-neutralizing activity compared to wild-type ACE2 dimer, with ~40-fold enhanced monomeric affinity toward SARS-CoV-2 RBD (Table 1) [
      • Chan K.K.
      • et al.
      Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2.
      ]. Although DMS provides large-scale datasets of single amino acid substitutions to reveal intrinsic protein properties, the limitation of this approach is the lack of established method to know which mutations can be combined synergistically. One solution is the use of computational modeling to structurally predict the effective combination without compromising protein stability or binding free energy [
      • Jing W.
      • Procko E.
      ACE2-based decoy receptors for SARS coronavirus 2.
      ]. The other is the machine learning-guided approach. Datasets from DMS and empirical affinity test for multiple mutations are used to train machine learning algorithm, and the resultant machine learning model predicts the affinity value for all possible combination of mutations [
      • Chu H.Y.
      • Wong A.S.L.
      Facilitating machine learning-guided protein engineering with smart library design and massively parallel assays.
      ,
      • Saito Y.
      • et al.
      Machine-learning-guided library design cycle for directed evolution of enzymes: the effects of training data composition on sequence space exploration.
      ].

      Computational design with growing sophistication

      There has been progress in computer-aided drug design using structure-based modeling and molecular dynamics simulation in various drug modalities including small compounds, peptides, and protein biologics [
      • Temml V.
      • Kutil Z.
      Structure-based molecular modeling in SAR analysis and lead optimization.
      ]. Following the emergence of the COVID-19 pandemic, many protein structures related to COVID-19 have been rapidly deposited in some virtual databases, such as the Protein Data Bank, helping drug development efforts. Pazgier and coworkers took advantage of high-resolution ACE2-RBD structures (6m0j [
      • Lan J.
      • et al.
      Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor.
      ] and 6vw1 [
      • Shang J.
      • et al.
      Structural basis of receptor recognition by SARS-CoV-2.
      ]) to design and generate a high-affinity ACE2 [
      • Chen Y.
      • et al.
      Engineered ACE2-Fc counters murine lethal SARS-CoV-2 infection through direct neutralization and Fc-effector activities.
      ]. Noncovalent intermolecular forces, such as van der Waals force, electrostatic interactions, hydrogen bonding, and hydrophobic interactions, play essential roles in determining the protein-binding force [
      • Gong X.
      • et al.
      Direct measurements of particle-surface interactions in aqueous solutions with total internal reflection microscopy.
      ]. On the basis of these noncovalent interactions of amino acid side chains at the interface between SARS-CoV-2 RBD and ACE2, they discovered that ACE2 mutant, M81 with L79F/M82Y/Q325Y substitutions obtained an ~10-fold increased affinity for the spike RBD over wild-type ACE2 (Table 1) [
      • Chen Y.
      • et al.
      Engineered ACE2-Fc counters murine lethal SARS-CoV-2 infection through direct neutralization and Fc-effector activities.
      ]. Also, Lu and coworkers performed structure-based computational virtual screening at the ACE2–RBD interface. Their algorithm identified S19W, T27W, and N330Y in ACE2 protein as key substitutions and their combinations achieved up to an eightfold increase in the affinity toward SARS-CoV-2 RBD over wild-type ACE2 (Table 1) [
      • Ye F.
      • et al.
      S19W, T27W, and N330Y mutations in ACE2 enhance SARS-CoV-2 S-RBD binding toward both wild-type and antibody-resistant viruses and its molecular basis.
      ].
      Molecular dynamics simulation can provide plentiful kinetic information and calculate thermodynamic properties for the protein–drug interactions. [
      • Decherchi S.
      • Cavalli A.
      Thermodynamics and kinetics of drug-target binding by molecular simulation.
      ]. Stadlmann and coworkers focused on glycan-mediated interactions between ACE2 and the spike protein [
      • Capraz T.
      • et al.
      Structure-guided glyco-engineering of ACE2 for improved potency as soluble SARS-CoV-2 decoy receptor.
