CN115485375A - Virus neutralization by soluble receptor fragments of the ACE-2 receptor - Google Patents

Abstract

The present invention refers to a soluble receptor fragment (SRF) of the ACE‑2 receptor, wherein the SRF comprises the peptidase domain (PD) of ACE‑2 or fragments and/or derivatives thereof. In addition, the present invention refers to the use of SRF according to the present invention in a method of treating the human or animal body by surgery or therapy, SRF as a vaccine or SRF for implementation on the human or animal body or for use in the treatment of human or animal body fluids or in diagnostic methods implemented on other materials. Furthermore, the present invention provides a method for treating and/or preventing viral infections, in particular viral infections caused by the family Coronaviridae, more particularly SARS-CoV, SARS-CoV-2, human coronavirus NL63 or SARS-CoV- 2. SRF caused by the method of viral infection. Finally, the invention relates to a method of capturing viral particles comprising the steps of providing immobilized SRF and contacting a liquid sample or fluid with the SRF under conditions that allow the SRF to bind the viral particles.

Description

Virus neutralization by soluble receptor fragments of the ACE-2 receptor

The present invention refers to soluble receptor fragments (SRF) of the ACE-2 receptor, wherein the SRF comprises the peptidase domain (PD) of ACE-2 or fragments and/or derivatives thereof. In addition, the present invention refers to the use of SRF according to the present invention in a method of treating the human or animal body by surgery or therapy, SRF as a vaccine or SRF for implementation on the human or animal body or for use in the treatment of human or animal body fluids or in diagnostic methods implemented on other materials. In addition, the present invention provides a method for treating and/or preventing viral infections, especially those caused by Coronaviridae, more particularly SARS-CoV, SARS-CoV-2, human coronavirus NL63 or SARS-CoV- 2. SRF caused by the method of viral infection. Finally, the invention relates to a method of capturing viral particles comprising the steps of providing immobilized SRF and contacting a liquid sample or fluid with the SRF under conditions that allow the SRF to bind the viral particles.

In general, viruses infect host cells by: (1) attaching to specific receptors on the target cell surface; (2) initiating viral uptake; (3) viral replication; and (4) producing native virus to (5) Keep releasing. Viral receptors on target cells can be proteins or carbohydrate or lipid structures.

Therefore, to combat viral infections, these steps can be targeted by various strategies to prevent or treat viral infections, and prevention also includes vaccination. Most of these strategies address the steps of the viral infection cycle after entry into the human body. Examples of this are the spike protein or the general cell entry mechanism as a target for therapeutic or vaccination strategies. Other therapeutic targets are, for example, essential viral enzymes required for proliferation, such as proteases or replicases. Most vaccination strategies involve the surface structure of the virus, typically proteins on the surface of the virus such as the spike or capsid proteins that can be used to trigger an immune response.

However, drug or vaccine development can be time-consuming and often unsuccessful. So, since many serious and even fatal problems occur after the virus enters the body, begins replicating in human cells, and thereafter spreads through the blood or lymphatic system, another option is to prevent viral ingestion into the body.

In order to prevent viral uptake, or at least to substantially reduce the viral load entering the body, soluble fragments of host cell receptors can be applied according to the invention, for example in wash solutions, to neutralize viral receptors and allow the neutralized Viruses are washed out from where they entered.

The leading example of a virus that has a large impact on humans is Sars-CoV-2, which causes COVID-19. SARS-CoV-2, or severe acute respiratory syndrome coronavirus 2, is the strain of the virus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). This virus belongs to the Coronaviridae family, a family of lipid-enveloped positive-sense RNA viruses. SARS-CoV-2 was previously known as the provisional name of 2019 novel coronavirus (2019-nCoV). SARS-CoV-2 is contagious in humans, and the World Health Organization (WHO) has declared the ongoing pandemic of COVID-19 a public health emergency of international concern.

The main route of transmission of Sars-CoV-2 in humans appears to be droplet infection. In theory, contact transmission, mainly around infected persons, is also possible. However, the main mode of transmission is droplet infection, through the absorption of droplets with a diameter of less than 5 μm produced by coughing and sneezing by other people through the mucous membranes of the nose, mouth, and possibly the eyes.

For Sars-CoV-2, the trimeric spike glycoprotein (S protein) on the surface of the virion is responsible for mediating receptor recognition and membrane fusion. During viral infection, the S protein is cleaved into S1 and S2 subunits. Subunit S1 contains the receptor-binding domain (RBD), which directly binds to the peptidase domain of angiotensin-converting enzyme 2 (ACE-2), while subunit S2 is responsible for membrane fusion. When S1 binds to the host receptor ACE-2, another cleavage site on S2 is exposed and cleaved by host proteases, a process critical for viral infection. The S protein of Sars-CoV-2 also utilizes ACE2 for host infection. The structure of the S protein of Sars-CoV-2 was also processed, showing that the extracellular domain of the S protein of Sars-CoV-2 binds with the peptidase domain of ACE-2 with high affinity, that is, a dissociation constant (Kd) of ~15nM. Combined (Wrapp et al.–2020).

In fact, ACE2 is the entry point for more coronaviruses, including at least SARS-CoV, SARS-CoV-2, and human coronavirus NL63, into cells.

Angiotensin-converting enzyme 2 (ACE-2), an enzyme attached to cell membranes in the lungs, arteries, heart, kidneys, and intestines, is part of the renin-angiotensin system that maintains homeostasis of blood pressure as well as fluid and salt in the body balance. ACE-2 lowers blood pressure by hydrolyzing angiotensin II, a vasoconstrictor, to angiotensin (1-7), a vasodilator. ACE-2 antagonizes the activity of the associated angiotensin-converting enzyme (ACE) by reducing the amount of angiotensin II and increasing angiotensin (1-7), making it a promising drug target for the treatment of cardiovascular diseases.

ACE-2 is a zinc-containing metalloenzyme located on the surface of endothelial cells and other cells. The ACE-2 protein consists of an N-terminal peptidase M2 (carboxypeptidase) domain and a C-terminal collectin kidney amino acid transporter domain, which is nonstructural. The C-terminal domain of ACE-2 is a membrane anchor, which results in the exposure of the enzymatically active domain on the cell surface of lung and other tissues. The peptidase domain consists of two subdomains (I and II) that surround the active site and are connected by an alpha-helix (Towler et al.-2004). The N-terminal subdomain I also contains zinc ions. The extracellular and transmembrane domains of ACE-2 are naturally cleaved, and the resulting soluble protein is released into the blood and eventually excreted in the urine.

There are many strategies to combat Sars-CoV-2 at basically all stages of the virus life cycle. These strategies are summarized in the review by Zhang et al. (2020). These strategies summarize various treatment options as well as attempts at vaccination. Most therapeutic strategies address the steps of the viral life cycle after entry into human cells. But there are also attempts aimed at activating the immune system by vaccinating with inactivated viruses or parts of viruses, or preventing cellular entry by blocking the interaction of the viral protein S with its cellular receptor ACE-2. In more detail, the above review summarizes the hypothetical replication cycle of SARS-CoV-2 as follows: SARS-CoV-2 uses the spike protein to bind to the ACE2 receptor on the cell surface, which subsequently triggers endocytosis. When the viral nucleoprotein capsid is released into the cytoplasm, the coated positive-strand genomic RNA [(+) gRNA] serves as a template to translate the polypeptide chain, and the polypeptide chain is cleaved into non-structural proteins, including RNA-dependent RNA polymerase. A single negative-strand RNA [(-)gRNA] synthesized from a (+)gRNA template is used to make more copies of the viral RNA. Subgenomic RNA (sgRNA) is synthesized by discontinuous transcription from a (+) gRNA template, which then encodes viral structures and accessory proteins, and is subsequently assembled with newly synthesized viral RNA to form new virions. Nascent virions are then transported to the plasma membrane in secretory vesicles and released by exocytosis. In addition, the review describes possible targets for the following anti-COVID-19 drugs: RhACE2, convalescent plasma, and the JAK inhibitor Baricitinib, which can inhibit the spike protein on the surface of SARS-CoV-2 from interacting with cells. Binding of surface-expressed ACE2. Lopinavir/Ritonavir and Favipiravir can inhibit the proteolysis of polypeptide chains. Remdesivir inhibits RNA-dependent RNA polymerase. EIDD-2801 inhibits the replication of SARS-CoV-2. iNO and zinc inhibit the replication of SARS-CoV-2. Vitamin D may induce antimicrobial peptides to reduce SARSCoV-2 replication. Ivermectin can effectively block the growth of SARS-CoV-2. Baricitinib can block the passage of SARS-CoV-2 into cells by inhibiting AAK1-mediated endocytosis. CQ and HCQ inhibit the virus/cell fusion process. LHQW and IFN can block the viral replication process (RNA transcription, protein translation and post-translational modification). Abbreviations: AAK1, aptamer-associated kinase 1; CQ, chloroquine; ER, endoplasmic reticulum; HCQ, hydroxychloroquine sulfate; IFN, interferon; iNO, inhaled nitric oxide; JAK, janus kinase; LHQW, lianhua Qingwen; rhACE2, recombinant human angiotensin-converting enzyme 2; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

In addition to the aforementioned treatment or vaccination concepts, another strategy is to reduce the viral load in the mouth and nose by rinsing the mouth and nose with an appropriate inactivating solution, which inactivates and washes off the virus. Animal experiments have shown that short-term exposure to the virus is not enough to make people sick. This is an interesting choice because it appears that a certain viral load is necessary to infect a new host. Gargling or rinsing with an inactivating solution can be used to prevent infection in people who have short-term contact with infected patients, such as doctors, nurses, etc. Alternatively, the inactivating solution can be used to reduce the viral load in the nose and mouth of highly infectious patients to reduce the risk of virus transmission, for example during medical procedures where masks cannot be used.

Strategies to block viral entry have focused on antibodies or soluble recombinant human ACE-2 receptor. Monteil et al. (2020) expressed a large extracellular fragment of ACE-2 (amino acids 1–740), which, when purified, prevented viral entry into different cells. Others could show that fusion proteins consisting of these extracellular ACE-2 domains with the Fc portion of human IgG1 are suitable for neutralizing the receptor-binding domain of the Sars-CoV-2 spike protein in vitro (Lei et al.–2020 ).

However, the use of soluble human recombinant ACE-2 protein in COVID-19 therapy is not without risks, as ACE-2 therapy is expected to result in depletion of angiotensin II, so attention must be paid to the effects on blood pressure and renal function (Alhenc-Gelase et al– 2020).

Therefore, new and alternative concepts for inactivating viruses such as Sars-CoV-2 are needed.

Therefore, the problem to be solved by the present invention is to provide new methods that can be used to inactivate and neutralize viruses, such as Coronaviridae, especially SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2 including Any of its mutants, such as variants from the British lineage B.1.1.7, B.1.351 from South Africa, B.1.617 from India, or variant B.1.1.28.1 from Brazil, may be present in the cells of virus-infected subjects Before. Furthermore, the problem addressed by the present invention was to provide novel means which are able to reduce the viral load without significant side effects, such as effects on blood pressure and kidney function. Furthermore, the problem to be solved by the present invention is to provide new methods for detecting virus particles and for washing liquid samples or fluids in which the viral load is reduced. Therefore, the problem to be solved by the present invention is to provide prophylactic and/or therapeutic tools to stop the infection process of Coronaviridae such as SARS-CoV-2 by preventing the virus from entering human cells. A further problem of the present invention is to provide prophylactic and therapeutic means useful for any variation of the Coronaviridae family, in particular for SARS-CoV, SARS-CoV-2, human coronavirus NL63 or SARS-CoV-2, including those derived from Variants of British lineage B.1.1.7, B.1.351 from South Africa, B.1.617 from India or variant B.1.1.28.1 from Brazil.

The problems underlying the invention are solved by the subject-matter defined in the claims.

The following figures are used to illustrate the present invention.

Figure 1 shows the binding of three SRF variants to the spike protein of Sars-CoV-2 immobilized on a biacore chip. Binding was observed for all three proteins tested, with variant 3_ACE2_19-615_E375Q showing the highest binding response. Variant 4_ACE_2_19-103;301-365 was bound less efficiently and variant 9_ACE2_19-615 was the least efficient. Samples 1-3 measured were buffer; sample 4 was variant (3)_ACE2_19-615_E375Q; sample 5 was variant (4)_ACE_2_19-103; 301-365; sample 6 was variant (9)_ACE2_19-615.

Figure 2 shows the neutralization of Sars-Cov-2 virus particles by adding SRF. Neutralization was tested with 80pfu/ml. Variant 9_ACE2_19-615 showed a small reduction already at 1 nM and a 50% reduction at 250 nM. Application of the variant 3_ACE2_19-615_E375Q in the neutralization assay resulted in a measurable reduction of approximately 40% at all concentrations tested.

Figure 3 shows the neutralization of Sars-Cov-2 virus particles by adding SRF. Neutralization was tested with 40pfu/ml. Variant 9_ACE2_19-615, showed 50% inhibition at 250 nM and complete inhibition at 1.5 μM. Application of variant 3_ACE2_19-615_E375Q, over a concentration range of 250 nM to 1 μM, resulted in a significant reduction. At 1.5 μM and 2 μM, almost complete reduction was observed.

Figure 4 shows the neutralization of Sars-Cov-2 UK variant (B.1.1.7) virus particles by addition of SRF. Neutralization was tested with 80pfu/ml. Variant 9_ACE2_19-615 (A) and variant 3_ACE2_19-615_E375Q (B) already showed a large reduction in pfu at 250 nM and a complete reduction at 1 μM.

The term "SARS-CoV-2" as used herein refers to severe acute respiratory syndrome coronavirus 2, the strain of the virus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). This virus belongs to the Coronaviridae family, a family of lipid-enveloped positive-sense RNA viruses. SARS-CoV-2 was previously known as the provisional name of 2019 novel coronavirus (2019-nCoV). As the virus exhibits mutations, the term "SARS-CoV-2" as used herein preferably refers to all variants of SARS-CoV-2 described herein, but also to all variants exhibiting a single mutation or multiple mutations or combinations of mutations , but is still named SARS-CoV-2.

As used herein, the terms "SARS-CoV-2 variant from British lineage B.1.1.7", "SARS-CoV-2 variant B.1.351 from South Africa" or "SARS-CoV-2 variant from Brazil Variant B.1.1.28.1" preferably refers to SARS-CoV-2 variants B.1.1.7, B.1.351 and B.1.28.1 as described eg in Singh et al. (2021).

The term "SARS-CoV-2 B.1.617 from India" as used herein preferably refers to variants with D111D, G142D, L452R, E484Q, D614G and P681R mutations in the Spike protein described by Cherian et al. (2021). body.

The term "ACE-2" as used herein refers to angiotensin converting enzyme 2 (ACE-2). It is an enzyme attached to cell membranes, such as those in the lungs, arteries, heart, kidneys, and intestines, and is part of the renin-angiotensin system that maintains blood pressure homeostasis and fluid and salt retention in the body balance. ACE-2 lowers blood pressure by hydrolyzing angiotensin II, a vasoconstrictor, to angiotensin (1-7), a vasodilator. ACE-2 antagonizes the activity of the associated angiotensin-converting enzyme (ACE) by reducing the amount of angiotensin II and increasing angiotensin (1-7), making it a promising drug target for the treatment of cardiovascular diseases. ACE-2 is a zinc-containing metalloenzyme located on the surface of endothelial cells and other cells. The ACE-2 protein consists of an N-terminal peptidase M2 (carboxypeptidase) domain and a C-terminal collectin kidney amino acid transporter domain, which is nonstructural. The C-terminal domain of ACE-2 is a membrane anchor, which results in the exposure of the enzymatically active domain on the cell surface of lung and other tissues. The peptidase domain consists of two subdomains (I and II) that surround the active site and are connected by an alpha-helix (Towler et al.-2004). The two subdomains are defined as follows (Towler et al.-2004): subdomain I containing the N-terminus and zinc, which consists of residues 19-102, 290-397 and 417-430; subdomain II containing the C-terminus, It consists of residues 103-289, 398-416 and 431-615. The N-terminal subdomain I also contains zinc ions. The structure of ACE-2 and its peptidase and neck domains are preferably as described by Yan et al. (2020). It is preferred that ACE-2 comprises the amino acid sequence shown in SEQ ID NO:1. It preferably comprises a peptidase domain as shown in SEQ ID NO:3 or 6 and a neck domain as shown in SEQ ID NO:2.

