JP2023524862A - Viral Neutralization by Soluble Receptor Fragments of the ACE-2 Receptor - Google Patents

Abstract

The present invention provides a soluble receptor fragment (SRF) of the ACE-2 receptor, characterized in that said SRF comprises the peptidase domain (PD) of ACE-2 or fragments and/or derivatives thereof. Relating to Body Fragment (SRF). Further, the present invention relates to methods for treating the human or animal body by surgery or therapy, for use as vaccines administered to the human or animal body or for use in diagnostic methods, or for body fluids from the human or animal body. It relates to the SFRs of the present invention for use with other samples. Additionally, the present invention provides an SRF for use in a method of treating and/or preventing a viral infection, said viral infection in particular a viral infection caused by a virus of the coronavirus family, more particularly A viral infection caused by SARS coronavirus, SARS coronavirus-2, human coronavirus NL63, or SARS-CoV-2. Finally, the invention features the steps of providing immobilized SRF and contacting the liquid sample or fluid with the SRF under conditions that allow the SRF to bind to viral particles. , relates to methods for trapping viral particles. [Selection drawing] Fig. 3

Description

The present invention relates to soluble receptor fragments (SRFs) of the ACE-2 receptor, which SRFs include the peptidase domain (PD) of ACE-2 or fragments and/or derivatives thereof. Further, the present invention provides SRF according to the present invention for use in methods of surgically or therapeutically treating the human or animal body, or SRFs administered to the human or animal body as a vaccine or as a diagnostic method, or from the human or animal body. It relates to SRF for use with bodily fluids and other materials. Further, the present invention provides SRFs for use in methods of treating and/or preventing viral infections, particularly viral infections caused by the Coronaviridae family, especially SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SRFs for use in methods of treating and/or preventing viral infection by SARS-CoV-2 are provided. Finally, the present invention relates to a method for capturing viral particles, comprising the steps of providing immobilized SRF; and a step of contacting.

Generally, a virus is delivered to a host cell by (1) binding to a specific receptor on the surface of a target cell, (2) initiation of viral entry, (3) viral replication, (4) production of progeny virus, (5) Subsequent release, via infection. Viral receptors in target cells are protein or carbohydrate or lipid structures.

Therefore, to combat viral infections, targeting these steps using a variety of strategies can treat or prevent viral infections, which also includes vaccination. Most of these strategies address steps in the viral infection cycle after human entry. Examples of this are spike proteins or more generally cell entry mechanisms as therapeutic targets or vaccination strategies. Other therapeutic targets are, for example, essential viral enzymes required for growth, such as proteases and replication enzymes. Vaccine strategies primarily involve viral surface structures, usually viral surface proteins, such as spike and capsid proteins, which can be used to provoke an immune response.

However, the development of drugs and vaccines may take a long time and is often unsuccessful. Therefore, after a virus invades the human body, starts replicating in cells, and then spreads through the blood and lymphatic system, many serious or even fatal problems occur. Another option is to prevent the incorporation of

To prevent viral uptake, or at least to greatly reduce the amount of virus entering the human body, soluble fragments of host cell receptors can be applied, for example, to washing solutions according to the invention to neutralize the viral receptors and to It can allow the harmonized virus to be washed out of the point of entry.

A representative example of a virus that has a large impact on the human body is SARS-CoV-2, which causes COVID-19. SARS-CoV-2, or Severe Acute Respiratory Syndrome Coronavirus 2, is the viral strain that causes the respiratory illness known as coronavirus infection 2019 (COVID-19). The virus belongs to the Coronaviridae family, a family of lipid-enveloped positive-sense RNA viruses. SARS-CoV-2 was previously called the 2019 novel coronavirus (2019-nCoV). SARS-CoV-2 infects humans, and the World Health Organization (WHO) has designated the ongoing COVID-19 pandemic as a "Public Health Emergency of International Concern."

