CN115485375A

CN115485375A - Viral neutralization of soluble receptor fragments of the ACE-2 receptor

Info

Publication number: CN115485375A
Application number: CN202180032456.4A
Authority: CN
Inventors: S.米勒
Original assignee: Lysando AG
Current assignee: Lysando AG
Priority date: 2020-05-11
Filing date: 2021-05-11
Publication date: 2022-12-16
Also published as: JP2023524862A; WO2021228854A1; EP4150071A1

Abstract

The present invention is directed to a Soluble Receptor Fragment (SRF) of the ACE-2 receptor, wherein the SRF comprises the Peptidase Domain (PD) of ACE-2 or a fragment and/or derivative thereof. Furthermore, the present invention refers to the use of the SRF according to the present invention in a method for treating the human or animal body by surgery or therapy, as a vaccine or SRF for implementation on the human or animal body or for use in a diagnostic method to be implemented on body fluids or other materials from humans or animals. Furthermore, the present invention provides an SRF for use in a method of treating and/or preventing a viral infection, in particular a viral infection caused by the family Coronaviridae, more in particular a viral infection caused by SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2. Finally, the present invention relates to a method of capturing viral particles, the method comprising the steps of providing an immobilized SRF, and contacting a liquid sample or fluid with the SRF under conditions that allow the SRF to bind to viral particles.

Description

Viral neutralization of soluble receptor fragments of the ACE-2 receptor

The present invention is directed to a Soluble Receptor Fragment (SRF) of the ACE-2 receptor, wherein the SRF comprises the Peptidase Domain (PD) of ACE-2 or a fragment and/or derivative thereof. Furthermore, the invention refers to the use of the SRF according to the invention in a method for the treatment of the human or animal body by surgery or therapy, as a vaccine or SRF for implementation on the human or animal body or for use in a diagnostic method implemented on body fluids or other materials from humans or animals. Furthermore, the present invention provides an SRF for use in a method of treating and/or preventing a viral infection, in particular a viral infection caused by the family Coronaviridae, more in particular a viral infection caused by SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2. Finally, the present invention relates to a method of trapping a viral particle, the method comprising the steps of providing an immobilized SRF, and contacting a liquid sample or fluid with the SRF under conditions that allow the SRF to bind to the viral particle.

In general, viruses infect host cells by: (1) attachment to specific receptors on the surface of target cells; (2) initiation of viral uptake; (3) viral replication; and (4) generating a native virus to (5) continue the release. The viral receptor on the target cell may be a protein or a carbohydrate or lipid structure.

Thus, 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 deal with the steps of the viral infection cycle after entry into the human body. An example of this is the spike protein or 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. Vaccination strategies mostly involve the surface structure of the virus, typically proteins on the surface of the virus, such as spike proteins or capsid proteins, which can be used to trigger an immune response.

However, the development of drugs or vaccines can be time consuming and often unsuccessful. Thus, another option is to prevent the uptake of viruses into the human body, since many serious and even fatal problems occur after the virus enters the human body, begins to replicate within the human body's cells, and thereafter spreads through the blood or lymphatic system.

To prevent the uptake of viruses, or at least to substantially reduce the viral load into the human body, soluble fragments of host cell receptors may be used according to the invention, for example in a wash solution, to neutralize the virus receptors and allow the neutralized virus to be washed out of the entry site.

A dominant 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 a viral strain that causes a respiratory disease known as coronavirus disease 2019 (COVID-19). This virus belongs to the family of coronaviridae, a lipid-enveloped positive-sense RNA virus family. SARS-CoV-2 was previously referred to as the temporary name of the 2019 novel coronavirus (2019-nCoV). SARS-CoV-2 is contagious in humans, and the World Health Organization (WHO) has identified COVID-19, which is currently prevalent on a large scale, as a public health emergency of international concern.

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

For Sars-CoV-2, the trimeric spike glycoprotein (S protein) on the surface of the virion (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 a 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 host receptor ACE-2, another cleavage site on S2 is exposed and cleaved by host proteases, a process that is 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 treated, showing that the ectodomain of the S protein of Sars-CoV-2 binds to the peptidase domain of ACE-2 with high affinity, i.e., with a dissociation constant (Kd) of-15 nM (Wrapp et al. -2020).

In fact, ACE2 is the entry of more coronaviruses, including at least SARS coronavirus, SARS coronavirus-2 and human coronavirus NL63, into the cell.

Angiotensin converting enzyme 2 (ACE-2) is an enzyme that attaches to the membranes of the lung, arteries, heart, kidney and intestine and is part of the renin-angiotensin system, maintaining blood pressure homeostasis and fluid and salt balance in the body. ACE-2 lowers blood pressure by hydrolyzing angiotensin II, a vasoconstrictor, to angiotensin (1-7), a vasodilator. ACE-2 counteracts the activity of the relevant 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 non-structural. The C-terminal domain of ACE-2 is the membrane anchor, which results in exposure of the enzymatically active domain to the cell surface of the lung and other tissues. The peptidase domain consists of two subdomains (I and II) which 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 domain of ACE-2 is naturally cleaved from the transmembrane domain, 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 essentially all stages of the virus life cycle. These strategies are summarized in the review of 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. There have been some attempts to activate the immune system by vaccination with inactivated viruses or parts of viruses, or to prevent cellular entry by blocking the interaction of the viral protein S with its cellular receptor ACE-2. In more detail, the above comments summarize the hypothetical replication cycle for SARS-CoV-2 as follows: SARS-CoV-2 uses spike proteins to bind to ACE2 receptors on the cell surface, which then triggers endocytosis. Upon release of the viral nucleoprotein shell into the cytoplasm, the coated positive-stranded genomic RNA [ (+) gRNA ] serves as a template to translate the polypeptide chains, cleaving the polypeptide chains into non-structural proteins, including RNA-dependent RNA polymerases. Single minus-strand RNA [ (-) gRNA ] synthesized from the (+) gRNA template was used to replicate more copies of viral RNA. Subgenomic RNAs (sgrnas) are synthesized by discrete transcription from a (+) gRNA template, then encode viral structures and accessory proteins, and are subsequently assembled with newly synthesized viral RNA to form new virions. The nascent virions are then transported to the plasma membrane in secretory vesicles and released by exocrine action. In addition, this review describes the following possible targets for anti-COVID-19 drugs: rhACE2, convalescent plasma (convalescent plasma) and JAK inhibitor Baritinib (Baricitinib) can inhibit the binding of SARS-CoV-2 surface spike protein to cell surface expressed ACE 2. Lopinavir (Lopinavir)/Ritonavir (Ritonavir) and Favipiravir (Favipiravir) inhibit proteolysis of polypeptide chains. Reidesciclovir (Remdesivir) inhibits RNA-dependent RNA polymerase. EIDD-2801 inhibits the replication of SARS-CoV-2. iNO and zinc can inhibit the replication of SARS-CoV-2. Vitamin D may induce antimicrobial peptides to reduce replication of sarclov-2. Ivermectin (Ivermectin) effectively blocks the growth of SARS-CoV-2. Baratinib can block the passage of SARS-CoV-2 into cells by inhibiting AAK 1-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 modifications). Abbreviations: AAK1, aptamer-related kinase 1; CQ, chloroquine; ER, endoplasmic reticulum; HCQ, hydroxychloroquine sulfate; IFN, interferon; iNO, inhaled nitric oxide; JAK, janus kinase; LHQW, lianhua antipyretic; rhACE2, recombinant human angiotensin converting enzyme 2; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

In addition to the therapeutic or vaccination concepts described above, another strategy is to inactivate and wash away the virus by flushing the oro-nasal with an appropriate inactivation solution, thereby reducing the viral load in the oro-nasal. Animal experiments have shown that short term exposure to viruses is not sufficient to cause disease in humans. This is an interesting option, since it seems that a certain viral load is a necessary condition for infecting new hosts. Rinsing or rinsing with the inactivating solution can be used to prevent infection in persons who have short-term contact with infected patients, such as doctors, nurses, etc. In addition, the inactivation solution may be used to reduce the viral load in the nose of highly infected patients to reduce the risk of viral transmission, for example during medical procedures where a mask cannot be used.

Strategies to block viral entry have focused on antibodies or soluble recombinant human ACE-2 receptors. Monteil et al (2020) expressed a large extracellular fragment of ACE-2 (amino acids 1-740) which, when purified, prevented the virus from entering different cells. Others have shown 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 risk, as ACE-2 therapy is expected to lead to angiotensin II depletion and therefore attention must be paid to the effects on blood pressure and renal function (alenc-Gelas et al-2020).

Therefore, there is a need for new and alternative concepts for inactivating viruses (e.g., sars-CoV-2).

