WO2022012688A1

WO2022012688A1 - Ace2-ig fusion variants

Info

Publication number: WO2022012688A1
Application number: PCT/CN2021/106972
Authority: WO
Inventors: Guocai ZHONG; Yujun Li; Haimin WANG; Xiaojuan TANG; Danting MA; Yifei Wang
Original assignee: Shenzhen Bay Laboratory
Priority date: 2020-07-17
Filing date: 2021-07-17
Publication date: 2022-01-20

Abstract

The present invention provides a soluble ACE2-Ig fusion variant having an antibody-like configuration, which comprises an ACE2 domain, and an immunoglobulin domain comprising Fc region.

Description

ACE2-Ig Fusion Variants

Introduction

As of May 16 ^th 2021, the uncontrolled COVID-19 pandemic has already caused ～160 million confirmed infections and over 3.3 million documented deaths across 216 countries, according to World Health Organization’s online updates (https: //www. who. int/emergencies /diseases/novel-coronavirus-2019) . It is reported that ～80%of COVID-19 patients exhibit mild-to-moderate symptoms, while ～20%of them develop serious manifestations such as severe pneumonia, acute respiratory distress syndrome (ARDS) , sepsis and even death. So far, there are more than six prophylactic COVID-19 vaccines that have been authorized by different countries for emergency use. These include two mRNA vaccines (Pfizer-BioNTech, US; Moderna, US) , two inactivated vaccines (Sinopharm, China; Sinovac, China) , and two adenoviral vectored vaccines (Sputnik V, Russia; AstraZeneca-Oxford, UK) . Over 1 billion doses of these vaccines have been administered worldwide. There are also some convalescent patient-derived antibodies that have been authorized for emergency use by the US FDA, such as Regeneron’s antibody cocktail consisting of casirivimab (REGN10933) and imdevimab (REGN10987) , and Eli Lilly’s antibody cocktail consisting of etesevimab (LY-CoV016) and bamlanivimab (LY-CoV555) . So far, all the SARS-CoV-2 vaccines and antibody therapeutics in clinical use were developed on the basis of the prototype SARS-CoV-2 strain.

Two subgroups of coronaviruses, including alphacoronaviruses (e.g. swine acute diarrhea syndrome coronavirus, SADS-CoV) and betacoronaviruses (e.g. severe acute respiratory syndrome coronavirus, SARS-CoV) , infect mammals and have broad host ranges spanning bats, rodents, domestic animals, and humans (1-7) . Bats are considered to be the natural reservoir hosts for a number of pathogenic human coronaviruses, including SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV) , HCoV-NL63, and HCoV-229E (6, 8) . The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) , the etiological agent of coronavirus disease 2019 (COVID-19) (8-10) , shares 79.5%genome sequence identity to SARS-CoV and 96.2%genome sequence identity to Bat-CoV RaTG13, a coronavirus detected in bat species Rhinolophus affinis, indicating that bats are also likely a reservoir host for SARS-CoV-2 (8-10) . Multiple SARS-CoV-2-related coronaviruses with 85.5%to 92.4%genome sequence similarity to SARS-CoV-2 have been identified in Malayan pangolins (Manis Javanica) recently (11-13) . Two of these pangolin coronaviruses, called as Pangolin-CoV-2020 (11) and GD Pangolin CoV (12) , have 90.23%and 92.4%genome sequence similarity to SARS-CoV-2, respectively, and have only one amino acid different from SARS-CoV-2 within the spike protein receptor binding motif (RBM) region. Based on these findings, pangolins have been proposed as an intermediate host or another natural host of SARS-CoV-2 (11-13) . Domestic animals have been shown to play key roles as intermediate hosts, such as camels for MERS-CoV and camelids for HCoV-229E, to transmit pathogenic coronaviruses from bats to humans (6) . Indeed, we recently found that SARS-CoV-2 can use human ACE2 and a wide range of animal-ACE2 orthologs, but not mouse ACE2, for cell entry. But a single amino-acid change within the spike receptor-binding domain (RBD; Q498H, Q498Y, or N501Y) could be sufficient to confer SARS-CoV-2 the ability to utilize mouse ACE2.

Receptor binding is a critical determinant for the host range of coronaviruses. SARS-CoV-2 utilizes ACE2, the SARS-CoV receptor, as an essential cellular receptor to infect cells. Our and other researchers’ studies show that SARS-CoV-2, SARS-CoV, a pangolin coronavirus, and a bat coronavirus utilize human ACE2 and its orthologs of diverse domestic animals, including camels, cattle, horses, goats, sheep, pigs, dogs, cats, and rabbits, for cell entry, suggesting that these animals might be able to serve as intermediate hosts to spread these viruses, and therapeutics targeting viral particle-ACE2 interaction may be used to protect from SARS-CoV-2, SARS-CoV, and SARS-CoV-like viruses that emerge over the course of the pandemic.

SARS-CoV-2 is a single-stranded RNA virus with moderate mutation and recombination frequencies. A number of spontaneous and selection-pressure-driven mutations of SARS-CoV-2 genome have been identified in viral variants that emerged during the course of the pandemic. With more and more SARS-CoV-2 variants being identified to carry diverse spike mutations within the RBD region, it’s possible that some of the spike mutations might alter the host range of the virus, or compromise the efficacy of vaccines or neutralizing antibodies developed on the basis of the prototype SARS-CoV-2 strain.

To better control the spreading of SARS-CoV-2 variants that emerge over the course of the pandemic, it is urgent to develop broadly anti-SARS-CoV-2 vaccines and therapeutics.

Summary of the Invention

The present invention focuses on a soluble ACE2-Ig fusion variant having an antibody-like configuration, which comprises an ACE2 domain, and an immunoglobulin domain comprising a Fc region. The experimental data of the present invention show that the said soluble ACE2-Ig fusion variant could be used to protect from SARS-CoV-2 and some other SARS-like viruses that might spillover into humans in the future.

In the first place, the present invention provides a soluble ACE2-Ig fusion variant having an antibody-like configuration, which comprises an ACE2 domain, and an immunoglobulin domain.

In some embodiments, the immunoglobulin domain comprises Fc region.

In some embodiments, the immunoglobulin domain comprises in order from N-to C-terminus a unit of CH2 and CH3 regions.

In some embodiments, the CH2 and CH3 regions are CH2 and CH3 regions of an IgG or IgA form.

One unit of CH2 and CH3 regions comprises two of CH2 fragments and two of CH3 fragments of an IgG or IgA.

Preferably, the IgG or IgA are human IgG or IgA.

In some embodiments, the ACE2 domain is a truncated ACE2 domain, wherein one truncated ACE2 domain comprises two truncated ACE2 or one truncated ACE2.

In some embodiments, the truncated ACE2 is ACE2 truncated at its residue within residues 720-740 or 615. Preferably, the truncated ACE2 is ACE2 truncated at its

residue

740 or 615.

In some embodiments, the truncated ACE2 has one or more mutation sites.

In some embodiments, the mutation site is selected from T27Y, D30E and L79I.

In some embodiments, one truncated ACE2 domain comprising two truncated ACE2 is linked to the immunoglobulin domain. Specifically, one truncated ACE2 domain comprising two truncated ACE2 is linked to a hinge region of N-terminus of CH2 region or a linker of C-terminus of CH3 region of the immunoglobulin domain. Thereby, a bivalent soluble ACE2-Ig fusion variant is obtained.

Preferably, the truncated ACE2 domain has two ACE2 truncated at its residue within residues 720-740, more preferably, truncated at its residue 740. Preferably, the ACE2 truncated at its residue within residues 720-740, more preferably, truncated at its residue 740 has a mutation of D30E. Preferably, the ACE2 truncated at its residue within residues 720-740, more preferably, truncated at its residue 740, has mutations of T27Y and D30E. Preferably, the ACE2 truncated at its residue within residues 720-740, more preferably, truncated at its residue 740, has a mutation of L79I. Preferably, the ACE2 truncated at its residue within residues 720-740, more preferably, truncated at its residue 740, has mutations of D30E and L79I. Preferably, the ACE2 truncated at its residue within residues 720-740, more preferably, truncated at its residue 740, has mutations of T27Y, D30E and L79I.

In some embodiments, two truncated ACE2 domains comprising two truncated ACE2 respectively are linked to the immunoglobulin domain. Specifically, two truncated ACE2 domains, comprising two truncated ACE2 respectively, are linked to a hinge region of N-terminus of CH2 regions and a linker of C-terminus of CH3 regions of the immunoglobulin domain respectively. Alternatively, one truncated ACE2 domain, comprising two truncated ACE2, is linked to the hinge region of N-terminus of CH2 regions, and two truncated ACE2 within the other truncated ACE2 domain are linked to two linkers of C-terminus of CH3 regions respectively. Thereby, a tetravalent soluble ACE2-Ig fusion variant is obtained.

Preferably, two ACE2 truncated at its residue within residues 720-740, more preferably, truncated at its residue 740, are linked to the hinge region of N-terminus of CH2 regions, and two ACE2 truncated at its residue 615 are linked to the linkers of C-terminus of CH3 regions. Preferably, the truncated ACE2 has a mutation of D30E.

Preferably, two ACE2 truncated at its residue within residues 720-740, more preferably, truncated at its residue 740, are linked to the hinge region of N-terminus of CH2 regions, and two ACE2 truncated at its residue within residues 720-740, more preferably, truncated at its residue 740, are linked to the linkers of C-terminus of CH3 regions. Preferably, the truncated ACE2 has a mutation of D30E.

Part of the hinge and/or residues 720-740 of truncated ACE2 may be replaced by a synthetic linker molecule. For example, up to 10 residues at the N-terminus of the immunoglobulin Fc region are replaced by a synthetic flexible linker.

Preferably, the synthetic flexible linker in the present invention has a general formula (GaSb) c, where a is an integer of 2～4, b is an integer of 0～2, and c is an integer of 0～7.

In some embodiments, residues 720-740 of the truncated ACE2 or a part thereof are used as the linker per se.

In some embodiments, the immunoglobulin domain further comprises CH1 and CL regions of an antibody.

In some embodiments, the immunoglobulin domain comprises both light chain constant region and heavy chain constant region.

In some embodiments, two truncated ACE2 domains comprising two truncated ACE2 respectively are linked to the immunoglobulin domain. Specifically, two truncated ACE2 domains comprising two truncated ACE2 respectively are linked to the linkers of N-terminus of CH1 and CL regions. Alternatively, two truncated ACE2 domains comprising two truncated ACE2 respectively are linked to four linkers of N-terminus of CH1 and CL regions. Alternatively, two truncated ACE2 domains comprising two truncated ACE2 respectively are linked to four linkers of N-terminus of light chain constant region and heavy chain constant region. Thereby, a tetravalent soluble ACE2-Ig fusion variant is obtained.

Preferably, the ACE2 is truncated at its residue within residues 720-740, more preferably, truncated at its residue 740. Preferably, the truncated ACE2 has a mutation of D30E. Preferably, the truncated ACE2 has mutations of D30E and L79I. Preferably, the truncated ACE2 has mutations of T27Y, D30E and L79I.

In some embodiments, one or more ACE2 domains comprising one truncated ACE2 may be added to the said tetravalent soluble ACE2-Ig fusion variant via one or more linkers. For example, one or more ACE2 domains comprising one truncated ACE2 may be added to C-terminus of CH3 region, or C-terminus of CL region.

In some embodiments, three truncated ACE2 domains comprising two truncated ACE2 respectively are linked to the immunoglobulin domain. Specifically, two truncated ACE2 domains comprising two truncated ACE2 respectively are linked to the linkers of N-terminus of CH1 and CL regions, alternatively, two truncated ACE2 domains comprising two truncated ACE2 respectively are linked to the four linkers of N-terminus of CH1 and CL regions, alternatively, two truncated ACE2 domains comprising two truncated ACE2 respectively are linked to the four linkers of N-terminus of light chain constant region and heavy chain constant region; and one truncated ACE2 domain comprising two truncated ACE2 is linked to a linker of C-terminus of CH3 regions of the immunoglobulin domain. Alternatively, two truncated ACE2 within one truncated ACE2 domain are linked to two linkers of C-terminus of CH3 regions respectively. Alternatively, two truncated ACE2 within one truncated ACE2 domain are linked to two linkers of C-terminus of CL regions respectively. Thereby, a hexavalent soluble ACE2-Ig fusion variant is obtained.

In some embodiments, four truncated ACE2 domains comprising two truncated ACE2 respectively are linked to the immunoglobulin domain. Specifically, two truncated ACE2 domains comprising two truncated ACE2 respectively are linked to the linkers of N-terminus of CH1 and CL regions, two truncated ACE2 comprising two truncated ACE2 respectively within one truncated ACE2 domain are linked to two linkers of C-terminus of CH3 regions respectively, and two truncated ACE2 within one truncated ACE2 domain are linked to two linkers of C-terminus of CL regions respectively. Thereby, an octavalent soluble ACE2-Ig fusion variant is obtained.

Part of the hinge and/or residues 720-740 of truncated ACE2 may be replaced by a synthetic linker molecule. For example, up to 30 residues at the N-terminus of the CH1 or CL region are replaced by a synthetic flexible linker.