The term "peptidase domain of ACE-2" or "PD of ACE-2" as used herein refers to the peptidase domain of ACE-2. ACE-2 PD lacks the neck domain of ACE-2. Preferably, the PD of ACE-2 has the amino acid sequence shown in SEQ ID NO:3 or 6. The PD of ACE-2 may contain alpha helices, beta sheets and/or beta turns. The PD of ACE-2 has an active site. The amino acid residues of the PD of ACE-2 listed in the table below are involved in the activity.

Residues involved in binding (refer to the PD sequence shown in SEQ ID NO:3).

The term "soluble" herein in relation to PD or SRF preferably means that the protein is present at a concentration of about 1 μg/ml to 1000 μg/ml in a physiological buffer, for example PBS at a temperature of about 20°C to about 40°C. in solution. More preferably, it is present in solution at a concentration of at least about 2 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml, 30 μg/ml, 40 μg/ml, 50 μg/ml, 60 μg/ml, 70 μg/ml, 80 μg/ml, 90 μg/ml, 100 μg/ml, 110 μg/ml, 120 μg/ml, 150 μg/ml or 200 μg/ml.

The term "polypeptide" or "protein" as used herein especially refers to a polymer of amino acids linked in a specific sequence by peptide bonds. The amino acid residues of polypeptides can be altered by covalent attachment of various groups, such as carbohydrates and phosphates. Other substances may be more loosely associated with the polypeptide, such as heme or lipids, resulting in conjugated polypeptides, which are also encompassed by the term "polypeptide" or "protein" as used herein. The term "polypeptide" or "protein" encompasses embodiments of polypeptides or proteins exhibiting selective modifications commonly used in the art, such as biotinylation, acetylation, pegylation, modification of amino, SH or carboxyl groups Chemical changes (e.g. protecting groups), etc. The term "polypeptide" or "protein" as used herein is not limited to a specific length of a polymeric chain of amino acids, but typically a polypeptide or protein will exhibit a length of greater than about 50 amino acids, greater than about 100 amino acids, or even greater than about 150 amino acids . Typically, but not necessarily, a typical polypeptide of the invention will not exceed about 850 amino acids in length. The term "polypeptide" or "protein" as used herein may also encompass dimeric fusions of polypeptides or proteins. In the case of dimeric fusions, typical polypeptides of the invention are generally no more than about 1600 or 1700 amino acids in length.

The term "derivative" as used herein refers to an amino acid sequence exhibiting one or more additions, deletions, insertions and/or substitutions and/or combinations thereof, compared to the respective reference sequence. This includes, for example, combinations of deletion/insertion, insertion/deletion, deletion/addition, addition/deletion, insertion/addition, addition/insertion, and the like. However, those skilled in the art will understand that the occurrence of an amino acid residue at a certain position in a derivative sequence that differs from the amino acid residue present at the same respective position in the reference sequence is not, for example, a deletion and subsequent insertion at the same position. combination, but a substitution as defined herein. Conversely, if a combination of one or more additions, deletions, insertions and substitutions is mentioned herein, then this refers to combinations of changes at various positions in the sequence, such as N-terminal additions and deletions within the sequence. Such derived sequences will exhibit a level of sequence identity, preferably at least 60%, such as at least 75%, at least 80%, at least 85%, at least 90%, At least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. Preferred derivatives are fragments of the parent molecule (eg a given SEQ ID NO) that retain the activity of the parent molecule, ie exhibit generally the same activity as the respective parent molecule if not otherwise stated. However, the activity may be the same, higher or lower than the respective parent molecule. Also preferred derivatives are those resulting from conservative amino acid substitutions within the parent sequence (eg, a given SEQ ID NO), which also retain a general level of activity of the parent molecule.

As used herein, the term "% sequence identity" must be understood as follows: two sequences to be compared are aligned such that there is a maximum relatedness between the sequences. This may include inserting "gaps" in one or both sequences to improve the degree of alignment. The % identity can then be determined over the entire length of each compared sequence (a so-called global alignment), which applies in particular to sequences of the same or similar length, or over a shorter, defined length (so-called local alignment), which is more suitable for sequences of unequal length. In the above cases, an amino acid sequence having, for example, at least 95% "sequence identity" to the query amino acid sequence is intended to mean that the subject amino acid sequence may include changes of up to 5 amino acids per 100 amino acids of the query amino acid sequence , the sequence of the target amino acid sequence is consistent with the query amino acid. In other words, up to 5% (5 out of 100) of the amino acid residues in the subject sequence may be inserted or substituted with another amino acid or deleted in order to obtain an amino acid sequence that is at least 95% identical to the query amino acid sequence. Methods of comparing the identity and homology of two or more sequences are well known in the art. The percentage at which two sequences are identical can be determined by using a mathematical algorithm. A preferred, but not limiting, example of a mathematical algorithm that can be used is the algorithm of Karlin et al. (1993) PNAS USA, 90:5873-5877. Such algorithms are integrated in the BLAST family of programs, such as the BLAST or NBLAST programs (see also Altschul et al., 1990, J. Mol. Biol. 215, 403-410 or Altschul et al. (1997), Nucleic Acids Res, 25: 3389-3402), accessible through the NCBI home page on the World Wide Web at ncbi.nlm.nih.gov) and FASTA (Pearson (1990), Methods Enzymol. 83, 63-98; Pearson and Lipman (1988), Proc. Natl. Acad . Sci. U.S. A85, 2444-2448.). Sequences that are to some extent identical to other sequences can be recognized by these programs. In addition, programs in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux et al, 1984, Nucleic Acids Res., 387-395), such as the BESTFIT and GAP programs, can be used to determine the % identity between two polypeptide sequences . BESTFIT uses the "local homology" algorithm of (Smith and Waterman (1981), J. Mol. Biol. 147, 195-197.) to find the best single region of similarity between two sequences. Where it is referred to herein as an amino acid sequence having a certain degree of sequence identity to a reference sequence, then said sequence differences are preferably due to conservative amino acid substitutions. Preferably, such sequences retain the activity of the reference sequence, eg, although possibly at a slower rate. Furthermore, where it is referred to herein as having "at least" a certain percentage of sequence identity, then preferably 100% sequence identity is not encompassed.

"Conservative amino acid substitutions," as used herein, can occur within a group of amino acids that have sufficiently similar physicochemical properties that substitutions between members of the group will preserve the biological activity of the molecule (see, for example, Grantham, R. (1974 ), Science 185, 862-864). In particular, conservative amino acid substitutions are preferably amino acids from the same class of amino acids (e.g., basic amino acids, acidic amino acids, polar amino acids, amino acids with aliphatic side chains, positively or negatively charged side chains, aromatic amino acid group, amino acid whose side chain can enter a hydrogen bridge, such as a side chain with a hydroxyl function, etc.). In this example, a conservative substitution refers to the substitution of a basic amino acid residue (Lys, Arg, His) for another basic amino acid residue (Lys, Arg, His), and an aliphatic amino acid residue (Gly, Ala, Val, Leu, Ile) for another aliphatic amino acid residue, an aromatic amino acid residue (Phe, Tyr, Trp) for another aromatic amino acid residue, serine for threonine, and isoleucine for Leucine. More conservative amino acid exchanges are known to those skilled in the art.

The term "deletion" as used herein preferably refers to the lack of 1, 2, 3, 4, 5 (or even more than 5) contiguous amino acid residues. Derivatives of the invention may exhibit one, two or more such deletions.

The term "insertion" as used herein preferably refers to the additional presence of 1, 2, 3, 4, 5 (even more than 5) consecutive amino acid residues in the derivative sequence compared to the respective reference sequence. Derivatives of the invention may exhibit one, two or more such insertions.

The term "addition" as used herein preferably refers to the additional presence of 1, 2, 3, 4, 5 (even more than 5) contiguous amino acid residues at the N-terminal and/or C-terminal of the derivative sequence compared to the respective reference sequence .

The term "substitution" as used herein refers to the presence of an amino acid residue at a certain position in a derivative sequence, which is different from the amino acid residue present or absent at the corresponding position in the reference sequence. Derivatives of the present invention may exhibit 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more Such supersedes. As noted above, it is preferred that such substitutions be conservative substitutions.

The term "mutation" of the Coronaviridae, SARS-CoV, SARS-CoV-2, human coronavirus NL63 or SARS-CoV-2 refers to any mutant of any of the above viruses, including British lineage B.1.1.7, South African B.1.351 variant, Indian variant of B.1.617 or Brazilian variant of B.1.1.28.1. In particular the Coronaviridae family, the term "mutation" of SARS-CoV, SARS-CoV-2, Human Coronavirus NL63 or SARS-CoV-2 refers to any mutation of the respective virus. The mutation may comprise a single mutation or multiple mutations. Said mutations may occur in the spike protein and/or another viral protein and/or other viral proteins. Mutations can occur in any part of the virus and can be silent mutations that have no effect or non-silent mutations that have an effect on viral properties such as host cell binding, replication, virus stability, etc.

The term "infection" as used herein preferably means that a virus has entered a host cell, especially a mammalian or human host cell, and is ready to replicate.

The term "host cell" as used herein preferably refers to any eukaryotic, animal, mammalian or human cell. It further preferably refers to any eukaryotic, animal, mammalian or human cell having an ACE-2 receptor. In particular, the term "host cell" as used herein refers to human, cat, tiger, poultry and mouse cells with ACE-2 receptors and cells capable of being infected by SARS-CoV-2/COVID-19 or any mutation or variant thereof All host cells were infected as described by Stout et al. (2020).

As used herein, the term "tag" refers to an amino acid sequence, usually fused to or contained within another amino acid sequence in the art, for a) increasing the expression of the entire amino acid sequence or polypeptide, b) promoting the expression of the entire amino acid sequence or polypeptide. The purification of the amino acid sequence or polypeptide c) facilitates the immobilization of the entire amino acid sequence or polypeptide, and/or d) facilitates the detection of the entire amino acid sequence or polypeptide. Examples of tags are His Tag, such as His5-Tag, His6-Tag, His7-Tag, His8-Tag, His9-Tag, His10-Tag, His11-Tag, His12-Tag, His16-Tag and His20-Tag, Strep- Tag, Avi-Tag, Myc-Tag, GST-Tag, JS-Tag, Cysteine-Tag, FLAG-Tag, HA-Tag, Thioredoxin or Maltose Binding Protein (MBP), CAT, GFP, YFP Wait. Those skilled in the art will know of a large number of tags suitable for different technical applications. For example, tags may render such labeled polypeptides suitable for antibody binding or other technical applications, eg in different ELISA assay formats.

The term "comprising" as used herein should not be construed as being limited to the meaning of "consisting of" (ie excluding the presence of other material additions). Conversely, "comprising" means that optional additional substances may be present. The term "comprising" encompasses "consisting of" (i.e. excluding the presence of additions of other substances) and "comprising but consisting of" (i.e. requiring the presence of additions of other substances) embodiments specifically contemplated within its scope, the former is more preferred.

The abbreviation "aa" as used herein preferably refers to the term "amino acid", especially in reference to the position of certain amino acids in a particular amino acid sequence. For example, "aa 19-103 of SEQ ID NO:3" refers to the amino acid sequence from the 19th to the 103rd amino acid residue in the amino acid sequence according to SEQ ID NO:3.

In a first object of the present invention, it is contemplated to provide a soluble receptor fragment (SRF) of the ACE-2 receptor, wherein the SRF comprises the peptidase domain (PD) of ACE-2. Alternatively, the SRF according to the invention comprises fragments and/or derivatives of the PD of ACE-2, in particular one, two or more fragments of the PD of ACE-2. If a SRF according to the invention comprises two or more fragments, the term "fragment combination of two or more fragments" or simply "fragment combination" may be used in this disclosure to describe this embodiment. In a further preferred embodiment of the invention, the SRF comprises a derivative of the PD of ACE-2 or a derivative of a fragment or combination of fragments of the PD of ACE-2. In a preferred embodiment of the invention, the SRF consists of the PD of ACE-2 or one, two or more fragments thereof or a derivative of the PD of ACE-2 or fragments thereof. In a further preferred embodiment, the SRF comprises or consists of PD of ACE-2 or one, two or more fragments thereof or derivatives of PD of ACE-2 or fragments thereof, wherein the active site is passed through one or more inactivated by a mutation.

Fragments, fragment combinations and/or derivatives of PD of ACE-2 of SRF according to the present invention have substantially the same binding affinity or characteristics as ACE-2 with the receptor binding cleft of viral spike protein, especially viral spike protein S. The wild-type full-length PD is the same or higher. Essentially, this means that the binding affinity or properties of the receptor binding cleft of the Spike protein of the same virus range from about 70% to about 150% of that of the wild-type full-length PD of ACE-2 as shown in SEQ ID NO:3. range, more preferably from about 80% to about 130% or from about 90% to about 120%. In a preferred embodiment of the present invention, the ACE that effectively binds to the receptor binding cleft of the viral spike protein S of SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2 or any mutation thereof Any fragment, combination of fragments and/or derivative of the PD of -2 is a fragment, combination of fragments or derivative of SRF according to the invention. If the binding affinity of the tested derivative, fragment or combination of fragments is at least 50%, 60%, 70%, 80% of the binding affinity of (9)_ACE2_19-615 according to SEQ ID NO: 12 as tested in the Examples herein , 90%, 100%, 110% or 120%, then the combination can be considered effective.

The inventors of the present invention have surprisingly found that the SRF according to the invention binds to the receptor binding cleft of the Spike protein of viruses, in particular to the receptor binding cleft of the Spike protein S of viruses of the Coronaviridae family. In a preferred embodiment, the SRF binds to the receptor binding cleft of the Spike protein S of the virus of SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2. In a particularly preferred embodiment, the SRF binds to the receptor binding cleft of the Spike protein S of the virus of SARS-CoV-2. By the combination described above, the virus is prevented from entering the host cell, preferably a human cell. As a result, the virus cannot start replicating and, therefore, the host cell (such as a human cell) cannot produce more virus particles.

Preferably, the PD of ACE-2 of the SRF according to the present invention is a soluble expressed isolated PD of ACE-2 or a soluble expressed isolated fragment, a combination of fragments and/or a derivative thereof. Using fragments or combinations of fragments of the full-length PD of ACE-2 can lead to increased solubility of SRF. The fragment or combination of fragments is preferably designed by deleting one, two, three, four, five or more structural elements of PD of ACE-2 that are essential for PD to interact with the viral spike protein , especially the binding activity of the receptor-binding cleft of the virus's spike protein S is not important. Structural elements of the PD of ACE-2 are, for example, those that are dispensable for the formation of alpha helices, beta sheets and beta turns. Alternatively or additionally, the solubility of the PD of ACE-2 or a derivative or fragment thereof may be increased by 1, 2, 3, 4, 5 or more mutations such as insertions, substitutions, deletions or additions. Alternatively or additionally, the solubility of the PD of ACE-2 or a derivative or fragment thereof may be increased by removing glycosylation sites.