The problem to be solved by the present invention is therefore to provide a new method useful for inactivating and neutralizing viruses, such as the coronaviridae family, in particular SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2 including any mutant thereof, such as a variant from british lineage b.1.1.7, b.1.351 in south africa, b.1.617 in india or b.1.1.28.1 from brazil, possibly before the virus infects cells of a subject. Furthermore, the problem to be solved by the present invention is to provide new means which enable a reduction of the viral load without causing significant side effects, such as effects on blood pressure and renal function. Furthermore, the problem to be solved by the present invention is to provide a new method for detecting virus particles and washing a liquid sample or fluid, wherein the viral load in the liquid sample or fluid is reduced. The problem underlying the present invention is therefore to provide a preventive and/or therapeutic tool for stopping the infection process of the coronaviridae family, such as SARS-CoV-2, by preventing the virus from entering the cells of the human body. It is a further problem of the present invention to provide prophylactic and therapeutic measures which are useful for any variation of the family coronaviridae, in particular for SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, including variants from the British lineage B.1.1.7, B.1.351 in south Africa, B.1.617 in India or B.1.1.28.1 from Brazil.

The problem to which the invention relates is solved by the subject matter defined in the claims.

The following figures are provided to illustrate the present invention.

FIG. 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 the variant 3_ACE2_19-615 _E375Qshowing the highest binding reaction. Variant 4_ACE _2_19-103;301-365 had a lower binding efficiency and the variant 9_ACE2_19-615 had the lowest binding efficiency. Samples 1-3 measured were buffers; sample 4 was variant (3) _ ACE2_19-615_e375q; sample 5 is variant (4) _ ACE _2 \ -19-103; 301-365; sample 6 was variant (9) _ ACE2_19-615.

FIG. 2 shows the neutralization of Sars-Cov-2 virions by addition of SRF. Neutralization was tested with 80 pfu/ml. The variant 9_ACE2_19-615 showed already a small reduction at 1nM and a 50% reduction at 250 nM. The use of the variant 3 v u ACE2 u 19-615 u E375Q in the neutralization assay resulted in a measurable reduction of about 40% at all concentrations tested.

FIG. 3 shows the neutralization of Sars-Cov-2 virions by addition of SRF. Neutralization was tested with 40 pfu/ml. The variant 9, ACE2, 19-615, showed 50% inhibition at 250nM and complete inhibition at 1.5. Mu.M. The use of the variant 3_ACE2_19-615_E375Q, at a concentration range of 250nM to 1. Mu.M, resulted in a significant reduction. At 1.5. Mu.M and 2. Mu.M, an almost complete reduction was observed.

FIG. 4 shows the neutralization of Sars-Cov-2 British variant (B.1.1.7) virus particles by addition of SRF. Neutralization was tested with 80 pfu/ml. Variant 9/19/615 (A) and variant 3/19/615/375Q (B) already showed a substantial reduction in pfu at 250nM and a complete reduction at 1. Mu.M.

The term "SARS-CoV-2" as used herein refers to Severe acute respiratory syndrome coronavirus 2, which is a viral strain that causes a respiratory disease known as coronavirus disease 2019 (COVID-19). This virus belongs to the family of coronaviridae, a family of lipid-enveloped positive-sense RNA viruses. SARS-CoV-2 was previously referred to as the temporary name of the 2019 novel coronavirus (2019-nCoV). As the virus shows mutations, the term "SARS-CoV-2" as used herein preferably refers to all variants of SARS-CoV-2 as described herein, but also to all variants showing a single mutation or a combination of multiple mutations or mutations, but still being named SARS-CoV-2.

The terms "SARS-CoV-2 variant from the British lineage B.1.1.7", "SARS-CoV-2 variant from south Africa B.1.351" or "SARS-CoV-2 variant from Brazil B.1.1.28.1" as used herein preferably refer to the SARS-CoV-2 variants B.1.1.7, B.1.351 and B.1.28.1 as described, for example, in Singh et al (2021).

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

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 attached to membranes of the lung, arteries, heart, kidney and intestine, and is part of the renin-angiotensin system, maintaining blood pressure homeostasis and fluid and salt balance in the body. ACE-2 lowers blood pressure by hydrolyzing angiotensin II, a vasoconstrictor, to angiotensin (1-7), a vasodilator. ACE-2 counteracts the activity of the relevant 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 non-structural. The C-terminal domain of ACE-2 is the membrane anchor, which results in exposure of the enzymatically active domain to the cell surface of the lung and other tissues. The peptidase domain consists of two subdomains (I and II) which surround the active site and are connected by an alpha helix (Towler et al-2004). These two subdomains are defined as follows (Towler et al-2004): subdomain I containing the N-terminus and zinc, consisting of residues 19-102, 290-397 and 417-430; subdomain II containing the C-terminus, consisting 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). Preferably, ACE-2 comprises the amino acid sequence shown as 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. The PD of ACE-2 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 comprise alpha helices, beta sheets, and/or beta turns. The PD of ACE-2 has an active site. The amino acid residues of PD of ACE-2 listed in the following table are related to activity.

Residues involved in binding (see PD sequence shown in SEQ ID NO: 3).

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

The term "polypeptide" or "protein" as used herein, refers in particular to a polymer of amino acids linked in a specific sequence by peptide bonds. The amino acid residues of a polypeptide can be altered by covalent attachment of various groups, such as carbohydrates and phosphates. Other substances may be more loosely associated with a polypeptide, such as heme or a lipid, to produce conjugated polypeptides, which are also encompassed by the terms "polypeptide" or "protein" as used herein. The term "polypeptide" or "protein" encompasses embodiments of polypeptides or proteins that exhibit selective modifications commonly used in the art, such as biotinylation, acetylation, pegylation, chemical changes of amino, SH, or carboxyl groups (e.g., protecting groups), and the like. The term "polypeptide" or "protein" as used herein is not limited to a particular length of the polymeric chain of amino acids, but typically a polypeptide or protein will exhibit a length of more than about 50 amino acids, more than about 100 amino acids, or even more 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 comprise a dimeric fusion of the polypeptide or protein. In the case of dimer 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, as 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, one skilled in the art will understand that an amino acid residue that differs from the amino acid residue that occurs at the same position in each of the reference sequences occurs at a position in the derivative sequence, not, for example, a combination of a deletion and a subsequent insertion at the same position, but a substitution as defined herein. Conversely, if a combination of one or more additions, deletions, insertions, and substitutions is referred to herein, it refers to a combination of changes at various positions in the sequence, such as additions at the N-terminus and deletions within the sequence. Such derivative sequences will exhibit a certain 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% with the respective reference sequence, e.g. a given SEQ ID NO. Preferred derivatives are fragments of the parent molecule (e.g. a given SEQ ID NO), which retain the activity of the parent molecule, i.e. exhibit the same activity at the general level as the respective parent molecule if not otherwise indicated. However, the activity may be the same, higher or lower than the respective parent molecule. Also preferred derivatives are those that result from conservative amino acid substitutions within the parent sequence (e.g. a given SEQ ID NO), again retaining the activity of the parent molecule at a general level.

As used herein, the term "% sequence identity" must be understood as follows: the two sequences to be compared are aligned to maximize the correlation between the sequences. This may include inserting "gaps" in one or both sequences to increase the degree of alignment. The% identity can then be determined over the entire length of each compared sequence (so-called global alignment), which is particularly applicable to sequences of the same or similar length, or over shorter, determined lengths (so-called local alignment), which is more applicable to sequences of different lengths. In the above cases, an amino acid sequence having, for example, "sequence identity" of at least 95% to a query amino acid sequence is intended to mean that the sequence of the amino acid sequence of interest is identical to the query amino acid except that the amino acid sequence of interest may include changes of up to 5 amino acids per 100 amino acids of the query amino acid sequence. In other words, in order to obtain an amino acid sequence having at least 95% identity to the query amino acid sequence, up to 5% (5 out of 100) amino acid residues may be inserted or substituted with another amino acid or deleted in the target sequence. Methods of comparing the identity and homology of two or more sequences are well known in the art. The percentage of identity of two sequences can be determined by using a mathematical algorithm. Preferably, but not by way of limitation, examples of mathematical algorithms that can be used are the algorithms of Karlin et al, (1993) PNAS USA, 90. Such algorithms are integrated in BLAST series programs, such as 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; 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 identified by these programs. In addition, the programs in 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% identity between two polypeptide sequences. BESTFIT uses the "local homology" algorithm (Smith and Waterman (1981), J.mol.biol.147, 195-197.) to find the best single region of similarity between two sequences. If reference is made herein to an amino acid sequence having a certain degree of sequence identity to a reference sequence, then the sequence differences are preferably due to conservative amino acid substitutions. Preferably, such sequences retain the activity of the reference sequence, e.g., although perhaps at a slower rate. Furthermore, if sequences are referred to herein as "at least" having a certain percentage of sequence identity, then 100% sequence identity is preferably not encompassed.