Preferably, the synthetic flexible linker has a general formula (GaSb) c, where a is an integer of 2～4, b is an integer of 0～2, and c is an integer of 0～7.

In some embodiments, one or more truncated ACE2 domain comprising one truncated ACE2 may be further linked to the said tetravalent soluble ACE2-Ig fusion variant, the said hexavalent soluble ACE2-Ig fusion variant, or the said octavalent soluble ACE2-Ig fusion variant.

In the present invention, the ACE2 domain is from human beings, or domestic animals, including camels, cattle, horses, goats, sheep, pigs, dogs, cats, or rabbits.

In the present invention, the immunoglobulin domain is from human beings, or domestic animals, including camels, cattle, horses, goats, sheep, pigs, dogs, cats, mouse, or rabbits.

In the present invention, it is shown that SARS-CoV-2, SARS-CoV, a pangolin coronavirus, and a bat coronavirus utilize human ACE2 and its orthologs of diverse domestic animals, including camels, cattle, horses, goats, sheep, cats, and rabbits, for cell entry, suggesting that these animals might be able to serve as intermediate hosts to spread these viruses, and a wide range of ACE2 orthologs are capable of supporting cell entry of the four tested viruses: SARS-CoV-2, SARS-CoV, a pangolin coronavirus, and a bat coronavirus. The present invention also indicates that humans and the said domestic animals could be generally susceptible to infections of the four distinct coronaviruses.

In the present invention, it is shown that (1) in contrast to an early isolate WHU01, the variants of concern (VOC) B. 1.351, and P. 1 were able to bind and use mouse ACE2 for entry, suggesting that these variants may have gained ability to infect mouse; and (2) the B. 1.1.7 pseudovirus was also found able to utilize mouse ACE2 for entry, but the binding between B. 1.1.7 RBD and mouse ACE2 was very weak, reflecting the sensitivity difference of the two assays for discriminating weak and strong interactions. Mice are vaccine-inaccessible rodent species that have large population size and could access the territories of both humans and domestic animals. The fact that the B. 1.351 and P. 1 variants have gained ability to infect mice raises the possibility of wild rodents becoming a second SARS-CoV-2 reservoir, adding one more concerning factor to these variants. In the present invention, it is shown that multiple variants, especially the VOCs B. 1.1.7, B. 1.351, and P. 1, have significantly increased affinity to cattle and pig ACE2 proteins, in contrast to the previous study, wherein it was shown that cattle and pigs are not or only weakly susceptible to experimental infection with SARS-CoV-2. Cattle and pigs are extremely important livestock animals which serve as two major sources of meat for humans.

In the present invention, it is shown that compared to the early isolate WHU01, all the tested variants carrying mutations in the RBD region showed partial resistance to at least one of the four antibodies (etesevimab/LY-CoV016, bamlanivimab/LY-CoV555, casirivimab/REGN10933, and imdevimab/REGN10987) ) that constitute two antibody cocktails under Emergency Use Authorization (EUA) from the U.S. FDA., suggesting that these variants might be products of host immunity-driven viral evolution. The VOCs B. 1.351 and P. 1 show significant resistance to three out of four tested antibodies, suggesting the possibility of these variants evading natural or vaccine-induced immunity.

In the present invention, it is shown that variants carrying immune evasion-associated RBD mutations also have enhanced RBD affinity to certain animal orthologs of ACE2, as exemplified by the above findings about the mink-associated Y453F variants and the three VOCs, B. 1.1.7, B. 1.351, and P. 1. This means that immune evasion and potential host-range alteration are highly linked events for SARS-CoV-2. Because of the huge size of SARS-CoV-2 infected population and high prevalence of the VOCs worldwide, those ‘dual-potential’ mutations are extremely concerning. They might promote cross-species transmission of the virus from humans to rodents (e.g. mice) or domestic animals (e.g. cattle or pigs) , forming secondary SARS-CoV-2 reservoirs that further drive the virus to evolve and facilitate wider-spread transmission of mutated viruses.

The soluble ACE2-Ig fusion variant in the present invention is a broad-spectrum anti-SARS-CoV-2 drug candidate. It is shown in the present invention that the soluble ACE2-Ig fusion variant is broadly effective against all the tested SARS-CoV-2 variants, including all the four VOCs (B. 1.1.7, B. 1.351, P. 1, and B. 1.617) designated by the WHO. Extensively mutating the ACE2 residues near the RBD-binding interface should be avoided.

In the present invention, it is shown that while SARS-CoV-2 variants showed significantly reduced or completely loss of sensitivity to neutralizing antibodies, most of them showed increased (up to ～15-fold) sensitivity to soluble ACE2-Ig fusion variant in the present invention. This might be explained by increased affinity of SARS-CoV-2 variant to human ACE2 or increased accessibility of the RBDs of SARS-CoV-2 variants. Specifically, it is observed a 4.7-fold, 2.9-fold, 2.0-fold and 1.6-fold increase in the affinity of human ACE2 to the RBDs of the mink-associated Y453F variant, the B. 1.1.7 variant, the B. 1.351 variant, and the P. 1 variant, respectively. The B. 1.258 is found with increased affinity to human ACE2. Therefore, it is suggested that SARS-CoV-2 is evolving toward better utilization of ACE2 as a receptor, either through increasing RBD affinity to ACE2, or through better exposure of its RBDs, or through both. It further indicates that ACE2 is still likely an essential receptor for SARS-CoV-2.

ACE2 also serves as a cellular receptor for a number of other coronaviruses, including SARS-CoV, HCoV-NL63, Pangolin-CoV-2020, Bat-CoV RaTG13, and some other SARS-like CoVs found in bats14, 16, 59. The soluble ACE2-Ig fusion variant in the present invention is therefore a promising broadly anti-coronavirus drug candidate that might be used to treat and prevent infection of any SARS-CoV-2 variant that emerges over the course of the pandemic. The soluble ACE2-Ig fusion variant in the present invention might also be a good alternative anti-SARS-CoV-2 agent for the populations who are not responsive to, or don’t have access to any prophylactic vaccines.

The present invention further shows that a soluble ACE2-Ig fusion variant, which carries a D30E mutation and has ACE2 truncated at its residue 740 but not 615, potently neutralizes entry of all the four distinct coronavirus pseudotypes, and its neutralization potency against pseudotypes of these related but diverse viruses, as well as SARS-CoV-2 live virus. Among the tested soluble ACE2-Ig variants, all the 740-version variants showed significantly better potency than the 615-version variants. In addition, D30E mutants of both 615-and 740-version proteins showed improved potency over the corresponding wildtypes.

The mechanism of the D30E-mediated improvement is likely that the mutation enhances the salt-bridge interaction between the residue 30 of the ACE2 and residue 417 of SARS-CoV-2 and Bat-CoV RaTG13 RBDs. The present invention shows that the 740-D30E variant of ACE2-Ig fusion is a broadly neutralizing immunoadhesin against SARS-CoV-2, SARS-CoV, Pangolin-CoV-2020, and Bat-CoV RaTG13.

A further improved fusion variant, that is multivalent soluble ACE2-Ig fusion variant, which has an antibody-like configuration, neutralizes SARS-CoV-2 pseudotype and live virus with more than 10-fold higher potency than the prototype ACE2-Ig. These data demonstrate that multivalent soluble ACE2-Ig fusion variant could be used to protect from SARS-CoV, SARS-CoV-2 and some other SARS-like viruses that might spillover into humans in the future. The multivalent soluble ACE2-Ig fusion variant is a bivalent soluble ACE2-Ig fusion variant having two soluble truncated ACE2, a tetravalent soluble ACE2-Ig fusion variant having four soluble truncated ACE2, a hexavalent soluble ACE2-Ig fusion variant having six soluble truncated ACE2, or an octavalent soluble ACE2-Ig fusion variant having eight soluble truncated ACE2. Preferably, the multivalent soluble ACE2-Ig fusion variant is a hexavalent soluble ACE2-Ig fusion variant having six soluble truncated ACE2 or an octavalent soluble ACE2-Ig fusion variant having eight soluble truncated ACE2.

Two new variants, ACE2-Ig-v3.1 and ACE2-Ig-v4.1, which have an antibody like configuration and are tetravalent ACE2-Ig fusion variant and hexavalent ACE2-Ig fusion variant respectively, show the most pronounced improvements over the bivalent 740-D30E ACE2-Ig-v1.1 fusion variant, suggesting that they are markedly improved ACE2-Ig fusion variants as potent entry inhibitors against SARS-CoV, SARS-CoV-2 and some other SARS-like viruses that might spillover into humans in the future.

The soluble ACE2-Ig fusion variant of the present invention may be prepared using any suitable method.

The multivalent soluble ACE2-Ig fusion variant of the present invention comprises substantially non-covalent inter-domain interactions.

In the second place, the present invention provides a method for inhibiting infection of SARS-CoV, SARS-CoV-2 and some other SARS-like viruses using ACE2 as an essential cellular receptor in a subject, comprising administrating to the subject a pharmaceutical composition comprising a pharmaceutically effective amount of the soluble ACE2-Ig fusion variant in the first place and a pharmaceutically acceptable carrier. In some embodiments, the route of administration of the pharmaceutical composition may be oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal.

The SARS-CoV-2 includes SAR-CoV-2 variants, including but not limited to WHU01; the variant D614G; the variant B. 1.258 that carries an N439K signature mutation in the RBD region; a mink-associated variant that carries a Y453F signature mutation in the RBD region; the variant B. 1.1.7, also called 501Y. V1 that has an N501Y signature mutation in the RBD region, and carries seven additional mutations in the remaining region of the spike protein; the variant B. 1.351, also called 501Y. V2 that has three signature amino-acid substitutions K417N-E484K-N501Y in the RBD region and a number of additional mutations in the remaining regions of the spike protein; the variant P. 1, also called 501Y. V3, that has three signature substitutions K417T-E484K-N501Y in the RBD region, similar to the RBD mutations of the B. 1.351 variant, and seven additional lineage-defining amino-acid substitutions in the remaining regions of the spike protein; the variant B. 1.617, which has L452R-E484Q signature double mutations in the RBD.

It is demonstrate in the present invention that all of the twelve SARS-CoV-2 variants tested are potently neutralized by the soluble ACE2-Ig fusion variant in the present invention, demonstrating that the soluble ACE2-Ig fusion variant in the present invention is a broad-spectrum anti-SARS-CoV-2 agent.

Compared to the early isolate WHU01, most circulating variants, including the VOCs B. 1.1.7, B. 1.351 and P. 1, showed significantly increased (up to ～15-fold) neutralization sensitivity to ACE2-Ig-v1, ACE2-Ig-v1.1 and ACE2-Ig-v3.1, the constructs that carry a wild-type or a D30E-mutated ACE2 domain.

It is shown in the present invention that although the variant B. 1.617 shows an almost complete resistance to the neutralizing antibody LY-CoV555 and a partial resistance to the antibody REGN10933, the three tested soluble ACE2-Ig fusion variants in the present invention all potently neutralized it, The variant B. 1.617, which has L452R-E484Q signature double mutations in the RBD, is causing uncontrolled outbreaks in India and has spread to multiple countries.

It is demonstrate in the present invention that ACE2-Ig is a good drug candidate against diverse SARS-CoV-2 variants that emerge over the course of the pandemic.

In the third place, the present invention provides a method for treating or preventing diseases associated with infection of SARS-CoV, SARS-CoV-2 and some other SARS-like viruses using ACE2 as an essential cellular receptor in a subject, comprising administrating to the subject a pharmaceutical composition comprising a pharmaceutically effective amount of the soluble ACE2-Ig fusion variant in the first place and a pharmaceutically acceptable carrier. In some embodiments, the route of administration of the pharmaceutical composition may be oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal.

Preferably, the diseases include but are not limited to pneumonia, severe acute respiratory infection, renal failure, heart failure, acute respiratory distress syndrome (ARDS) , liver injury, intestinal disease or severe acute respiratory syndrome.

In the fourth place, the present invention provides use of the soluble ACE2-Ig fusion variant in the first place in the manufacturing a medicament for inhibiting infection of SARS-CoV, SARS-CoV-2 and some other SARS-like viruses using ACE2 as an essential cellular receptor in a subject.

In the fifth place, the present invention provides use of the soluble ACE2-Ig fusion variant in the first place in the manufacturing a medicament for treating or preventing diseases associated with infection of SARS-CoV, SARS-CoV-2 and some other SARS-like viruses using ACE2 as an essential cellular receptor.

The subject in the present invention may be human beings, or domestic animals, including camels, cattle, horses, goats, sheep, dogs, cats, mice, rats and rabbits.

In the present invention, the terms ACE2-Ig constructs, ACE2-Ig and the soluble ACE2-Ig fusion variant are used interchangeably.

The invention encompasses all combination of the particular embodiments recited herein.

Brief Description of the Drawings

Figure 1 shows that a wide range of ACE2 orthologs support binding to RBD proteins of SARS-CoV-2, Pangolin-CoV-2020, Bat-CoV RaTG13, and SARS-CoV.