Droplet infection is thought to be the primary route of human transmission of Sars-CoV-2. Theoretically, contact transmission, primarily around an infected person, is also possible. However, the main route of infection is droplet infection, in which droplets with a diameter of less than 5 μm generated when coughing or sneezing are absorbed by the other person through mucous membranes such as the nose, mouth, and in some cases the eyes.

In SARs-CoV-2, the trimeric spike glycoprotein (S protein) on the surface of the virus particle plays a role in mediating receptor recognition and membrane fusion. Upon viral infection, the S protein is cleaved into S1 and S2 subunits. The S1 subunit contains a receptor-binding domain (RBD) that directly binds the peptidase domain of angiotensin-converting enzyme 2 (ACE-2), while the S2 subunit mediates membrane fusion. I am in charge. Binding of S1 to the host receptor ACE-2 exposes another cleavage site on S2 for cleavage by host proteases, a process critical to viral infection. The S protein of Sars-CoV-2 also utilizes the infected host's ACE2. The structure of the Sars-CoV-2 S protein has also been elucidated, and the extracellular domain of the Sars-CoV-2 S protein binds to the peptidase domain of ACE-2 with high affinity, a dissociation constant (Kd) of ~15 nM. shown to bind (Wrapp et al.-2020).

Indeed, ACE2 functions as a cell entry portal for additional coronaviruses, including at least SARS coronavirus, SARS coronavirus-2, human coronavirus NL63, and others.

Angiotensin-converting enzyme 2 (ACE-2) is an enzyme expressed in the cell membranes of the lungs, arteries, heart, kidneys, intestines, etc. constitutes a part. ACE2 lowers blood pressure by hydrolyzing angiotensin II (one of the vasoconstrictors) to angiotensin (1-7) (one of the vasodilators). ACE-2 counteracts the activity of the associated angiotensin-converting enzyme (ACE) by decreasing the amount of angiotensin II and increasing angiotensin (1-7), thus showing promise for treating cardiovascular disease. drug target.

ACE-2 is a zinc-containing metalloenzyme present on the surface of endothelium and other cells. The ACE-2 protein consists of an N-terminal peptidase M2 (carboxypeptidase) domain and a non-structural, C-terminal corecrine renal amino acid transporter domain. The C-terminal domain of ACE-2 is a membrane anchor, which exposes the enzymatically active domain on the cell surface of lung and other tissues. This peptidase domain is composed of two subdomains (I and II) that surround the active site and are connected by an α-helix (Towler et al.-2004). The N-terminal subdomain I also contains a zinc ion. The extracellular domain of ACE-2 is spontaneously cleaved from the transmembrane domain and the resulting soluble protein is released into the bloodstream and ultimately excreted in the urine.