      ]. In silico modeling and molecular dynamics simulations of fully glycosylated proteins have suggested that the spike–ACE2 complex formation is disrupted by glycans attached at N90 and N322 of ACE2. Also, the substitution of T92Q or N322Q to remove the respective glycans resulted in up to twofold increased affinity compared to wild-type ACE2. Full enzymatic deglycosylation further increased affinity for the spike RBD, as well as neutralization activity against SARS-CoV-2 (Table 1). Islam and coworkers also conducted computational mutagenesis and subsequent molecular dynamics for free-energy calculations on ACE2-SARS-CoV2/RBD interaction. The resultant ACE2 mutant, FFWF composed of S19F/T27F/K31W/N330F substitutions, achieved ninefold affinity enhancement over the wild-type ACE2 [
      • Havranek B.
      • et al.
      Computationally designed ACE2 decoy receptor binds SARS-CoV-2 spike (S) protein with tight nanomolar affinity.
      ].
      Multiple sequence alignment (MSA) is generally the alignment of three or more biological protein or DNA sequences, which infers the homology and the evolutionary relationships between the sequences [
      • Kapli P.
      • et al.
      Phylogenetic tree building in the genomic age.
      ]. Farzan and coworkers focused on the ACE2 protein sequences of horseshoe bats, because horseshoe bats are candidate reservoirs of SARS-CoV-2 and their spike RBD may have evolved to bind to horseshoe bat ACE2 [
      • Mou H.
      • et al.
      Mutations derived from horseshoe bat ACE2 orthologs enhance ACE2-Fc neutralization of SARS-CoV-2.
      ]. Computational MSA of horseshoe-bat ACE2 revealed five consensus residues, Q24E/T27K/H34S/N49E/N90D. These residues were introduced in human ACE2, and they showed enhanced binding and neutralization efficacy (Table 1) [
      • Mou H.
      • et al.
      Mutations derived from horseshoe bat ACE2 orthologs enhance ACE2-Fc neutralization of SARS-CoV-2.
      ].
      Some other studies have combined multiple approaches to maximize the ACE2 binding toward SARS-CoV-2 RBD. Wells and coworkers combined computational modeling with sequential directed evolution based on yeast surface display [
      • Glasgow A.
      • et al.
      Engineered ACE2 receptor traps potently neutralize SARS-CoV-2.
      ]. They determined important amino acid side chains for binding of ACE2 to SARS-CoV-2 spike RBD by computational alanine scanning on the binding interface. Further free-energy calculations and computational saturation mutagenesis generated ACE2 mutant, CVD293 harboring K31F/H34I/E35Q substitutions. Directed evolution following computational design revealed another mutant, CVD313 with K31F/N33D/H34S/E35Q substitutions, which exhibited binding affinity and virus neutralization efficacy comparable to 3N39v2 [
      • Higuchi Y.
      • et al.
      Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.
      ] and sACE2v2.4 [
      • Chan K.K.
      • et al.
      Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2.
      ].
      Currently, computational design generates only ACE2 mutants with weaker affinity to SARS-CoV-2 RBD compared with cell-based mutagenesis, and requires further fine adjustment from wet laboratory experiments [
      • Higuchi Y.
      • et al.
      Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.
      ,
      • Sims J.J.
      • et al.
      Intranasal gene therapy to prevent infection by SARS-CoV-2 variants.
      ,
      • Chan K.K.
      • et al.
      Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2.
      ,
      • Chen Y.
      • et al.
      Engineered ACE2-Fc counters murine lethal SARS-CoV-2 infection through direct neutralization and Fc-effector activities.
      ,
      • Ye F.
      • et al.
      S19W, T27W, and N330Y mutations in ACE2 enhance SARS-CoV-2 S-RBD binding toward both wild-type and antibody-resistant viruses and its molecular basis.
      ,
      • Havranek B.
      • et al.
      Computationally designed ACE2 decoy receptor binds SARS-CoV-2 spike (S) protein with tight nanomolar affinity.
      ,
      • Glasgow A.
      • et al.
      Engineered ACE2 receptor traps potently neutralize SARS-CoV-2.
      ]. However, the field of protein structure prediction and design is rapidly evolving, and new technologies based on deep learning that trains artificial neural network to approximate complicated unknown functions in a high-dimensional abstract space [
      • Ding W.
      • et al.
      Protein design via deep learning.
      ], are expected to play a major role in the near future.

      Fc fusion to manipulate pharmacokinetics, immunological activity, and avidity effect