"conservative amino acid substitutions," as used herein, may occur in a group of amino acids that have sufficiently similar physicochemical properties that substitutions between members of the group will retain the biological activity of the molecule (see, e.g., grantham, r. (1974), science 185, 862-864). In particular, conservative amino acid substitutions are preferably substitutions in which the amino acid is from the same class of amino acids (e.g., basic amino acids, acidic amino acids, polar amino acids, amino acids having aliphatic side chains, amino acids having positively or negatively charged side chains, amino acids having aromatic side chains, amino acids having side chains that can enter hydrogen bridges, side chains having a hydroxyl function, etc.). In this example, conservative substitution means substitution of a basic amino acid residue (Lys, arg, his) for another basic amino acid residue (Lys, arg, his), substitution of an aliphatic amino acid residue (Gly, ala, val, leu, ile) for another aliphatic amino acid residue, substitution of an aromatic amino acid residue (Phe, tyr, trp) for another aromatic amino acid residue, substitution of serine for threonine, and substitution of isoleucine for leucine. More conservative amino acid exchanges are known to those skilled in the art.

The term "deletion" as used herein preferably means the absence of 1, 2, 3, 4, 5 (or even more than 5) consecutive amino acid residues in the derived sequence compared to the corresponding reference sequence, whether within the sequence or at the N-or C-terminus. The derivatives of the invention may exhibit one, two or more such deletions.

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

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

The term "substitution" as used herein refers to the presence of an amino acid residue at a position in the derivative sequence that is different from the amino acid residue present or absent at the corresponding position in the reference sequence. The derivatives of the 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 substitutions. As mentioned above, it is preferred that such substitutions are conservative substitutions.

The term "mutation" of coronaviridae, SARS coronavirus-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 africa b.1.351 variant, indian b.1.617 variant or brazil b.1.1.28.1 variant. In particular, the term "mutation" of SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2 in the family of Coronaviridae refers to any mutation of the respective virus. The mutation may comprise a single mutation or a plurality of mutations. The mutation may occur in the spike protein and/or another viral protein and/or other viral proteins. The mutation may occur in any part of the virus, and may be a silent mutation that has no effect or a non-silent mutation that has an effect on a property of the virus, such as host cell binding, replication, virus stability, etc.

The term "infection" as used herein preferably means that the virus has entered a host cell, in particular a mammalian or human host cell, and is ready for replication.

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 having ACE-2 receptors and all host cells capable of being infected with SARS-CoV-2/COVID-19 or any mutation or variant thereof, as described in Stout et al (2020).

As used herein, the term "tag" refers to an amino acid sequence, typically fused to or contained within another amino acid sequence in the art, for a) increasing expression of the entire amino acid sequence or polypeptide, b) facilitating purification of the entire amino acid sequence or polypeptide, c) facilitating immobilization of the entire amino acid sequence or polypeptide, and/or d) facilitating detection of the entire amino acid sequence or polypeptide. Examples of tags are His tags 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, and the like. Those skilled in the art will be aware of a large number of labels suitable for different technical applications. For example, the tag may render such labeled polypeptides suitable for antibody binding or other technical applications, e.g., in different ELISA assay formats.

The term "comprising" as used herein should not be understood as being limited to the meaning of "consisting of 823030; i.e. excluding the presence of other additions. Conversely, "comprising" means that optional additional substances may be present. The term "comprising" encompasses embodiments specifically contemplated within its scope of "consisting of 823030composition" (i.e. excluding the presence of other substance additions) and "comprising but consisting of (i.e. requiring the presence of other substance additions), the former being more preferred.

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

In a first object of the 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 a fragment and/or derivative of PD of ACE-2, in particular one, two or more fragments of PD of ACE-2. If an SRF according to the present 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 fragment or fragment 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 a fragment thereof. In a further preferred embodiment, the SRF comprises or consists of PD of ACE-2 or one, two or more fragments thereof or a derivative of PD of ACE-2 or a fragment thereof, wherein the active site is inactivated by one or more mutations.

The binding affinity or properties of the fragments, fragment combinations and/or derivatives of PD of ACE-2 of the SRF according to the invention to the receptor binding cleft of a viral spike protein, in particular viral spike protein S, are essentially the same or higher than the wild-type full-length PD of ACE-2. Essentially, this means that the binding affinity or property to the receptor binding cleft of the same viral spike protein is in the range of about 70% to about 150%, more preferably about 80% to about 130% or about 90% to about 120% of the wild-type full length PD of ACE-2 as shown in SEQ ID No. 3. In a preferred embodiment of the invention, any fragment, fragment combination and/or derivative of PD of ACE-2 that effectively binds to the viral spike protein S of SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, or any mutated receptor binding cleft thereof is a fragment, fragment combination or derivative of an SRF according to the invention. Binding is considered effective if the binding affinity of the derivative, fragment or combination of fragments tested is at least 50%, 60%, 70%, 80%, 90%, 100%, 110% or 120% of the binding affinity of (9) _ ACE2_19-615 according to SEQ ID NO 12 as tested in the examples herein.

The inventors of the present invention have surprisingly found that the SRF according to the present invention binds to the receptor-binding cleft of the viral spike protein, in particular to the receptor-binding cleft of the spike protein S of viruses of the family coronaviridae. In a preferred embodiment, SRF binds to the receptor-binding cleft of spike protein S of a virus of SARS coronavirus, SARS coronavirus-2, human coronavirus NL63, or SARS-CoV-2. In a particularly preferred embodiment, SRF binds to the receptor-binding cleft of spike protein S of the virus of SARS-CoV-2. By the above binding, the virus is prevented from entering the host cell, preferably a human cell. As a result, the virus cannot begin to replicate and, therefore, the host cell (e.g., human cell) cannot produce more viral particles.

Preferably, the PD of ACE-2 of the SRF according to the invention is a soluble expression isolated PD of ACE-2 or a soluble expression isolated fragment, combination of fragments and/or derivatives thereof. Use of fragments or combinations of fragments of full-length PD of ACE-2 may result in increased solubility of SRF. The fragments or fragment combinations are preferably designed by deleting one, two, three, four, five or more structural elements of the PD of ACE-2, which structural elements are not important for the binding activity of PD to the receptor binding cleft of a viral spike protein, in particular of the spike protein S of a virus. The structural elements of the PD of ACE-2 are, for example, those that are not necessary for the formation of alpha helices, beta sheets and beta turns. Alternatively or additionally, the solubility of 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 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 a viral spike protein, particularly the receptor binding cleft of the viral spike protein S and inhibits binding of the spike protein S to the host cell.

Preferably, the SFR comprises a fragment or first fragment of a PD of ACE-2 that is 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 in length. Preferably, the SRF comprises a PD fragment of ACE-2 of at most 614, 615, 616, 617, 618, 619, 620, 582, 583, 584, 585, 586, 587, 595, 596, 597, 598, 599, or 600 amino acid residues in length. In a preferred embodiment, the SFR comprises a fragment or first fragment of the PD of ACE-2, which is about 80 to about 600 amino acids in length, more preferably about 84 to about 597 amino acids.

If the SFR according to the invention comprises two or more fragments of 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 a length of about 50 to about 150, about 60 to about 130 amino acids, or about 64 to about 125 amino acid residues. Particularly preferred fragments have a length of 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 a preferred embodiment of the invention, the SRF comprises at least SEQ ID NO:3, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all 14 of the Sars-CoV-2 contact residues Q24, T27, F28, D30, K31, H34, E35, E37, D38, Y41, Q42, L79, 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 invention, the SRF comprises at least 5, 6, 7, 8, 9, 10, 11, 12 or all 13 of the Sars-CoV-2 contact residues Q24, T27, F28, K31, H34, E37, D38, Y41, Q42, L45, L79, M82, Y83 of SEQ ID NO 3.

In a further preferred embodiment of the invention, the SRF comprises SEQ ID NO:34-43, 3, 4, 5, 6, 7, 8, 9 or all 10 alpha-helical structures of a PD of ACE-2 or a derivative thereof. The derivative of any one of the amino acid sequences according to 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 alpha-helical structure. These deletions may be within a given amino acid sequence and/or at one or both ends. SEQ ID NO:34, preferably comprising at least the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 8, 9, 10, or all 11 of the Sars-CoV-2 contact residues Q24, T27, F28, D30, K31, H34, E35, E37, D38, Y41, and Q42. SEQ ID NO:40 preferably comprises the amino acid sequence of SEQ ID NO:3, sars-CoV-2 contact residue N330 and/or SEQ ID NO:3

contacts

1, 2 or 3 of residues Q325, E329 and N330.