In a particularly preferred embodiment of the invention, the SFR may comprise any fragment of the PD of ACE-2 that binds to the receptor binding cleft of the viral Spike protein, in particular the viral Spike protein S, and inhibits the Spike protein S binding to host cells.

Preferably, the SFR comprises a fragment or the first fragment of the PD of ACE-2 having a length of at least 80, 81, 82, 83, 84, 85, 110, 111, 112, 113, 114, 580, 581, 582, 583 , 584, 593, 594, 595, 596 or 597 amino acid residues. Preferably, the SRF comprises a PD fragment of ACE-2 up to a length of 614, 615, 616, 617, 618, 619, 620, 582, 583, 584, 585, 586, 587, 595, 596, 597, 598, 599 or 600 amino acid residues. In preferred embodiments, the SFR comprises a fragment or first fragment of the PD of ACE-2, which is about 80 to about 600 amino acids, more preferably about 84 to about 597 amino acids in length.

If the SFR according to the invention comprises two or more fragments of the PD of ACE-2, the first fragment preferably has a length of about 80 to 120 amino acid residues, or a length of about 84 to about 114 amino acid residues . Particularly preferred fragments have 80, 81, 82, 83, 84, 85, 86, 87, 110, 111, 112, 113, 114, 115 or 116 amino acid residues.

If the SFR according to the invention comprises a second fragment of the PD of ACE-2, said second fragment preferably has from about 50 to about 150, from about 60 to about 130 amino acids, or from about 64 to about 125 amino acid residues. length. Particularly preferred fragments have 60, 61, 62, 63, 64, 65, 66, 67, 82, 83, 84, 85, 86, 87, 120, 121, 122, 123, 124, 125, 126, 127 or 128 amino acid residues in length.

In a preferred embodiment of the invention, the SRF comprises at least Sars-CoV-2 contact residues Q24, T27, F28, D30, K31, H34, E35, E37, D38, Y41, Q42, L79, 5, 6, 7, 8, 9, 10, 11, 12, 13 or all 14 of M82, Y83. Preferably, the SRF comprises at least 60%, 70%, 80% or 90% of the residues listed above.

In a further preferred embodiment of the present invention, the SRF comprises at least Sars-CoV-2 contact residues Q24, T27, F28, K31, H34, E37, D38, Y41, Q42, L45, L79, M82 of SEQ ID NO:3 , 5, 6, 7, 8, 9, 10, 11, 12 or all 13 of Y83.

In a further preferred embodiment of the present invention, the SRF comprises 3, 4, 5, 6, 7, 8, 9 or all 10 alphas of the PD of ACE-2 shown in SEQ ID NO: 34-43 or derivatives thereof - Helical structure. A derivative according to any one of the amino acid sequences of SEQ ID NO: 34-43 preferably comprises 1, 2, 3, 4 or 5 deletions or mutations, provided that the derivative is still capable of forming an α-helical structure. These deletions may be within the given amino acid sequence and/or at one or both ends. A derivative of SEQ ID NO: 34, preferably comprising at least 5 of the Sars-CoV-2 contact residues Q24, T27, F28, D30, K31, H34, E35, E37, D38, Y41 and Q42 of SEQ ID NO: 3 , 6, 7, 8, 9, 10 or all 11. The derivative of SEQ ID NO: 40 preferably comprises Sars-CoV-2 contact residue N330 of SEQ ID NO: 3 and/or 1, 2 or 3.

In a preferred embodiment of the invention, SRF comprises at least the following α-helical structure:

- as shown in SEQ ID NO: 34, aa 21-53 of SEQ ID NO: 3 or a derivative thereof as defined above.

- as shown in SEQ ID NO:35, aa 56-80 of SEQ ID NO:3 or its derivatives as defined above, and

- as shown in SEQ ID NO: 36, aa 91-100 of SEQ ID NO: 3 or a derivative thereof as defined above.

Examples of such SFRs are SFRs comprising an amino acid sequence as set forth in SEQ ID NO: 8 or 13, also as set forth in SEQ ID NO: 4-14, 17-22, 25-27 and 29-31. In a further preferred embodiment of the present invention, SRF additionally comprises a further α-helical structure, ie aa 110-129 of SEQ ID NO: 3, as shown in SEQ ID NO: 37 or a derivative thereof as defined above. Examples of such SFRs are SFRs comprising an amino acid sequence as set forth in SEQ ID NO: 9 or 14, also as set forth in SEQ ID NO: 4-6, 10, 12 and 17-19.

If the SRF of the present invention comprises a second fragment of the PD of ACE-2, said second fragment preferably comprises the following α-helical structure:

- as shown in SEQ ID NO:39, aa 304-318 of SEQ ID NO:3 or a derivative thereof as defined above, and/or

- as shown in SEQ ID NO: 40, aa 325-330 of SEQ ID NO: 3 or a derivative thereof as defined above.

An example of such a second fragment is shown in SEQ ID NO:15. Examples of SRFs comprising such second fragments are shown in SEQ ID NOs: 4-7, 10-12, 17-22 and 25-27. In a further preferred embodiment of the invention, the second fragment of SRF comprises an α-helical structure, ie aa 366-385 of SEQ ID NO:3, as shown in SEQ ID NO:41 or a derivative thereof as defined above. An example of such a second fragment is shown in SEQ ID NO:23. Examples of SRFs comprising such second fragments are shown in SEQ ID NOs: 4-6, 10-12, 17-22, 25-27 and 29-31.

In a further preferred embodiment of the invention, the second segment of SRF comprises the following α-helical structure:

- as shown in SEQ ID NO:42, aa 400-412 of SEQ ID NO:3 or its derivatives as defined above, and/or

- as shown in SEQ ID NO:43, aa 415-421 of SEQ ID NO:3 or a derivative thereof.

An example of such a second fragment is the amino acid sequence shown in SEQ ID NO:28. Examples of SRFs comprising such second fragments are shown in SEQ ID NOs: 4-6, 10, 12, 17-19, 25-27 and 29-31.

In a preferred embodiment of the invention, the SRF further comprises 1, 2, 3, 4, 5 or 6 of the Sars-CoV-2 contact residues N330, K353, G354, D355, R357, R393 of SEQ ID NO:3 indivual.

In a further preferred embodiment of the invention, the SRF comprises 1, 2, 3, 4, 5 of the Sars-CoV-2 contact residues Q325, E329, N330, K353, G354, D355, R357 of SEQ ID NO:3 , 6 or 7.

In a particularly preferred embodiment, the SFR according to the invention comprises at least aa 19-103 of the PD of ACE-2 (with respect to the amino acid sequence shown in SEQ ID NO: 3). Therefore, the SFR according to the present invention at least comprises the amino acid sequence shown in SEQ ID NO: 8 or its derivatives. Said derivative preferably comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, and preferably at least contain the following α-helical structures:

- SEQ ID NO: 34 or a derivative thereof,

- SEQ ID NO: 35 or a derivative thereof, and

- SEQ ID NO: 36 or a derivative thereof.

The PD of ACE-2 of SRF according to the present invention or its fragments, fragment combinations and/or derivatives can be expressed in any suitable expression system, such as in Baculovirus (Baculovirus) cells, HEK293 cells, CHO or other eukaryotic in cells. Alternatively, SRF proteins can be expressed in E. coli. To purify expressed SRF proteins, affinity tags such as His-Tag or Strep-Tag or other standard purification techniques can be used.

Therefore, in a preferred embodiment of the invention, the SRF according to the invention may comprise a tag, preferably an affinity tag, such as His-Tag, Strep-Tag, MBP-tag or any other tag used in standard purification techniques.

In a further preferred embodiment of the invention, the SRF may additionally comprise methionine as a translation initiation signal.

The PD of ACE-2 preferably has an amino acid sequence according to SEQ ID NO:3 or 6. In a preferred embodiment of the invention, the SRF consists of an amino acid sequence according to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6 or a fragment, combination of fragments or derivatives of the PD of ACE-2 or fragments thereof or SRF containing the above. An SRF consisting of or comprising an amino acid sequence according to SEQ ID NO: 4 is a preferred example of an SRF consisting of or comprising a fragment of an amino acid sequence according to SEQ ID NO: 3, since it lacks the amino acid of SEQ ID NO: 3 1-18. An example of an SRF comprising the sequence shown in SEQ ID NO:4 is an SRF consisting of or comprising the sequence shown in SEQ ID NO:12. In addition to the sequence shown in SEQ ID NO: 4, the SRF having the sequence shown in SEQ ID NO: 12 also includes methionine as a translation initiation signal and a tag, namely His-tag.

A further preferred example of an SRF consisting of or comprising an amino acid sequence according to SEQ ID NO: 6 is a SRF consisting of or comprising an amino acid sequence fragment according to SEQ ID NO: 3, since it consists of amino acid 2 of SEQ ID NO: 3 -615 composition, thus lacking amino acid 1 of SEQ ID NO:3, ie lacking methionine as a translation initiation signal.

A further preferred example of an SRF consisting of or comprising an amino acid sequence according to SEQ ID NO: 8 is a fragment of an amino acid sequence according to SEQ ID NO: 3, since SEQ ID NO: 8 consists of SEQ ID NO Composition of amino acids 19-103 of :3, thus lacking amino acids 1-18 and 104-615 of SEQ ID NO:3. An example of an SRF comprising the sequence shown in SEQ ID NO:8 is an SRF consisting of or comprising the sequence shown in SEQ ID NO:13. The SRF having the sequence shown in SEQ ID NO: 13 contains, in addition to the sequence shown in SEQ ID NO: 8, methionine as a translation initiation signal and a tag, namely His-tag.

A further preferred example of a SRF consisting of or comprising an amino acid sequence according to SEQ ID NO: 9 is a fragment of an amino acid sequence according to SEQ ID NO: 3, since SEQ ID NO: 9 comprises SEQ ID NO :3 amino acids 19-132, thus lacking the amino acids 1-18 and 133-615 of SEQ ID NO:3. An example of an SRF comprising the sequence shown in SEQ ID NO:9 is an SRF consisting of or comprising the sequence shown in SEQ ID NO:14. In addition to the sequence shown in SEQ ID NO:9, the SRF having the sequence shown in SEQ ID NO: 14 also includes methionine as a translation initiation signal and a tag, namely His-tag.

An SRF consisting of or comprising an amino acid sequence according to SEQ ID NO: 17 is a further preferred example of an SRF consisting of or comprising a fragment of an amino acid sequence according to SEQ ID NO: 3, since SEQ ID NO: 17 consists of SEQ ID NO: 3 amino acids 19-602, thus lacking the amino acids 1-18 and 603-615 of SEQ ID NO:3. An example of an SRF comprising a derivative of a fragment as shown in SEQ ID NO: 17 is an SRF consisting of or comprising a sequence shown in SEQ ID NO: 18 or 19. The SRF having the sequence shown in SEQ ID NO: 18 has a Glu375Gln substitution, ie, a glutamic acid to glutamine substitution at position 375 of the sequence shown in SEQ ID NO: 3, ie, position 357 of SEQ ID NO: 18. In addition to the sequence shown in SEQ ID NO: 18, the SRF having the sequence shown in SEQ ID NO: 19 also includes methionine as a translation initiation signal and a tag, ie, His-tag.

In a further preferred embodiment of the present invention, the SRF comprises more than one fragment of the amino acid sequence according to SEQ ID NO:3, i.e. a combination of two, three or more fragments of the amino acid sequence according to SEQ ID NO:3 . In a particularly preferred embodiment, the SRF according to the invention comprises two fragments of the amino acid sequence according to SEQ ID NO:3. The fragments of such a combination of fragments may be directly associated with each other, or they may be linked together via a linker. The linker should be flexible enough to allow binding to a subunit of a trimeric spike or a subunit of a different spike trimer. The linker may be a linker already contained in an ACE-2 receptor junctional structural module, like an alpha-sheet in a full-length protein. Alternatively, it may be a linker designed to be stable, flexible enough to allow the variant to fold into a body-like structure, and resistant to cleavage by proteases to increase the half-life of the variant. Such linkers are preferably about 5 to about 50 amino acid residues, about 10 to about 30 amino acid residues or about 15 to 25 amino acid residues in length.

In a preferred embodiment, the SRF according to the present invention comprises a fragment combination of the first and second fragments of SEQ ID NO: 3, wherein the first fragment is selected from the amino acid sequence of SEQ ID NO: 8 or derivatives thereof, the second Fragments are selected from the amino acid sequences of SEQ ID NO: 15, 23, 28 and 32 or their respective derivatives. In a further preferred embodiment, the SRF according to the present invention comprises a fragment combination of the first and second fragments of SEQ ID NO:3, wherein the first fragment is selected from the amino acid sequence shown in SEQ ID NO:9 or derivatives thereof , the second fragment is selected from the amino acid sequences shown in SEQ ID NO: 15, 23, 28 and 32 or their respective derivatives. In a further preferred embodiment, the second fragment according to SEQ ID NO: 23, 28 and 32 comprises the substitution Glu375Gln, ie glutamic acid is replaced by glutamine at position 375 of the sequence shown in SEQ ID NO:3.

Preferably, the first fragment and the second fragment of the combination of fragments are linked by a linker selected from the amino acid sequences shown in SEQ ID NO: 16, 24 and 33.

In a preferred embodiment, the SRF according to the invention comprises a fragment combination of the first and second fragments of SEQ ID NO:3, wherein the first fragment consists of the amino acid sequence according to SEQ ID NO:8 or a derivative thereof, The second fragment consists of the amino acid sequence according to SEQ ID NO: 15 or a derivative thereof. In a further preferred embodiment, said first and second fragments are connected by a linker having an amino acid sequence according to SEQ ID NO:16. In a further preferred embodiment, the SRF comprises or consists of the amino acid sequence according to SEQ ID NO: 7 or a derivative thereof. An example of an SRF comprising the sequence shown in SEQ ID NO:7 is an SRF consisting of or comprising the sequence shown in SEQ ID NO:11. The SRF having the sequence shown in SEQ ID NO: 11 contains, in addition to the sequence shown in SEQ ID NO: 7, methionine as a translation initiation signal and a tag, namely His-tag.

In a further preferred embodiment, the SRF according to the present invention comprises a combination of fragments as shown in SEQ ID NO: 20, 21, 22, 25, 26, 27, 29, 30, 31 or each derivative thereof.

Derivatives of SEQ ID NO: 3-14, 17-22, 25-27, 29-31 preferably refer to derivatives having at least 60, 70, 80, 90, 95 or 98% sequence identity to a given sequence, wherein Said derivative preferably comprises at least 5, 6, 7, 8, 9 of Q24, T27, F28, D30, K31, H34, E35, E37, D38, Y41, Q42, L79, M82, Y83 of SEQ ID NO.3 , 10, 11, 12, 13 or 14, and at least the following alpha-helical structures:

- SEQ ID NO: 34 or a derivative thereof,

- SEQ ID NO: 35 or a derivative thereof, and

- SEQ ID NO: 36 or a derivative thereof.

A derivative of SEQ ID NO: 34-43 preferably refers to a derivative comprising 1, 2, 3, 4 or 5 deletions or mutations, such as substitutions, especially conservative amino acid substitutions, provided that the derivative is still capable of forming α - Helical structure.

Preferably, the SRF according to the invention does not comprise further domains or sequences of ACE2, or at least 10, at least 15, at least 20, at least 30 , at least 40, at least 50, at least 60 or at least 70 contiguous amino acids. In particular, the SFR according to the present invention preferably does not comprise the sequence or further domain of aa 620-805 of ACE2 shown in SEQ ID NO: 1, or at least 10, at least 15, at least 20, at least 30, at least 40, at least 50. A portion of at least 60 or at least 70 contiguous amino acids.