      Fusion with the antibody Fc domain is a major modification to enhance drug concentration in target tissues and therapeutic effects. Many biologically active proteins or peptides have short serum half-lives due to their constant cellular uptake and subsequent lysosomal degradation, mainly in endothelial cells [
      • Kontermann R.E.
      Strategies for extended serum half-life of protein therapeutics.
      ]. Human IgG antibody has a half-life of ~21 days. Its Fc domain allows it to bind to the neonatal Fc receptor (FcRn) in a pH-dependent manner. In pinocytosis-mediated IgG internalization and subsequent endosomal sorting, binding of Fc to FcRn at endosomal pH (5.5–6.0) prevents IgG from being directed to the endolysosome for degradation and enables its sorting to the plasma membrane. Upon fusion with the plasma membrane, IgG is released from FcRn at extracellular pH 7.4 and recycled back into circulation, thus prolonging its serum half-life [
      • Huang C.
      Receptor-Fc fusion therapeutics, traps, and MIMETIBODY technology.
      ,
      • Lee C.H.
      • et al.
      An engineered human Fc domain that behaves like a pH-toggle switch for ultra-long circulation persistence.
      ]. For ACE2 decoys, IgG1-Fc fusion extended the half-life from ~3 to ~30 h, and enabled treatment of COVID-19 in animal models [
      • Higuchi Y.
      • et al.
      Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.
      ,
      • Haschke M.
      • et al.
      Pharmacokinetics and pharmacodynamics of recombinant human angiotensin-converting enzyme 2 in healthy human subjects.
      ,
      • Zhang L.
      • et al.
      Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants.
      ].
      Also, IgG-Fc domain engages with Fcγ receptor (FcγR) family expressed on effector leukocytes and triggers the clearance of viral particles, cytotoxic elimination of virus-infected cells, and induction of antiviral T cell responses [
      • Yamin R.
      • et al.
      Fc-engineered antibody therapeutics with improved anti-SARS-CoV-2 efficacy.
      ]. These Fc- and FcγR-mediated functions of antibody are termed Fc-effector function [
      • van Erp E.A.
      • et al.
      Fc-mediated antibody effector functions during respiratory syncytial virus infection and disease.
      ]. The importance of Fc-effector function is especially recognized in tumor immunotherapy [
      • DiLillo D.J.
      • Ravetch J.V.
      Differential Fc-receptor engagement drives an anti-tumor vaccinal effect.
      ] and some mAbs already approved for clinical use are engineered to enhance Fc-effector function for efficient elimination of cancer cells [
      • van der Horst H.J.
      • et al.
      Fc-engineered antibodies with enhanced Fc-effector function for the treatment of B-cell malignancies.
      ]. In case of virus diseases, FcγR interaction also has a risk of antibody-dependent enhancement (ADE) of virus infection to FcγR-positive immune cells, which has been, in fact, observed during secondary infection with dengue virus [
      • Narayan R.
      • Tripathi S.
      Intrinsic ADE: the dark side of antibody dependent enhancement during dengue infection.
      ]. However, there are studies that indicate that there is no definite risk of ADE of SARS-CoV-2 infection and that Fc-effector function is important in the therapeutic and prophylactic administration of mAbs. Mouse studies have shown that mAbs containing mutated Fc lacking ability to bind to FcγR lose therapeutic efficacy and that mutations enhancing Fc-effector function further promote therapeutic effects of SARS-CoV-2 neutralizing mAbs [
      • Yamin R.
      • et al.
      Fc-engineered antibody therapeutics with improved anti-SARS-CoV-2 efficacy.
      ,
      • Bournazos S.
      • et al.
      Fc-optimized antibodies elicit CD8 immunity to viral respiratory infection.
      ,
      • Winkler E.S.
      • et al.
      Human neutralizing antibodies against SARS-CoV-2 require intact Fc effector functions for optimal therapeutic protection.
      ]. Even in the prophylaxis of virus infection that mainly requires neutralizing activity [
      • Winkler E.S.
      • et al.
      Human neutralizing antibodies against SARS-CoV-2 require intact Fc effector functions for optimal therapeutic protection.
      ,
      • Ju B.
      • et al.
      Human neutralizing antibodies elicited by SARS-CoV-2 infection.
      ], another study has reported that mAbs with mutations enhancing Fc-effector function improve prevention of COVID-19, similar to therapeutic administration [
      • Yamin R.
      • et al.
      Fc-engineered antibody therapeutics with improved anti-SARS-CoV-2 efficacy.
      ].
      Substitution of critical amino acids in Fc to modulate binding to specific receptors have been applied in clinical mAbs against COVID-19 considering the risk of ADE of virus infection, even though mouse studies and SARS-CoV-2 vaccine efficacy have disproved it. Etesevimab and cilgavimab (AZD1061) have LALA (L234A/L235A) and TM (L234F/L235E/P331S) mutations, respectively, and both mutations inhibit the binding of Fc to FcγR [
      • Corti D.
      • et al.
      Tackling COVID-19 with neutralizing monoclonal antibodies.
      ]. Also, to extend the half-life of COVID-19 mAbs, sotrovimab (VIR-7831) and tixagevimab (AZD8895) contain FcRn-oriented LS (M428L/N434S) and YTE (M252Y/S254T/T256) mutations, respectively, [
      • Lee C.H.
      • et al.
      An engineered human Fc domain that behaves like a pH-toggle switch for ultra-long circulation persistence.
      ,
      • Corti D.
      • et al.
      Tackling COVID-19 with neutralizing monoclonal antibodies.
      ]. One open question in this area is whether enhancement of Fc-effector function or FcRn orientation is more suitable for COVID-19 therapy and prophylaxis. Another question is whether these Fc mutations have synergistic effects on antiviral efficacy. VIR-7832 is a derivative of sotrovimab (VIR-7831) and carries FcRn-oriented LS (M428L/N434S) mutation, as well as GAALIE (G236A/ A330L/I332E) mutation to enhance Fc-effector function [
      • Cathcart A.L.
      • et al.
      The dual function monoclonal antibodies VIR-7831 and VIR-7832 demonstrate potent in vitro and in vivo activity against SARS-CoV-2.
      ]. VIR-7832 is currently being evaluated in a Phase 2 study, directly comparing it with its parent, sotrovimab (VIR-7831), to assess the potential synergistic benefit of Fc-effector function and FcRn-related half-life extension in humans (NCT04746183).
      Fusing IgG-Fc domain to other proteins also enhances avidity, that is the overall binding strength of protein interaction, due to bivalent target binding domain. Further increasing the valency of target binding domain by making quadrivalent and hexavalent sACE2-Fc proved beneficial in achieving more efficient neutralization [
      • Li Y.
      • et al.
      SARS-CoV-2 and three related coronaviruses utilize multiple ACE2 orthologs and are potently blocked by an improved ACE2-Ig.
      ,
      • Miller A.
      • et al.
      A super-potent tetramerized ACE2 protein displays enhanced neutralization of SARS-CoV-2 virus infection.
      ]. Trivalent sACE2 is also effective when spatially aligned to the spike trimer [
      • Guo L.
      • et al.
      Engineered trimeric ACE2 binds viral spike protein and locks it in “Three-up” conformation to potently inhibit SARS-CoV-2 infection.
      ,
      • Xiao T.
      • et al.
      A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent.
      ]. These multivalent approaches are promising but these benefits need to be weighed against disadvantages such as low production yield.