Figure 2 shows protein sequence alignment of the seventeen ACE2 orthologs.

Figure 3 shows ACE2 ortholog-mediated binding to RBD proteins of SARS-CoV-2 and three related coronaviruses.

Figure 4 shows surface staining of 293T cells transfected with each of the sixteen ACE2 orthologs or a vector plasmid control.

Figure 5 shows that ACE2 ortholog-mediated cell entry of SARS-CoV-2, Bat-CoV RaTG13, and SARS-CoV.

Figure 6 shows that ACE2 orthologs of humans and most domestic mammals, including camels, cattle, horses, goats, sheep, cats, and rabbits, support entry of all the tested coronavirus pseudotypes and SARS-CoV-2 live virus.

Figure 7 shows soluble ACE2 (ACE2-Ig) variants and their potency of blocking SARS-CoV-2 Δfurin pseudovirus infection.

Figure 8 shows RBD-Fc and ACE2-Ig fusion variants used in neutralization assays of Figs. 7 and 9.

Figure 9 shows the D30E mutation improves 740-Ig’s neutralization activity against SARS-CoV-2 and Bat-CoV RaTG13 pseudoviruses but not Pangolin-RBD/BJ01 or SARS-CoV pseudovirus.

Figure 10 shows flow cytometry data supporting the D30E improvement of neutralization potency shown in Figs. 7 and 9.

Figure 11 shows ACE2-Ig fusion variants developed in the present invention.

Figure 12 shows improving ACE2-Ig’s neutralization potency by introducing mutations to soluble ACE2 domains.

Figure 13 shows that ACE2-Ig fusion variants containing tetravalent ACE2 have higher neutralization potency over ACE2-Ig-v1.1.

Figure 14 shows that ACE2-Ig variants of antibody-like configurations significantly outperform ACE2-Ig-v1.1.

Figure 15 shows that ACE2-Ig-v3.1 potently neutralizes SARS-CoV-2 live virus.

Figure 16 shows that ACE2-Ig variant containing hexavalent or octavalent ACE2 significantly outperforms ACE2-Ig-v1.1 but not ACE2-Ig-v3.1.

Figure 17 shows ACE2-Ig-v3.4 and ACE2-Ig-v3.5 significantly outperform ACE2-Ig-v3.1.

Figure 18 shows SARS-CoV-2 spike variants and related mutations investigated.

Figure 19 shows Western blot detection of ACE2 and SARS-CoV-2 spike proteins.

Figure 20 shows sequences of eight animal-ACE2 orthologs investigated in this study are aligned,

Figure 21 shows Animal-ACE2 tropism of SARS-CoV-2-variant pseudoviruses.

Figure 22 shows interaction kinetics of forty RBD-ACE2 pairs.

Figure 23 shows SDS-PAGE images of purified recombinant Fc fusion proteins used in Figure 21.

Figure 24 shows neutralization of SARS-CoV-2 variants by recombinant ACE2 proteins.

Figure 25 shows the effects of K417N, K417T, E484K, N501Y mutations on the ability of SARS-CoV-2 pseudovirus to utilize mouse ACE2.

Figure 26 shows neutralization sensitivity of SARS-CoV-2 variants to four monoclonal antibodies in clinical use.

Figure 27 shows interaction kinetics of twenty-four RBD-antibody pairs.

Figure 28 shows interactions between SARS-CoV-2 RBD and four monoclonal antibodies.

Figure 29 shows SDS-PAGE images of purified recombinant ACE2-Ig proteins used in Figure 30.

Figure 30 shows neutralization sensitivity of SARS-CoV-2 variants to eight ACE2-Ig constructs.

Figure 31 shows distribution of the mutated residues of the eight ACE2-Ig constructs.

Figure 32 shows neutralization sensitivity of the B. 1.617 pseudovirus to antibodies and ACE2-Ig constructs.

Figure 33 shows that ACE2-Ig-v1.1-related constructs with ACE2 truncated at its residue within residues 720-740 inhibit SARS-CoV-2 pseudovirus infection with similar efficacy.

Figure 34 shows that ACE2-Ig-v3.1-related constructs with ACE2 truncated at its residue within residues 720-740 inhibit SARS-CoV-2 pseudovirus infection with similar efficacy.

Figure 35 shows the neutralization efficacy of ACE2-Ig-v3.1-related linker variants with a synthetic flexible linker of (GGGGS) c, where c is an integer of 0～7.

Description of Particular Embodiments of the Invention

The descriptions of particular embodiments and examples are provided by way of illustration and not by way of limitation. Those skilled in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.

Materials and methods

1. Ethics statement

All experiments involving SARS-CoV-2 live virus infections were approved (No. QS2020050050) by the ethics committee of Shenzhen Center for Disease Control and Prevention (SZCDC) and the ethics committee of Shenzhen Bay Laboratory. All SARS-CoV-2 live virus experiments were performed in the biosafety level 3 facility at SZCDC.

2. Cells

293T cells and Vero cells were kindly provided by Stem Cell Bank, Chinese Academy of Sciences, confirmed mycoplasma-free by the provider, and maintained in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies) at 37 ℃ in a 5%CO ₂-humidified incubator. Growth medium was supplemented with 2 mM Glutamax-I (Gibco, Cat. No. 35050061) , 100 μM non-essential amino acids (Gibco, Cat. No. 11140050) , 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, Cat. No. 15140122) , and 10%FBS (Gibco, Cat. No. 10099141C) . 293T-based stable cells expressing human ACE2 were maintained under the same culture condition as 293T, except that 3 μg/mL of puromycin was added to the growth medium. 293F cells for recombinant protein production were generously provided by Dr. Yu J. Cao (School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School) and maintained in SMM 293-TII serum-free medium (Sino Biological, Cat. No. M293TII) at 37 ℃, 8%CO ₂, in a shaker incubator at 125 rpm.

3. Plasmids

DNA fragments encoding C-terminally S-tagged ACE2 orthologs were synthesized in pUC57 backbone plasmid by Sangon Biotech (Shanghai, China) . These fragments were then cloned into pQCXIP plasmid (Clontech) between SbfI and NotI restriction sites. DNA fragments encoding spike proteins of SARS-CoV-2 WHU01 (GenBank: MN988668.1) , SARS-CoV-1 BJ01 (GenBank: AY278488.2) , Pangolin-CoV (National Genomics Data Center: GWHABKW00000000) (11) , and Bat-CoV RaTG13 (GenBank: MN996532) (8) , were synthesized by the Beijing Genomic Institute (BGI, China) and Sangon Biotech (Shanghai, China) and then cloned into pcDNA3.1 (+) plasmid or pCAGGS plasmid between EcoRI and XhoI restriction sites. Plasmids encoding recombinant RBD-Ig or ACE2-Ig fusion variants were generated by cloning each of the gene fragments into a pCAGGS-based mouse-IgG2a or human IgG1 Fc fusion protein expression plasmid between NotI and BspEI sites. The retroviral reporter plasmids encoding a Gaussia luciferase or a green fluorescent protein (GFP) reporter gene were constructed by cloning the reporter genes into pQCXIP plasmid (Clontech) , respectively.

DNA fragment encoding spike protein of SARS-CoV-2 WHU01 (GenBank: MN988668.1) was synthesized by the Beijing Genomic Institute (BGI, China) and then cloned into pCAGGS plasmid between EcoRI and XhoI restriction sites. Plasmids encoding SARS-CoV-2 spike variants were generated according to the in-fusion cloning protocol. To facilitate SARS-CoV-2 pseudovirus production, spike sequences for WHU01 and all the variants investigated in this study all contain a furin-cleavage site mutation (ΔPRRA) . We had shown in our previous study that the ΔPRRA mutation does not affect SARS-CoV-2 cross-species receptor usage or neutralization sensitivity. The retroviral reporter plasmids encoding a Gaussia luciferase reporter gene were constructed by cloning the reporter genes into the pQCXIP plasmid (Clontech) . DNA fragments encoding C-terminally S-tagged ACE2 orthologs were synthesized in pUC57 backbone plasmid by Sangon Biotech (Shanghai, China) . These fragments were then cloned into pQCXIP plasmid (Clontech) between SbfI and NotI restriction sites. Plasmids encoding recombinant RBD and soluble ACE2 variants were generated by cloning each of the gene fragments into a pCAGGS-based mouse-IgG2a or human IgG1 Fc fusion protein expression plasmid between NotI and BspEI sites. DNA fragments encoding heavy and light chains of anti-SARS-CoV-2 antibodies were synthesized by Sangon Biotech (Shanghai, China) and then cloned into a pCAGGS plasmid. Four antibodies (LY-CoV016, LY-CoV-555, REGN10933, and REGN10987) that constitute two antibody cocktails authorized by the U.S. FDA for emergency use were included in this study.

4. Pseudovirus Titration.

Pseudovirus titer were determined by TCID ₅₀ followed a previous protocol. Viruses were diluted 100 times as a working solution and then serially diluted in a 1/2 log10 manner. Human ACE2 expressed HeLa cells were infected with those diluted viruses in 96-well plates. Culture supernatants were refreshed every 12 hours and loaded to a Gaussia luciferase assay at 48 hours post infection. TCID ₅₀ of each pseudovirus was calculated by the Reed-Muench method.

5. Western Blot to detect spike protein (C9-tag) and MLV P30 protein in SARS-CoV-2 pseudovirus-containing supernatant.

Pseudovirus-containing supernatants (8000 TCID50 of each pseudovirus) were mixed with SDS-PAGE loading buffer and boiled at 95℃ for 10min. Spike protein (C9-tag) on the surface of pseudovirus was detected using a mouse anti-C9-tag monoclonal antibody 1D4 (Invitrogen, Cat. No. MA1-722) , and an HRP-conjugated goat anti-mouse IgG Fc secondary antibody (Invitrogen, Cat. No. 31437) . MLV P30 protein within the pseudovirus capsid was detected using a rabbit anti-MLV-P30 polyclonal antibody (Origene, Cat. No. AP33447PU-N) and an HRP-conjugated goat anti-rabbit IgG Fc secondary antibody (Invitrogen, Cat. No. 31463) .

6. IgG Fc fusion protein production and purification

293F cells at the density of 6×10 ⁵ cells/mL were seeded into 100 mL SMM 293-TII serum-free medium (Sino Biological, Cat. No. M293TII) one day before transfection. Cells were then transfected with 100 μg plasmid in complex with 250 μg PEI MAX 4000 (Polysciences, Inc, Cat. No. 24765-1) . Cell culture supernatants were collected at 48 to 72 hours post transfection. Recombinant Fc fusion proteins are purified using Protein A Sepharose CL-4B (GE Healthcare, Cat. No. 17-0780-01) , eluted with 0.1 M citric acid at pH 4.5 and neutralized with 1M Tris-HCl at pH 9.0. Buffers were then exchanged to PBS and proteins were concentrated by 30 kDa cut-off Amicon Ultra-15 Centrifugal Filter Units (Millipore, Cat. No. UFC903096) .

7. Flow cytometry for detecting interactions of RBD-Ig proteins with cell surface ACE2 orthologs

293T cells were seeded at 20%density in 48-well plates at 12-15 hours before transfection. Cells in each well were then transfected with 0.5 μL of lipofectamine 2000 (Life Technologies, Cat. No. 11668019) in complex with 200 ng of plasmid encoding one of the sixteen ACE2 orthologs or a D30E mutant of the human ACE2. Culture medium was changed at 6 hours after transfection. Cells were then detached with 5 mM EDTA (Life Technologies, Cat. No. 15575020) at 36 hours post transfection. Cells were then stained with 5 μg/mL RBD-Ig proteins at 37℃ for 10 min, washed three times, and then stained with 2 μg/mL Alexa488 conjugated goat anti-mouse IgG secondary antibody (Invitrogen, Cat. No. A-11001) at room temperature for 20 min. After another three washes, cells were analyzed by Attune NxT flow cytometer (Thermo Fisher) and signals of 10,000 FSC/SSC gated cells were collected for each sample.

8. Western Blot to detect S-tagged ACE2 (ACE2-S-tag) or C9-tagged spike (Spike-C9-tag) expression in 293T cells

293T cells were seeded at 20%density in 6-well plates at 12-15 hours before transfection. Cells in each well were then transfected with 2 μg of plasmid in complex with 5 μL of lipofectamine 2000 (Life Technologies, Cat. No. 11668019) . Thirty-six hours after transfection, cells were lysed and 10 μg of total protein were used for western blot. ACE2-S-tag expression was detected by using 6.2, a mouse anti-S-tag monoclonal antibody (Invitrogen, Cat. No. MA1-981) , and an HRP-conjugated goat anti-mouse IgG Fc secondary antibody (Invitrogen, Cat. No. 31437) . Beta-actin was used as an internal control. Spike-C9-tag expression was then detected by using 1D4, a mouse anti-C9-tag monoclonal antibody (Invitrogen, Cat. No. MA1-722) , and the HRP-conjugated goat anti-mouse IgG Fc secondary antibody (Invitrogen, Cat. No. 31437) .