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

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

Aa 56-80 of SEQ ID NO.3 or derivatives thereof as defined above, as shown in SEQ ID NO. 35, and

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

An example of such SFRs is a SFR comprising the sequence as set forth in SEQ ID NO:8 or 13, as also shown in SEQ ID NO:4-14, 17-22, 25-27 and 29-31. In a further preferred embodiment of the invention, the SRF additionally comprises a further alpha-helical structure, i.e. the sequence of SEQ ID NO:3 aa 110-129 of SEQ ID NO:37 or a derivative thereof as defined above. An example of such SFRs is a SFR comprising the sequence as set forth in SEQ ID NO:9 or 14, also as shown in SEQ ID NO:4-6, 10, 12 and 17-19.

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

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

Aa 325-330 of SEQ ID NO.3 or derivatives thereof as defined above, as shown in SEQ ID NO. 40.

Examples of such second fragments are shown in SEQ ID NO:15, in (b). Examples of SRFs comprising such second fragments are shown in SEQ ID NO:4-7, 10-12, 17-22 and 25-27. In a further preferred embodiment of the invention, the second fragment of SRF comprises the alpha-helical structure, aa 366-385 of SEQ ID NO.3, a derivative thereof as shown in SEQ ID NO. 41 or as defined above. Examples of such second fragments are 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 fragment of SRF comprises the following α -helical structure:

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

Aa 415-421 of SEQ ID NO.3 or derivatives thereof, as shown in SEQ ID NO. 43.

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 NO: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 Sars-CoV-2 contact residues N330, K353, G354, D355, R357, R393 of SEQ ID NO. 3.

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

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 as SEQ ID NO: 3). Thus, the SFR according to the invention comprises at least the amino acid sequence shown as SEQ ID NO 8 or a derivative thereof. The derivative preferably comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of the residues Q6, T9, F10, D12, K13, H16, E17, E19, D20, Y23, Q24, L61, M64, Y65 of SEQ ID No.8, and preferably at least comprises the following α -helical structure:

34 or a derivative thereof,

35 or a derivative thereof, and

36 or a derivative thereof.

The PD of ACE-2 or a fragment, combination of fragments and/or derivative thereof of an SRF according to the invention may be expressed in any suitable expression system, such as in Baculovirus (Baculovirus) cells, HEK293 cells, CHO or other eukaryotic cells. Alternatively, the SRF protein may be expressed in E.coli. For purification of the expressed SRF protein, affinity tags such as His-Tag or Strep-Tag or other standard purification techniques may be used.

Thus, 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 translation initiation signal.

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 a fragment, a combination of fragments or derivatives or fragments thereof according to the amino acid sequence of SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 6 or PD of ACE-2 or SRF comprises the above. Consisting of a sequence according to SEQ ID NO:4 or an SRF comprising the sequence consists of an amino acid sequence according to SEQ ID NO:3 or an SRF comprising the same, because it lacks the amino acid sequence of SEQ ID NO:3, amino acids 1-18. Comprises a nucleotide sequence as set forth in SEQ ID NO:4 is the sequence represented by SEQ ID NO:12 or an SRF comprising the same. Has the sequence shown in SEQ ID NO:12 except that the SRF comprises the sequence shown in SEQ ID NO:4, methionine and a tag, namely His-tag, as translation initiation signals.

A further preferred example of an SRF consisting of or comprising the amino acid sequence according to SEQ ID No. 6 is an SRF consisting of or comprising a fragment of the amino acid sequence according to SEQ ID No.3, as it consists of amino acids 2 to 615 of SEQ ID No.3 and thus lacks amino acid 1 of SEQ ID No.3, i.e. lacks methionine as translation initiation signal.

Consisting of a sequence according to SEQ ID NO:8 is a SRF consisting of or comprising the amino acid sequence according to SEQ ID NO:3 or an SRF comprising the same, since the amino acid sequence of SEQ ID NO:8 is represented by SEQ ID NO:3, and thus lacks the amino acid sequence of SEQ id no:3 amino acids 1-18 and 104-615. Comprises the amino acid sequence of SEQ ID NO: an example of an SRF of the sequence shown by SEQ ID NO:13 or an SRF comprising the same. Has the sequence shown in SEQ ID NO:13 in addition to the SRF comprising 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 an SRF consisting of or comprising the amino acid sequence according to SEQ ID NO 9 is a further preferred example consisting of or comprising a fragment of the amino acid sequence according to SEQ ID NO 3, since SEQ ID NO 9 comprises amino acids 19 to 132 of SEQ ID NO 3 and thus lacks amino acids 1 to 18 and 133 to 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. The SRF having the sequence shown in SEQ ID NO. 14 comprises methionine as a translation initiation signal and a tag, his-tag, in addition to the sequence shown in SEQ ID NO. 9.

Consisting of a sequence according to SEQ ID NO:17 is an SRF consisting of or comprising the amino acid sequence according to SEQ ID NO:3 or an SRF comprising the same, since the amino acid sequence of SEQ ID NO:17 consists of SEQ ID NO:3, and thus lacks the amino acid sequence of SEQ ID NO:3 amino acids 1-18 and 603-615. An example of an SRF comprising a derivative of the fragment shown as SEQ ID NO. 17 is an SRF consisting of or comprising the sequence shown as SEQ ID NO. 18 or 19. Has the sequence shown in SEQ ID NO:18 has a substitution of Glu375Gln, i.e. in SEQ ID NO:3, i.e. at position 375 of the sequence shown in SEQ ID NO: a glutamic acid to glutamine substitution at position 357 of 18. Has the sequence shown in SEQ ID NO:19 except that the SRF comprises the sequence shown in SEQ ID NO:18, methionine and a tag, his-tag, are included as translation initiation signals.

In a further preferred embodiment of the 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 bound to 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 to a subunit of a different spike trimer. The linker may be a linker already comprised in the ACE-2 receptor linking building block like the alpha-sheet in the 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 from about 5 to about 50 amino acid residues, from about 10 to about 30 amino acid residues or from about 15 to 25 amino acid residues in length.

In a preferred embodiment, the SRF according to the invention comprises SEQ ID NO:3, wherein the first fragment is selected from the group consisting of SEQ ID NOs: 8 or a derivative thereof, and a second fragment is selected from the group consisting of SEQ ID NO: 15. 23, 28 and 32 or respective derivatives thereof. In a further preferred embodiment, the SRF according to the invention comprises a combination of fragments of the first and second fragment of SEQ ID NO.3, wherein the first fragment is selected from the amino acid sequence shown in SEQ ID NO. 9 or a derivative thereof and the second fragment is selected from the amino acid sequences shown in SEQ ID NO. 15, 23, 28 and 32 or a derivative of each thereof. In a further preferred embodiment, the amino acid sequence according to SEQ ID NO: 23. 28 and 32 comprises the substitution Glu375Gln, i.e. in SEQ ID NO:3 to glutamine at position 375 of the sequence shown in figure 3.

Preferably, the first fragment and the second fragment of the combination of fragments are represented by a sequence selected from the group consisting of SEQ ID NOs: 16. 24 and 33, respectively.

In a preferred embodiment, the SRF according to the invention comprises a combination of fragments of the first and second fragment 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 and the second fragment consists of the amino acid sequence according to SEQ ID NO. 15 or a derivative thereof. In a further preferred embodiment, the first and second fragments are linked 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 an 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 comprises methionine and a tag, his-tag, as a translation initiation signal in addition to the sequence shown in SEQ ID NO. 7.

In a further preferred embodiment, the SRF according to the invention comprises a sequence as set forth in SEQ ID NO: 20. 21, 22, 25, 26, 27, 29, 30, 31 or derivatives thereof.

SEQ ID NO:3-14, 17-22, 25-27, 29-31 preferably refers to a derivative 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, 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 at least the following alpha-helical structure:

34 or a derivative thereof,

35 or a derivative thereof, and

36 or a derivative thereof.

The derivatives of SEQ ID NO 34-43 preferably mean derivatives which comprise 1, 2, 3, 4 or 5 deletions or mutations, for example substitutions, in particular conservative amino acid substitutions, with the proviso that the derivatives are still able to form an alpha-helical structure.

Preferably, the SRF according to the invention does not comprise further domains or sequences of ACE2, or a portion of 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 consecutive amino acids thereof, other than any fragment or derivative of PD of ACE-2 as described herein. In particular, the SFR according to the invention preferably does not comprise the sequence or further domain of aa 620-805 of ACE2 as depicted in SEQ ID NO 1 or a part of 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 consecutive amino acids thereof.

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

methionine as 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 without the further domains or sequences of ACE2 as defined above.