      Dual aspects of ACE2 enzymatic activity on drugs for COVID-19

      ACE2 enzymatic activity has both the potential of lung protection and risk of adverse effects. Considering the therapeutic potential of rhACE2 for ARDS via conversion of proinflammatory Ang II to Ang 1–7, the enzymatic activity of the ACE2 decoy is expected to ameliorate lung inflammatory injury due to SARS-CoV-2 infection. Although there is extensive evidence to support the efficacy of rhACE2 in animal models of ARDS [
      • Zhang H.
      • Baker A.
      Recombinant human ACE2: acing out angiotensin II in ARDS therapy.
      ,
      • Kuba K.
      • et al.
      Angiotensin-converting enzyme 2 (ACE2) in the pathogenesis of ARDS in COVID-19.
      ], Phase 2 trials have found no improvement in clinical outcomes of ventilated ARDS patients. It is possible that the dosage of rhACE2 may have been insufficient to show beneficial effects [
      • Zhang H.
      • Baker A.
      Recombinant human ACE2: acing out angiotensin II in ARDS therapy.
      ]. If ACE2 decoy were to go to the clinic, the estimated dose of ACE2 decoy for COVID-19 would be ~tenfold higher than that used in ARDS clinical trials [
      • Higuchi Y.
      • et al.
      Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.
      ,
      • Khan A.
      • et al.
      A pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome.
      ]. This dose may be sufficient to show protective effects against lung inflammation [
      • Zhang L.
      • et al.
      An engineered ACE2 decoy receptor can be administered by inhalation and potently targets the BA.1 and BA.2 omicron variants of SARS-CoV-2.
      ]; however, enzymatic activity could also lead to hemodynamic instability and unexpected adverse effects due to overconversion of Ang II to Ang 1–7. One approach to resolve the excessive enzymatic activity is the elimination of ACE2 metallopeptidase activity. Disruption of ACE2 zinc-binding motif, responsible and critical for its metallopeptidase activity inactivates it. Following this concept, H374N/H378N substitution was used in some studies; however, ACE2 without zinc-binding motif becomes structurally unstable [
      • Lei C.
      • et al.
      Neutralization of SARS-CoV-2 spike pseudotyped virus by recombinant ACE2-Ig.
      ,
      • Glasgow A.
      • et al.
      Engineered ACE2 receptor traps potently neutralize SARS-CoV-2.
      ,
      • Zhang Z.
      • et al.
      Potent prophylactic and therapeutic efficacy of recombinant human ACE2-Fc against SARS-CoV-2 infection in vivo.
      ,
      • Ferrari M.
      • et al.
      Characterization of a novel ACE2-based therapeutic with enhanced rather than reduced activity against SARS-CoV-2 variants.
      ]. Another strategy is introducing a mutation in ACE2 to block the binding with its substrate, Ang II. Wells and coworkers successfully designed a ACE2 decoy with H345L mutation. This mutation preserved ACE2 decoy thermostability and clearly blocked Ang II binding [
      • Glasgow A.
      • et al.
      Engineered ACE2 receptor traps potently neutralize SARS-CoV-2.
      ]. The introduction of a disulfide bond with S128C/V343C substitutions in ACE2 can also obstruct ACE2 substrate binding by closing the binding pocket [
      • Higuchi Y.
      • et al.
      Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.
      ]. The resultant ACE2 closed conformation not only completely eliminates enzymatic activity, but also greatly improves thermostability, which is a desirable attribute for biotherapeutics.