9. Production of reporter retroviruses pseudotyped with coronavirus spike proteins or VSV-G

MLV retroviral vector-based coronavirus-spike and VSV-G pseudotypes were produced using a previously described protocol (23) with some modifications. 293T cells were seeded at 30%density in 150 mm dish at 12-15 hours before transfection. Cells were then transfected with 67.5 μg of polyethylenimine (PEI) Max 40,000 (Polysciences, Inc, Cat. No. 24765-1) in complex with 11.25 μg of plasmid encoding a coronavirus spike protein or VSV-G, 11.25 μg of plasmid encoding murine leukemia virus (MLV) Gag and Pol proteins, and 11.25 μg of a pQCXIP-based GFP or luciferase reporter plasmid. Eight hours after transfection, cell culture medium was refreshed and changed to growth medium containing 2%FBS (Gibco, Cat. No. 10099141C) and 25 mM HEPES (Gibco, Cat. No. 15630080) . Cell culture supernatants were collected at 36-48 hours post transfection, spun down at 3000×g for 10 min, and filtered through 0.45 μm filter units to remove cell debris. Coronavirus spike-pseudotyped viruses were then concentrated 10 times at 2000×g using 100 kDa cut-off Amicon Ultra-15 Centrifugal Filter Units (Millipore. Cat. No. UFC910024) .

10. Pseudovirus infection of 293T cells expressing ACE2 orthologs

293T cells were seeded at 20%density in poly-lysine pre-coated 48-well plates 12-15 hours before transfection. Cells in each well were then transfected with 0.5 μL of lipofectamine 2000 (Life Technologies, Cat. No. 11668019) in complex with 200 ng of a vector control plasmid or a plasmid encoding one of the sixteen ACE2 orthologs. Cell culture medium was refreshed at 6 hours post transfection. Additional 18 hours later, cells in each well were infected with 50 μL of SARS-CoV-2 wildtype pseudovirus (10×concentrated) , 20 μL of SARS-CoV-2 ΔFurin pseudovirus (10×concentrated) , 50 μL of Bat-CoV RaTG13 pseudovirus (10×concentrated) , 10 μL of SARS-CoV-1 pseudovirus (10×concentrated) , or 10 μL of VSV-G pseudovirus diluted in 200 μL of culture medium containing 2%FBS (Gibco, Cat. No. 10099141C) . Culture medium was refreshed at 2 hours post pseudovirus infection and the medium was refreshed every 12 hours. For the luciferase reporter virus-infected cells, cell culture supernatants were collected and subjected to a Gaussia luciferase assay at 48 hours post infection. The GFP reporter virus-infected cells were stained with Hoechst 33342 (Invitrogen, Cat. No. H3570) and subjected to fluorescent microscopy (IX73 microscope, Olympus) at 48 hours post infection.

11. Gaussia luciferase luminescence flash assay

To measure Gaussia luciferase expression, 20 μL of cell culture supernatant of each sample and 100 μL of assay buffer containing 4 μM coelenterazine native (Biosynth Carbosynth, Cat. No. C-7001) were added to one well of a 96-well black opaque assay plate (Corning, Cat. No. 3915) , and measured with the Tristar5 multifunctional microplate reader (Berthold Technologies) for 0.1 second/well.

12. Production and Purification of ACE2-Ig protein and SARS-CoV-2 antibodies.

293F cells at the density of 6 × 10 ⁵ cells/mL were seeded into 100 mL SMM 293-TII serum-free medium (Sino Biological, Cat. No. M293TII) one day before transfection. Cells were then transfected with 100 μg plasmid in complex with 250 μg PEI MAX 4000 (Polysciences, Inc, Cat. No. 24765-1) . Cell culture supernatants were collected at 48 to 72 hours post transfection. Human IgG1 Fc-containing proteins were purified using Protein A Sepharose CL-4B (GE Healthcare, Cat. No. 17-0780-01) , eluted with 0.1 M citric acid at pH 4.5 and neutralized with 1 M Tris-HCl at pH 9.0. Buffers were then exchanged to PBS and proteins were concentrated by 30 kDa cut-off Amicon Ultra-15 Centrifugal Filter Units (Millipore, Cat. No. UFC903096) .

13. Biolayer interferometry (BLI) assay.

The BLI assays were performed on a Fortebio Octet RED384 instrument, with the temperature and shaking speed at 30 ℃ and 1000 rpm respectively. ACE2-hFc constructs were diluted to 5 μg/mL in 1× assay buffer containing 150 mM NaCl, 0.1%Tween-20, 10mM HEPES and 0.1%BSA (pH 7.4) , and used as ligands for the assays. RBD-mFc constructs were serially diluted to 100nM, 50nM, 25 nM, 12.5 nM, and 6.25 nM in the 1× assay buffer. Each experiment group started with a 10 min warm-up for pre-hydration of AHC biosensors, followed by cycles of baseline (60 s) , loading (60 s) , baseline2 (60s) , association (100 s) , dissociation (600 s) and regeneration plus neutralization (30 s) . A 1: 1 Langmuir binding model was applied for data processing. All fitted diagrams (global fit) display the entire association window and the first 200 s (or 100 s only for house mouse and Chinese rufous horseshoe bat ACE2-related assays) of dissociation phase.

14. Coronavirus pseudovirus neutralization assay

Coronavirus spike protein-pseudotyped luciferase reporter viruses were pre-diluted in DMEM (2%FBS, heat-inactivated) containing titrated amounts of RBD-Ig or ACE2-Ig fusion variant proteins. An Fc fusion protein of an anti-influenza HA antibody, F10-scFv (25) , was used as a control protein here. Virus-inhibitor mixtures were then added to ACE2-expressing 293T or HeLa cells in poly-lysine (Sigma, Cat. No. P4832-50ml) pre-coated 96-well plates and incubated overnight at 37 ℃. Cells were then washed with serum-free medium and incubated in 150 μL of DMEM (2%FBS) at 37 ℃. Cell culture supernatants were collected for Gaussia luciferase assay at 48 h post infection.

15. SARS-CoV-2 live virus isolation

SARS-CoV-2 RNA RT-qPCR-positive oropharyngeal swab samples were filtered through 0.45 μm filter units and diluted in DMEM supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, Cat. No. 15140122) . Samples were then inoculated to Vero cells in a 6-well plate and incubated at 37 ℃ for 1 h. Cells were then washed once with PBS and added with fresh DMEM containing 2%FBS, 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, Cat. No. 15140122) . Cells were then cultured at 37℃ and observed daily under microscope for cytopathic effect. The culture supernatant was examined for the presence of SARS-CoV-2 virus by qRT-PCR. The cells were sequentially stained with 1: 200 diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, Cat. No. 40588-T62) at 37℃ for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, Cat. No. A-11011) at 37℃ for 20 min, and 0.5 μg/ml of DAPI (Sigma-Aldrich, Cat. No. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope, Olympus) . Virus titer, tissue culture infectious dose (TCID50) , was determined by infecting Vero cells with 1/2 log10 diluted virus and calculated by the Reed-Muench method (30) .

16. SARS-CoV-2 live virus infection of ACE2 ortholog-expressing 293T cells

293T cells expressing one of the sixteen ACE2 orthologs were inoculated with SARS-CoV-2 live virus at 800 TCID50 and incubated for 1 h at 37 ℃. Cells were then washed with serum-free medium and incubated in 150 μL of DMEM (2%FBS) at 37 ℃ for additional 24 h. Cells were then fixed with 4%paraformaldehyde in PBS, permeabilized with 0.5%Triton X-100, and sequentially stained with 1: 200 diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, Cat. No. 40588-T62) at 37℃ for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, Cat. No. A-11011) at 37℃ for 20 min, and 0.5 μg/ml of DAPI (Sigma-Aldrich, Cat. No. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope, Olympus) .

17. SARS-CoV-2 live virus neutralization by ACE2-Ig fusion variants

SARS-CoV-2 live virus at 800 TCID ₅₀ were pre-diluted in DMEM (2%FBS, heat-inactivated) containing titrated amounts of ACE2-Ig variant proteins and incubated at 37℃ for 1 h. Virus-inhibitor mixtures were then added to ACE2-expressing 293T or HeLa cells in poly-lysine pre-coated 48-well plates and incubated for 1 h at 37 ℃. Cells were then washed with serum-free medium and incubated in 400 μL of DMEM (2%FBS) at 37 ℃ for 24 to 40 hours. Cells were then fixed for immunofluorescence staining of viral nucleocapsid proteins as described above.

18. Spike variants and related mutations investigated in this study

We first cloned eleven spike genes of seven SARS-CoV-2 natural variants. They include a spike gene of an early isolate WHU01, representing the prototype SARS-CoV-2 variant, a spike gene of the well-studied D614G variant, a spike gene of the B. 1.258 variant that carries an N439K signature mutation in the RBD region, two spike genes of a mink-associated variant that carries a Y453F signature mutation in the RBD region, and six spike genes of three variants of concern (VOCs) , B. 1.1.7, B. 1.351, and P. 1 (Figure 18A) . The VOC B. 1.1.7, also called 501Y. V1, was first identified in the UK in late summer to early autumn 2020, then spread rapidly across the UK, and now has been identified in at least 114 countries. B. 1.1.7 has an N501Y signature mutation in the RBD region, and carries seven additional mutations in the remaining region of the spike protein. Two spike sequences, one that only carries the N501Y mutation and the other that carries all the spike mutations, were used in the following studies (Figure 18A) . The VOC B. 1.351, also called 501Y. V2, was first detected in South Africa from samples collected in early August, then rapidly became dominant locally, and now has been identified in at least 68 countries. B. 1.351 variant has three signature amino-acid substitutions (K417N-E484K-N501Y) in the RBD region and a number of additional mutations in the remaining regions of the spike protein. Similarly, two spike sequences, one that only carries the K417N-E484K-N501Y mutations and the other that carries all the spike mutations, were used in the following studies (Figure 18A) . The VOC P. 1, also called 501Y. V3, emerged in Brazil around mid-November 2020, and now has rapidly spread to at least 37 countries. P. 1 variant has three signature substitutions (K417T-E484K-N501Y) in the RBD region, similar to the RBD mutations of the B. 1.351 variant. It also carries seven additional lineage-defining amino-acid substitutions in the remaining regions of the spike protein. Again, two spike sequences, one that only carries the K417T-E484K-N501Y mutations and the other that carries all the spike mutations, were used in the following studies (Figure 1A) . We then further cloned a non-natural spike variant, Y453F-E484K-Q498H triple mutant, that could evolve from the early isolate WHU01 through acquiring only three missense nucleotide mutations in the RBD, or from a Y453F or an E484K variant through acquiring two missense nucleotide mutations (Figure 18A) . All the residues associated with the above mentioned RBD mutations are indicated in the structure of human ACE2 in complex with the RBD of a prototype SARS-CoV-2 variant (Figure 18B) . Figure 18A: twelve spike variants investigated in this study are illustrated; Spike mutations associated with each variant are indicated; Figure 18B shows that interactions between SARS-CoV-2 RBD and ACE2 involve a large number of contact residues (PDB accession no. 6M0J) ; the ACE2 residues in less than

from RBD atoms are shown; all SARS-CoV-2 variant-associated RBD mutations investigated in this study are shown and labelled.

The variant B. 1.617, which has L452R-E484Q signature double mutations in the RBD, is causing uncontrolled outbreaks in India and has spread to multiple countries. We therefore generated a spike variant that carries the L452R-E484Q mutations, and tested neutralization sensitivity of this variant.

19. Data collection and analysis

All the experiments were repeated 2 to 4 times. All infection assays of Figure 6 were independently performed by two different people and all the data are reproducible in different hands. Key neutralization assays of Figures 7 and 9 were independently performed by two or three different people and all the data are reproducible in different hands. Attune NxT Software (Thermo Fisher) was used to collect and analyze flow cytometry data. Image Lab Software (Bio-Rad) was used to collect SDS-PAGE and Western-Blot image data. Cell Sens Software (Olympus) was used to collect fluorescent microscopy data. ICE Software (Berthold Technologies) was used to collect luciferase assay data. GraphPad Prism 6.0 software was used for figure preparation and statistical analyses.

20. Statistical analysis

Data expressed as mean values ± s.d. Statistical analyses were performed using two-sided two-sample Student’s t-test using GraphPad Prism 6.0 software when applicable. Differences were considered significant at P < 0.01.