Therefore, the term "comprising" related to the definition of SFR according to the present invention means that, in addition to any fragment or derivative of PD comprising ACE-2 described herein, the SFR may especially comprise one or more of the following components :

-Methionine as a translation initiation signal

- one or more tags

- one or more joints

- proteins, antibodies or fragments thereof, other compounds or molecules as further defined below

But no further domains or sequences of ACE2 as defined above.

In a preferred embodiment of the invention, the SFR according to the invention does not contain more than 14, 18, more preferably more than 31, 40, 50, 60 , 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600 amino acids. Preferably, none of these additional amino acids comprises the sequence or domain of ACE2 as defined above.

The inventors of the present invention have surprisingly found that viruses of the family Coronaviridae such as Sars-CoV-2 can be inactivated or neutralized with the soluble receptor fragment (SRF) of the ACE-2 receptor. These SRFs comprise or consist of a soluble expressed isolated peptidase domain (PD) of ACE-2 or its fragments, combinations of fragments or only derivatives thereof, where the neck domain is described in the structure of Yan et al. (2020) was cracked. Therefore, the SFR according to the present invention does not comprise the amino acid sequence shown in SEQ ID NO:2. In order to avoid the impact on blood pressure and renal function, the active site of PD is preferably inactive, because the goal according to the present invention is to block the receptor binding cleft of Coronaviridae such as Sars-CoV-2 Spike protein S, without affecting Significant effects on blood pressure or renal function. Furthermore, in order to prevent viral uptake, or at least substantially reduce the viral load entering the human body, the inventors have found that soluble fragments of host cell receptors, in particular SRF according to the invention, can be applied in the cleaning solution according to the invention, For neutralization of viral receptors, it is preferred to wash out the neutralized virus from the point of entry.

Therefore, in a highly preferred embodiment of the invention, the derivative, fragment and/or combination of fragments of PD is PD, a fragment of PD or a combination of fragments, wherein the active site of PD is preferably passed through 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more mutations, such as insertions, additions, deletions or substitutions inactivating. In particular, the activity of PD to hydrolyze angiotensin II (a vasoconstrictor) to angiotensin (1-7) (a vasodilator) was inactivated by the mutation. The inactivated PD, inactivated PD fragment or combination of inactivated fragments preferably has an activity of hydrolyzing angiotensin II to angiotensin which is reduced by about 60% compared to active PD, active PD fragment or combination of active fragments to about 100%, more preferably from about 70% to about 100%, from about 80% to about 100% or from about 90% to about 100%.

In a preferred embodiment, the PD of the SRF according to the invention is inactivated by 1, 2, 3, 4, 5 or more suitable mutations, insertions, additions, deletions or substitutions of amino acids as listed in the table below . Suitable mutations are those suitable to inactivate PD. Suitable mutations can be readily determined by those skilled in the art, for example by routine cloning techniques.

In a particularly preferred embodiment of the invention, the SRF according to the invention comprises a PD of ACE-2 or a fragment, a combination of fragments or a derivative thereof, with a substitution at position 375 as shown in SEQ ID NO: 3, in particular Glu375Gln Substitution is preferred. Said substitution preferably results in inactivation of the active site of PD. In a more preferred embodiment, the SRF comprises a fragment of an inactivated PD as shown in SEQ ID NO: 5, or fragments, combinations of fragments and/or derivatives thereof. An example of an SRF comprising the sequence shown in SEQ ID NO:5 is an SRF consisting of or comprising the sequence shown in SEQ ID NO:10. The SRF having the sequence shown in SEQ ID NO: 10 contains, in addition to the sequence shown in SEQ ID NO: 5, methionine as a translation initiation signal and a tag, ie, Histag. Further examples of SRFs according to the invention comprising an inactivated PD of ACE-2, an inactivated fragment thereof or a combination of inactivated fragments thereof are shown as SEQ ID NOs: 18, 19, 21, 22, 26, 27, 30 and 31 .

The SRFs having the sequences shown in SEQ ID NO: 21, 26 and 30 each have a Glu375Gln substitution, i.e. at position 375 of the sequence shown in SEQ ID NO: 3, ie at position 175 of SEQ ID NO: 21 and 26 and SEQ ID NO: At position 144 of 30, glutamic acid was substituted with glutamine.

In a further preferred embodiment, the PD of the SRF according to the present invention comprises, in addition to the mutation at position 375, in particular the substitution of Glu375Gln, one, two, three, four or five further mutations, as shown in the table above Insertions, additions, deletions or substitutions of amino acids listed in .

According to the use of SRF in the present invention, SRF and PD of SRF, fragments, combinations of fragments or derivatives thereof, respectively, are preferably capable of dimerization. Dimerization can increase the stability of SRF and/or can increase the binding affinity for protein S. Alternatively, the PD of SRF, fragments, combinations of fragments or derivatives thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20 or more mutations, such as insertions, additions, deletions or substitutions, monomerize PD. Monomerization can increase the rate of distribution in solution.

According to the present invention, the solution containing SRF is used as, for example, mouthwash or nasal cavity rinsing solution, and the binding pockets of the free Coronaviridae such as Sars-CoV-2's spike protein (for example, protein S) present in these parts of the human body will be absorbed by SRF. Combined, no longer able to infect the subject's cells. These solutions can also be applied prophylactically to prevent infection in advance. SRF according to the present invention can also be used therapeutically to inactivate virus particles at the site of infection in the human body without significant effects of SRF on blood pressure and kidney function.

Alternatively, SRFs can be immobilized on beads or other surfaces to capture virus particles, for example by creating an affinity effect to improve washing, thereby facilitating the washout of virus particles from the infected area.

Alternatively, SRFs can be immobilized on beads or other surfaces to capture viral particles, e.g. to improve diagnostic quality, or washout efficacy. This also includes the use of dialysis applications, where SRFs are coupled to dialysis membranes or beads to filter out viral particles.

Alternatively, SRF can be immobilized or fused to proteins, such as the Fc portion of IgG1, for in vivo applications.

Alternatively, SRFs can be immobilized or fused to proteins for in vivo applications to dimerize PDs and thus enhance affinity.

Thus, in a preferred embodiment of the invention, the SRF is immobilized, bound, coupled or attached to a bead, membrane such as a dialysis membrane or other surface. In a further preferred embodiment, the SRF is immobilized, bound, coupled, linked or fused to a protein, antibody, antibody fragment, eg the Fc portion of IgGl or other compound or molecule.

For example, SRFs according to the present invention may additionally contain tags, linkers, proteins, compounds or molecules, which may facilitate the stability, solubility, distribution and half-life of SRFs in the human body or other characteristics required to address their respective applications. In general, SRFs can be modified to meet the challenges of the application addressed, as can known therapeutic proteins (e.g. summarized in the review by Dellas et al. (2021)). For example, the SRF may additionally contain maltose binding protein (MBP) to increase the stability and/or solubility of the SRF, or an Fc fusion to enable dimerization.

Accordingly, the present invention also provides SRF according to all embodiments of the invention described above for use in a method of treatment of the human or animal body by surgery or therapy, as a vaccine or as SRF for administering on the human or animal body Or for use in diagnostic methods performed on bodily fluids or other materials from humans or animals.

More particularly, the present invention provides SRF according to all embodiments of the invention described above for use in a method of treating and/or preventing a viral infection in a subject, in particular a viral infection caused by the family Coronaviridae, more In particular, SARS-CoV, SARS-CoV-2, human coronavirus NL63 or SARS-CoV-2 including any mutants thereof, such as from British lineage B.1.1.7, South African B.1.351, Indian B. Viral infections caused by variants of 1.617 or variant B.1.1.28.1 from Brazil.

In particular, SRFs according to the invention are useful for therapeutic and and/or are particularly effective in preventing viral infection in a subject. To test whether any potential mutations or variants allow host cell receptor binding, particularly ACE-2 receptor binding, affinity measurements can be performed on a Biacore chip or by MicroScale thermophoresis, as described in the examples below. If the result of such a test is a stronger binding than the standard deviation of the negative control, the tested virus can be considered to be of the family Coronaviridae, SARS-CoV, SARS-CoV-2, human coronavirus NL63, or SARS-CoV-2 Mutants or variants which can effectively treat or prevent viral infection by the SRF according to the invention.

Prevention of viral infection with a SRF according to the invention may involve inactivating or neutralizing the virus, in particular by blocking the binding pocket of the virus' Spike protein, more specifically by binding the SRF to the virus' binding protein. Preferably, the prevention of viral infection by SRF according to the present invention may comprise inactivating or neutralizing the virus, in particular by blocking the binding pocket of the virus' Spike protein (Protein S), more in particular by binding the SRF to the virus Protein S binding.

The replication of viruses is also known to mutate, or mutate. These mutations may or may not have an effect on viral proteins. Viral receptor proteins also mutate, but only mutations that still allow host cell receptor binding are replicated. This means that the binding pocket of the viral receptor protein is relatively stable to mutations that affect binding to the host cell, otherwise the binding to the host cell may be lost and viruses with these mutations will not be replicated. Humans are made up of 100 trillion cells. Naturally, these cells would not have mutated a biologically functional receptor to avoid viral infection. Thus, host cell para-receptors, especially human cell para-receptors, are highly conserved. Thus, coronaviruses are under great pressure not to have mutations in their host cell receptors or spike proteins that do not recognize host cell receptors with significant affinity. From British pedigree B.1.1.7 (the spike mutation N501Y (which has a higher affinity for ACE-2), plus other mutations in the spike) or B.1.351 from South Africa (multiple mutations in the spike protein, Although variants including K417N, E484K, N501Y) have mutations in the spike protein, the spike protein still binds to the host cell receptor ACE-2, so the SRF according to the present invention is also effective for the above variants.

The SRF according to the present invention provides a blocking tool, which can block the spike protein of the virus and make the virus lose its ability to infect. Pretending that the SRF is the lock of the human cell, what to look for is the key for the virus to interface with it. Once the virus is docked to SRF, it cannot infect human cells and therefore cannot replicate. Inactivated virus, ie virus docked with SRF according to the present invention, will be destroyed by the immune system. The SRFs according to the invention, in particular the enzymatically inactive SRFs which do not affect the blood pressure system according to the invention, do not interact with living cells and thus protect the natural microbiota of humans.

The subject to be treated is preferably a human or an animal, especially a mammal, most preferably a human.

In a preferred embodiment, the SRF used according to the invention is administered in an amount sufficient to reduce the viral load capable of infecting cells of the subject and/or inactivate viral particles at the site of infection in the subject. The effective amount of the compound to be administered can be readily determined by those skilled in the art by methods familiar to physicians and clinicians during preclinical and clinical trials.

In a further preferred embodiment, the SRF used according to the invention is fused to a protein, wherein the fused SRF is administered in an amount sufficient to increase the affinity by allowing dimerization of the PD.

According to all embodiments of the present invention, an effective amount of SRF for use in methods of treating and/or preventing viral infections can be administered to a subject in need thereof by many well-known methods of administering pharmaceutical compounds. The compounds may be administered topically or systemically, with locally administration being preferred. The route of administration may be nasal, oral, intraocular, topical, systemic, intravenous, inhalation, injection or locally or any other suitable route of administration. Therefore, the SRF used according to the invention is preferably administered nasally, orally, intraocularly, topically, systemically, intravenously or by wound irrigation.

A further aspect of the invention refers to a pharmaceutical composition or medical product comprising a SRF according to all embodiments of the invention together with pharmaceutically or physiologically acceptable excipients and/or carriers.

The pharmaceutical compositions according to the invention may additionally contain one or more conventional additives. Some examples of such additives include solubilizers, such as glycerin; antioxidants, such as benzalkonium chloride, benzyl alcohol, chloretone, or chlorobutanol; and/or isotonic agents.

The pharmaceutical composition or medical product can be formulated as tablets, lozenges, candies, drops, chewing gum, lollipops, sprays, especially nasal, buccal, mouth, throat or wound sprays, rinses, especially nasal , oral, wound, or eye rinses, injections, balms, ointments, eye drops, or mouth or throat rinses.

As mentioned above, the administration of the SRF according to the invention and/or the pharmaceutical composition according to the invention may especially be formulated or designed to prevent the uptake of viruses, or at least reduce the viral load into the human body. Thus, the application is preferably performed to allow for a wash effect or as a wash to neutralize the virus receptors, preferably allowing the neutralized virus to be washed out from the point of entry.

Furthermore, the invention also provides a pharmaceutical pack comprising one or more compartments, wherein at least one compartment comprises a SRF according to the invention or a pharmaceutical composition according to the invention.

In another specific embodiment of the present invention, the SRF according to the present invention and/or the pharmaceutical composition of the present invention are used for the manufacture of medicines for the treatment or prevention of viral infections, especially those caused by Coronaviridae, more particularly by Caused by SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, most preferably by SARS-CoV-2 including any mutants thereof, e.g. from British lineage B.1.1.7, South African B .1.351, variants of B.1.617 from India, or variants B.1.1.28.1 from Brazil.

In a specific embodiment of the present invention, the SRF according to the present invention and/or the pharmaceutical composition of the present invention are used as a medicament for the treatment or prevention of viral infections, especially viral infections caused by Coronaviridae, more particularly SARS Caused by a coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, most preferably by SARS-CoV-2 including any mutants thereof, such as from British lineage B.1.1.7, South African B. 1.351, a variant of B.1.617 from India or a viral infection caused by variant B.1.1.28.1 from Brazil.

In a further aspect of the present invention is a method of treating or preventing viral infection by administering or applying an effective amount of the SRF according to the present invention or the pharmaceutical composition according to the present invention to a subject, especially to a human or an animal, in particular by A viral infection caused by the family Coronaviridae, more particularly by SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, most preferably by SARS-CoV-2 including any mutants thereof, Viral infections such as variants from British lineage B.1.1.7, B.1.351 from South Africa, B.1.617 from India, or variant B.1.1.28.1 from Brazil.

In a further aspect, the invention provides a method of capturing viral particles, the method comprising:

a) providing a SRF according to the present invention, wherein the SRF is immobilized, bound, coupled or attached to a bead, a column or column material thereof, a membrane or other surface, and

b) contacting the liquid sample or fluid with the SRF of step a) under conditions that allow the binding of the SRF to the viral particles.

The virus particle is preferably a virus particle or a whole virus, preferably of the family Coronaviridae, more particularly a virus selected from the group consisting of: SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2 Include any mutations thereof, such as variants from British pedigree B.1.1.7, B.1.351 from South Africa, B.1.617 from India or variant B.1.1.28.1 from Brazil.

In preferred embodiments, the method of capturing viral particles is a method of detecting captured viral particles. The detection method additionally comprises the step of detecting captured viral particles. This detection can be performed by detection of captured virus particles. Alternatively, the method may comprise the steps of eluting the viral particles and detecting the eluted viral particles. Furthermore, the method of capturing viral particles and/or the method of detecting captured viral particles may comprise further steps, such as one or more washing steps.

In a further preferred embodiment, the method of capturing viral particles is a method of washing a liquid sample or fluid, wherein the viral load in the liquid sample or fluid is reduced due to the capture of viral particles. The washing method may be adapted to neutralize the virus receptor, preferably allowing neutralized virus to be washed out.

For example, the method can be used in dialysis to reduce the viral load in a subject's bodily fluids. In particular, washing neutralizes the virus receptor, preferably washing the neutralized virus out of the body fluid of the subject. Alternatively or additionally, this prevents the subject's cells from taking up the virus, or at least substantially reduces the viral load entering the body. In order to do this, the SRF according to the invention is preferably immobilized, bound, coupled or attached to a dialysis membrane, the fluid being a body fluid such as whole blood, plasma or a blood fraction.

Some particularly preferred embodiments of the invention are summarized in items 1 to 24 below.