      Broad-spectrum efficacy against SARS-CoV-2 variants and sarbecoviruses

      As with most neutralizing antibodies, the main therapeutic target for ACE2 decoys is blocking the interaction of virus with native ACE2. In contrast to therapeutic mAbs, the concern of viral escape is expected to be low for decoy receptors. Virus mutants escaping from decoy receptors would have limited binding affinity toward the native ACE2 receptors, which results in impaired infectivity. This idea is supported by a recent study where ACE2 decoy receptors showed no signs of emerging escape mutants when treated at sparing concentration during long-term SARS-CoV-2 culture [
      • Higuchi Y.
      • et al.
      Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.
      ]. Although many existing mAbs showed less neutralization efficacy against omicron, ACE2 decoy versions fused with IgG1-Fc (3N39v4, 3J320v2, and sACE2v2.4) exhibited similar neutralization activity against the original Wuhan strain and multiple variants, including omicron in cultured cells [
      • Ikemura N.
      • et al.
      An engineered ACE2 decoy neutralizes the SARS-CoV-2 Omicron variant and confers protection against infection in vivo.
      ]. Comprehensive mapping by DMS of ACE2 and the spike RBD is a useful tool not only to improve binding affinity, but also to identify escape mutations from immunized sera and neutralizing agents [
      • Chan K.K.
      • et al.
      An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants.
      ,
      • Chan K.K.
      • et al.
      Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2.
      ,
      • Starr T.N.
      • et al.
      Deep mutational scanning of SARS-CoV-2 receptor binding domain reveals constraints on folding and ACE2 binding.
      ,
      • Starr T.N.
      • et al.
      Prospective mapping of viral mutations that escape antibodies used to treat COVID-19.
      ,
      • Greaney A.J.
      • et al.
      Antibodies elicited by mRNA-1273 vaccination bind more broadly to the receptor binding domain than do those from SARS-CoV-2 infection.
      ]. DMS of SARS-CoV-2 spike RBD to evaluate escape from the 3N39v4 decoy revealed that L455Y and N487Q mutations in the RBD partially reduced the neutralization efficacy of both 3N39v4 and wild-type ACE2 decoys. However, the 3N39v4 decoy preserved functional neutralization against viruses harboring these mutations in the RBD, indicating that no single-residue mutation in RBD achieved complete escape from engineered ACE2 decoy receptors, unlike in the case of mAbs [
      • Ikemura N.
      • et al.
      An engineered ACE2 decoy neutralizes the SARS-CoV-2 Omicron variant and confers protection against infection in vivo.
      ].
      In theory, ACE2 decoys would neutralize other sarbecoviruses that use ACE2 as an entry receptor. In fact, 3N39v4 decoy shows therapeutic efficacy against both SARS-CoV-1 and SARS-CoV-2 clades, including pangolin CoV (GD-1 and GX-P5L) and bat CoV (RaTG13, RsSHC014, and WIV1) [
      • Ikemura N.
      • et al.
      An engineered ACE2 decoy neutralizes the SARS-CoV-2 Omicron variant and confers protection against infection in vivo.
      ]. sACE2v2.4 decoy also exhibit a better KD value for the RBD of some sarbecoviruses than wild-type ACE2 decoy does [
      • Chan K.K.
      • et al.
      An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants.
      ]. These results indicate the potential efficacy of engineered ACE2 receptor decoys against future zoonotic coronavirus diseases [
      • Rappazzo C.G.
      • et al.
      Broad and potent activity against SARS-like viruses by an engineered human monoclonal antibody.
      ].