Examples

Example 1. A wide range of ACE2 orthologs support binding to RBD proteins of SARS-CoV-2, Pangolin-CoV-2020, Bat-CoV RaTG13, and SARS-CoV-1

The RBD of SARS-CoV-2, like that of SARS-CoV-1, mainly relies on residues in its RBM to bind ACE2 (Fig 1A) . Some of these residues are also conserved in the RBDs of Pangolin-CoV-2020 and Bat-CoV RaTG13 (Fig 1B) . Through analyzing ACE2 residues that are within

from SARS-CoV-2 RBD atoms, we found that these residues are highly conserved across the sixteen analyzed ACE2 orthologs, including human ACE2 and ACE2 orthologs of fifteen domestic and wild animals (Figs 1A and C, Fig 2, and Table 1) , indicating that a substantial proportion of these orthologs might be able to bind RBDs of these diverse but related coronaviruses. We then constructed expression plasmids for the sixteen ACE2 orthologs and expressed mouse IgG2 Fc fusion proteins of the RBDs of SARS-CoV-2 WHU01, Pangolin-CoV-2020, Bat-CoV RaTG13, and SARS-CoV BJ01 (Fig 3A) . These RBD proteins were then used to perform surface staining of 293T cells transfected with each of the sixteen ACE2 orthologs or a vector plasmid control (Figs 3B-3D, and Fig 4) . All the RBD proteins showed binding to a number of ACE2 orthologs. Unexpectedly, although the SARS-CoV-2 and SARS- CoV RBDs differ significantly in the RBM region and ACE2-contact residues, both RBDs bound to eleven ACE2 orthologs with ten identical ones. Moreover, the Pangolin-CoV-2020 RBD, which differs from SARS-CoV-2 RBD with only one amino acid within the RBM region, kept nine SARS-CoV-2 RBD-interacting ACE2 orthologs and gained three additional interacting ones, including that of rat, mouse and chicken. Then the RBD of Bat-CoV RaTG13 showed a binding profile significantly different and narrower than the other three RBDs. Note that human ACE2 and ACE2 orthologs of some domestic animals, including that of camel, cattle, horse, goat, sheep, cat, and rabbit, support efficient binding to all the four tested RBDs, suggesting that these ACE2 orthologs might be generally functional for supporting cell entry of the four tested viruses.

Table 1. Species and accession numbers of ACE2 orthologs

Example 2. A wide range of ACE2 orthologs can support entry of the four coronaviruses

To evaluate spike protein-mediated entry of these coronaviruses, we first tested multiple conditions for establishing a robust SARS-CoV-2 pseudovirus reporter assay (Figs 5A and 5B) . We found that, although the transmembrane serine protease TMPRSS2 slightly enhanced the pseudovirus-produced reporter signals under certain conditions, the most robust signals were produced by the reporter retroviruses pseudotyped with a SARS-CoV-2 spike variant that had the ‘PRRAR’ furin site mutated to a single arginine residue. Then reporter retroviruses pseudotyped with six different spike proteins, including a wildtype SARS-CoV-2 spike (SARS-CoV-2 WHU01 pp) , a furin site deletion mutant of SARS-CoV-2 spike (SARS-CoV-2 Δfurin pp) , a wildtype SARS-CoV spike (SARS-CoV BJ01 pp) , a SARS-CoV spike carrying the Pangolin-CoV-2020 RBD (Pangolin-RBD/BJ01 pp) , a wildtype Bat-CoV spike (Bat-CoV RaTG13 pp) , and a Bat-CoV spike carrying the Pangolin-CoV-2020 RBD (Pangolin-RBD/RaTG13 pp) , were respectively used to infect 293T cells expressing each of the sixteen ACE2 orthologs. A VSV-G-pseudotyped reporter retrovirus whose entry is independent of ACE2 was used as a control virus. As expected, all the orthologs that supported RBD binding were also functional on supporting pseudovirus infection. Again, ACE2 orthologs of humans and most domestic mammals, including camels, cattle, horses, goats, sheep, cats, and rabbits, supported entry of all the tested pseudoviruses (Fig 5C and Figs 6A-6G) . The ability of these ACE2 orthologs to support SARS-CoV-2 infection were further confirmed by infection assays using SARS-CoV-2 live virus (Fig 6H) . These data indicate that humans and these domestic animals could be generally susceptible to infections of the four distinct coronaviruses.

Example 3. Recombinant RBD-Ig and ACE2-Ig proteins potently block SARS-CoV-2 entry

Recombinant RBD and soluble ACE2 proteins have been shown to potently block SARS-CoV entry (22, 23) . To investigate whether similar approaches could also be applied to SARS-CoV-2, we first produced mouse IgG2a Fc fusion proteins of RBD (RBD-Ig) and soluble ACE2 (ACE2-Ig) variants (Fig 7A) . Specifically, the RBD variants include wildtype RBDs of SARS-CoV, Pangolin-CoV-2020, and SARS-CoV-2, and four mutants of SARS-CoV-2 RBD (Figs 3A and 8A) . The ACE2-Ig variants include human ACE2 truncated at its residue 615 (615-wt) (human ACE2 truncated at its residue 615 as shown in SEQ ID NO: 1) and 740 (740-wt) (human ACE2 truncated at its residue 740 as shown in SEQ ID NO: 3) , respectively, and mutants of the 615-and 740-version ACE2-Ig proteins (Fig 8B) . We then evaluated these proteins for their potency of blocking SARS-CoV-2 Δfurin pseudovirus infection (Figs 7B and 7C) . Among all the RBD-Ig variants, the Y505W mutant of SARS-CoV-2 RBD showed modestly improved potency over the wildtype, and wildtype RBD of Pangolin-CoV-2020 showed the best neutralization activity among all the tested RBDs (Fig 7B) . Among the tested ACE2-Ig variants, interestingly, all the 740-version variants showed significantly better potency than the 615-version variants (two-tailed two-sample t-test, p<0.001; Fig 7C) . In addition, D30E mutants of both 615- (human ACE2 truncated at its residue 615 having a mutation of D30E as shown in SEQ ID NO: 2) and 740-version (human ACE2 truncated at its residue 740 having a mutation of D30E as shown in SEQ ID NO: 4) proteins showed improved potency over the corresponding wildtypes, and the 740-D30E variant outperformed all the RBD-Ig and ACE2-Ig variants.

Example 4. The 740-D30E variant of ACE2-Ig fusion is a broadly neutralizing immunoadhesin against SARS-CoV-2, SARS-CoV-1, Pangolin-CoV-2020, and Bat-CoV RaTG13

The 740-wt (human ACE2 truncated at its residue 740 as shown in SEQ ID NO: 3) and 740-D30E (human ACE2 truncated at its residue 740 having a mutation of D30E as shown in SEQ ID NO: 4) variants of ACE2-Ig fusion for their neutralization activities against SARS-CoV-2, SARS-CoV, Pangolin-CoV-2020, and Bat-CoV RaTG13 pseudotypes (Figs 9A-9E) were further tested. Interestingly, the D30E mutation improves 740-Ig’s neutralization activity against SARS-CoV-2 and Bat-CoV RaTG13 pseudoviruses (Figs 9A, 9B and 9D) but not Pangolin-RBD/BJ01 or SARS-CoV pseudovirus (Figs 9C and 9E) . Consistently, cell-surface ACE2 carrying this D30E mutation binds SARS-CoV-2 RBD better than the wildtype ACE2 does, and this enhanced binding was not observed with SARS-CoV RBD (Fig 10) . The D30 residue of human ACE2 was consistently found to form a salt-bridge interaction with the K417 residue of SARS-CoV-2 RBD in multiple released structures of SARS-CoV-2 RBD in complex with ACE2 (19, 20) (Fig 9F) . The residue 417 of Bat-CoV RaTG13 is also a lysine, while the same residue is an arginine for Pangolin-CoV-2020 RBD and a valine for SARS-CoV RBD. Thus, the mechanism of the D30E-mediated improvement is likely that the mutation enhances the salt-bridge interaction between the residue 30 of the ACE2 and residue 417 of SARS-CoV-2 and Bat-CoV RaTG13 RBDs. ACE2-Ig-v1.1 comprises two truncated ACE2-740-D30E (human ACE2 truncated at its residue 740 having a mutation of D30E as shown in SEQ ID NO: 4) . These data suggest that the 740-D30E variant of ACE2-Ig fusion (ACE2-Ig-v1.1) is a broadly neutralizing immunoadhesin against SARS-CoV-2, SARS-CoV, Pangolin-CoV-2020, and Bat-CoV RaTG13.

Example 5. Improving ACE2-Ig’s neutralization potency by introducing mutations to soluble ACE2 domains.

As shown in Figs 11 and 12, four ACE2-Ig fusion variants: ACE2-Ig-v1, ACE2-Ig-v1.1, ACE2-Ig-v1.2, and ACE2-Ig-v1.3 are constructed. ACE2-Ig-v1 comprises two truncated ACE2-740 (human ACE2 truncated at its residue 740 without mutation as shown in SEQ ID NO: 3) . ACE2-Ig-v1.2 comprises two truncated ACE2-740-T27Y-D30E (human ACE2 truncated at its residue 740 having mutations of T27Y and D30E as shown in SEQ ID NO: 5) . ACE2-Ig-v1.3 comprises two truncated ACE2-740-L79I (human ACE2 truncated at its residue 740 having a mutation of L79I as shown in SEQ ID NO: 6) . Figure 12A shows diagrams of ACE2-Ig fusion variants characterized in the following studies, and Figure 12B shows that human ACE2-expressing 293T cells were infected with SARS-CoV-2 pseudotype in the presence of the indicated ACE2-IgG fusion proteins at the indicated concentrations. An unrelated Ig protein was used as a control. Viral entry was measured by luciferase reporter expression at 48 hours post infection and percentage-of-infection (Infection%) values were calculated. Data points represent mean ± s.d. of three biological replicates. Obviously, ACE2-Ig-v1.1, ACE2-Ig-v1.2, and ACE2-Ig-v1.3 indicate better inhibition effects than ACE2-Ig-v1.

Example 6. ACE2-Ig variants containing tetravalent ACE2 have higher neutralization potency over ACE2-Ig-v1.1

Fig. 13A shows diagrams of ACE2-Ig fusion variants: ACE2-Ig-v1.1, ACE2-Ig-v2, and ACE2-Ig-v2.1 characterized in the following studies, and Fig. 13B shows that human ACE2-expressing 293T cells were infected with SARS-CoV-2 pseudotype in the presence of the indicated IgG Fc fusion proteins at the indicated concentrations. ACE2-Ig-v2 comprises two truncated ACE2-740-D30E (human ACE2 truncated at its residue 740 having a mutation of D30E as shown in SEQ ID NO: 4) , and two truncated ACE2-615-D30E (human ACE2 truncated at its residue 615 having a mutation of D30E as shown in SEQ ID NO: 2) . ACE2-Ig-v2.1 comprises four truncated ACE2-740-D30E (human ACE2 truncated at its residue 740 having a mutation of D30E as shown in SEQ ID NO: 4) . An unrelated Ig protein was used as a control. Viral entry was measured by luciferase reporter expression at 48 hours post infection and percentage-of-infection (Infection%) values were calculated. Data points represent mean ± s.d. of three biological replicates.

Obviously, ACE2-Ig-v2 and ACE2-Ig-v2.1 indicate better inhibition effects than ACE2- Ig-v1.1.

Example 7. ACE2-Ig variants of antibody-like configurations significantly outperform ACE2-Ig-v1.1

Fig. 14A shows diagrams of ACE2-Ig fusion variants: ACE2-Ig-v1.1, ACE2-Ig-v3.1, and ACE2-Ig-v4.1 characterized in the following studies, and Figs. 14B-14E show Human ACE2-expressing 293T (B, D, and E) or HeLa (C) cells were infected with SARS-CoV-2 (B and C) , SARS-CoV (D) , or Pangolin-CoV-2020 (E) pseudotype in the presence of the indicated IgG Fc fusion proteins at the indicated concentrations. ACE2-Ig-v3.1 comprises four truncated ACE2-740-D30E (human ACE2 truncated at its residue 740 having a mutation of D30E as shown in SEQ ID NO: 4) . ACE2-Ig-v4.1 comprises four truncated ACE2-740-D30E (human ACE2 truncated at its residue 740 having a mutation of D30E as shown in SEQ ID NO: 4) , and two truncated ACE2-615-D30E (human ACE2 truncated at its residue 615 having a mutation of D30E as shown in SEQ ID NO: 2) . Viral entry was measured by luciferase reporter expression at 48 hours post infection and percentage-of-infection (Infection%) values were calculated. Data points represent mean ± s.d. of three biological replicates. ACE2-Ig-v3.1 and ACE2-Ig-v4.1 have estimated IC ₅₀ values of 40-70 pM and IC ₉₀ values of 250-730 pM against SARS-CoV-2, representing >10-fold improvement on IC ₅₀ and >25-fold improvement on IC ₉₀ compared to that of soluble ACE2-Ig-v1.1 (Figs 14B) . These data demonstrate that ACE2-Ig-v3.1 and ACE2-Ig-v4.1 is a markedly improved ACE2-Ig variant as a potent entry inhibitor against SARS-CoV-2 virus.

Obviously, ACE2-Ig-v3.1 and ACE2-Ig-v4.1 significantly outperform ACE2-Ig-v1.1.

Example 8. ACE2-Ig-v3.1 potently neutralizes SARS-CoV-2 live virus

Human ACE2-expressing HeLa cells were infected with SARS-CoV-2 live virus at 800 TCID ₅₀ in the presence of a control Ig protein, ACE2-Ig-v1, or ACE2-Ig-v3.1 at the indicated concentrations. Cells were then fixed and stained with rabbit anti-SARS-CoV-2 nucleocapsid (NP) polyclonal antibody for fluorescence microscopy at 24 hours post infection. Note that, as shown in Fig. 15, ACE2-Ig-v3.1 at 0.16 μg/mL showed a more potent inhibition of SARS-CoV-2 live virus infection than ACE2-Ig-v1 at 0.8 μg/mL. Moreover, ACE2-Ig-v3.1 at 0.8 μg/mL (1.85 nM) completely abolished viral NP signal. Obviously, ACE2-Ig-v3.1 indicates significantly better inhibition effects than ACE2-Ig-v1.