CLAIMS 1. Soluble receptor fragment (SRF) of the ACE-2 receptor, wherein the SFR comprises the peptidase domain (PD) of ACE-2 or fragments and/or derivatives thereof.

2. The SRF according to item 1, wherein the SRF binds to the receptor binding cleft of the spike protein of a virus, in particular to the family Coronaviridae, more particularly to SARS-CoV, SARS-CoV-2, human-CoV-NL63 or SARS-CoV-2. CoV-2 includes any of its mutants, such as the spike protein of viruses from British lineage B.1.1.7, South African B.1.351, Indian B.1.617, or Brazilian variant B.1.1.28.1 The receptor-binding cleft of S binds.

3. SRF according to item 1 or 2, wherein fragments and/or derivatives thereof comprise one, two or more fragments of the PD of ACE-2 and/or one, two or more of the PD of ACE-2 derivatives of fragments.

4. The SRF according to any one of the preceding, wherein its fragments and/or derivatives comprise 3, 4, 5, 6, 7, 8 of the PD of ACE-2 shown in SEQ ID NO: 34-43 or derivatives thereof , 9 or all 10 α-helical structures, in particular wherein said derivative comprises 1, 2, 3, 4 or 5 deletions or mutations, provided that the derivative is still capable of forming an α-helical structure.

5. The SRF according to any one of the preceding, wherein its fragments and/or derivatives at least comprise the Sars-CoV-2 contact residues Q24, T27, F28, D30, K31, H34, E35, E37, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of D38, Y41, Q42, L79, M82, Y83.

6. The SRF according to any of the preceding items, wherein its fragments and/or derivatives at least comprise Sars-CoV contact residues Q24, T27, F28, K31, H34, E37, D38, Y41, Q42, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of L45, L79, M82, Y83.

7. The SRF according to any one of the preceding, wherein its fragment and/or derivative at least comprise 1, 2 of the Sars-CoV-2 contact residues N330, K353, G354, D355, R357, R393 of SEQ ID NO:3 , 3, 4, 5 or 6 pieces.

8. The SRF according to any one of the preceding, wherein its fragment and/or derivative at least comprise 1, 2 of the Sars-CoV contact residues Q325, E329, N330, K353, G354, D355, R357 of SEQ ID NO:3 , 3, 4, 5, 6 or 7.

9. The SRF according to any of the preceding items, wherein fragments thereof and/or derivatives thereof at least comprise the following α-helical structures:

- SEQ ID NO: 34 or a derivative thereof,

- SEQ ID NO: 35 or a derivative thereof, and

- SEQ ID NO: 36 or a derivative thereof.

10. The SRF according to any one of the foregoing, wherein its fragments and/or derivatives at least comprise an amino acid sequence according to SEQ ID NO:8 or a derivative thereof, which has at least 60, 70, 80 of SEQ ID NO.8 , 90, 95 or 98% sequence identity, particularly wherein said derivative comprises residues Q6, T9, F10, D12, K13, H16, E17, E19, D20, Y23, Q24, At least 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of L61, M64, and Y65, and preferably at least include the following α-helical structures:

- SEQ ID NO: 34 or a derivative thereof.

- SEQ ID NO: 35 or a derivative thereof, and

- SEQ ID NO: 36 or a derivative thereof.

11. The SRF according to any one of the preceding, wherein the SRF comprises a fragment combination of two fragments of the PD of ACE-2 or a derivative thereof, in particular wherein the first fragment is selected from the amino acid sequence of SEQ ID NO: 8 and 9 or Derivatives, the second fragment is selected from the amino acid sequence of SEQ ID NO: 15, 23, 28 and 32 or each derivative thereof.

12. The SRF according to any one of the preceding, wherein the SRF comprises an inactivated PD of ACE-2 or a derivative thereof or an inactivated fragment or combination of fragments of the PD of ACE-2, in particular wherein the inactivated PD, derivative, fragment or Fragment combinations contain mutations such as insertions, additions, deletions or substitutions at one or more of the following positions:

13. The SRF according to any of the preceding items, wherein the SRF comprises an amino acid sequence according to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 or derivatives and/or fragments thereof, in particular Wherein SRF comprises according to SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO :27, SEQ ID NO:29, SEQ ID NO:30, the amino acid of SEQ ID NO:31, or have at least 60, 70, 70, Derivatives with 80, 90, 95 or 98% sequence identity, provided that said derivatives comprise at least Q24, T27, F28, D30, K31, H34, E35, E37, D38, Y41, Q42 of SEQ ID NO:3 , 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of , L79, M82, Y83, and alpha-helical structures containing at least the following:

- SEQ ID NO: 34 or a derivative thereof,

- SEQ ID NO: 35 or a derivative thereof, and

- SEQ ID NO: 36 or a derivative thereof.

14. The SRF according to any one of the preceding, wherein the SRF is immobilized, bound, coupled, linked or fused to a protein, antibody, antibody fragment, eg the Fc portion of IgGl, or other compound or molecule.

15. The SRF according to any one of the preceding, wherein the SRF is immobilized, bound, coupled or attached to a bead, a membrane, such as a dialysis membrane, a column or column material or other surface.

16. SRF according to any one of the preceding, for use in a method of treating the human or animal body by surgery or therapy, as a vaccine or for its administration on the human or animal body or for use in the treatment of human or animal body fluids or in diagnostic methods implemented on other materials.

17. SRF according to any one of items 1 to 14, for use in the treatment and/or prophylaxis of viral infections, in particular caused by Coronaviridae, more in particular by SARS-CoV, SARS-CoV-2, Human Corona Virus NL63 or SARS-CoV-2 including any mutants thereof, such as variants from British lineage B.1.1.7, South African B.1.351, Indian B.1.617 or variant B.1.1.28.1 from Brazil cause method of virus infection.

18. SRF for use according to item 17, wherein preventing viral infection comprises inactivating or neutralizing the virus, in particular by blocking the binding pocket of the spike protein, more in particular by binding the SRF to a binding protein of the virus, more in particular By blocking the binding pocket of the Spike protein (Protein S) of the Coronaviridae family, more particularly by binding SRF to the binding protein S of the virus.

19. SRF for use according to item 17 or 18, wherein the SRF is administered in an amount sufficient to reduce the viral load of a virus capable of infecting cells of the subject and/or inactivate viral particles at the site of infection in the subject.

20. SRF for use according to any one of items 17 to 19, wherein the SRF is formulated for nasal, oral, intraocular, topical, systemic, intravenous or wound irrigation administration or administration by inhalation or injection.

21. A pharmaceutical composition or medical product comprising SRF for use according to any one of 1 to 14 and pharmaceutically or physiologically acceptable excipients and/or carriers, in particular wherein the pharmaceutical composition or medical product is formulated Tablets, lozenges, bonbons, drops, chewing gum, lollipops, sprays, especially nasal, oral, oral , throat or wound sprays, rinsing solutions, especially nasal, oral, wound or eye rinsing solutions, injection solutions, balms, ointments, eyedrops or mouth Or a throat wash.

22. A method of capturing viral particles, the method comprising:

a) provide an SRF according to item 14 or 15, and

b) contacting the liquid sample or fluid with the SRF of step a) under conditions that allow the binding of the SRF to the virus particle.

23. The method according to item 22, wherein the method is a method of detecting captured viral particles, and wherein the method additionally comprises the step of detecting captured viral particles.

24. The method according to item 22, wherein the method is a method of washing a liquid sample or fluid, wherein the viral load in the liquid sample or liquid is reduced due to the capture of the virus particles, in particular wherein the SFR is immobilized, bound, Coupled or attached to a dialysis membrane and the fluid is a bodily fluid such as whole blood, plasma or blood fractions.

The following examples illustrate the invention but are not to be considered as limiting. It should be understood that the detailed description and specific examples disclosed herein, while indicating particular embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art. personnel is evident from this description.

Example 1--expression and purification of SRF and spike protein in baculovirus cells

SRF (according to SEQ ID NO: 4, with or without Strep-tag) was expressed in the baculovirus system (Sf9) according to the manufacturer's protocol (BD Biosciences). Briefly, 2 μg of SRF (according to SEQ ID NO:4) encoding plasmid was mixed with 0.5 μg of BD BaculoGold linearized baculovirus DNA, and 1 ml of BD BaculoGold Transfection Buffer B was added after 5 minutes at room temperature. This mixture was added dropwise to Sf9 insect cells (ATCC) pre-coated with 1 ml of transfection buffer A, and then the cells were incubated at 28 °C for 4 h and further transfected in fresh SF-900II SFM (Invitrogen) cells for an additional 4 days. After 4 days, the supernatant was collected and used to infect the cells, followed by a 3-day culture period for virus amplification. The cell culture supernatant of the 4th cycle was collected for purification of SRF (according to SEQ ID NO: 4) protein.

SRF (according to SEQ ID NO: 5, with or without Strep-tag) was expressed in the baculovirus system (Sf9) according to the manufacturer's protocol (BD Biosciences). Briefly, 2 μg of SRF (according to SEQ ID NO:5) encoding plasmid was mixed with 0.5 μg of BD BaculoGold linearized baculovirus DNA, and 1 ml of BD BaculoGold Transfection Buffer B was added after 5 minutes at room temperature. This mixture was added dropwise to Sf9 insect cells (ATCC) pre-coated with 1 ml of transfection buffer A, and then the cells were incubated at 28 °C for 4 h and further transfected in fresh SF-900II SFM (Invitrogen) cells for an additional 4 days. After 4 days, the supernatant was collected and used to infect the cells, followed by a 3-day culture period for virus amplification. The cell culture supernatant of the fourth cycle was collected for purification of SRF (SEQ ID NO:5) protein.

Spike protein S with His tag was obtained from Sino Biological 40592-V08B-1. Strep-tag variants were cloned into expression vectors and soluble expressed in baculovirus SF9 cells to allow proper glycosylation. Purify via Strep-Tag or other standard purification techniques.

Example 2 - Measuring SRF Binding to Spike Protein S in an Elisa Assay

Microplates coated with SRF (according to SEQ ID NO:4) were incubated with soluble Spike S (Strep-tagged). This was followed by three washes with PBS-buffer to wash away unbound Spike protein. Protein S binding to immobilized SRF (according to SEQ ID NO:4) was then quantified using streptavidin-conjugated horseradish peroxidase. Binding of protein S to immobilized SRF (according to SEQ ID NO:4) was successfully measured.

Protein S coated microplates were incubated with SRF (according to SEQ ID NO:5) and additionally included Strep tag. Unbound SRF (according to SEQ ID NO: 5) was then washed away by three washes with PBS-buffer. SRF (according to SEQ ID NO:5) bound to immobilized protein S was then quantified using streptavidin-conjugated horseradish peroxidase. Binding of SRF (according to SEQ ID NO:5) to immobilized protein S was successfully measured.

Example 3 - Affinity Measurements on Biacore Chips - Part 1

Strep-tagged SRF was captured using a sensor chip SA pre-immobilized with streptavidin. Spike protein S was then injected and binding to immobilized SRF (according to SEQ ID NO:4) was measured. A blank flow cell was used to correct for binding reactions. The surface plasmon resonance (SPR) method was used on the BIAcore system to measure the binding affinity of SRF. Binding of protein S to immobilized SRF (according to SEQ ID NO:4) was successfully measured, whereas when protein S was incubated with soluble SRF (according to SEQ ID NO:4) and injected into the Biacore system, binding to immobilized SRF (according to SEQ ID NO:4) was detected. Binding according to SEQ ID NO:4) was significantly reduced. Injection of protein S into a Biacore chip with immobilized SRF (according to SEQ ID NO:4) after incubation at higher concentrations with soluble SRF (according to SEQ ID NO:4) resulted in no detectable binding .

Embodiment 4--virus inactivation test. Treatment of Vero E6 cells with SRF

Seed Vero E6 cells in 48-well plates in DMEM containing 10% FBS. 24 hours after seeding, SRF was mixed with different concentrations of virus (1:1) in DMEM (0% FBS) at 37°C in a final volume of 100 ml per well. After 30 min, Vero-E6 was infected with SRF or a mixture of SRF/SARS-CoV-2 for 1 h and then washed, or without washing for 15 h, the cells were washed 3 times with PBS and then added to 500 ml of a new complete culture supplemented with SRF base. 15 hours after infection, the supernatant was removed, washed 3 times with PBS, then lysed with Trizol, and analyzed by qRT-PCR to detect viral RNA. Virus-infected Vero E6 cells were used as a positive control. Vero E6 cells infected with SRF only served as a negative control. Mixing SRF and SARS-CoV-2 before infecting Vero E6 cells showed a significant reduction in infectivity. Application of SRF (according to SEQ ID NO:4) or SRF (according to SEQ ID NO:5) did not result in significant differences.

Embodiment 5--Expression and purification of SRF and spike protein in HEK293 cells

The gene encoding SRF comprises the amino acid sequence according to SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:4, SEQ ID NO:8 and SEQ ID NO:9, and the above gene is cloned into the standard expression vector pTZ (Trenzyme ) for transient expression in the supernatant of mammalian HEK293 cells. For translation and purification reasons, the above amino acid sequences were supplemented with His tag and methionine as translation initiation signal, respectively. Therefore, the coding sequence including methionine as translation initiation signal, secretion signal and His-tag actually used for cloning is as follows:

-(3)_ACE2_19-615_E375Q, as shown in SEQ ID NO:10 (comprising the amino acid sequence shown in SEQ ID NO:5),

-(4)_ACE_2_19-103; 301-365, as shown in SEQ ID NO:11 (comprising the amino acid sequence shown in SEQ ID NO:7),

-(9)_ACE2_19-615, as shown in SEQ ID NO: 12 (comprising the amino acid sequence shown in SEQ ID NO: 4).

-(1)_ACE2_19-103, as shown in SEQ ID NO:13 (comprising the amino acid sequence shown in SEQ ID NO:8), and

-(8)_ACE2_19-132, as shown in SEQ ID NO: 14 (comprising the amino acid sequence shown in SEQ ID NO: 9).

Protein expression was carried out in shake flasks with the target protein in the cell culture supernatant. The supernatant was collected after expression, and the target protein was purified by His-tag affinity chromatography.

Constructs (1) and (8) were expressed as MBP fusions by fusing the respective constructs to MBP via a short linker. The coding sequence includes methionine as translation initiation signal, secretion signal, MBP, short linker and His tag. Protein expression was carried out in shake flasks with the target protein in the cell culture supernatant. The supernatant was collected after expression, and the target protein was purified by His-tag affinity chromatography.

Example 6 - Affinity measurement on a Biacore chip - Part II

SARS-CoV-2 spike protein S1 (aa14-683), His-Avi-Tag recombinant protein (Invitrogen, Catalog#RP-87681) and three variants of SRF ((3)_ACE2_19-615_E375Q, 1.24mg/ml (4)_ACE_2_19-103; 301-365, 1 mg/ml; (9)_ACE2_19-615, 0.74 mg/ml) were performed on Cytiva's Biacore X100 using the Biotin CAPture kit on the sensor chip CAP. The buffer is PBS, pH 7.4.

After equilibration and conditioning of the Biacore system, the CAP chip was coated with Biotin CAPture reagent.

Immobilization of spike protein S1 on flow cell one yielded ~100 RU (ligand concentration 0.43 μΜ; contact time: 420 s; stationary phase: 300 s).

The relative response to binding was measured with the highest concentration of ACE-2 variant without reference to flow cell two due to non-specific reference binding (contact time: 120 s; dissociation time: 600 s; regeneration not required). Three priming cycles were performed with buffer.