      Antiviral effects of ACE2 decoy receptors in animal models

      Pharmacokinetics and tissue absorption are important factors affecting in vivo therapeutic efficacy. The half-life of rhACE2 (i.e., non-Fc fusion) is reported to be ~3 h in humans [
      • Haschke M.
      • et al.
      Pharmacokinetics and pharmacodynamics of recombinant human angiotensin-converting enzyme 2 in healthy human subjects.
      ,
      • Huentelman M.J.
      • et al.
      Structure-based discovery of a novel angiotensin-converting enzyme 2 inhibitor.
      ]. However, fusion with wild-type IgG1-Fc and LALA-mutated Fc extended the half-life to 30–145 h in mice [
      • Higuchi Y.
      • et al.
      Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.
      ,
      • Ferrari M.
      • et al.
      Characterization of a novel ACE2-based therapeutic with enhanced rather than reduced activity against SARS-CoV-2 variants.
      ,
      • Iwanaga N.
      • et al.
      ACE2-IgG1 fusions with improved in vitro and in vivo activity against SARS-CoV-2.
      ]. Lung distribution was also sufficient to achieve effective neutralization [
      • Higuchi Y.
      • et al.
      Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.
      ,
      • Iwanaga N.
      • et al.
      ACE2-IgG1 fusions with improved in vitro and in vivo activity against SARS-CoV-2.
      ]. Wild-type ACE2 decoys composed of wild-type or LALA-mutated Fc protect lungs from the original Wuhan strain in both prophylactic pre-infection and therapeutic post-infection regimens in hamsters [
      • Ferrari M.
      • et al.
      Characterization of a novel ACE2-based therapeutic with enhanced rather than reduced activity against SARS-CoV-2 variants.
      ], as well as K18-hACE2 transgenic or Ad5-hACE2 transduced mice (Table 1) [
      • Iwanaga N.
      • et al.
      ACE2-IgG1 fusions with improved in vitro and in vivo activity against SARS-CoV-2.
      ]. The 3N39v4 decoy also exhibits therapeutic efficacy in hamsters infected with Wuhan [
      • Higuchi Y.
      • et al.
      Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.
      ], as well as omicron [
      • Ikemura N.
      • et al.
      An engineered ACE2 decoy neutralizes the SARS-CoV-2 Omicron variant and confers protection against infection in vivo.
      ] strains. sACE2v2.4 decoy was evaluated in epithelium-specific human ACE2 expressing (K18 hACE2 transgenic) mice that exhibited severe lung edema and death upon SARS-CoV-2 infection [
      • Oladunni F.S.
      • et al.
      Lethality of SARS-CoV-2 infection in K18 human angiotensin-converting enzyme 2 transgenic mice.
      ]. Prophylactic and therapeutic administration of sACE2v2.4 decoy improved pneumonia and survival from Wuhan and gamma strain infections (Table 1) [
      • Zhang L.
      • et al.
      Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants.
      ]. sACE2v2.4 and 3N39v4 decoys exhibited antiviral effects at a dose of 10–20 mg/kg in rodent models of COVID-19, similar to therapeutic antibodies [
      • Baum A.
      • et al.
      REGN-COV2 antibodies prevent and treat SARS-CoV-2 infection in rhesus macaques and hamsters.
      ,
      • Kreye J.
      • et al.
      A therapeutic non-self-reactive SARS-CoV-2 antibody protects from lung pathology in a COVID-19 hamster model.
      ,
      • Su W.
      • et al.
      Neutralizing monoclonal antibodies that target the spike receptor binding domain confer Fc receptor-independent protection against SARS-CoV-2 infection in Syrian hamsters.
      ]. Considering the successful clinical outcome of mAbs [
      • Weinreich D.M.
      • et al.
      REGEN-COV antibody combination and outcomes in outpatients with Covid-19.
      ,
      • Gottlieb R.L.
      • et al.
      Effect of bamlanivimab as monotherapy or in combination with etesevimab on viral load in patients with mild to moderate COVID-19: a randomized clinical trial.
      ,
      • Boggiano C.
      • et al.
      Update on and future directions for use of anti-SARS-CoV-2 antibodies: National Institutes of Health summit on treatment and prevention of COVID-19.
      ], ACE2 decoy receptors are also expected to have sufficient efficacy to treat COVID-19.
      Improved pharmacokinetics of ACE2 decoys has the potential to ameliorate lung inflammation due to effective conversion of Ang II to Ang 1–7 by sustained ACE2 enzymatic activity. A recent study demonstrated additional therapeutic benefits of the ACE2 bioactive form in the lethal COVID-19 model of K18-hACE2 transgenic mice [
      • Zhang L.
      • et al.
      An engineered ACE2 decoy receptor can be administered by inhalation and potently targets the BA.1 and BA.2 omicron variants of SARS-CoV-2.
      ]. However, the superiority of bioactive form should be comprehensively evaluated considering parameters that include structural thermostability and risks of unexpected adverse effects.
      Therapeutic mAbs are typically administered by intravenous or intramuscular injection, but such systemic delivery is inefficient in achieving optimal drug concentration at local sites of infection. Liquid aerosol inhalation is a promising noninvasive strategy to enhance efficacy against respiratory infections like COVID-19. The major challenge of inhalation is the short half-life, 16–24 h for inhaled mAbs in humans [
      • Parray H.A.
      • et al.
      Inhalation monoclonal antibody therapy: a new way to treat and manage respiratory infections.
      ]. Even though the clearance rate is fast, as shown in a study by Kobie and coworkers using a hamster model of COVID-19, the inhalation delivery route achieved ~100-fold more efficient delivery to the lungs than intravenous administration did. The beneficial therapeutic effect was observed in a single inhaled dose of only 0.6 mg/kg [
      • Piepenbrink M.S.
      • et al.
      Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters.
      ]. Inhalation of engineered ACE2 decoy receptors exhibited similar efficacy with an estimated dose of ~0.5 mg/kg in mice [
      • Zhang L.
      • et al.
      An engineered ACE2 decoy receptor can be administered by inhalation and potently targets the BA.1 and BA.2 omicron variants of SARS-CoV-2.
      ]. Since the aerosol drug delivery rate to lungs is ~50% in humans [
      • Piepenbrink M.S.
      • et al.
      Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters.
      ,
      • Kleinstreuer C.
      • et al.
      Targeted drug-aerosol delivery in the human respiratory system.
      ], inhalation is expected to have dose-sparing benefits. Moreover, it is convenient for self-administration on an outpatient basis or at home; preventing hospitalization to conserve critical hospital resources, especially during waves of infection. Several Phase 1 studies have already demonstrated the safety and tolerability of mAbs as inhaled aerosols [
      • van der Straten K.
      • et al.
      Optimization of anti-SARS-CoV-2 neutralizing antibody therapies: roadmap to improve clinical effectiveness and implementation.
      ,
      • Syed Y.Y.
      Regdanvimab: first approval.
      ]. Based on these considerations, it is reasonable to argue that engineered ACE2 decoy receptor should also be developed as an inhaled formulation to treat COVID-19.