In a preferred embodiment of the invention, the SFR according to the invention does not comprise 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 other than any fragment or derivative of the PD of ACE-2 as described herein. Preferably, none of these additional amino acids comprise 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 Soluble Receptor Fragments (SRF) of the ACE-2 receptor. These SRFs comprise or consist of a soluble expressed isolated Peptidase Domain (PD) of ACE-2 or a fragment, combination of fragments or just derivatives thereof, wherein the neck domain described in the structure of Yan et al (2020) is cleaved off. Thus, SFRs according to the invention do not comprise SEQ ID NO:2, or a pharmaceutically acceptable salt thereof. To avoid an influence on blood pressure and kidney function, the active site of PD is preferably inactive, since it is an object according to the present invention to block the receptor binding cleft of the coronaviridae, such as Sars-CoV-2 spike protein S, without having a significant influence on blood pressure or kidney function. Furthermore, to prevent the uptake of viruses, or at least to substantially reduce the viral load into 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 a wash solution according to the invention for neutralizing virus receptors, preferably capable of washing out the neutralized virus from the entry site.

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

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, such as insertions, additions, deletions or substitutions of amino acids as listed in the table below. Suitable mutations are those suitable for inactivating PD. Suitable mutations can be readily determined by those skilled in the art, for example, by conventional cloning techniques.

In a particularly preferred embodiment of the invention, the SRF according to the invention comprises PD of ACE-2 or a fragment, combination of fragments or derivative thereof, having the amino acid sequence of SEQ ID NO: the substitution at position 375 shown in 3, in particular the substitution Glu375Gln, is preferred. The substitution preferably results in inactivation of the active site of PD. In a more preferred embodiment, the SRF comprises a fragment of inactivated PD as shown in SEQ ID NO. 5 or a fragment, combination of fragments and/or derivative thereof. Comprises the amino acid sequence of SEQ ID NO:5 is the sequence represented by SEQ ID NO:10 or an SRF comprising the same. Has the sequence of SEQ ID NO:10 except that the SRF comprises the sequence shown in SEQ ID NO:5, methionine as a translation initiation signal and a tag, namely His tag. 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 the shown SEQ ID NO: 18. 19, 21, 22, 26, 27, 30 and 31.

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

In a further preferred embodiment, the PD of the SRF according to the invention comprises one, two, three, four or five further mutations, such as insertions, additions, deletions or substitutions of the amino acids listed in the above table, in addition to the mutation at position 375, in particular the substitution of Glu375 Gln.

According to the use of the SRF of the invention, the SRF and the PD, fragments, combinations of fragments or derivatives thereof, respectively, of the SRF are preferably capable of dimerizing. Dimerization may increase the stability of the SRF and/or may increase binding affinity for protein S. Alternatively, the PD of SRF, a fragment, combination of fragments, or derivative thereof, comprises 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, that monomerizes the PD. The monomerization may increase the distribution speed in the solution.

Using the SRF containing solution according to the invention as e.g. oral rinse or nasal rinse, the binding pocket of free spike proteins (e.g. protein S) of the family coronaviridae, e.g. Sars-CoV-2, present in these parts of the human body will be bound by the SRF and no longer be able to infect the cells of the subject. These solutions can also be applied prophylactically to prevent infection prematurely. The SRF according to the invention may also be used in therapy to inactivate viral particles at the site of infection in the human body, without the SRF having a significant effect on blood pressure and renal function.

Alternatively, the SRF may be immobilized on beads or other surfaces to capture viral particles, for example by creating an affinity effect to improve the wash effect, thereby facilitating the washing of viral particles out of the infected area.

Alternatively, SRF may be immobilized on beads or other surfaces to capture viral particles, for example to improve diagnostic quality, or wash effectiveness. This also includes the use of dialysis applications where SRF is coupled to dialysis membranes or beads to filter out viral particles.

Alternatively, the SRF may be immobilized or fused to a protein, such as the Fc portion of IgG1, for use in humans.

In addition, SRF can be immobilized or fused to a protein for application in humans to dimerize PD, thereby increasing 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 fragment, such as the Fc portion of IgG1 or other compound or molecule.

For example, the SRF according to the present invention may additionally comprise tags, linkers, proteins, compounds or molecules, which may facilitate stability, solubility, distribution and half-life in the human body or other characteristics needed to address their respective applications. In general, SRF can be modified to address the challenges of the treated application, as with known therapeutic proteins (e.g., as summarized in the review of Dellas et al (2021)). For example, the SRF may additionally comprise Maltose Binding Protein (MBP) to increase the stability and/or solubility of the SRF, or an Fc fusion to achieve dimerization.

Thus, the present invention also provides SRF according to all of the above embodiments of the invention for use in a method of treatment of the human or animal body by surgery or therapy, as a vaccine or SRF for use in a method of diagnosis carried out on the human or animal body or on body fluids or other materials from the human or animal body.

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 coronavirus family, more particularly a viral infection caused by SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, including any mutant thereof, such as a variant from british lineage b.1.1.7, b.1.351 in south africa, b.1.617 in india or b.1.1.28.1 from brazil.

In particular, SRFs according to the present invention are particularly effective for treating and/or preventing viral infection in a subject if the viral mutation or variant still allows host cell receptor binding, in particular wherein the mutated virus or viral variant still binds ACE-2 receptors of potential host cells. To test whether any potential mutation or variant allows host cell receptor binding, in particular ACE-2 receptor binding, affinity measurements can be performed on Biacore chips or by MicroScale thermophoresis, as described in the examples below. If such test results are a stronger binding than the standard deviation of the negative control, the tested virus may be considered a mutant or variant of coronaviridae, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, which may be effectively treated by an SRF according to the invention or prevent a viral infection by an SRF according to the invention.

Prevention of viral infection with an SRF according to the invention may comprise inactivation or neutralization of the virus, particularly by blocking the binding pocket of the spike protein of the virus, more particularly by binding the SRF to the binding protein of the virus. Preferably, the prevention of viral infection by the SRF according to the present invention may comprise inactivation or neutralization of the virus, in particular by blocking the binding pocket of the spike protein (protein S) of the virus, more in particular by binding the SRF to the binding protein S of the virus.

It is well known that the replication of viruses also produces mutations or mutations. These mutations may or may not have an effect on the viral proteins. Viral receptor proteins are also mutated, but only those mutations that still allow host cell receptor binding will replicate. This means that the binding pocket of the viral receptor protein is relatively stable to mutations that affect binding to the host cell, which might otherwise be lost, and viruses with these mutations will not be replicated. A human consists of 100 trillion cells. Naturally, these cells do not mutate a biologically functional receptor in order to avoid viral infection. Thus, docking receptors for host cells, particularly human cells, are highly conserved. Thus, coronaviruses have great pressure to make their host cell receptors or spike proteins free of mutations that do not recognize host cell docking receptors with significant affinity. Variants from british lineage b.1.1.7 (spike mutation N501Y (which has a higher affinity for ACE-2), plus other mutations of the spike) or b.1.351 from south africa (multiple mutations of the spike protein, including K417N, E484K, N501Y) although there are mutations in the spike protein, still bind to the host cell docking receptor ACE-2, so SRF according to the invention is also effective against the above variants.

The SRF according to the invention provides a blocking means which blocks the spike protein of the virus, rendering the virus non-infectious. Assuming that the SRF is a lock for human cells, a key to which the virus is docked is sought. Once the virus docks with SRF, it cannot infect human cells and therefore cannot replicate. Inactivated virus, i.e. virus that interfaces with SRF according to the invention, will be destroyed by the immune system. The SRF according to the present invention, particularly the SRF without enzyme activity that does not affect the blood pressure system according to the present invention, does not interact with living cells, and thus can protect the natural microbiota of humans.

The subject to be treated is preferably a human or an animal, in particular 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 to inactivate viral particles at the site of infection in the subject. Effective amounts of the compounds to be administered can be readily determined by those skilled in the art during preclinical and clinical trials by methods familiar to physicians and clinicians.

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 affinity by allowing dimerization of PD.

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

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

The pharmaceutical composition according to the invention may additionally contain one or more conventional additives. Some examples of such additives include solubilizing agents, such as glycerol; antioxidants, such as benzalkonium chloride, benzyl alcohol, chlorobutanol (chlorobutanol) or chlorobutanol (chlorobutanol); and/or an isotonic agent.

The pharmaceutical composition or medical product may be formulated as a tablet, lozenge, confectionery, drop, chewing gum, lollipop, spray, in particular nasal, oral, mouth, throat or wound spray, irrigation fluid, in particular nasal, oral, wound or eye irrigation fluid, injection, balm, ointment, eyedrop or mouth or throat cleanser.

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 to reduce the viral load into the human body. Thus, the application is preferably performed to allow a washing effect or as a washing solution to neutralize the virus receptor, preferably to allow the neutralized virus to be washed out of the entry site.