The result is: (3)_ACE2_19-615_E375Q 443.6RU

(4)_ACE_2_19-103; 301-365, 1mg/ml 352.0RU

(9)_ACE2_19-615 55.1RU

cycle frequency relative response buffer 1 -6 buffer 2 -5.6 buffer 3 -5.2 (3)_ACE2_19-615_E375Q 4 443.6 (4)_ACE_2_19-103; 301-365 5 352 (9)_ACE2_19-615 6 55.1

result:

All three proteins tested bound to the spike protein of Sars-CoV-2 immobilized on a biocore chip, with variant (3)_ACE2_19-615_E375Q showing the highest binding response. Variant (4)_ACE_2_19-103;301-365 had lower binding efficiency and variant (9)_ACE2_19-615 had the lowest binding efficiency. Even though (9)_ACE2_19-615 showed the lowest binding affinity compared with other constructs, its binding to SARS-CoV-2 spike protein S1 was clearly confirmed.

Example 7 - Measuring Binding Affinity by MicroScale Thermophoresis

MicroScale thermophoresis was used to measure the affinity between the Sars-CoV-2 spike protein and the respective target proteins. In principle, the binding affinity of two molecules is measured by detecting changes in fluorescence signal due to IR-laser-induced temperature changes. The range of variation of the fluorescent signal is related to the binding of the ligand to the fluorescent target. Therefore, it enables quantitative analysis of molecular interactions in solution at the microliter scale with high sensitivity.

Fluorescently label the spike protein:

SARS CoV 2 spike protein S1 (aa14 683), His tag, Avi tag; Invitrogen, Cat. Number: RP 87681, the stock solution concentration in 1x PBS, pH 7.4 buffer is 10.0 μM.

Dilute the target protein into labeling buffer 1x PBS pH 7.4, 0.005% Tween to achieve a labeling concentration of 9.7 μM. Introduce DMSO into the labeling buffer as the dye stocks are prepared in DMSO. The labeling concentration of the dye was 20.1 μM (dye:protein molar ratio 3:1). Red NHS 650 ^{second generation} was used as fluorescent dye. Labeling was performed at 25°C for 30 minutes. After labeling, unbound dye is removed by a gel filtration step and the target protein is taken into the final buffer. Add 0.005% Tween 20 to reduce protein sticking/sticking and improve protein recovery. The concentration of labeled target protein is determined by measuring the fluorescence of the dye. Total protein concentration and protein recovery were determined by fluorescence contrast measurements using a Tycho NT.6 instrument. When labeling, approximately 2 fluorescent tags are introduced per protein.

Combined measurements:

Measurement setup for variant: (9) ACE 2 19-615

Target: SARS CoV 2 Spike S1, used at constant 10nM

Ligand: (9) ACE 2 19-615, starting at 4.73 μΜ, titrated in 16 1:1 dilution steps.

Instrument: Monolith NT.115Pico

Buffer: 1x PBS pH 7.4, 0.005% Tween 20

result: K_D amplitude S/N Experiment 1 114nM -35.4 19.6 Experiment 2 192nM -31.0 24.6

Measurement Setup (3) ACE 2 19-615 E375Q

Target: SARS CoV 2 Spike S1, used at constant 10nM

Ligands: (3) ACE 2 19-615 E375Q, starting at 7.92 μM, titrated in 16 1:1 dilution steps.

Instrument: Monolith NT.115Pico

Buffer: 1x PBS pH 7.4, 0.005% Tween 20

result: K_D amplitude S/N Experiment 1 23.7nM -39.0 13.0 Experiment 2 23.1nM -37.9 16.8

Measurement Setup (4)_ACE_2_19-103; 301-365

Target: SARS CoV 2 Spike S1, used at constant 10nM

Ligands: (4)_ACE_2_19-103;301-365, titrated in 16 1:1 dilution steps starting from 22.9 μΜ.

Instrument: Monolith NT.115Pico

Buffer: 1x PBS pH 7.4, 0.005% Tween 20

result: K_D amplitude S/N Experiment 1 42.8nM -35.0 14.6 Experiment 2 43.3nM -29.0 13.4

Measurement setup (1) ACE2 19-103 fused with MBP

Target: SARS CoV 2 Spike S1, used at constant 10nM

Ligands: (1) ACE2 19-103 fused to MBP, titrated in 16 1:1 dilution steps starting from 1.26 μM.

Instrument: Monolith NT.115Pico

Buffer: 1x PBS pH 7.4, 0.005% Tween 20

result: K_D amplitude S/N Experiment 1 4.94nM -24.8 15.9 Experiment 2 2.70nM -28.2 9.1

Measurement setup (8) ACE2 19-132 fusion with MBP

Target: SARS CoV 2 Spike S1, used at constant 10nM

Ligands: (8) ACE2 19-132 fused to MBP, titrated in 16 1:1 dilution steps starting from 1.29 μM.

Instrument: Monolith NT.115Pico

Buffer: 1x PBS pH 7.4, 0.005% Tween 20

result: K_D amplitude S/N Experiment 1 1.74nM -23.2 26.2 Experiment 2 2.39nM -19.7 15.1

result:

The variants (8) ACE2 19-132 fused to MBP and (1) ACE2 19-103 fused to MBP showed the best binding affinities. The other three variants had lower binding efficiency, variant (3)_ACE2_19-615_E375Q had stronger binding than (4)_ACE_2_19-103;301-365 (former ACE2_19-103), variant (9)_ACE2_19-615 The binding force is the lowest. Even though (9)_ACE2_19-615 showed the lowest binding affinity compared to other constructs, its binding to SARS CoV2 spike S1 was clearly confirmed.

Example 8--SARS-CoV-2 (B.3 line) and recombinant SRF variants neutralize plaque assay

Material:

DMEM medium: Thermo Fishe41965039

FBS: Pan Biotech #P30-3702

Penicillin/Streptomycin: Sigma #P4333

OptiPro-SFM: Thermo Fisher#12309019

DPBS (without Ca/Mg): Thermo Fisher 14190144

Carboxymethylcellulose: Sigma#C5013

MEM: Pan Biotech #P03-0710

NaHCO3: Roth#HN01.1

Paraformaldehyde: AppliChem#141328.1212

Crystal Violet (C.I.42555): Merck#1159400025

On day 0, 1.25× ^{10 5} VeroE6 cells/well were plated into a 24-well plate and cultured at 37° C. and 5% CO ₂ for 24 hours. On day 1, serial dilutions of recombinant SRF variants were prepared as follows:

Stock solution concentration:

Variant_19-615_E375Q: Mw=70, 6kDa, c=1, 24mg/ml→17, 563μM

Variant_19-615: Mw=70, 6kDa, c=0, 74 mg/ml → 10, 481 μM

SRF variants were thawed on ice. Preparations were performed in OptiPro-SFM (serum-free medium) in three sets of 120 [mu]l dilutions of SRF variants in round bottom 96-well plates. An additional 12 wells were filled with 120 μl OptiPro-SFM as a control and several 120 μl vehicle control samples were prepared in PBS in OptiPro-SFM. The board is tightly sealed without air bubbles.

virus dilution

In the BSL-3 laboratory, an aliquot of in vitro propagated SARS-CoV- ² (line B.3, isolated February 2020, 1.8x106 PFU/ml) was thawed and pressed in OptiPro-SFM A pre-dilution was performed at 1:2250 (1:4500 final dilution, 80 PFU/200 μl). 120 μl of this dilution is required per well for both the protein dilution and the positive control.

SRF variant-virus co-incubation

Pour the 1:2250 virus dilution into the reservoir. Add 120 μl of virus dilution to each SRF variant dilution well and positive control well with a multichannel pipette. Seal the plate and incubate for 1 hr at 37 °C and 5% _CO .

Infect VeroE6 cells

Media was removed from up to 6 wells of a 24-well plate using a vacuum pump or P1000 at a time, and 200 μl of SRF variant dilution/virus mix, virus (positive control) or OptiProSFM (negative control) was added. After completing the first 24-well plate, start the timer to count and place the plate back in the incubator. Proceed in this way until all samples from the 96-well plate have been added to the cells. Whenever a board is completed, the time on the timer is recorded. Cells were incubated at 37°C for 1 hour.

Change media after infection

Mix 1.5% (w/v) carboxymethylcellulose (autoclaved) and 2x MEM (sterile filtered, containing 2x Pen/Strep, 4% FBS, and _NaHCO3 to ensure pH) 1:1 , and preheat in a water bath. After the post virus addition timer reached 1 hour, the supernatant of each 24-well plate was aspirated (by vacuum or P1000) and replaced with 1 ml of carboxymethylcellulose-MEM/well. Return the plate to the incubator and incubate for 72 hours.

On day 4, cells were fixed and stained. Remove a partial volume of supernatant from each well with vacuum or a 10 ml serological pipette without touching or near the bottom surface of the well. Immerse the entire 24-well plate in 6% formaldehyde in a fixation/shipping container and cover the 24-well plate. The plates were left at room temperature for 30 minutes. Wipe down the exterior of the container with a disinfectant and remove the container from the BSL-3. Open the container and pour as much formaldehyde back into the container as possible. Each well was rinsed 3 times with tap water. Then, add 1% crystal violet staining solution to each well, cover and incubate at room temperature for 30 minutes. Use the funnel to pour the crystal violet back into the bottle, rinse the plate with tap water until the drained water is no longer blue, and allow the plate and lid to air dry completely. Plaques were counted in each well.

result:

(9)_ACE2_19-615(40PFU)

unprocessed 250nM 500nM 750nM 1μM 1.5μM 2μM 18 15 15 4 6 1 1 twenty two 17 12 13 4 1 3 twenty four 16 7 6 7 1 2 twenty four twenty two average 22,0 16,0 11, 3 7,7 5,7 1,0 2,0

(3)_ACE2_19-615_E375Q(40PFU)

unprocessed 250nM 500nM 750nM 1μM 1.5μM 2μM 18 17 twenty four 7 2 1 4 29 14 11 11 11 5 0 35 17 8 8 7 2 30 29 average 28, 2 15,5 17,3 8,7 7,0 4,3 2,0

(9)_ACE2_19-615(80PFU)

unprocessed 1nM 25nM 100nM 250nM 500nM 1μM 86 74 87 75 59 46 twenty four 85 80 83 80 61 56 32 87 88 70 81 60 58 35 90 95 average 88,6 80,7 80,0 78,7 60,0 53,3 30,3

(3)_ACE2_19-615_E375Q(80PFU)

unprocessed 1nM 25nM 100nM 250nM 500nM 1μM 87 75 81 78 62 53 46 96 87 75 86 69 64 39 106 83 82 80 68 67 32 96 88 average 94,6 81,7 79,3 81,3 66,3 61,3 39,0

As shown in the above table and Figure 2 and Figure 3, the results are shown as follows:

Neutralization tests were performed with 80 pfu/ml: variant 4_ACE_2_19-103; 301-365 showed no significant reduction in pfu at all concentrations tested. Variant 9_ACE2_19-615 showed a small reduction already at 1 nM and a 50% reduction at 250 nM. Application of variant 3_ACE2_19-615_E375Q in the neutralization assay resulted in a measurable reduction of about 40% at all concentrations tested.

Neutralization test with 40pfu/ml:

Variant 4_ACE_2_19-103;301-365 did not show a significant reduction in pfu at all concentrations tested. Variant 9_ACE2_19-615, showed 50% inhibition at 250 nM and complete inhibition at 1,5 μM. Application of variant 3_ACE2_19-615_E375Q resulted in a significant reduction over a concentration range of 250 nM to 1 μM. At 1, 5 μM and 2 μM there was almost complete reduction.

Example 9 - Neutralizing plaque assay of SARS-CoV-2 UK variant (B.1.1.7) and recombinant SRF variant

Such as Rambaut, A., Loman, N., Pybus, O., Barclay, W., Barrett, J., Carabelli, A., Connor, T., Peacock, T., Robertson, D.L., and Volz, E. (2020), the neutralizing plaque assay performed with the recombinant SRF variant described in Example 8 was a variant of SARS-CoV-2, the SARS-CoV-2 UK variant (B.1.1.7 ) to carry out. Preliminary genomic characterization of a UK-emergent SARS-CoV-2 line, defined by a new set of spike mutations.

result:

(9)_ACE2_19-615(80 PFU)

unprocessed 250nM 500nM 750nM 1μM 1.5μM 2μM 83 11 3 2 3 0 2 90 12 4 3 0 0 1 83 4 5 1 1 0 0 99 87 average 88,4 9,0 4,0 2,0 1, 3 0,0 1,0

(3)_ACE2-19-615_E375Q(80PFU)

unprocessed 250nM 500nM 750nM 1μM 1.5μM 2μM 81 14 3 4 2 1 1 98 11 6 4 1 3 1 91 14 3 0 1 0 1 77 83 average 86,0 13,0 4,0 2,7 1, 3 1, 3 1,0

As shown in Figure 4 and the table above, variant 9_ACE2_19-615 and variant 3_ACE2_19-615_E375Q already showed a large reduction at 250 nM and a complete reduction at 1 μM.

references

Alhenc-Gelas F, Drueke TB. Kidney Int. 2020Apr 14.pii:S0085-2538(20)30401-4

Daniel Wrapp, Nianshuang Wang, Kizzmekia S. Corbett, Jory A. Goldsmith, Ching-Lin Hsieh, Olubukola Abiona, Barney S. Graham, Jason S. McLellan, Science, 2020 Mar 13;367(6483):1260–1263.

Jiancheng Zhang, Bing Xie and Kenji Hashimoto, Current status of potential therapeutic candidates for the COVID-19crisis, Brain, Behavior, and Immunity, online available since 22April 2020, https://doi.org/10.1016/j.bbi.2020.04.046

Lei C, Qian K, Li T, Zhang S, Fu W, Ding M, Hu S. Nat Commun. 2020Apr 24;11(1):2070.

Monteil V, Kwon H, Prado P, Hagelkrüys A, Wimmer RA, Stahl M, Leopoldi A, Garreta E, Hurtado Del Pozo C, Prosper F, Romero JP, Wirnsberger G, Zhang H, Slutsky AS, Conder R, Montserrat N, Mirazimi A, Penninger JM.Cell.2020Apr 17.pii:S0092-8674(20)30399-8

Nikki Dellas, Joyce Liu, Rachel C. Botham, Gjalt W. Huisman, Adapting protein sequences for optimized therapeutic efficacy, Current Opinion in Chemical Biology, Volume 64, 2021, Pages 38-47, ISSN 1367-5931.

Sarah Cherian, Varsha Potdar, Santosh Jadhav, Pragya Yadav, NiveditaGupta, Mousmi Das, Soumitra Das, Anurag Agarwal, Sujeet Singh, Priya Abraham, Samiran Panda, Shekhar Mande, Renu Swarup, Balram Bhargava, Rajesh Bhushan, NICteam, INSACOvergent Consortium evo of SARS-CoV-2 spike mutations, L452R, E484Q and P681R, in the second wave of COVID-19 in Maharashtra, IndiabioRxiv 2021.

Singh, J.; Samal, J.; Kumar, V.; Sharma, J.; Agrawal, U.; Ehtesham, N.Z.; of New SARS-CoV-2 Variants B.1.1.7, B.1.351 and B.1.1.28.1: Clinical, Diagnostic, Therapeutic and Public Health Implications. Viruses 2021, 13, 439. (2021).

Stout AE, André NM, Jaimes JA, Millet JK, Whittaker GR. Coronaviruses incats and other companion animals: Where does SARS-CoV-2/COVID-19 fit? Vet Microbiol. 2020 Aug;247:108777.