      Concluding remarks and future perspectives

      Advances in innovative technologies for therapeutic antibody development in the past 20 years accelerated the development and use of mAbs against SARS-CoV-2 in clinical settings within the first year of the COVID-19 pandemic. Cocktails of mAbs successfully neutralized earlier SARS-CoV-2 variants; however, the novel omicron variant exhibits extensive evasion of vaccines and therapeutic mAbs, even in cocktail form, due to the unprecedented evolution in the spike. ACE2 decoy receptors are expected to be free from complete virus escape, which is supported by evidence from results of DMS of the spike RBD and SARS-CoV-2 mutagenesis with sparing ACE2 decoy doses. Studies discussed in this review have shown that engineered ACE2 decoy receptors neutralize omicron and other sarbecoviruses that use ACE2 as an entry receptor, indicating broad efficacy against future variants and zoonotic coronavirus diseases.
      Although the safety of rhACE2 for ARDS has been verified in Phase 1 human clinical trial, engineered ACE2 decoy receptors against COVID-19 would require higher doses than were used in clinical trials for ARDS, raising concerns about adverse effects. These concerns need to be addressed to gain clinical use approval (see Outstanding questions). One of them is overconversion of Ang II to Ang 1–7 due to excessive enzymatic activity of higher dose of ACE2 decoy receptors, which could induce hemodynamic dysregulation or unexpected adverse effects. One approach to avoid this concern is the elimination of enzymatic activity of ACE2 decoy receptors. Among the proposed options to modulate ACE2 enzymatic activity, Ang-II-binding pocket closure by disulfide bond formation benefits biologic use because it not only blocks the enzymatic activity but also significantly enhances thermostability [
      • Higuchi Y.
      • et al.
      Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.
      ].
      For concerns about drug toxicity, immunogenicity and off-target binding to endogenous proteins are the main issues that must be investigated. Mutations in ACE2 protein have the risk of stimulating host immunity toward the drug and crossreactive responses to host tissues. Fortunately, immunogenicity can be predicted by T cell activation experiments and computational analysis [
      • Paul S.
      • et al.
      Major histocompatibility complex binding, eluted ligands, and immunogenicity: benchmark testing and predictions.
      ], and high-risk mutations can be excluded from engineered ACE2 decoy receptors in advance. Also, the controversies surrounding the use of the modified Fc domain, expected dose, and the optimal route of drug administration has been partially resolved by studies that have shown the advantage of enhancing Fc-effector function and aerosol inhalation in COVID-19 disease models. Comprehensive screening for engineered ACE2 decoy receptors binding to endogenous proteins, especially membrane proteins, will help to assess the risk of off-target effects. ACE2 decoy receptors are a promising modality against current and future coronavirus diseases. It is crucial that studies continue to accumulate clinical data in various conditions to optimize the antiviral effects of engineered ACE2 decoy receptors and translation to the clinic.
      Is the elimination of ACE2 enzymatic activity required for preventing hemodynamic dysregulation and other unexpected effects?
      What is the best approach to eliminate ACE2 enzymatic activity in terms of efficiency, antigenicity, and structural stability?
      Which Fc modulation is more suitable for COVID-19 therapy and prophylaxis, Fc-effector enhancement or FcRn orientation?
      Whether Fc-effector enhancement and FcRn orientation have synergistic effects on antiviral efficacy?
      Is liquid aerosol inhalation more suitable to treat COVID-19 in terms of therapeutic efficacy, dose-sparing effects, and drug accessibility?
      Can high affinity decoy receptors be applied for other virus diseases once virus receptors are identified?