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

In another specific embodiment of the invention, the SRF according to the invention and/or the pharmaceutical composition of the invention is used for the manufacture of a medicament for the treatment or prevention of a viral infection, in particular caused by the family coronaviridae, more in particular by SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, most preferably by SARS-CoV-2 including any mutant thereof, such as a variant from british lineage b.1.1.7, b.1.351 in south africa, b.1.617 in india or b.1.1.28.1 in brazil.

In a particular embodiment of the invention, the SRF according to the invention and/or the pharmaceutical composition of the invention is used as a medicament for the treatment or prevention of viral infections, in particular viral infections caused by the family coronaviridae, more in particular by SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, most preferably by SARS-CoV-2 including any mutant thereof, such as a variant from british lineage b.1.1.7, b.1.351 in south africa, b.1.617 in india or b.1.1.28.1 in brazil.

In a further aspect of the invention is a method of treating or preventing a viral infection, in particular a viral infection caused by the coronavirus family, more particularly SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, most preferably SARS-CoV-2 including any mutant thereof, such as a variant from british lineage b.1.1.7, b.1.351 in south africa, b.1.617 in india or b.1.1.28.1 from brazil, by administering or applying an effective amount of an SRF according to the invention or a pharmaceutical composition according to the invention to a subject, in particular to a human or an animal.

In a further aspect, the present invention provides a method of trapping a viral particle, the method comprising:

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

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

The viral particles are preferably viral particles or whole viruses, preferably of the family coronaviridae, more particularly viruses selected from the group consisting of: SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2 comprises any mutation thereof, such as a variant from British lineage B.1.1.7, B.1.351 in south Africa, B.1.617 in India or B.1.1.28.1 from Brazil.

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

In a further preferred embodiment, the method of capturing virus 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 virus particles. The method of washing may be adapted to neutralize the virus receptor, preferably to allow washing out of the neutralized virus.

For example, the method may be used for dialysis to reduce the viral load in a body fluid of a subject. In particular, the washing neutralizes the virus receptor, preferably being capable of washing the neutralized virus out of the body fluid of the subject. Alternatively or additionally, this may prevent the cells of the subject from taking up the virus, or at least may substantially reduce the viral load into the human body. 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 present invention are summarized in the following items 1 to 24.

A Soluble Receptor Fragment (SRF) of the ACE-2 receptor, wherein the SFR comprises the Peptidase Domain (PD) of ACE-2 or a fragment and/or derivative thereof.

2. The SRF according to item 1, wherein the SRF binds to the receptor-binding cleft of a viral spike protein, in particular to the coronaviridae, more in particular to SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, including any mutant thereof, such as the receptor-binding cleft of spike protein S of a virus from British lineage B.1.1.7, B.1.351 in south Africa, B.1.617 in India, or variant B.1.1.28.1 in Brazil.

3. The SRF according to

item

1 or 2, wherein the fragment and/or derivative thereof comprises one, two or more fragments of PD of ACE-2 and/or a derivative of one, two or more fragments of PD of ACE-2.

4. The SRF according to any of the preceding, wherein the fragment and/or derivative thereof comprises SEQ ID NO:34-43, or 3, 4, 5, 6, 7, 8, 9 or all 10 alpha-helical structures of a PD of ACE-2 or a derivative thereof, particularly wherein the derivative comprises 1, 2, 3, 4 or 5 deletions or mutations, provided that the derivative is still capable of forming alpha-helical structures.

5. The SRF according to any preceding claim, wherein the fragment and/or derivative thereof comprises at least 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 of SEQ ID NO 3.

6. The SRF according to any preceding claim, wherein the fragment and/or derivative thereof comprises at least 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the Sars-CoV contact residues Q24, T27, F28, K31, H34, E37, D38, Y41, Q42, L45, L79, M82, Y83 of SEQ ID NO 3.

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

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

9. SRF according to any of the preceding claims, wherein the fragment and/or derivative thereof comprises at least the following α -helix structure:

34 or a derivative thereof,

35 or a derivative thereof, and

36 or a derivative thereof.

10. SRF according to any of the preceding, wherein a fragment and/or derivative thereof comprises at least an amino acid sequence according to SEQ ID No.8 or a derivative thereof having at least 60, 70, 80, 90, 95 or 98% sequence identity with SEQ ID No.8, in particular wherein said derivative comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of the residues Q6, T9, F10, D12, K13, H16, E17, E19, D20, Y23, Q24, L61, M64, Y65 of SEQ ID No.8, and preferably comprises at least the following alpha-helical structure:

34 or a derivative thereof.

35 or a derivative thereof, and

36 or a derivative thereof.

11. The SRF according to any of the preceding, wherein the SRF comprises a fragment combination of two fragments of PD of ACE-2 or a derivative thereof, in particular wherein the first fragment is selected from the group consisting of SEQ ID NO:8 and 9 or a derivative thereof, and a second fragment selected from the group consisting of SEQ ID NOs: 15. 23, 28 and 32 or respective derivatives thereof.

12. The SRF according to any 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 PD of ACE-2, in particular wherein the inactivated 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:

13. the SRF according to any one of the preceding, 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 the SRF comprises an amino acid sequence 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, SEQ ID NO 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 an alpha helix structure comprising at least the following:

34 or a derivative thereof,

35 or a derivative thereof, and

36 or a derivative thereof.

14. The SRF according to any of the preceding, wherein the SRF is immobilized, bound, coupled, linked or fused to a protein, antibody fragment, such as the Fc portion of IgG1, or other compound or molecule.

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

16. 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 a body fluid or other material from the human or animal body.

17. The SRF according to any of items 1 to 14 for use in a method of treating and/or preventing a viral infection, in particular caused by the coronaviridae family, more in particular caused by SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, including any mutant thereof, such as a variant from british lineage b.1.1.7, b.1.351 in south africa, b.1.617 in india or b.1.1.28.1 from brazil.

18. The 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 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.

19. The SRF for use according to clause 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 infection in the subject.

20. The SRF for use according to any 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. Pharmaceutical composition or medical product comprising an SRF for use according to any one of claims 1 to 14 and a pharmaceutically or physiologically acceptable excipient and/or carrier, in particular wherein the pharmaceutical composition or medical product is formulated as a tablet (tablet), lozenge (lozeng), candy (bonbon), drop (drop), chewing gum (chewing gum), lollipop (lollipop), spray (spray), in particular nasal, oral, mouth, throat or wound spray, irrigation fluid (irrigation fluid), in particular nasal, oral, wound or eye irrigation fluid, injection fluid (injection fluid), balm (balm), ointment (ointment), eye drop (eyedrop) or mouth or throat wash (wash).

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

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

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

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 fluid is reduced due to the capture of the viral particles, in particular wherein the SFRs are immobilized, bound, coupled or attached to a dialysis membrane, and the fluid is a body fluid, such as whole blood, plasma or a blood fraction.

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, while indicating specific 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 from this description.

Example 1 expression and purification of SRF and spike proteins in baculovirus cells

SRF (according to SEQ ID NO:4, with or without Strep-tag) was expressed in the baculovirus system (Sf 9) according to the manufacturer's protocol (BD Biosciences). Briefly, 2. Mu.g of SRF (according to SEQ ID NO: 4) encoding plasmid was mixed with 0.5. Mu.g of BD Baculogold linearized baculovirus DNA, and after standing for 5 minutes at room temperature, 1ml of BD Baculogold transfection buffer B was added. This mixture was added dropwise to Sf9 insect cells (ATCC) previously covered with 1ml of transfection buffer A, and then the cells were cultured at 28 ℃ for 4 hours, and the transfected cells were further cultured in fresh SF-900II SFM (Invitrogen) for an additional 4 days. After 4 days, the supernatant was collected to infect cells, and a 3-day culture cycle was performed for virus amplification. The 4 th cycle cell culture supernatant was collected for purification of the 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 (Sf 9) according to the manufacturer's protocol (BD Biosciences). Briefly, 2. Mu.g of SRF (according to SEQ ID NO: 5) encoding plasmid was mixed with 0.5. Mu.g of BD Baculogold linearized baculovirus DNA, left to stand at room temperature for 5 minutes and then 1ml of BD Baculogold transfection buffer B was added. This mixture was added dropwise to Sf9 insect cells (ATCC) previously covered with 1ml of transfection buffer A, and then the cells were cultured at 28 ℃ for 4 hours, and the transfected cells were further cultured in fresh SF-900II SFM (Invitrogen) for an additional 4 days. After 4 days, the supernatant was collected to infect cells, and a 3-day culture cycle was performed for virus amplification. The 4 th cycle cell culture supernatant was collected for purification of SRF (SEQ ID NO: 5) protein.