Example 9. ACE2-Ig variant containing hexavalent or octavalent ACE2 significantly outperforms ACE2-Ig-v1.1 but not ACE2-Ig-v3.1

Fig 16A shows diagrams of soluble ACE2-Ig fusion variants: ACE2-Ig-v1.1, ACE2-Ig-v3.1, ACE2-Ig-v4.1, ACE2-Ig-v5.1 and ACE2-Ig-v6.1 characterized in the following studies. ACE2-Ig-v5.1 comprises four truncated ACE2-740-D30E (human ACE2 truncated at its residue 740 having a mutation of D30E as shown in SEQ ID NO: 4) , and two truncated ACE2-615-D30E (human ACE2 truncated at its residue 615 having a mutation of D30E as shown in SEQ ID NO: 2) . ACE2-Ig-v6.1 comprises four truncated ACE2-740-D30E (human ACE2 truncated at its residue 740 having a mutation of D30E as shown in SEQ ID NO: 4) , and four truncated ACE2-615-D30E (human ACE2 truncated at its residue 615 having a mutation of D30E as shown in SEQ ID NO: 2) . Fig 16B shows human ACE2-expressing 293T cells were infected with SARS-CoV-2 pseudotype in the presence of the indicated ACE2-Ig fusion proteins at the indicated concentrations. Viral entry was measured by luciferase reporter expression at 48 hours post infection and percentage-of-infection (Infection%) values were calculated. Data points represent mean ± s.d. of three biological replicates.

ACE2-Ig variant containing hexavalent (ACE2-Ig-v4.1 and ACE2-Ig-v5.1) or octavalent (ACE2-Ig-v6.1) ACE2 significantly outperforms ACE2-Ig-v1.1 but not ACE2-Ig-v3.1.

Example 10. ACE2-Ig-v3.4 and ACE2-Ig-v3.5 significantly outperform ACE2-Ig-v3.1

Fig. 17A shows diagrams of ACE2-Ig fusion variants: ACE2-Ig-v1.1, ACE2-Ig-v3.1, ACE2-Ig-v3.4 and ACE2-Ig-v3.5 characterized in the following studies. Fig. 17B shows that human ACE2-expressing 293T cells were infected with SARS-CoV-2 pseudotype in the presence of the indicated ACE2-IgG fusion proteins at the indicated concentrations. ACE2-Ig-v3.4 comprises four truncated ACE2-740-D30E-L79I (human ACE2 truncated at its residue 740 having mutations of D30E and L79I as shown in SEQ ID NO: 7) . ACE2-Ig-v3.5 comprises four truncated ACE2-740-T27Y-D30E-L79I (human ACE2 truncated at its residue 740 having mutations of T27Y, D30E and L79I as shown in SEQ ID NO: 8) . Viral entry was measured by luciferase reporter expression at 48 hours post infection and percentage-of-infection (Infection%) values were calculated. Data points represent mean ± s.d. of three biological replicates.

ACE2-Ig-v3.4 and ACE2-Ig-v3.5 significantly outperform ACE2-Ig-v3.1.

Example 11. Pseudoviruses of the B. 1.1.7, B. 1.351, and P. 1 variants displayed patterns of animal-ACE2 tropism distinct from the patterns of other variants

To evaluate ACE2 ortholog-mediated viral entry of the above-mentioned SARS-CoV-2 variants, we produced luciferase reporter retroviruses pseudotyped with one of these different spike variants (Figure 19A) . These reporter pseudoviruses were used to infect 293T cells expressing each of eight ACE2 orthologs (Figures 19B and 20, Table 2) , including the human ACE2 and ACE2 orthologs of cattle (Bos taurus) , pig (Sus scrofa domesticus) , cat (Felis catus) , rabbit (Oryctolagus cuniculus) , bat (Rhinolophus sinicus isolate Rs-3357) , rat (Rattus norvegicus) , and mouse (Mus musculus) . Parallel infection experiments using 293T cells transfected with an empty vector plasmid were included as controls. Consistent with our previous report ¹⁴, the early isolate WHU01 efficiently infected 293T cells expressing most of the tested ACE2 orthologs except for that of rat and mouse (Figure 21A) . While a similar pattern was also observed with the variants D614G, Y453F, and B. 1.258 (Figures 21B, 21C, and 21G) , distinct patterns were observed with the remaining variants. The mink-associated variant Y453F-F486L-N501T showed a modest ability to use rat ACE2 for entry (Figure 21D) . More significant changes were observed with the variants B. 1.1.7, B. 1.351, P. 1, and Y453F-E484K-Q498H, all of which showed ability to use ACE2 orthologs of rat and mouse for entry (Figures 21E, 21F, and 21H-L) . In addition, the variants B. 1.351 and P. 1 showed complete loss of the ability to use bat ACE2 for entry (Figures 21H-K) . These data suggest that the mutations acquired by different variants might have changed SARS-CoV-2 RBD affinity to different ACE2 orthologs.

Figure 19. Western blot detection of ACE2 and SARS-CoV-2 spike proteins. (A) MLV retroviral vector-based pseudoviruses of the indicated SARS-CoV-2 variants were validated for the presence of spike protein (C9-tag) and MLV-Gag p30 protein. (B) 293T cells transfected with the indicated ACE2 genes (S-tagged) were collected at 48 hours post transfection. Expression levels of the indicated ACE2 proteins were detected using Western Blot.

Figure 20. Sequences of eight animal-ACE2 orthologs investigated in this study are aligned, with only residues in less than

from RBD atoms shown here. Species and accession numbers of the amino-acid sequences of these ACE2 orthologs are provided in Table S1. The numbering of the aligned sequences is based on human ACE2 protein, and the residues different from the corresponding ones in human ACE2 are highlighted in gray.

Figure 21. Animal-ACE2 tropism of SARS-CoV-2-variant pseudoviruses. (A-L) 293T cells expressing one of the eight indicated ACE2 orthologs were infected with each of twelve pseudoviruses corresponding to the twelve SARS-CoV-2 spike variants shown in Figure 1. ACE2-mediated pseudovirus entry was measured by a luciferase reporter expression at 48 hours post infection. Data shown are representative of three independent experiments performed by two different people with similar results, and data points represent mean ± s.d. of three biological replicates.

Table 2. Species and accession numbers of the amino-acid sequences of the eight ACE2 orthologs investigated in this study.

Example 12. Y453F mutation enhances RBD affinity to human, cattle, pig, and cat ACE2 proteins

It is sought to quantitatively study the ability of the variants to utilize various animal orthologs of ACE2. The extracellular domains of the eight ACE2 orthologs and the RBD domains of five spike variants, including WHU01, Y453F, B. 1.1.7, B. 1.351 and P. 1, were expressed in 293F cells as immunoglobulin Fc fusion proteins. Purified ACE2 and RBD recombinant proteins were then used as immobilized receptors and analytes, respectively, in a bio-layer interferometry (BLI) -based assay to measure the interaction kinetics of forty RBD-ACE2 pairs (Figures 22 and 23; Tables 3 and 4) . When the RBD of early SARS-CoV-2 isolate WHU01 was tested against different ACE2 proteins, strong interactions were observed for human, cattle, pig, cat, and rabbit ACE2 proteins, with human ACE2 showing the highest affinity to this RBD (Figure 22 and Table 3) . No any binding signal was detected for mouse ACE2 (Figure 22H) . We then compared the kinetics data of each RBD variants to the data of the WHU01 variant. We observed that the Y453F mutation increased RBD affinity to human ACE2 by ～4.7-fold (Figure 22A and Table 3) , consistent with a very recent report ⁴². In addition, cattle, pig, and cat ACE2 proteins, respectively, were also found with >2-fold higher affinities to the Y453F RBD than that to the WHU01 RBD (Figures 22B-D, and Table 3) .

Figure 22-continued. Interaction kinetics of forty RBD-ACE2 pairs. (A-H) A bio-layer interferometry (BLI) -based assay was used to measure the kinetics of RBD-ACE2 interactions. Recombinant RBD proteins of five SARS-CoV-2 variants, including WHU01, B. 1.298, B. 1.1.7, B. 1.351, and P. 1, were sequentially used as analytes to measure their interactions with recombinant ACE2 proteins of eight species, including human (A) , cattle (B) , pig (C) , cat (D) , rabbit (E) , bat (F) , rat (G) , mouse (H) . The thick lines are the binding sensorgrams for each analyte at 100 nM, 50 nM, 25 nM, 12.5 nM, or 6.25 nM. The thin lines show fits of the data to a 1: 1 Langmuir binding model (global fit) . The affinity (K _D, nM) data for all the forty RBD-ACE2 pairs are summarized in Table 3. Full kinetics data, including the on-rate (k _a, M ^-1s ^-1) , off-rate (k _dis, s ^-1) , and affinity (K _D, nM) for each interaction, are provided in Table 4.

Figure 23. SDS-PAGE images of purified recombinant Fc fusion proteins used in Figure 21. (A) Recombinant Fc fusions of animal ACE2 proteins. (B) Recombinant Fc fusions of SARS-CoV-2 variant RBDs.

Table 3. The affinities (K _D, nM) and BLI response values (in parentheses; nm) of the forty RBD-ACE2 interaction pairs

Table 4. Interaction kinetics data, including the BLI Response (nm) at 100 nM analyte, on-rate (k _a, M ^-1s ^-1) , off-rate (k _dis, s ^-1) , and affinity (K _D, nM) , of the forty ACE2-RBD pairs

Example 13. N501Y mutation enhances RBD affinity to most of the tested ACE2 proteins, especially cattle and pig ACE2 proteins

In the case of the B. 1.1.7 RBD which has a N501Y signature mutation, we observed 2.9-fold increase in the RBD affinity to human ACE2 (Figure 22A and Table 3) . This is consistent with the binding data from other reports and the increased infectivity of the B. 1.1.7 variant. Structural studies show that the Tyr501 residue of the B. 1.1.7 RBD inserts into a cavity at the binding interface and forms a perpendicular π–π stacking interaction with the Tyr41residue of ACE2. Perhaps because of a same mechanism, the B. 1.1.7 RBD also displayed significantly increased affinity to all the other tested ACE2 proteins, except for bat ACE2 that has a His41 rather than a Tyr41 (Figures 22B-H and 20; Table 3) . More pronounced affinity increase was observed with cattle ACE2 (5.0-fold) and pig ACE2 (6.1-fold) , making the affinities of cattle and pig ACE2 proteins to B. 1.1.7 RBD slightly higher than that of human ACE2 to WHU01 RBD (Figures 22B, C, and Table 3) . Only very weak binding signals were detected for mouse ACE2, though the B. 1.1.7 pseudovirus efficiently utilized mouse ACE2 for entry (Figures 21E, F, and 22H) . Consistent with the binding data, recombinant cattle and pig ACE2 proteins, respectively, neutralized B. 1.1.7 pseudovirus ～17-and ～80-fold more efficient than neutralizing WHU01 pseudovirus, while recombinant human ACE2 protein only showed a 4-fold difference (Figure 23A-C) . Recombinant mouse ACE2 protein at the tested concentrations did not show any neutralization of either WHU01 or B. 1.1.7 pseudovirus (Figure 23D) .

Figure 24. Neutralization of SARS-CoV-2 variants by recombinant ACE2 proteins. (A-D) Recombinant Fc fusions of human ACE2 (A) , cattle ACE2 (B) , pig ACE2 (C) , and mouse ACE2 (D) were tested for their neutralization potencies against pseudoviruses of five SARS-CoV-2 variants, including WHU01, Y453F, B. 1.1.7, B. 1.351, and P. 1. HeLa-hACE2 cells were infected with each of the five pseudoviruses in the presence of an ACE2 protein at the indicated concentrations. Viral entry was measured by luciferase reporter expression at 48 hours post infection. Luminescence values observed at each concentration were divided by the values observed at concentration zero to calculate percentage-of-infection (Infection%) values. Data shown are representative of two independent experiments performed by two different people with similar results. Data points represent mean ± s.d. of three biological replicates.