Numerous strategies exist to deal with Sars-CoV-2, essentially at all stages of the viral life cycle. These policies are summarized, for example, in a review paper by Zhang et al. (2020). These policies summarize vaccination efforts in addition to various treatment options. Most of these therapeutic strategies address steps in the viral life cycle after entry into human cells. However, several attempts have aimed to prevent cell entry, either by activating the immune system by vaccination with inactivated virus or parts of the virus, or by viral protein S and cell receptors. There are also attempts to prevent cell entry by inhibiting its interaction with body ACE-2. More specifically, the review paper summarizes the hypothetical replication cycle of SARS-CoV-2 as follows. Thus, SARS-CoV-2 uses a spike protein to bind to the ACE2 receptor on the cell surface, followed by endocytosis. When the viral nucleocapsid is released into the cytoplasm, the encapsulated positive-strand genomic RNA [(+)gRNA] is used as a template for translation of the polypeptide chain, which is cleaved into nonstructural proteins, including RNA-dependent RNA polymerases. be. By employing a single negative-strand RNA [(-) gRNA] synthesized from a (+) gRNA template, more viral RNA is replicated. Subgenomic RNAs (sgRNAs) are synthesized by discontinuous transcription from a (+)gRNA template, encode viral structural and accessory proteins, and then associate with newly synthesized viral RNA to form new viruses. form particles. This nascent virus particle is then transported to the plasma membrane in a secretory vesicle and released by exocytosis. In addition, review papers describe the following potential targets for anti-COVID-19 drugs: RhACE2, convalescent plasma, and the JAK inhibitor baricitinib may weaken the binding of the spike protein on the surface of SARS-CoV-2 to ACE2 expressed on the cell surface. Lopinavir/ritonavir and favipiravir inhibit proteolysis of polypeptide chains. Remdesivir inhibits RNA-dependent RNA polymerase. EIDD-2801 may inhibit replication of SARS-CoV-2. iNO and zinc may inhibit replication of SARS-CoV-2. Vitamin D may inhibit SARS-CoV-2 replication by inducing antimicrobial peptides. Ivermectin can effectively inhibit the growth of SARS-CoV-2. Baricitinib can block passage of SARS-CoV-2 entering cells by inhibiting AAK1-mediated endocytosis. CQ and HCQ inhibit the virus/cell fusion process. LHQW and IFN can block viral replication processes (RNA transcription, protein translation, post-translational modifications).
Abbreviations:
AAK1, adapter-associated kinase 1,
CQ, chloroquine,
ER, endoplasmic reticulum;
HCQ, hydroxychloroquine sulfate,
IFNs, interferons,
iNO, inhaled nitric oxide,
JAK, Janus kinase,
LHQW, Renka Seion,
rhACE2, recombinant human angiotensin-converting enzyme 2,
SARS-CoV-2, severe acute respiratory syndrome coronavirus type 2.

In addition to the treatment or vaccination concepts described above, another strategy is to reduce the viral load in the mouth and nose by rinsing with a suitable inactivating solution that inactivates and cleanses the virus. is. Animal studies have shown that short-term exposure to the virus is not enough to cause illness. This is an interesting option as it appears that a certain amount of virus is required to infect a new host. Rinsing or rinsing with an inactivating solution can be used to prevent transmission to persons who have had short-term contact with infected patients, such as doctors and nurses. In some cases, the use of inactivating solutions to reduce the viral load in the mouth and nose of highly infectious patients can reduce the risk of viral transmission, for example in medical procedures where masks cannot be used. .

Strategies to block viral entry have focused on antibodies or soluble recombinant human ACE-2 receptor. The authors of Monteil et al. (2020) were able to express and purify a large extracellular fragment of ACE-2 (amino acids 1-740) to block viral entry into various cells. We were able to show that fusion proteins composed of these extracellular ACE-2 domains with the Fc region of human IgG1 were suitable for neutralizing the receptor-binding domain of the Sars-CoV-2 spike protein in vitro. (Non-Patent Literature Lei et al.-2020).

However, the use of human soluble ACE2 recombinant protein in COVID-19 is not without risk, as ACE-2 treatment is thought to result in angiotensin II deficiency, thus affecting blood pressure and renal function. caution should be exercised (Alhenc-Gelas et al - 2020).

Therefore, there is a need for new and alternative approaches to inactivating viruses such as Sars-CoV-2.

Therefore, the problem to be solved by the present invention is a virus of the Coronaviridae family, in particular a virus such as SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, which is a mutant strain derived from the United Kingdom. A certain B. 1.1.7 lineage, a mutant strain B. from South Africa. 1.351, B. . 1.617 mutant, or the B. 1. To provide a novel means that can be used to inactivate and neutralize viruses containing any variants, such as the 1.1.28.1 variant, possibly prior to infecting test cells. is. Furthermore, another problem to be solved by the present invention is to provide a novel means capable of reducing the amount of virus without serious side effects such as effects on blood pressure and renal function. A further problem to be solved by the present invention is to provide a novel method for detecting viral particles and washing a liquid sample or fluid, characterized in that the viral load is reduced. . Therefore, the problem to be solved by the present invention is a prophylactic and/or therapeutic method for stopping the infectious process by coronavirus family viruses such as SARS-CoV-2 by preventing viral entry into human cells. It's about providing tools. A further problem underlying the present invention is any mutant strain of a virus of the coronavirus family, in particular SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, for example a mutant from the United Kingdom. The strain B. 1.1.7 lineage, a mutant strain B. from South Africa. 1.351, B. . 1.617 mutant, or the B. It is to provide preventive and/or therapeutic means useful for all variants, including the 1.1.28.1 variant.