The His tag-bearing spike protein S was obtained from Sino Biological 40592-V08B-1. The Strep-tag variants were cloned into expression vectors and expressed soluble in baculovirus SF9 cells to allow for correct glycosylation. Purification is carried out via Strep-Tag or other standard purification techniques.

Example 2 measurement of binding of SRF to spike protein S in Elisa assay

The SRF (according to SEQ ID NO: 4) coated microplates were incubated with soluble spike protein S (Strep-labeled). Three washes with PBS-buffer were then performed to wash away unbound spike protein. Protein S bound to the immobilized SRF (according to SEQ ID NO: 4) was then quantified with streptavidin-conjugated horseradish peroxidase. The binding of protein S to the immobilized SRF (according to SEQ ID NO: 4) was successfully measured.

The protein S coated microplates were incubated with SRF (according to SEQ ID NO: 5) and additionally contained Strep tag. Subsequently, three washes with PBS-buffer were performed to wash away unbound SRF (according to SEQ ID NO: 5). The SRF (according to SEQ ID NO: 5) bound to the immobilized protein S was then quantified with streptavidin-conjugated horseradish peroxidase. Binding of SRF (according to SEQ ID NO: 5) to immobilized protein S was successfully measured.

Example 3- -affinity measurement on Biacore chip- -first part

Strep-labeled SRF was captured with a sensor chip SA of streptavidin pre-immobilized. Spike protein S was then injected and binding to the immobilized SRF (according to SEQ ID NO: 4) was measured. Binding reactions were corrected with blank flow cells. Surface Plasmon Resonance (SPR) methods were used in 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, a significant decrease in binding to immobilized SRF (according to SEQ ID NO: 4) was detected. After incubation of protein S with soluble SRF (according to SEQ ID NO: 4) at higher concentrations, it was injected into a Biacore chip with immobilized SRF (according to SEQ ID NO: 4) resulting in NO detectable binding.

Example 4 virus inactivation assay. Treatment of Vero E6 cells with SRF

Vero E6 cells were seeded in 48-well plates in DMEM containing 10% FBS. SRF was mixed with different concentrations of virus (1. After 30 minutes Vero-E was infected with SRF or SRF/SARS-CoV-2 containing mixture for 1 hour and then washed, or 15 hours without washing, cells were washed 3 times with PBS and then 500ml of new complete medium supplemented with SRF was added. 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. Vero E6 cells infected with virus served as positive control. Vero E6 cells infected with SRF alone served as negative control. Mixing SRF and SARS-CoV-2 before infection of Vero E6 cells showed a significant reduction in infectivity. The use of SRF (according to SEQ ID NO: 4) or SRF (according to SEQ ID NO: 5) did not result in a significant difference.

Example 5 expression and purification of SRF and spike proteins 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, which was cloned into the standard expression vector pTZ (Trenzyme) to be suitable for transient expression in the supernatant of mammalian HEK293 cells. For translational and purification reasons, the above amino acid sequences were supplemented with His tag and methionine as translation initiation signals, respectively. Thus, the coding sequence actually used for cloning, which includes methionine as a translation initiation signal, a secretion signal and His-tag, 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 shown as SEQ ID NO:11 (comprising the amino acid sequence shown as 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).

The expression of the protein is carried out in shake flasks, the target protein being in the cell culture supernatant. After expression, the supernatant was collected 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. The expression of the protein is carried out in shake flasks, the target protein being in the cell culture supernatant. After expression, the supernatant was collected and the target protein was purified by His-tag affinity chromatography.

Example 6- -measurement of affinity on Biacore chip- -second part

SARS-CoV-2 spike protein S1 (aa 14-683), his-Avi-Tag recombinant protein (Invitrogen, catalog # RP-87681) and the three variants of SRF ((3) _ ACE2_ 19-615/u E375Q,1.24mg/ml; (4) _ ACE2 \19-103, 301-365,1mg/ml; (9) _ ACE2_19-615, 0.74mg/ml) were performed on Cytiva' S Biacore X100 using the Biotin CAPture kit on sensor chip CAP. The buffer was PBS, pH 7.4.

After the Biacore system was equilibrated and adjusted, the CAP chips were coated with Biotin CAPture reagents.

The spike protein S1 was immobilized on flow cell one, yielding-100 RU (ligand concentration 0.43. Mu.M; contact time: 420S; stationary phase: 300S).

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

The result was (3) _ ACE2_19-615_E375Q 443.6RU

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

(9)_ACE2_19-615 55.1RU

Circulation of	Number of times	Relative reaction
			Buffer solution	1	-6
Buffer solution	2	-5.6
			Buffer solution	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

As a result:

all three proteins tested bound to the spike protein of Sars-CoV-2 immobilized on a biocore chip, with the variant (3) _ ACE2_19-615 _ei375qshowing the highest binding response. Variant (4) _ ACE _2 \, 19-103;301-365 had a lower binding efficiency and the variant (9) _ ACE2_19-615 had the lowest binding efficiency. Even though (9) _ ACE2_19-615 showed the lowest binding affinity compared to the other constructs, its binding to SARS-CoV-2 spike protein S1 was clearly demonstrated.

Example 7 measurement of binding affinity by MicroScale thermophoresis

The affinity between the spike protein of Sars-CoV-2 and the respective target protein was measured by MicroScale thermophoresis. In principle, the binding affinity of two molecules is measured by detecting the change in fluorescence signal due to the IR-laser induced temperature change. The range of change in the fluorescent signal is related to the binding of the ligand to the fluorescent target. Therefore, it can quantitatively analyze molecular interactions in solution on a microliter scale with high sensitivity.

Fluorescent labeling of spike proteins:

SARS CoV 2 spike protein S1 (aa 14 683), his tag, avi tag; invitrogen, cat. Number: RP 87681, stock concentration 10.0. Mu.M in 1xPBS, pH 7.4 buffer.

The target protein was diluted into labeling buffer 1x PBS pH 7.4, 0.005% Tween to reach a labeling concentration of 9.7 μ M. DMSO was introduced into the labeling buffer as the dye stock was prepared in DMSO. The labeling concentration of the dye was 20.1. Mu.M (dye: protein molar ratio 3. Use of Red NHS 650 ^{Second oneSubstitute for Chinese traditional medicine} As a fluorescent dye. Labeling was performed at 25 ℃ for 30 minutes. After labeling, unbound dye is removed by a gel filtration step and the target protein is brought into the final buffer. Tween 20 was added at 0.005% to reduce protein adhesion/cohesion and improve protein recovery. The concentration of the 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. For labeling, approximately 2 fluorescent tags were introduced per protein.

And (3) binding measurement:

measurement setup of variants: (9) ACE 2-615

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

Ligand: (9) ACE 2-615, starting from 4.73 μ M, titrated with 16 dilution steps of 1.

The instrument comprises the following steps: monolith NT.115Pico

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

As a result:	K _D	amplitude of vibration	S/N
				Experiment
1	114nM	-35.4	19.6
				Experiment 2	192nM	-31.0	24.6

Measurement settings (3) ACE 2-615 E375Q

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

Ligand: (3) ACE 2-615 e375q, starting from 7.92 μ M, was titrated with 16 dilution steps of 1.

The instrument comprises: monolith NT.115Pico

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

As a result:	K _D	amplitude of vibration	S/N
				Experiment
1	23.7nM	-39.0	13.0
				Experiment 2	23.1nM	-37.9	16.8

Measurement settings (4) _ ACE _2 \ -19-103; 301-365

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

Ligand: (4) _ ACE _2 \, 19-103;301-365, titration was started from 22.9 μ M with 16 dilution steps of 1.

The instrument comprises: monolith NT.115Pico

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

As a result:	K _D	amplitude of vibration	S/N
				Experiment
1	42.8nM	-35.0	14.6
				Experiment 2	43.3nM	-29.0	13.4

Measurement setup (1) ACE 2-103 is fused with MBP

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

Ligand: (1) ACE 2-103 fused to MBP in 16 1: the dilution step of 1 starts with a titration of 1.26. Mu.M.

The instrument comprises the following steps: monolith NT.115Pico

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

As a result:	K _D	amplitude of vibration	S/N
				Experiment
1	4.94nM	-24.8	15.9
				Experiment 2	2.70nM	-28.2	9.1

Measurement setup (8) ACE 2-132 fusing with MBP

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

Ligand: (8) ACE 2-132 was fused to MBP and titrated from 1.29 μ M with 16 dilution steps of 1.