      Acknowledgments

      This work was supported by the Japan Agency for Medical Research and Development (AMED), Research Program on Emerging and Re-emerging Infectious Diseases under JP 21fk0108465 (A.H., J.T., and T.O.), Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research) under JP21am0101108 (D.M.S) and JP21am0101075(2527) (J.T.).

      Declaration of interests

      A.H., J.T., and T.O. are the inventors on a patent filed by Kyoto Prefectural University of Medicine and Osaka University (ACE2 mutant protein, PCT/JP2021/031372).

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      Glossary

      Acute respiratory distress syndrome (ARDS)
      a life-threatening respiratory failure characterized by rapid onset of widespread inflammation in the lungs. Excessive fluid impairs gas exchange and induces symptoms including shortness of breath (dyspnea), rapid breathing (tachypnea), and bluish skin coloration (cyanosis).
      Affinity
      strength of interaction at a singular binding site.
      Angiotensin-converting enzyme 2 (ACE2)
      a zinc metallopeptidase that catalyzes the hydrolysis of Ang II into Ang 1–7. This reaction counteracts the RAS and negatively regulates vasoconstriction and vascular permeability. ACE2 is expressed on the cell surface and some ACE2 is cleaved by ADAM17 to be released as a soluble form of ACE2. Both SARS-CoV-1 and SARS-CoV-2 use ACE2 as a cell-entry receptor.
      Avidity
      overall strength of protein–protein interaction with more than one biding site.
      Clade
      group of biological taxa or species that includes all the evolutionary descendants of a common ancestor.
      Decoy receptors
      soluble version of receptors that bind to viruses competitively with native receptors on host cells and inhibit viruses infection.
      Deep mutational scanning (DMS)
      a method to comprehensively understand how amino acid substitutions at each position in a protein of interest affect its function. By combining protein mutagenesis, high-throughput functional analysis, and deep sequencing, the functional impact of a huge number of mutations can be quantitatively evaluated.
      Disulfide bond
      bridges formed between cysteine residues in proteins. This fundamental building block for the molecular architecture can play a key role in basic biological processes as well as stabilizing protein structure. The formation of a disulfide bond by two sulfur atoms of spatially proximal cysteines is processed by a two-electron oxidation from reduced sulfhydryl groups of cysteines (S-H) to the oxidized cystine (S-S) residue.
      Fc
      homodimeric domain consisting of the C-terminal half of the immunoglobulin heavy chain. Fc modulates antibodies efficacy by effector functions such as antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity through the interaction with Fcγ receptors on immune cells and the complement protein C1q.
      Receptor binding domain (RBD)
      key domain within a spike protein monomer that binds to the cell surface ACE2. Binding of the RBD to ACE2 induces a conformational change in the spike protein, leading to virus–cell membrane fusion followed by infection.
      Spike glycoprotein
      trimeric structure found on the outside of the virus particle and gives coronaviruses their crown-like appearance. It has two subunits, of which S1 binds to ACE2 and S2 facilitates membrane fusion to complete entry into host cells.
      Valency
      total number of binding sites involved in protein–protein interaction.