The further problem underlying the invention is solved by the subject matter of protection defined in the claims.

The following drawings are intended to illustrate the invention.

FIG. 1 depicts the binding of three SRF variants to the spike protein of Sars-CoV-2 immobilized on a Biacore chip. Binding to all three proteins tested was observed, with mutant 3_ACE2_19-615_E375Q showing the greatest binding response. Mutants 4_ACE_2_19-103; 301-365 showed low binding efficiency and mutant 9_ACE2_19-615 showed the lowest binding. Measurement samples 1-4 are buffer solutions, sample 4 is mutant (3)_ACE2_19-615_E375Q, sample 5 is mutant (4)_ACE_2_19-103; 301-365, sample 6 is mutant (9)_ACE2_19- was 615. FIG. 2 depicts neutralization of Sars-Cov-2 virus particles by SRF addition. Neutralization tests were performed at 80 pfu/ml. Mutant 9_ACE2_19-615 showed a small reduction already at 1 nM and a 50% reduction at 250 nM. The use of mutant 3_ACE2_19-615_E375Q in neutralization assays resulted in measurable reductions at all concentrations tested, up to approximately 40% reduction. FIG. 3 depicts neutralization of Sars-Cov-2 virus particles by SRF addition. Neutralization tests were performed at 40 pfu/ml. Mutant 9_ACE2_19-615 showed 50% inhibition at 250 nM and complete inhibition at 1.5 μM. Use of mutant 3_ACE2_19-615_E375Q resulted in a large reduction in the concentration range of 250 nM to 1 μM. Nearly complete reduction was observed at 1.5 μM and 2 μM. FIG. 4 depicts neutralization of Sars-Cov-2 UK mutant (B.1.1.7) virus particles by addition of SRF. Neutralization tests were performed at 80 pfu/ml. Mutant 9_ACE2_19-615 (A) and mutant 3_ACE2_19-615_E375Q (B) both showed significant pfu reduction already at 250 nM and complete reduction at 1 μM.

As used herein, the term "SARS-CoV-2" refers to Severe Acute Respiratory Syndrome Virus 2, the viral strain that causes the respiratory disease known as coronavirus infection 2019 (COVID-19). . The virus belongs to the Coronaviridae family, which is a family of lipid-enveloped positive-sense RNA viruses. SARS-CoV-2 was previously known under the tentative name 2019 novel coronavirus (2019-nCoV). Due to the observed occurrence of mutations in viruses, the term "SARS-CoV-2" as used herein preferably only refers to all variants of SARS-CoV-2 described. but also all variants exhibiting either a single mutation, multiple mutations, or a combination of mutations, also referred to as SARS-CoV-2.

As used herein, "SARS-CoV-2 variant B.1.1.7 from British lineage", "SARS-CoV-2 South African variant (B.1.351)" or "SARS-CoV- The term "B.2 Brazil Mutant (B.1.1.28.1)" preferably refers to the SARS-CoV-2 mutant B.2 brazilian mutant, eg as described in Singh et al. 1.1.7, B. 1.351 and B. It refers to 1.1.28.1.

"SARS-CoV-2 B.1.617 from India" as used herein preferably has mutations D111D, G142D, L452R, E484Q, It refers to mutants with D614G and P681R.