Example 14. The RBDs of B. 1.351 and P. 1 have increased affinities to cattle, pig, and mouse ACE2 proteins

In the case of the RBDs of B. 1.351 and P. 1 which share the E484K-N501Y signature mutations, almost identical patterns were observed (Figure 22) . Consistent with the pseudovirus infection data (Figures 21H-K) , the B. 1.351 and P. 1 RBDs showed a complete loss of interaction with bat ACE2, and a clear gain of interaction with mouse ACE2 (Figures 22F and 22H) . Consistently, when mouse ACE2 protein was tested in the pseudovirus neutralization assays, B. 1.351 and P. 1 variants could be neutralized by this protein (Figure 24D) . Additional pseudovirus infection experiments showed that every RBD mutation of the B. 1.351 and P. 1 variants contribute to the utilization of mouse ACE2 for entry, with the N501Y mutation showed the most prominent effect (Figure 25) . A Y41A single mutation in mouse ACE2 significantly impaired infectivity of all the single-spike-mutation pseudoviruses, but not the triple-spike-mutation pseudoviruses, while Y41A-H353A double mutation in mouse ACE2 significantly impaired infectivity of all the pseudoviruses, but much less pronounced effect was observed with the triple-spike-mutation pseudoviruses (Figure 25) . These data suggest that the B. 1.351 and P. 1 RBDs gain binding to mouse ACE2 through the mutated RBD residues forming multiple new interactions with ACE2.

Compared to WHU01 RBD, B. 1.351 and P. 1 RBDs respectively also showed 2.9-and 2.7-fold higher affinities to cattle ACE2, and 3.4-and 2.9-fold higher affinities to pig ACE2 protein, possibly because of the shared N501Y mutation in these two variants (Figures 22B and C) . Consistent with these binding data, recombinant cattle and pig ACE2 proteins, respectively, neutralized both the B. 1.351 and P. 1 pseudoviruses significantly more efficiently than neutralizing WHU01 pseudovirus (Figures 24B and C) .

Figure 25. The effects of K417N, K417T, E484K, N501Y mutations on the ability of SARS-CoV-2 pseudovirus to utilize mouse ACE2. (A-G) SARS-CoV-2 pseudoviruses carrying the indicated spike mutations were used to infect 293T cells expressing human ACE2 or a mouse ACE2 mutant. 293T cells transfected with an empty vector were used as a control.

ACE2-mediated pseudovirus entry was measured by a luciferase reporter expression at 48 hours post infection. Luminescence values were divided by the values observed with human ACE2 to calculate Relative Infection (%) values. Data shown are representative of three independent experiments performed by two different people with similar results, and data points represent mean ± s.d. of three biological replicates. (H) Expression levels of the ACE2 constructs used in the above experiments were detected using Western blotting.

Example 15. Spike mutations cause escape from potent neutralizing antibodies in clinical use

We then performed pseudovirus neutralization assays to evaluate sensitivity of these diverse SARS-CoV-2 variants to four therapeutic antibodies in clinical use (etesevimab/LY-CoV016, bamlanivimab/LY-CoV555, casirivimab/REGN10933, and imdevimab/REGN10987) . While both the early strain WHU01 and the D614G variant were highly sensitive to all the four antibodies, all the RBD-mutated variants showed partial or complete resistance to at least one antibody (Figure 26) . Specifically, both of the variants carrying a Y453F mutation showed strong resistance to REGN10933 (Figures 26C and 26D) . The B. 1.258 variant that carries an N439K mutation showed partial resistance to REGN10987 (Figure 26G) . It is of note that the VOCs B. 1.351 and P. 1 showed strong resistance to REGN10933, and complete resistance to both LY-CoV016 and LY-CoV555, the two components of an antibody-cocktail therapy authorized for emergency use by the U.S. FDA (Figures 26H-K) . In addition, the spike Y453F-E484K-Q498H triple mutation made the pseudovirus partially resistant to all the four tested antibodies (Figure 26L) .

Figure 26. Neutralization sensitivity of SARS-CoV-2 variants to four monoclonal antibodies in clinical use. (A-L) Twelve SARS-CoV-2 variant pseudoviruses were tested for their neutralization sensitivity to four monoclonal antibodies (LY-CoV016/etesevimab, LY-CoV555/bamlanivimab, REGN10933/casirivimab, REGN10987/imdevimab) that constitute two antibody cocktails authorized by the U.S. FDA for emergency use. HeLa-hACE2 cells, a stable cell line that overexpresses human ACE2, were infected with each of the twelve pseudoviruses in the presence of the indicated neutralizing antibodies at the indicated concentrations. Viral entry was measured by luciferase reporter expression at 48 hours post infection. Luminescence values observed at each concentration were divided by the values observed at concentration zero to calculate percentage-of-infection (Infection%) values. Data shown are representative of two independent experiments performed by two different people with similar results, and data points represent mean ± s.d. of three biological replicates.

Example 16. The impact of RBD mutations on antibody affinity

We then performed BLI assays to quantitatively measure the kinetics of antibody-RBD interactions (Figure 27, and Table 4) . The binding data are fully consistent with the pseudovirus neutralization data. Specifically, the mink-associated Y453F mutation decreased RBD affinity to REGN10933 (Figure 27C) . The B. 1.1.7-signature mutation N501Y decreased RBD binding to LY-CoV016 (Figure 27A) . The RBDs of VOCs B. 1.351 and P. 1 showed significantly decreased binding to REGN10933, weakly-detectable interaction with LY-CoV016, and complete loss of interaction with LY-CoV555 (Figures 27A-C) . Then the Y453F-E484K-Q498H triple mutation compromised RBD affinity to all the four antibodies (Figures 27A-D) . Consistent with these binding data, analyzing structural data of these antibodies in complex with the original RBD revealed contact of LY-CoV016 with RBD residues Lys417, LY-CoV555 with RBD residues Glu484 and Phe486, REGN10933 with RBD residues Lys417, Tyr453, Glu484, and Phe486, and REGN10987 with RBD residues Asn439 and Gln498 (Figure 28) . These data demonstrate that SARS-CoV-2 variants could easily develop resistance to neutralization antibodies or even antibody cocktails in clinical use, highlighting the necessity of developing broad-spectrum anti-coronavirus agents.

Figure 27. Interaction kinetics of twenty-four RBD-antibody pairs. (A-D) The BLI-based assay was used to measure the kinetics of RBD-antibody interactions. Recombinant RBD proteins of six SARS-CoV-2 spike variants, including WHU01, B. 1.298, B. 1.1.7, B. 1.351, P. 1, and Y453F-E484K-N501 were sequentially used as analytes to measure their interactions with four monoclonal antibodies, including LY-CoV016 (A) , LY-CoV555 (B) , REGN10933 (C) , and REGN10987 (D) . The thick lines are the binding sensorgrams for each analyte at 100 nM, 50 nM, 25 nM, 12.5 nM, or 6.25 nM. The thin lines show fits of the data to a 1: 1 Langmuir binding model (global fit) . The affinity (K _D, nM) for each interaction is indicated in the insets of the figures. Full kinetics data, including the on-rate (k _a, M ^-1s ^-1) , off-rate (k _dis, s ^-1) , and affinity (K _D, nM) for each interaction, are provided in Table 4.

Figure 28. Interactions between SARS-CoV-2 RBD and four monoclonal antibodies. (A-D) Structures of SARS-CoV-2 RBD in complex with LY-CoV016 (A; PDB accession no. 7C01) , LY-CoV555 (B; PDB accession no. 7MKG) , REGN10933 (C; PDB accession no. 6XDG) , or REGN10987 (D; PDB accession no. 6XDG) are analyzed. Antibody residues in less than

from RBD atoms and RBD residues associated with SARS-CoV-2 mutations are shown as sticks. SARS-CoV-2 mutation-associated RBD residues that have direct contact with antibody residues or reside within

from antibody atoms are shown as spheres and labelled.

Table 4. Interaction kinetics data, including the BLI Response (nm) at 100 nM analyte, on-rate (k _a, M ^-1s ^-1) , off-rate (k _dis, s ^-1) , and affinity (K _D, nM) , of the twenty-four mAb-RBD pairs

Example 17. Spike mutations change sensitivity of SARS-CoV-2 variants to different ACE2-Ig constructs

ACE2-Ig, a recombinant Fc fusion protein of soluble human ACE2, is a potent SARS-CoV-2 entry inhibitor that has been reported in multiple studies. We evaluated in vitro neutralization sensitivity of the twelve SARS-CoV-2 variants to eight representative ACE2-Ig constructs described in these studies (Figure 29) , including three constructs (ACE2-Ig-v1, ACE2-Ig-v1.1, ACE2-Ig-v3.1) , one construct from Chan et al (ACE2-Ig-Chan-v2.4) , and four constructs from Glasgow et al (ACE2-Ig-Glasgow-293, ACE2-Ig-Glasgow-310, ACE2-Ig-Glasgow-311, ACE2-Ig-Glasgow-313) . Because we found that the CLD domain of human ACE2 has an ～20-fold contribution to ACE2-Ig’s neutralization activity against SARS-CoV-2 pseudoviruses, we used CLD-containing soluble ACE2 domains for all the constructs tested in this study. We got three interesting findings here (Figures 30) . First, all the twelve SARS-CoV-2 variants were potently neutralized by all the eight ACE2-Ig constructs, demonstrating that ACE2-Ig is a broad-spectrum anti-SARS-CoV-2 agent. Second, the mink-associated variant Y453F-F486L-N501T showed partial resistance to ACE2-Ig-Glasgow-310 and ACE2-Ig-Glasgow-313, but clearly increased sensitivity to ACE2-Ig-v1, ACE2-Ig-v1.1 and ACE2-Ig-v3.1, the constructs that are not heavily mutated in the spike-binding interface of the soluble ACE2 domain (Figures 30A-C, F, H, and 31) . These data demonstrate that extensively mutating the ACE2 residues near the RBD-binding interface to increase ACE2-Ig’s neutralizing potency might compromise the protein’s neutralization breadth. Third, compared to the early isolate WHU01, most circulating variants, including the VOCs B. 1.1.7, B. 1.351 and P. 1, showed significantly increased (up to ～15-fold) neutralization sensitivity to ACE2-Ig-v1, ACE2-Ig-v1.1 and ACE2-Ig-v3.1, the constructs that carry a wild-type or a D30E-mutated ACE2 domain (Figures 30A-C) . These data suggest that SARS-CoV-2 evolves toward better utilization of ACE2 as a receptor. This is consistent with structural studies showing that spike trimers of multiple SARS-CoV-2 variants, including a mink-associate variant and the VOCs B. 1.1.7, B. 1.1.28, and B1.351, have higher propensity to adopt ‘RBD-up’ or open state than the D614G variant does.

During preparation of this manuscript, the variant B. 1.617, which has L452R-E484Q signature double mutations in the RBD, is causing uncontrolled outbreaks in India and has spread to multiple countries. We therefore generated a spike variant that carries the L452R-E484Q mutations, and tested neutralization sensitivity of this variant. Although this variant also showed an almost complete resistance to the neutralizing antibody LY-CoV555 and a partial resistance to the antibody REGN10933, the three tested ACE2-Ig constructs all potently neutralized it (Figure 32) . These data demonstrate that ACE2-Ig is a good drug candidate against diverse SARS-CoV-2 variants that emerge over the course of the pandemic.

Figure 29. SDS-PAGE images of purified recombinant ACE2-Ig proteins used in Figure 30.

Figure 30. Neutralization sensitivity of SARS-CoV-2 variants to eight ACE2-Ig constructs. (A-H) Eight recombinant ACE2-Ig protein constructs, including three from our recent study (ACE2-Ig-v1, ACE2-Ig-v1.1, ACE2-Ig-v3.1; A-C) , one from Chan et al (ACE2-Ig-Chan-v2.4; D) ; and four from Glasgow et al (ACE2-Ig-Glasgow-293, ACE2-Ig-Glasgow-310, ACE2-Ig-Glasgow-311, ACE2-Ig-Glasgow-313; E-H) , were tested for their neutralization potencies against the twelve SARS-CoV-2 variant pseudoviruses. HeLa-hACE2 cells were infected with each of the twelve pseudoviruses in the presence of the indicated ACE2-Ig proteins at the indicated concentrations. Viral entry was measured by luciferase reporter expression at 48 hours post infection. Luminescence values observed at each concentration were divided by the values observed at concentration zero to calculate percentage-of-infection (Infection%) values. Data shown are representative of three independent experiments performed by two different people with similar results, and data points represent mean ± s.d. of three biological replicates.

Figure 31. Distribution of the mutated residues of the eight ACE2-Ig constructs. Interactions between SARS-CoV-2 RBD and ACE2 involve a large number of contact residues (PDB accession no. 6M0J) . All SARS-CoV-2 variant-associated RBD mutations investigated in this study are shown as sticks. ACE2-residues mutated in any of the eight ACE2-Ig constructs are shown as spheres and labelled. Asp30 was mutated to a Glu30 in the ACE2-Ig-v1.1 and ACE2-Ig-v3.1 constructs. Thr27 and Leu79 were chosen for mutation by both Chan et al and Glasgow et al. Asn330 was the third residue mutated in ACE2-Ig-Chan-v2.4 by Chan et al. The remaining labelled spheres were the residues mutated in the four ACE2-Ig constructs (ACE2-Ig-Glasgow-293, ACE2-Ig-Glasgow-310, ACE2-Ig-Glasgow-311, ACE2-Ig-Glasgow-313) developed by Glasgow et al.