Towler P, Staker B, Prasad SG, Menon S, Tang J, Parsons T, Ryan D, Fisher M, Williams D, Dales NA, Patane MA, Pantoliano MW. J Biol Chem. 2004 Apr 23;279(17):17996 -8007

Yan, R. et al. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science 367, 1444–1448 (2020).

sequence listing

<110> Lysando AG

<120> Virus neutralization by soluble receptor fragments of the ACE-2 receptor

<130> LYS-054 PCT

<140> unknown

<141> 2021-05-11

<150>EP20173886.1

<151> 2020-05-11

<160> 43

<170> PatentIn version 3.5

<210> 1

<211> 805

<212> PRT

<213> Homo sapiens full length ACE-2

<400> 1

Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala

1 5 10 15

Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe

20 25 30

Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp

35 40 45

Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn

50 55 60

Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala

65 70 75 80

Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln

85 90 95

Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys

100 105 110

Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser

115 120 125

Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu

130 135 140

Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu

145 150 155 160

Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu

165 170 175

Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg

180 185 190

Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu

195 200 205

Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu

210 215 220

Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu

225 230 235 240

His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile

245 250 255

Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly

260 265 270

Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys

275 280 285

Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala

290 295 300

Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu

305 310 315 320

Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro

325 330 335

Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly

340 345 350

Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp

355 360 365

Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala

370 375 380

Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe

385 390 395 400

His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys

405 410 415

His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn

420 425 430

Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly

435 440 445

Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe

450 455 460

Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met

465 470 475 480

Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr

485 490 495

Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe

500 505 510

Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala

515 520 525

Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile

530 535 540

Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu

545 550 555 560

Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala

565 570 575

Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe

580 585 590

Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr

595 600 605

Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu

610 615 620

Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met

625 630 635 640

Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu

645 650 655

Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val

660 665 670

Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro

675 680 685

Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile

690 695 700

Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn

705 710 715 720

Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln

725 730 735

Pro Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val

740 745 750

Ile Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg

755 760 765

Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile

770 775 780

Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Thr Asp Asp

785 790 795 800

Val Gln Thr Ser Phe

805

<210> 2

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Neck domain of human ACE-2 (616-726)

<400> 2

Gln Ser Ile Lys Val Arg Ile Ser Leu Lys Ser Ala Leu Gly Asp Lys

1 5 10 15

Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr Leu Phe Arg Ser Ser Val

20 25 30

Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys Val Lys Asn Gln Met Ile

35 40 45

Leu Phe Gly Glu Glu Asp Val Arg Val Ala Asn Leu Lys Pro Arg Ile

50 55 60

Ser Phe Asn Phe Phe Val Thr Ala Pro Lys Asn Val Ser Asp Ile Ile

65 70 75 80

Pro Arg Thr Glu Val Glu Lys Ala Ile Arg Met Ser Arg Ser Arg Ile

85 90 95

Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser Leu Glu Phe Leu Gly Ile

100 105 110

<210> 3

<211>615

<212> PRT

<213> Artificial sequence

<220>

<223> Peptidase domain of human ACE-2 (1-615)

<400> 3

Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala

1 5 10 15

Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe

20 25 30

Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp

35 40 45

Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn

50 55 60

Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala

65 70 75 80

Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln

85 90 95

Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys

100 105 110

Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser

115 120 125

Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu

130 135 140

Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu

145 150 155 160

Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu

165 170 175

Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg

180 185 190

Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu

195 200 205

Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu

210 215 220

Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu

225 230 235 240

His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile

245 250 255

Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly

260 265 270

Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys

275 280 285

Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala

290 295 300

Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu

305 310 315 320

Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro

325 330 335

Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly

340 345 350

Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp

355 360 365

Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala

370 375 380

Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe

385 390 395 400

His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys

405 410 415

His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn

420 425 430

Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly

435 440 445

Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe

450 455 460

Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met

465 470 475 480

Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr

485 490 495

Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe

500 505 510

Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala

515 520 525

Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile

530 535 540

Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu

545 550 555 560

Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala

565 570 575

Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe

580 585 590

Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr

595 600 605

Asp Trp Ser Pro Tyr Ala Asp

610 615

<210> 4

<211> 597

<212> PRT

<213> Artificial sequence

<220>

<223> Peptidase domain fragment of human ACE-2 (19-615)

<400> 4

Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly

35 40 45

Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Met

50 55 60

Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln

65 70 75 80

Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg Pro

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly Asn

305 310 315 320

Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly

325 330 335

Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys Asn

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala Asp

595

<210> 5

<211> 597

<212> PRT

<213> Artificial sequence

<220>

<223> Fragment of the peptidase domain of human ACE-2 with Glu375Gln substitution (19-615)

<400> 5

Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly

35 40 45

Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Met

50 55 60

Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln

65 70 75 80

Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg Pro

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly Asn

305 310 315 320

Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly

325 330 335

Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Gln Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys Asn

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala Asp

595

<210> 6

<211>614

<212> PRT

<213> Artificial sequence

<220>

<223> Peptidase domain of human ACE-2 (2-165)

<400> 6

Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala Ala

1 5 10 15

Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn

20 25 30

His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn

35 40 45

Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala

50 55 60

Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln

65 70 75 80

Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu

85 90 95

Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser

100 105 110

Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr

115 120 125

Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu

130 135 140

Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg

145 150 155 160

Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg

165 170 175

Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala

180 185 190

Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val

195 200 205

Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp

210 215 220

Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His

225 230 235 240

Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser

245 250 255

Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg

260 265 270

Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro

275 280 285

Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln

290 295 300

Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro

305 310 315 320

Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly

325 330 335

Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys

340 345 350

Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe

355 360 365

Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr

370 375 380

Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His

385 390 395 400

Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His

405 410 415

Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu

420 425 430

Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr

435 440 445

Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys

450 455 460

Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys

465 470 475 480

Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr

485 490 495

Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile

500 505 510

Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu

515 520 525

Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser

530 535 540

Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly

545 550 555 560

Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys

565 570 575

Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr

580 585 590

Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp

595 600 605

Trp Ser Pro Tyr Ala Asp

610

<210> 7

<211> 165

<212> PRT

<213> Artificial sequence

<220>

<223> Fragment combinations of the peptidase domain of ACE-2 (19-103;301-365)

<400> 7

Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly

35 40 45

Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Met

50 55 60

Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln

65 70 75 80

Ala Leu Gln Gln Asn Gly Ser Ala Gly Ser Ala Ala Gly Gly Ser Ala

85 90 95

Gly Ser Ala Ala Gly Ala Trp Asp Ala Gln Arg Ile Phe Lys Glu Ala

100 105 110

Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn Met Thr Gln Gly Phe

115 120 125

Trp Glu Asn Ser Met Leu Thr Asp Pro Gly Asn Val Gln Lys Ala Val

130 135 140

Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly Asp Phe Arg Ile Leu

145 150 155 160

Met Cys Thr Lys Val

165

<210> 8

<211> 85

<212> PRT

<213> Artificial sequence

<220>

<223> Fragment of the peptidase domain of ACE-2 (19-103)

<400> 8

Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly

35 40 45

Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Met

50 55 60

Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln

65 70 75 80

Ala Leu Gln Gln Asn

85

<210> 9

<211> 114

<212> PRT

<213> Artificial sequence

<220>

<223> Fragment of the peptidase domain of ACE-2 (19-132)

<400> 9

Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly

35 40 45

Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Met

50 55 60

Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln

65 70 75 80

Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val

<210> 10

<211>610

<212> PRT

<213> Artificial sequence

<220>

<223> (3)_ACE2_19-615_E375Q

<400> 10

Met Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn

1 5 10 15

His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn

20 25 30

Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala

35 40 45

Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln

50 55 60

Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu

65 70 75 80

Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser

85 90 95

Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr

100 105 110

Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu

115 120 125

Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg

130 135 140

Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg

145 150 155 160

Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala

165 170 175

Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val

180 185 190

Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp

195 200 205

Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His

210 215 220

Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser

225 230 235 240

Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg

245 250 255

Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro

260 265 270

Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln

275 280 285

Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro

290 295 300

Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly

305 310 315 320

Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys

325 330 335

Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe

340 345 350

Leu Thr Ala His His Gln Met Gly His Ile Gln Tyr Asp Met Ala Tyr

355 360 365

Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His

370 375 380

Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His

385 390 395 400

Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu

405 410 415

Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr

420 425 430

Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys

435 440 445

Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys

450 455 460

Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr

465 470 475 480

Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile

485 490 495

Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu

500 505 510

Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser

515 520 525

Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly

530 535 540

Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys

545 550 555 560

Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr

565 570 575

Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp

580 585 590

Trp Ser Pro Tyr Ala Asp Gly Gly Gly Ser His His His His His His His His

595 600 605

His His

610

<210> 11

<211> 178

<212> PRT

<213> Artificial sequence

<220>

<223> (4)_ACE_2_19-103;301-365

<400> 11

Met Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn

1 5 10 15

His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn

20 25 30

Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala

35 40 45

Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln

50 55 60

Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu

65 70 75 80

Gln Ala Leu Gln Gln Asn Gly Ser Ala Gly Ser Ala Ala Gly Gly Ser

85 90 95

Ala Gly Ser Ala Ala Gly Ala Trp Asp Ala Gln Arg Ile Phe Lys Glu

100 105 110

Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn Met Thr Gln Gly

115 120 125

Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly Asn Val Gln Lys Ala

130 135 140

Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly Asp Phe Arg Ile

145 150 155 160

Leu Met Cys Thr Lys Val Gly Gly Gly Ser His His His His His His His His

165 170 175

His His

<210> 12

<211>610

<212> PRT

<213> Artificial sequence

<220>

<223> (9)_ACE2_19-615

<400> 12

Met Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn

1 5 10 15

His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn

20 25 30

Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala

35 40 45

Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln

50 55 60

Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu

65 70 75 80

Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser

85 90 95

Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr

100 105 110

Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu

115 120 125

Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg

130 135 140

Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg

145 150 155 160

Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala

165 170 175

Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val

180 185 190

Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp

195 200 205

Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His

210 215 220

Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser

225 230 235 240

Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg

245 250 255

Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro

260 265 270

Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln

275 280 285

Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro

290 295 300

Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly

305 310 315 320

Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys

325 330 335

Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe

340 345 350

Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr

355 360 365

Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His

370 375 380

Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His

385 390 395 400

Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu

405 410 415

Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr

420 425 430

Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys

435 440 445

Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys

450 455 460

Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr

465 470 475 480

Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile

485 490 495

Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu

500 505 510

Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser

515 520 525

Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly

530 535 540

Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys

545 550 555 560

Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr

565 570 575

Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp

580 585 590

Trp Ser Pro Tyr Ala Asp Gly Gly Gly Ser His His His His His His His His

595 600 605

His His

610

<210> 13

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<223> (1)_ACE2_19-103

<400> 13

Met Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn

1 5 10 15

His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn

20 25 30

Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala

35 40 45

Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln

50 55 60

Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu

65 70 75 80

Gln Ala Leu Gln Gln Asn Gly Gly Gly Ser His His His His His His His His

85 90 95

His His

<210> 14

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> (8)_ACE2_19-132

<400> 14

Met Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn

1 5 10 15

His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn

20 25 30

Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala

35 40 45

Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln

50 55 60

Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu

65 70 75 80

Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser

85 90 95

Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr

100 105 110

Gly Lys Val Gly Gly Gly Ser His His His His His His His His His His His

115 120 125

<210> 15

<211> 65

<212> PRT

<213> Artificial sequence

<220>

<223> Fragment 301-365 of the peptidase domain (1-615) of human ACE-2

<400> 15

Ala Trp Asp Ala Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val

1 5 10 15

Ser Val Gly Leu Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met

20 25 30

Leu Thr Asp Pro Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala

35 40 45

Trp Asp Leu Gly Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val

50 55 60

Thr

65

<210> 16

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 1

<400> 16

Asn Gly Ser Ala Gly Ser Ala Ala Gly Gly Ser Ala Gly Ser Ala Ala

1 5 10 15

Gly

<210> 17

<211> 584

<212> PRT

<213> Artificial sequence

<220>

<223> Fragment of the peptidase domain of human ACE-2 (19-602)

<400> 17

Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly

35 40 45

Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Met

50 55 60

Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln

65 70 75 80

Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg Pro

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly Asn

305 310 315 320

Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly

325 330 335

Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys Asn

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser

580

<210> 18

<211> 584

<212> PRT

<213> Artificial sequence

<220>

<223> Fragment of the peptidase domain of human ACE-2 with Glu375Gln substitution (19-602)

<400> 18

Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly

35 40 45

Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Met

50 55 60

Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln

65 70 75 80

Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg Pro

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly Asn

305 310 315 320

Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly

325 330 335

Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Gln Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys Asn

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser

580

<210> 19

<211> 597

<212> PRT

<213> Artificial sequence

<220>

<223> (2)_ACE_19-602_E375Q

<400> 19

Met Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn

1 5 10 15

His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn

20 25 30

Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala

35 40 45

Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln

50 55 60

Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu

65 70 75 80

Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser

85 90 95

Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr

100 105 110

Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu

115 120 125

Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg

130 135 140

Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg

145 150 155 160

Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala

165 170 175

Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val

180 185 190

Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp

195 200 205

Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His

210 215 220

Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser

225 230 235 240

Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg

245 250 255

Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro

260 265 270

Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln

275 280 285

Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro

290 295 300

Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly

305 310 315 320

Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys

325 330 335

Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe

340 345 350

Leu Thr Ala His His Gln Met Gly His Ile Gln Tyr Asp Met Ala Tyr

355 360 365

Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His

370 375 380

Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His

385 390 395 400

Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu

405 410 415

Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr

420 425 430

Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys

435 440 445

Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys

450 455 460

Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr

465 470 475 480

Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile

485 490 495

Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu

500 505 510

Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser

515 520 525

Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly

530 535 540

Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys

545 550 555 560

Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr

565 570 575

Trp Leu Lys Asp Gln Asn Lys Asn Ser Gly Gly Gly Ser His His His

580 585 590

His His His His His His

595

<210> 20

<211> 187

<212> PRT

<213> Artificial sequence

<220>

<223> Fragment combinations of peptidase domains (19-103; 301-387)

<400> 20

Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly

35 40 45

Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Met

50 55 60

Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln

65 70 75 80

Ala Leu Gln Gln Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

85 90 95

Gly Gly Gly Ser Ala Trp Asp Ala Gln Arg Ile Phe Lys Glu Ala Glu

100 105 110

Lys Phe Phe Val Ser Val Gly Leu Pro Asn Met Thr Gln Gly Phe Trp

115 120 125

Glu Asn Ser Met Leu Thr Asp Pro Gly Asn Val Gln Lys Ala Val Cys

130 135 140

His Pro Thr Ala Trp Asp Leu Gly Lys Gly Asp Phe Arg Ile Leu Met

145 150 155 160

Cys Thr Lys Val Thr Met Asp Asp Phe Leu Thr Ala His His Glu Met

165 170 175

Gly His Ile Gln Tyr Asp Met Ala Tyr Ala Ala

180 185

<210> 21

<211> 187

<212> PRT

<213> Artificial sequence

<220>

<223> Fragment assembly of peptidase domain with Glu375Gln substitution (19-103; 301-387)