The term "ACE-2" as used herein refers to angiotensin converting enzyme 2 (ACE-2). It is an enzyme attached to the membranes of cells, for example in the lungs, arteries, heart, kidneys and intestines, and is part of the renin-angiotensin system that maintains blood pressure homeostasis and fluid and salt balance in the body. ACE2 lowers blood pressure by hydrolyzing angiotensin II (one of the vasoconstrictors) to angiotensin (1-7) (one of the vasodilators). ACE-2 counteracts the activity of the associated angiotensin-converting enzyme (ACE) by decreasing the amount of angiotensin II and increasing angiotensin (1-7), thus showing promise for treating cardiovascular disease. drug target. ACE-2 is a zinc-containing metalloenzyme present on the surface of endothelium and other cells. The ACE-2 protein consists of an N-terminal peptidase M2 (carboxypeptidase) domain and a non-structural C-terminal corecrine renal amino acid transporter domain. The C-terminal domain of ACE-2 is a membrane anchor, which exposes the enzymatically active domain on the cell surface of lung and other tissues. This peptidase domain is composed of two subdomains (I and II) that surround the active site and are linked by an α-helix (Towler et al.-2004). These two subdomains are defined as follows (Towler et al. - 2004): the N-terminal zinc-containing subdomain I, composed of residues 19-102, 290-397, and 417-430, and C-terminal containing subdomain II, composed of residues 103-289, 398-416, and 431-615. The N-terminal subdomain also contains a zinc ion. The structure of ACE-2 and its peptidase and neck domains are preferably as described in Yan et al. (2020). Preferably, ACE-2 includes the amino acid sequence shown in SEQ ID NO:1. It preferably consists of a peptidase domain shown in SEQ ID NO:3 or SEQ ID NO:6 and a neck domain shown in SEQ ID NO:2.

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

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

The term "soluble" as used herein in reference to PD or SRF preferably means that the protein is about It refers to dissolution at concentrations from 1 μg/ml to about 1000 mg/ml. More preferably, it is at least about 2 μg/mL, at least about 5 μg/mL, at least about 10 μg/ml, at least about 20 μg/ml, at least about 30 μg/ml, at least about 40 μg/ml, at least about 50 μg/ml, at least about 60 μg/ml, at least about 70 μg/ml, at least about 80 μg/ml, at least about 90 μg/ml, at least about 100 μg/ml, at least about 110 μg/ml, at least about 120 μg/ml, at least about 150 μg/ml or at least about 200 μg/ml It is dissolved at a concentration of ml.

The terms "polypeptide" or "protein" as used herein specifically refer to a polymer consisting of amino acids joined by peptide bonds in a specific sequence. Amino acid residues of polypeptides may be modified, for example, by covalent attachment of various groups such as carbohydrates and phosphates. Other substances may be more loosely associated with the polypeptide, such as heme or lipids, giving rise to composite polypeptides that are also included in the terms "polypeptide" or "protein" as used herein. The term "polypeptide" or "protein" includes polypeptides or proteins exhibiting optional modifications commonly used in the art, such as biotinylation, acetylation, PEGylation, or amino groups, SH Also included are embodiments involving polypeptides or proteins exhibiting groups, or chemical alterations of carboxyl groups (such as protecting groups), and the like. As used herein, the term "polypeptide" or "protein" is not limited to amino acid polymer chains of any particular length, although typically polypeptides or proteins are greater than about 50 amino acids in length, about Lengths greater than 100 amino acids, alternatively greater than about 150 amino acids are indicated. Typically, although not necessarily, a typical polypeptide according to the invention does not exceed about 850 amino acids in length. The term "polypeptide" or "protein" as used herein can also include dimer fusions of polypeptides or proteins. For dimeric fusions, typical polypeptides of the invention do not exceed about 1600 or 1700 amino acids in length.