Figure 32. Neutralization sensitivity of the B. 1.617 pseudovirus to antibodies and ACE2-Ig constructs. (A) The B. 1.617 pseudovirus that carries the L452R-E484Q spike mutations was tested for its neutralization sensitivity to the four monoclonal antibodies (LY-CoV016, LY-CoV555, REGN10933, REGN10987) that constitute two antibody cocktails authorized by the U.S. FDA for emergency use. HeLa-hACE2 cells, a stable cell line that overexpresses human ACE2, were infected with B. 1.617 pseudovirus in the presence of the indicated neutralizing antibodies at the indicated concentrations. Viral entry was measured by luciferase reporter expression at 48 hours post infection. Luminescence values observed at each concentration were divided by the values observed at concentration zero to calculate percentage-of-infection (Infection%) values. (B) Experiments similar to A, except that three ACE2-Ig constructs (ACE2-Ig-v1, ACE2-Ig-v1.1, and ACE2-Ig-v3.1) were used as the neutralizing agents against the B. 1.617 pseudovirus. Data shown are representative of two independent experiments performed by two different people with similar results, and data points represent mean ± s.d. of three biological replicates.

Example 18.

HeLa-hACE2 cells, a stable cell line that overexpresses human ACE2, were infected with SARS-CoV-2 pseudovirus in the presence of the indicated ACE2-Ig variants at the indicated concentrations. Viral entry was measured by luciferase reporter expression at 48 hours post infection. Luminescence values observed at each concentration were divided by the values observed at concentration zero to calculate percentage-of-infection (Infection%) values. Data shown are mean ± s.d. of three independent experiments.

Figure 33 shows ACE2-Ig-v1.1-related constructs with ACE2 truncated at its residue within residues 720-740 inhibit SARS-CoV-2 pseudovirus infection with similar efficacy. Figure 34 shows ACE2-Ig-v3.1-related constructs with ACE2 truncated at its residue within residues 720-740 inhibit SARS-CoV-2 pseudovirus infection with similar efficacy. Figure 35 shows neutralization efficacy of ACE2-Ig-v3.1-related linker variants with a synthetic flexible linker of (GGGGS) c, where c is an integer of 0～7.

Discussion:

In the present invention, it is found that SARS-CoV-2 and three other coronaviruses can utilize human ACE2 and ACE2 orthologs of multiple domestic and wild animals for cell entry, suggesting a need of developing prophylaxis and therapeutics that are broadly effective against these coronaviruses. The present invention identified three key improvements to ACE2-Ig, a candidate of broadly effective entry inhibitor that could treat an established SARS-CoV-2 infection or provide protection from a new infection. First, all the 740-version ACE2-Ig variants showed markedly enhanced neutralization potency over the 615-version variants. The reason for this is not very clear yet, but it’s possible that the Collectrin-like domain (CLD) , included in the 740 version, stabilizes an orientation of the ACE2 enzymatic domain favorable to S-protein binding, or that the CLD has an independent anti-SARS-CoV-2 activity. Second, likely because SARS-CoV-2 has adapted in animals (e.g. pangolins) whose ACE2 orthologs have a glutamic acid at position 30, the D30E mutation further improves the 740-version ACE2-Ig’s neutralization potency against SARS-CoV-2. Moreover, the D30E mutation also enables the protein to neutralize all the four distinct SARS-like coronaviruses. Third, by leveraging antibody structure’s valency and avidity advantage, we generated a further improved variants ACE2-Ig-v3, ACE2-Ig-v3.1, ACE2-Ig-v3.4, ACE2-Ig-v3.5, ACE2-Ig-v4.1, ACE2-Ig-v5.1, ACE2-Ig-v6.1 that has at least 10-fold higher anti-SARS-CoV-2 potency over the ACE2-Ig-v1.1. Considering that fusion with IgG Fc normally improves a biologic’s half-life and that we’ve markedly improved neutralization potency of ACE2-Ig, we therefore optimistically expect that the ACE2-Ig variants developed in this study can be used to provide an effective protection from SARS-CoV-2 and other SARS-like viruses that might spillover into humans in the future, as well as SARS-CoV-2 variants that emerge over the course of the current pandemic. In the present invention, we investigated animal-ACE2 usage and neutralization sensitivity of thirteen spike genes derived from eight SARS-CoV-2 variants, including the B. 1.1.7, B. 1.351, P. 1, and B. 1.617 variants. We found that, in contrast to a prototype SARS-CoV-2 variant, the B. 1.1.7, B. 1.351, and P. 1 variants of concern had significantly enhanced affinities to cattle, pig, and mouse ACE2 proteins, suggesting increased susceptibility of these species to these SARS-CoV-2 variants. We then observed that most variants were partially or completely resistant against at least one of four tested antibodies in clinical use, with B. 1.351 and P. 1 showing significant resistance to three of them. Nevertheless, eight ACE2-Ig constructs, a class of broad-spectrum anti-SARS-CoV-2 drug candidates, efficiently neutralized all variants. Moreover, most variants were found to be significantly more sensitive (up to ～15-fold) to ACE2-Ig neutralization than the prototype variant, suggesting that SARS-CoV-2 evolves toward better utilizing ACE2, and that ACE2-Ig is an attractive drug candidate for coping with SARS-CoV-2 mutations.

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Claims

A soluble ACE2-Ig fusion variant having an antibody-like configuration, which comprises an ACE2 domain, and an immunoglobulin domain, wherein the immunoglobulin domain comprises a Fc region.
The soluble ACE2-Ig fusion variant of claim 1, wherein the immunoglobulin domain comprises in order from N-to C-terminus a unit of CH2 and CH3 regions.
The soluble ACE2-Ig fusion variant of claim 1, wherein the CH2 and CH3 regions are CH2 and CH3 regions of an IgG or IgA form, preferably, the IgG or IgA is IgG or IgA from camels, cattle, horses, goats, sheep, pigs, dogs, cats, mice, rats, or rabbits.
The soluble ACE2-Ig fusion variant of claim 1, wherein the ACE2 domain is a truncated ACE2 domain, and one truncated ACE2 domain comprises two truncated ACE2 or one truncated ACE2.
The soluble ACE2-Ig fusion variant of claim 1, wherein the truncated ACE2 is ACE2 truncated at its residue within residues 720-740 or 615, preferably, truncated at its residue 740 or 615.
The soluble ACE2-Ig fusion variant of claim 1, wherein the truncated ACE2 has one or more mutation sites selected from T27Y, D30E and L79I.
The soluble ACE2-Ig fusion variant of claim 1, wherein one truncated ACE2 domain comprising two truncated ACE2 is linked to the immunoglobulin domain, alternatively, one truncated ACE2 domain comprising two truncated ACE2 is linked to a hinge region of N-terminus of CH2 region or a linker of C-terminus of CH3 region of the immunoglobulin domain, thereby, a bivalent soluble ACE2-Ig fusion variant is obtained.
The soluble ACE2-Ig fusion variant of claim 1, wherein two truncated ACE2 domains, comprising two truncated ACE2 respectively, are linked to the immunoglobulin domain, preferably, two truncated ACE2 domains, comprising two truncated ACE2 respectively, are linked to a hinge region of N-terminus of CH2 regions and a linker of C-terminus of CH3 regions of the immunoglobulin domain respectively; alternatively, one truncated ACE2 domain comprising two truncated ACE2 is linked to the hinge region of N-terminus of CH2 regions, and two truncated ACE2 within the other truncated ACE2 domain are linked to two linkers of C-terminus of CH3 regions respectively, thereby, a tetravalent soluble ACE2-Ig fusion variant is obtained.
The soluble ACE2-Ig fusion variant of claim 1, wherein the immunoglobulin domain further comprises CH1 and CL regions.
The soluble ACE2-Ig fusion variant of claim 1, wherein the immunoglobulin domain comprises both light chain constant region and heavy chain constant regions.
The soluble ACE2-Ig fusion variant of claim 1, wherein two truncated ACE2 domains, comprising two truncated ACE2 respectively, are linked to the immunoglobulin domain, preferably, two truncated ACE2 domains, comprising two truncated ACE2 respectively, are linked to the linkers of N-terminus of CH1 and CL regions; alternatively, two truncated ACE2 domains, comprising two truncated ACE2 respectively, are linked to the four linkers of N-terminus of CH1 and CL regions; alternatively, two truncated ACE2 domains, comprising two truncated ACE2 respectively, are linked to the four linkers of N-terminus of light chain constant region and heavy chain constant region, thereby, a tetravalent soluble ACE2-Ig fusion variant is obtained.
The soluble ACE2-Ig fusion variant of claim 11, wherein one or more ACE2 domains comprising one truncated ACE2 are added to the tetravalent soluble ACE2-Ig fusion variant via one or more linkers, preferably, one or more ACE2 domains comprising one truncated ACE2 are added to C-terminus of CH3 region, or C-terminus of CL region.
The soluble ACE2-Ig fusion variant of claim 1, wherein three truncated ACE2 domains, , comprising two truncated ACE2 respectively, are linked to the immunoglobulin domain, preferably, two truncated ACE2 domains, comprising two truncated ACE2 respectively, are linked to the linkers of N-terminus of CH1 and CL regions; alternatively, two truncated ACE2 domains, comprising two truncated ACE2 respectively, are linked to the four linkers of N-terminus of CH1 and CL regions; alternatively, two truncated ACE2 domains, comprising two truncated ACE2 respectively, are linked to the four linkers of N-terminus of light chain constant region and heavy chain constant region; and one truncated ACE2 domain, comprising two truncated ACE2, is linked to a linker of C-terminus of CH3 regions of the immunoglobulin domain; alternatively, two truncated ACE2 within one truncated ACE2 domain are linked to two linkers of C-terminus of CH3 regions respectively; alternatively, two truncated ACE2 within one truncated ACE2 domain are linked to two linkers of C-terminus of CL regions respectively, thereby, a hexavalent soluble ACE2-Ig fusion variant is obtained.
The soluble ACE2-Ig fusion variant of claim 1, wherein four truncated ACE2 domains, comprising two truncated ACE2 respectively, are linked to the immunoglobulin domain, preferably, two truncated ACE2 domains, comprising two truncated ACE2 respectively, are linked to the linkers of N-terminus of CH1 and CL regions, two truncated ACE2 within one truncated ACE2 domain are linked to two linkers of C-terminus of CH3 regions respectively, and two truncated ACE2 within one truncated ACE2 domain are linked to two linkers of C- terminus of CL regions respectively, thereby, an octavalent soluble ACE2-Ig fusion variant is obtained.
The soluble ACE2-Ig fusion variant of claim 1, wherein the ACE2 domain is from human beings, or domestic animals, including camels, cattle, horses, goats, sheep, pigs, dogs, cats, and rabbits.
The soluble ACE2-Ig fusion variant of any one of claim 7-8 and 11-14, wherein the linker is a synthetic flexible linker of up to 30 residues, preferably having a general formula (GaSb) c, where a is an integer of 2～4, b is an integer of 0～2, and c is an integer of 0～7.
The soluble ACE2-Ig fusion variant of claim 1, wherein residues 720-740 of the truncated ACE2 or a part thereof are used as the linker per se.
A method for inhibiting infection of SARS-CoV, SARS-CoV-2 and other SARS-like viruses using ACE2 as an essential cellular receptor in a subject, comprising administrating to the subject a pharmaceutical composition comprising a pharmaceutically effective amount of the soluble ACE2-Ig fusion variant in any one of claims 1-17 and a pharmaceutically acceptable carrier.
A method for treating or preventing diseases associated with SARS-CoV, SARS-CoV-2 and some other SARS-like viruses using ACE2 as an essential cellular receptor in a subject, comprising administrating to the subject a pharmaceutical composition comprising a pharmaceutically effective amount of the soluble ACE2-Ig fusion variant in any one of claims 1-17 and a pharmaceutically acceptable carrier.
The method of claim 18 or 19, wherein the route of administration of the pharmaceutical composition is selected from oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal and rectal.
The method of claim 19, wherein the diseases are selected from pneumonia, severe acute respiratory infection, renal failure, heart failure, acute respiratory distress syndrome (ARDS) , liver injury, intestinal disease and severe acute respiratory syndrome.
The method of claim 19, wherein SARS-CoV-2 includes the prototype and variants of SARS-CoV-2.
Use of the soluble ACE2-Ig fusion variant in any one of claims 1-17 in manufacturing a medicament for inhibiting infection of SARS-CoV, SARS-CoV-2 and other SARS-like viruses using ACE2 as an essential cellular receptor in a subject.
Use of the soluble ACE2-Ig fusion variant in any one of claims 1-17 in manufacturing a medicament for treating or preventing diseases associated with infection of SARS-CoV, SARS-CoV-2 and other SARS-like viruses using ACE2 as an essential cellular receptor.
The use of claim 24, wherein the diseases are selected from pneumonia, severe acute respiratory infection, renal failure, heart failure, acute respiratory distress syndrome (ARDS) , liver injury, intestinal disease and severe acute respiratory syndrome.
The use of any one of claims 23-25, wherein SARS-CoV-2 includes the prototype and variants of SARS-CoV-2.
The method of any one of claims 18-22, or the use of any one of claims 23-26, wherein the subject is human beings, or a domestic animal, selected from camel, cattle, horse, goat, sheep, pig, cat, dog, and rabbit, mouse and rat.