<400> 21

Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly

35 40 45

Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Met

50 55 60

Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln

65 70 75 80

Ala Leu Gln Gln Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

85 90 95

Gly Gly Gly Ser Ala Trp Asp Ala Gln Arg Ile Phe Lys Glu Ala Glu

100 105 110

Lys Phe Phe Val Ser Val Gly Leu Pro Asn Met Thr Gln Gly Phe Trp

115 120 125

Glu Asn Ser Met Leu Thr Asp Pro Gly Asn Val Gln Lys Ala Val Cys

130 135 140

His Pro Thr Ala Trp Asp Leu Gly Lys Gly Asp Phe Arg Ile Leu Met

145 150 155 160

Cys Thr Lys Val Thr Met Asp Asp Phe Leu Thr Ala His His Gln Met

165 170 175

Gly His Ile Gln Tyr Asp Met Ala Tyr Ala Ala

180 185

<210> 22

<211> 200

<212> PRT

<213> Artificial sequence

<220>

<223> (5)_ACE2_19-103;301-387_E375Q

<400> 22

Met Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn

1 5 10 15

His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn

20 25 30

Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala

35 40 45

Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln

50 55 60

Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu

65 70 75 80

Gln Ala Leu Gln Gln Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

85 90 95

Gly Gly Gly Gly Ser Ala Trp Asp Ala Gln Arg Ile Phe Lys Glu Ala

100 105 110

Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn Met Thr Gln Gly Phe

115 120 125

Trp Glu Asn Ser Met Leu Thr Asp Pro Gly Asn Val Gln Lys Ala Val

130 135 140

Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly Asp Phe Arg Ile Leu

145 150 155 160

Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu Thr Ala His His Gln

165 170 175

Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala Ala Gly Gly Gly Ser

180 185 190

His His His His His His His His His His His

195 200

<210> 23

<211> 87

<212> PRT

<213> Artificial sequence

<220>

<223> Fragment of the peptidase domain (1-615) of human ACE-2 301-387

<400> 23

Ala Trp Asp Ala Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val

1 5 10 15

Ser Val Gly Leu Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met

20 25 30

Leu Thr Asp Pro Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala

35 40 45

Trp Asp Leu Gly Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val

50 55 60

Thr Met Asp Asp Phe Leu Thr Ala His His Glu Met Gly His Ile Gln

65 70 75 80

Tyr Asp Met Ala Tyr Ala Ala

85

<210> 24

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 2

<400> 24

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 25

<211> 225

<212> PRT

<213> Artificial sequence

<220>

<223> Combination of fragments of the peptidase domain of ACE-2 (19-103; 301-425)

<400> 25

Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly

35 40 45

Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Met

50 55 60

Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln

65 70 75 80

Ala Leu Gln Gln Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

85 90 95

Gly Gly Gly Ser Ala Trp Asp Ala Gln Arg Ile Phe Lys Glu Ala Glu

100 105 110

Lys Phe Phe Val Ser Val Gly Leu Pro Asn Met Thr Gln Gly Phe Trp

115 120 125

Glu Asn Ser Met Leu Thr Asp Pro Gly Asn Val Gln Lys Ala Val Cys

130 135 140

His Pro Thr Ala Trp Asp Leu Gly Lys Gly Asp Phe Arg Ile Leu Met

145 150 155 160

Cys Thr Lys Val Thr Met Asp Asp Phe Leu Thr Ala His His Glu Met

165 170 175

Gly His Ile Gln Tyr Asp Met Ala Tyr Ala Ala Gln Pro Phe Leu Leu

180 185 190

Arg Asn Gly Ala Asn Glu Gly Phe His Glu Ala Val Gly Glu Ile Met

195 200 205

Ser Leu Ser Ala Ala Thr Pro Lys His Leu Lys Ser Ile Gly Leu Leu

210 215 220

Ser

225

<210> 26

<211> 225

<212> PRT

<213> Artificial sequence

<220>

<223> Combination of fragments of the peptidase domain of ACE-2 with Glu375Gln substitution (19-103; 301-425)

<400> 26

Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly

35 40 45

Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Met

50 55 60

Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln

65 70 75 80

Ala Leu Gln Gln Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

85 90 95

Gly Gly Gly Ser Ala Trp Asp Ala Gln Arg Ile Phe Lys Glu Ala Glu

100 105 110

Lys Phe Phe Val Ser Val Gly Leu Pro Asn Met Thr Gln Gly Phe Trp

115 120 125

Glu Asn Ser Met Leu Thr Asp Pro Gly Asn Val Gln Lys Ala Val Cys

130 135 140

His Pro Thr Ala Trp Asp Leu Gly Lys Gly Asp Phe Arg Ile Leu Met

145 150 155 160

Cys Thr Lys Val Thr Met Asp Asp Phe Leu Thr Ala His His Gln Met

165 170 175

Gly His Ile Gln Tyr Asp Met Ala Tyr Ala Ala Gln Pro Phe Leu Leu

180 185 190

Arg Asn Gly Ala Asn Glu Gly Phe His Glu Ala Val Gly Glu Ile Met

195 200 205

Ser Leu Ser Ala Ala Thr Pro Lys His Leu Lys Ser Ile Gly Leu Leu

210 215 220

Ser

225

<210> 27

<211> 238

<212> PRT

<213> Artificial sequence

<220>

<223> (6)_ACE2_19-103;301-425_E375Q

<400> 27

Met Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn

1 5 10 15

His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn

20 25 30

Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala

35 40 45

Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln

50 55 60

Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu

65 70 75 80

Gln Ala Leu Gln Gln Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

85 90 95

Gly Gly Gly Gly Ser Ala Trp Asp Ala Gln Arg Ile Phe Lys Glu Ala

100 105 110

Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn Met Thr Gln Gly Phe

115 120 125

Trp Glu Asn Ser Met Leu Thr Asp Pro Gly Asn Val Gln Lys Ala Val

130 135 140

Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly Asp Phe Arg Ile Leu

145 150 155 160

Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu Thr Ala His His Gln

165 170 175

Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala Ala Gln Pro Phe Leu

180 185 190

Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu Ala Val Gly Glu Ile

195 200 205

Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu Lys Ser Ile Gly Leu

210 215 220

Leu Ser Gly Gly Gly Ser His His His His His His His His His His His

225 230 235

<210> 28

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> Fragment 301-425 of the peptidase domain (1-615) of human ACE-2

<400> 28

Ala Trp Asp Ala Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val

1 5 10 15

Ser Val Gly Leu Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met

20 25 30

Leu Thr Asp Pro Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala

35 40 45

Trp Asp Leu Gly Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val

50 55 60

Thr Met Asp Asp Phe Leu Thr Ala His His Glu Met Gly His Ile Gln

65 70 75 80

Tyr Asp Met Ala Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala

85 90 95

Asn Glu Gly Phe His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala

100 105 110

Ala Thr Pro Lys His Leu Lys Ser Ile Gly Leu Leu Ser

115 120 125

<210> 29

<211> 194

<212> PRT

<213> Artificial sequence

<220>

<223> Fragment combinations of the peptidase domain of ACE-2 (19-103; 342-425)

<400> 29

Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly

35 40 45

Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Met

50 55 60

Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln

65 70 75 80

Ala Leu Gln Gln Asn Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu

85 90 95

Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala Val

100 105 110

Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly Asp Phe Arg Ile Leu

115 120 125

Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu Thr Ala His His Glu

130 135 140

Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala Ala Gln Pro Phe Leu

145 150 155 160

Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu Ala Val Gly Glu Ile

165 170 175

Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu Lys Ser Ile Gly Leu

180 185 190

Leu Ser

<210> 30

<211> 194

<212> PRT

<213> Artificial sequence

<220>

<223> Fragment assembly of the peptidase domain of ACE-2 with Glu375Gln substitution (19-103; 342-425)

<400> 30

Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly

35 40 45

Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Met

50 55 60

Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln

65 70 75 80

Ala Leu Gln Gln Asn Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu

85 90 95

Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala Val

100 105 110

Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly Asp Phe Arg Ile Leu

115 120 125

Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu Thr Ala His His Gln

130 135 140

Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala Ala Gln Pro Phe Leu

145 150 155 160

Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu Ala Val Gly Glu Ile

165 170 175

Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu Lys Ser Ile Gly Leu

180 185 190

Leu Ser

<210> 31

<211> 207

<212> PRT

<213> Artificial sequence

<220>

<223> (7)_ACE2_19-103;342-425_E375Q

<400> 31

Met Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn

1 5 10 15

His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn

20 25 30

Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala

35 40 45

Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln

50 55 60

Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu

65 70 75 80

Gln Ala Leu Gln Gln Asn Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

85 90 95

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala

100 105 110

Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly Asp Phe Arg Ile

115 120 125

Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu Thr Ala His His

130 135 140

Gln Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala Ala Gln Pro Phe

145 150 155 160

Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu Ala Val Gly Glu

165 170 175

Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu Lys Ser Ile Gly

180 185 190

Leu Leu Ser Gly Gly Gly Ser His His His His His His His His His His His

195 200 205

<210> 32

<211> 84

<212> PRT

<213> Artificial sequence

<220>

<223> Fragment 342-425 of the peptidase domain (1-615) of human ACE-2

<400> 32

Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly Asp Phe Arg

1 5 10 15

Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu Thr Ala His

20 25 30

His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala Ala Gln Pro

35 40 45

Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu Ala Val Gly

50 55 60

Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu Lys Ser Ile

65 70 75 80

Gly Leu Leu Ser

<210> 33

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 3

<400> 33

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu

1 5 10 15

Ala Ala Ala Lys Glu Ala Ala Ala Lys

20 25

<210> 34

<211> 33

<212> PRT

<213> Artificial sequence

<220>

<223> amino acids 21-53 of human ACE-2

<400> 34

Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn His Glu Ala

1 5 10 15

Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr Asn Thr

20 25 30

Asn

<210> 35

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> amino acids 56-80 of human ACE-2

<400> 35

Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly Asp Lys Trp Ser Ala

1 5 10 15

Phe Leu Lys Glu Gln Ser Thr Leu Ala

20 25

<210> 36

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> amino acids 91-100 of human ACE-2

<400> 36

Leu Thr Val Lys Leu Gln Leu Gln Ala Leu

1 5 10

<210> 37

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> amino acids 110-129 of human ACE-2

<400> 37

Glu Asp Lys Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr

1 5 10 15

Ile Tyr Ser Thr

20

<210> 38

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> amino acids 294-300

<400> 38

Thr Asp Ala Met Val Asp Gln

1 5

<210> 39

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> amino acids 304-318 of human ACE-2

<400> 39

Ala Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val

1 5 10 15

<210> 40

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> amino acids 325-330 of human ACE-2

<400> 40

Gln Gly Phe Trp Glu Asn

1 5

<210> 41

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> amino acids 366-385 of human ACE-2

<400> 41

Met Asp Asp Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr

1 5 10 15

Asp Met Ala Tyr

20

<210> 42

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> amino acids 400-412 of human ACE-2

<400> 42

Phe His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala

1 5 10

<210> 43

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acids 415-421 of human ACE-2

<400> 43

Pro Lys His Leu Lys Ser Ile

1 5

Claims (15)

1. A Soluble Receptor Fragment (SRF) of the ACE-2 receptor, wherein the SFR comprises the Peptidase Domain (PD) of ACE-2 or fragments and/or derivatives thereof.
2. 2. The SRF according to claim 1, wherein said SRF binds to the receptor-binding cleft of the viral spike protein, in particular to the coronaviridae family, more in particular to the SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, including any mutation thereof, such as the receptor-binding cleft of the spike protein S of a virus from British lineage B.1.1.7, B.1.351 from south Africa, B.1.617 from India or variant B.1.1.28.1 from Brazil.
3. 3. The SRF according to claim 1 or 2, wherein said fragment and/or derivative thereof comprises one, two or more fragments of the PD of ACE-2 and/or a derivative of one, two or more fragments of the PD of ACE-2.
4. 4. The SRF according to any of the preceding claims, wherein said fragment and/or derivative thereof comprises SEQ ID NO:34-43 of 3, 4, 5, 6, 7, 8, 9 or 10 of the alpha-helical structure of the PD of ACE-2 or a derivative thereof, and optionally

   (i) 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of Sars-CoV-2 contact residues Q24, T27, F28, D30, K31, H34, E35, E37, D38, Y41, Q42, L79, M82, Y83, and/or

   (ii) 3, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of Sars-CoV contact residues Q24, T27, F28, K31, H34, E37, D38, Y41, Q42, L45, L79, M82, Y83, and optionally, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of SEQ ID NO

   (iii) 3, 1, 2, 3, 4, 5 or 6 of Sars-CoV-2 contact residues N330, K353, G354, D355, R357, R393, and/or

   (iv) 3, 1, 2, 3, 4, 5, 6 or 7 of Sars-CoV contact residues Q325, E329, N330, K353, G354, D355, R357.
5. 5. The SRF according to any of the preceding claims, wherein said SRF comprises an inactive PD of ACE-2 or a derivative thereof or an inactive fragment or combination of fragments of PD of ACE-2, in particular wherein said inactive PD, derivative, fragment or combination of fragments comprises a mutation, such as an insertion, addition, deletion or substitution, at one or more of the following positions:

   

   
6. 6. the SRF of any preceding claim, wherein the SRF comprises an amino acid sequence according to SEQ ID NO 3, 4, 5 or 6 or a derivative and/or fragment thereof, in particular wherein the SRF comprises an amino acid sequence according to SEQ ID NO 7, 8, 9, 10, 11, 12, 13, 14, 17, 18, 19, 20, 21, 22, 25, 26, 27, 29, 30, 31 or a sequence identical to SEQ ID NO:3-14, 7-22, 25-27 or 29-31, with the proviso that the derivative comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of Q24, T27, F28, D30, K31, H34, E35, E37, D38, Y41, Q42, L79, M82, Y83 of SEQ ID No.3, and preferably the α -helical structure of:

   34 or a derivative thereof,

   35 or a derivative thereof, and

   36 or a derivative thereof.
7. 7. The SRF according to any of the preceding claims, wherein said SRF is immobilized, bound, coupled, linked or fused to a protein, antibody fragment, such as e.g. the Fc part of IgG1, or other compound or molecule.
8. 8. The SRF according to any of the preceding claims, wherein the SRF is immobilized, bound, coupled or attached on beads, membranes, such as dialysis membranes, columns or column materials or other surfaces.
9. 9. The SRF according to any preceding claim, for use in a method of treatment of the human or animal body by surgery or therapy, as a vaccine or for use in a diagnostic method carried out on the human or animal body or carried out on body fluids or other materials from the human or animal body.
10. 10. The SRF according to any of the claims 1 to 8, for use in a method of treating and/or preventing a viral infection, in particular caused by the family coronaviridae, more in particular by a SARS coronavirus, a SARS coronavirus-2, a human coronavirus NL63 or a SARS-CoV-2, including any mutation thereof, such as a variant from british lineage b.1.1.7, b.1.351 from south africa, b.1.617 from india or a variant b.1.1.28.1 from brazil, in particular wherein preventing a viral infection comprises inactivating or neutralizing the virus, in particular by blocking the binding pocket of the spike protein, more in particular by binding the SRF to the binding protein of the virus, more in particular by blocking the binding pocket of the spike protein (protein S) of the family coronaviridae, more in particular by binding the SRF to the binding protein S of the virus.
11. 11. The SRF for use according to claim 10, wherein the SRF is administered in an amount sufficient to reduce the viral load of a virus capable of infecting cells of a subject and/or inactivate viral particles at a site of infection within the subject.
12. 12. The SRF for use according to any one of claims 10 to 11, wherein the SRF is formulated for nasal, oral, intraocular, topical, systemic, intravenous or wound irrigation administration or administration by inhalation or injection.
13. 13. Pharmaceutical composition or medical product comprising the SRF according to any one of claims 1 to 7 and a pharmaceutically or physiologically acceptable excipient and/or carrier, in particular wherein the pharmaceutical composition or medical product is formulated as a tablet, lozenge, candy, drop, chewing gum, lollipop, spray, in particular nasal, oral, throat or wound spray, irrigation solution, in particular nasal, oral, wound or eye irrigation solution, injection solution, balm, ointment, eye drop or mouth or throat rinse.
14. 14. A method of capturing viral particles, the method comprising:

    a) Providing an SRF according to claim 7 or 8, and

    b) Contacting a liquid sample or fluid with the SRF of step a) under conditions that allow the SRF to bind to the viral particle.
15. 15. The method of claim 14, wherein the method is

    (i) A method for detecting said captured viral particles, wherein said method additionally comprises the step of detecting said captured viral particles, or

    (ii) Method for washing a liquid sample or fluid, wherein the viral load in the liquid sample or liquid is reduced due to the capture of the viral particles, in particular wherein the SFRs are immobilized, bound, coupled or attached on a dialysis membrane and the fluid is a body fluid such as whole blood, plasma or a blood fraction.

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