The term "derivative" as used herein refers to an amino acid sequence that exhibits one or more additions, deletions, insertions and/or substitutions and/or combinations thereof compared to the respective reference sequence. refers to This includes, for example, combinations of deletion/insertion, insertion/deletion, deletion/addition, addition/deletion, insertion/addition, addition/insertion, and the like. Those skilled in the art will appreciate that the presence of an amino acid residue at a particular position in a derivative sequence that is different from the amino acid residue present at each same position in a reference sequence is, for example, a deletion at the same position and its It will be understood that the permutations defined herein are not combinations of subsequent insertions, etc. Rather, when a combination of one or more additions, deletions, and substitutions is referred to, combinations of changes at different positions in the sequence are intended, e.g., additions at the N-terminus and is intended for the deletion of The sequences thus obtained exhibit a certain level of sequence identity, preferably at least 60%, such as at least 75%, at least 80%, with each reference sequence, eg, a given SEQ ID NO. , at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. Preferred derivatives are, unless stated otherwise, fragments of the parent molecule, e.g. a given SEQ ID NO, which retains the activity of the parent molecule, i.e. exhibiting at a general level the same activity as the respective parent molecule. . However, the above activities can be the same, higher or lower than the respective parent molecule. Also preferred are derivatives resulting from conservative amino acid substitutions within the parent sequence, eg, derivatives that retain the activity of the parent molecule at a general level for a given SEQ ID NO.

As used herein, "~% sequence identity" is understood as follows. That is, the two sequences to be compared are aligned so that the maximum correlation between the sequences is obtained. This may involve increasing the size of the alignment by inserting "gaps" in one or both sequences. The percent sequence identity may then be determined over the entire length of each of the sequences being compared (referred to as a global alignment), which is particularly suitable for sequences of the same or similar length, or short given A determination can be made over the length (called a local alignment), which is more suitable for sequences that vary in length. In this sense, an amino acid sequence that has, for example, at least 95% “sequence identity” to a query amino acid sequence will have at most 5 times the subject amino acid sequence for every 100 amino acids contained in the query amino acid sequence. It is intended that the sequence of the subject amino acid sequence is identical to the query sequence, except that it contains an amino acid modification of . In other words, to obtain an amino acid sequence with a sequence that has at least 95% identity to the query amino acid sequence, insert up to 5% (5 out of 100) amino acid residues into the subject sequence, or Other amino acids can be substituted or deleted. Methods for comparing the identity and homology of two or more sequences are well known in the art. The percent identity between two sequences can be determined using a mathematical algorithm or the like. A non-limiting example of a preferred mathematical algorithm that can be used is the algorithm of Kerlin et al. ((1993), PNASUSA, 90:5873-5877). Such algorithms are incorporated into the BLAST family of programs, such as the BLAST or NBLAST programs (Altschulet al., 1990, J. Mol. Biol. 215, 403-410 or Altschul et al. (1997), Nucleic Acids Res, 25:3389-3402), accessible from the NCBL home page (World Wide Web site "ncbi.nlm.nih.gov"), and FASTA (Pearson (1990), Methods Enzymol. 83, 63-98; Pearson and Lipman (1988), Proc. Natl. Acad. Sci. can be identified using these programs, as well as the program available in the Wisconsin Sequence Analysis Package version 9.1 (Devereux et al, 1984, Nucleic Acids Res. , 387-395), for example, the programs BESTFIT and GAP can be used to determine the percent identity of two polypeptide sequences.BESTFIT is (Smith and Waterman (1981), J. Mol. Biol. 147 , 195-197) to find the most similar single region between two sequences, where amino acids sharing a specific length of sequence identity with a reference sequence are used. When referring to sequences, the above differences in sequences preferably result from conservative amino acid substitutions Preferably, such sequences preferably exhibit the activity of the reference sequence, even at a slower rate, e.g. Furthermore, when reference is made herein to sequences sharing "at least" a certain percentage of sequence identity, 100% sequence identity is preferably not included.