The instrument comprises the following steps: monolith NT.115Pico

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

As a result:	K _D	amplitude of vibration	S/N
				Experiment
1	1.74nM	-23.2	26.2
				Experiment 2	2.39nM	-19.7	15.1

As a result:

the variants (8) ACE 2-132 fused to MBP and (1) ACE 2-103 fused to MBP showed the best binding affinity. The binding efficiency of the other three variants was lower, and the binding capacity of variant (3) _ ACE2_19-615 _E375Qwas stronger than that of (4) _ ACE _2_19-103;301-365 (pre-ACE 2_ 19-103), variant (9) _ ACE2_19-615, showed the lowest binding capacity. Even though (9) _ ACE2_19-615 showed the lowest binding affinity compared to the other constructs, its binding to SARS CoV 2 spike S1 was clearly confirmed.

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

Materials:

DMEM medium: thermo Fishe41965039

FBS:Pan Biotech#P30-3702

Penicillin/streptomycin: sigma # P4333

OptiPro-SFM:Thermo Fisher#12309019

DPBS (Ca/Mg-free): thermo Fisher 14190144

Carboxymethyl cellulose: 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, 1.25x10 ⁵ Plating of VeroE6 cells/well into 24-well plates, CO% ₂ And cultured for 24 hours. On day 1, serial dilutions of recombinant SRF variants were prepared as follows:

concentration of stock solution:

variant _19-615: e375q, mw =70, 6kDa, c =1, 24mg/ml → 17, 563 μ M

Variant _19-615 mw =70, 6kDa, c =0, 74mg/ml → 10, 481 μ M

SRF variants were thawed on ice. Three sets of 120. Mu.l SRF variant dilutions were prepared in OptiPro-SFM (serum free medium) in round bottom 96-well plates. An additional 12 wells were loaded with 120 μ l OptiPro-SFM as a control, and several 120 μ l vehicle control samples were prepared in OptiPro-SFM with PBS. The plate is tightly sealed and free of air bubbles.

Viral dilution

In BSL-3 laboratory, in vitro propagated SARS-CoV-2 (line B.3, isolated at 2 months 2020, 1.8x10 ⁶ PFU/ml) were thawed and pre-diluted in OptiPro-SFM at 1. Each well of protein dilution and positive control required 120 μ l of this dilution.

SRF variant-Virus Co-incubation

The virus dilution of 1A reservoir. Mu.l of virus dilution was added to each SRF variant dilution well and positive control well using a multichannel pipettor. Sealing plates at 37 ℃ and 5% CO ₂ And culturing for 1 hour.

Infection of VeroE6 cells

Media were removed from up to 6 wells of a 24-well plate at a time using a vacuum pump or P1000, and 200 μ l of SRF variant dilution/virus mixture, virus (positive control), or OptiProSFM (negative control) was added. After the first 24-well plate is completed, a timer is started to count and the plate is placed back in the incubator. This was done until all samples from the 96-well plate were added to the cells. The time on the timer is recorded each time a panel is completed. The cells were cultured at 37 ℃ for 1 hour.

Post-infection replacement of culture media

1.5% (w/v) carboxymethylcellulose (autoclaved) and 2 XMEM (sterile filtered, containing 2 XPen/Strep, 4% FBS and NaHCO) ₃ To ensure pH) was mixed as 1 and preheated in a water bath. After a timer of 1 hour after addition of the virus, the supernatant of each 24-well plate was aspirated (by vacuum or P1000) and replaced with 1ml of carboxymethyl cellulose-MEM per well. The plate was returned to the incubator and incubated for 72 hours.

On day 4, cells were fixed and stained. A partial volume of the supernatant was removed from each well with vacuum or a 10ml serological pipette, without touching or being close to the bottom surface of the well. The entire 24-well plate was immersed in 6% formaldehyde in a holding/shipping container and capped on the 24-well plate. The plates were left at room temperature for 30 minutes. The exterior of the container is wiped with sanitizer and the container is removed from the BSL-3. The vessel was opened and as much formaldehyde as possible was poured back into the vessel. Each well was rinsed 3 times with tap water. Then, 1% crystal violet staining solution was added to each well, the lid was closed, and the wells were incubated at room temperature for 30 minutes. The crystal violet was poured back into the bottle using a funnel, the plate was rinsed with tap water until the drained water was no longer blue, and the plate and lid were completely air dried. Plaques in each well were counted.

As a result:

(9)_ACE2_19-615(40PFU)

	untreated with	250nM	500nM	750nM	1μM	1.5μM	2μM
								18	15	15	4	6	1	1
	22	17	12	13	4	1	3
								24	16	7	6	7	1	2
	24
								22
Average	22，0	16，0	11，3	7，7	5，7	1，0	2，0

(3)_ACE2_19-615_E375Q(40PFU)

	Untreated	250nM	500nM	750nM	1μM	1.5μM	2μM
								18	17	24	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)

	Untreated	1nM	25nM	100nM	250nM	500nM	1μM
								86	74	87	75	59	46	24
	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)

	Untreated	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 out	94，6	81，7	79，3	81，3	66，3	61，3	39，0

As shown in the above table and fig. 2 and 3, the results are shown below:

neutralization tests were performed with 80 pfu/ml: variant 4_ACE _2_19-103;301-365 did not show significant pfu reduction at all concentrations tested. The variant 9_ACE2_19-615 showed already a small reduction at 1nM and a 50% reduction at 250 nM. The use of variant 3_ACE2_19-615 _E375Qin the neutralization assay resulted in a measurable reduction of about 40% at all concentrations tested.

Neutralization tests were performed with 40 pfu/ml:

variant 4_ACE _2_19-103;301-365 did not show a significant reduction in pfu at all concentrations tested. The variant 9_ACE2_19-615 showed 50% inhibition at 250nM and complete inhibition at 1, 5. Mu.M. The use of the variant 3_ACE2_19-615 _E375Qresulted in a significant reduction in the concentration range of 250nM to 1. Mu.M. At 1, 5 μ M and 2 μ M, the reduction was almost complete.

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

The assay for neutralizing plaques using the recombinant SRF variants described in example 8 was performed with a variant of SARS-CoV-2 UK variant (b.1.1.7), as described in Rambaut, a., loman, o., barclay, w., barrett, j., carabelli, a., connor, t., peacock, t., robertson, d.l., and Volz, e. (2020). The primary genomics characteristic of the SARS-CoV-2 line, emerging in the United kingdom, is defined by a novel set of spike mutations.

As a result:

(9)_ACE2_19-615(80 PFU)

	untreated with	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)

	Untreated	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 out	86，0	13，0	4，0	2，7	1，3	1，3	1，0

As shown in FIG. 4 and the above table, variant 9_ACE2_19-615 and variant 3_ACE2_19-615 _E375Qalready showed substantial reduction at 250nM and complete reduction at 1 μ M.

Reference to the literature

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、2020Mar 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、Nivedita Gupta、Mousmi Das、Soumitra Das、Anurag Agarwal、Sujeet Singh、Priya Abraham、Samiran Panda、Shekhar Mande、Renu Swarup、Balram Bhargava、Rajesh Bhushan、NIC team、INSACOG Consortium Convergent evolution of SARS-CoV-2 spike mutations、L452R、E484Q and P681R、in the second wave of COVID-19 in Maharashtra、India bioRxiv 2021.

Singh、J.；Samal、J.；Kumar、V.；Sharma、J.；Agrawal、U.；Ehtesham、N.Z.；Sundar、D.；Rahman、S.A.；Hira、S.；Hasnain、S.E.Structure-Function Analyses 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 in cats and other companion animals:Where does SARS-CoV-2/COVID-19 fitVet 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> Lai Sangduo Corp (Lysando AG)

<120> viral neutralization of soluble receptor fragments of 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> full-length ACE-2 for homo sapiens

<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 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 peptidase domain of human ACE-2 substituted with Glu375Gln (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 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> combination of fragments of peptidase domain of ACE-2 (19-103

<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 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> fragments of 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

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

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

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

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

115 120 125

<210> 15

<211> 65

<212> PRT

<213> Artificial sequence

<220>

<223> fragment 301-365 of 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> Joint 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 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 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 peptidase domain of human ACE-2 substituted with Glu375Gln (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 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

595

<210> 20

<211> 187

<212> PRT

<213> Artificial sequence

<220>

<223> combination of fragments of peptidase Domain (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> combination of fragments of peptidase domain substituted with Glu375Gln (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

195 200

<210> 23

<211> 87

<212> PRT

<213> Artificial sequence

<220>

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

<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> Joint 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> combinations of fragments of 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 peptidase domain of ACE-2 substituted with Glu375Gln (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

225 230 235

<210> 28

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> fragment 301-425 of 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> combination of fragments of 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> combination of fragments of peptidase domain of ACE-2 substituted with Glu375Gln (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

195 200 205

<210> 32

<211> 84

<212> PRT

<213> Artificial sequence

<220>

<223> fragment 342-425 of 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> Joint 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

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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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.