As used herein, a "conservative amino acid substitution" is a group of amino acids having sufficiently similar physicochemical properties such that substitutions between members of the group will preserve the biological activity of the molecule. (see, eg, Grantham, R. (1974), Science 185, 862-864). In particular, conservative amino acid substitutions are preferably those in which amino acids are substituted with amino acids of the same type (e.g., basic amino acids, acidic amino acids, polar amino acids, amino acids with aliphatic side chains, amino acids with positively or negatively charged side chains). , amino acids with aromatic groups in their side chains, side chains that can enter into hydrogen bonding, eg amino acids with side chains with hydroxyl functionality, etc.). Conservative substitutions are in the present case e.g. replacing a basic amino acid residue (Lys, Arg, His) with another basic amino acid residue (Lys, Arg, His), an aliphatic amino acid residue ( Gly, Ala, Val, Leu, Ile) with another aliphatic amino acid residue, replacing an aromatic amino acid residue (Phe, Tyr, Trp) with another aromatic amino acid residue, replacing threonine replacement by serine or leucine by isoleucine. Additional conservative amino acid exchanges will be known to those of skill in the art.

The term "deletion" as used herein preferably refers to 1, 2, 1, 2, or 1, 2, or 4 in the derived sequence compared to the respective reference sequence, either intrasequentially or at the N-terminus or C-terminus. Refers to a lack of 3, 4, 5 (or even more than 5) contiguous amino acid residues. Derivatives of the invention may exhibit one, two or more such deletions.

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

The term "addition" as used herein preferably refers to 1, 2, 3, 4, 5 ( or even more than 5) refers to the additional presence of contiguous amino acid residues.

The term "substitution" as used herein refers to the presence of an amino acid residue at a particular position in the derived sequence that is different from the amino acid residue that is or is not present at the corresponding position in the reference sequence. point to Derivatives of the invention can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , or more. As noted above, preferably such substitutions are conservative substitutions.

The term "mutation" for a virus of the coronavirus family, SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, refers to the mutant B. 1.1.7, the mutant B. from South Africa. 1.351, a mutant from India, B. 1.617 or the mutant B. Refers to any mutation for any of the above viruses, including 1.1.28.1. In particular, the term "mutation" of the coronavirus family of viruses, SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, refers to any mutation associated with the respective virus. The mutations may include both single mutations or multiple mutations. The mutations may occur in the spike protein and/or another viral protein and/or other viral proteins. The one or more mutations may be present in any part of the virus and may be silent mutations that have no effect, or non-affecting viral properties such as host cell binding, replication, or viral stability. It may be a silent mutation.

The term "infected" as used herein preferably refers to the virus entering a host cell, particularly a mammalian or human cell, and becoming ready to replicate.

The term "host cell" as used herein preferably refers to any eukaryotic, animal, mammalian or human cell. It also refers to any eukaryotic, animal, mammalian, or human cell that has an ACE-2 receptor. In particular, the term "host cell" as used herein refers to human, cat, tiger, chicken, and mouse cells that have the ACE-2 receptor and those described, for example, in Stout et al. (2020). All host cells that can be infected with SARS-CoV-2/COVID-19 or any mutants or variants thereof described herein, such as those described herein.

The term "tag" herein is typically used in the art to a) improve expression of the amino acid sequence or entire polypeptide, b) facilitate purification of the amino acid sequence or entire polypeptide, c) An amino acid sequence that is fused to or included in another amino acid sequence to facilitate immobilization of the amino acid sequence or the entire polypeptide and/or d) facilitate detection of the amino acid sequence or the entire polypeptide. point to 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, etc. . A person skilled in the art will know a vast number of tags suitable for various technical applications. The tag can, for example, render such tagged polypeptides suitable for antibody binding, eg, in various ELISA assay formats, or other technical applications.

The term "comprising" as used herein is not to be interpreted as being limited to the meaning of "consisting of" (ie excluding the presence of additional others). Rather, "including" implies that there may optionally be additional things. The term “comprising” includes “consisting of” (i.e., excluding the presence of additional others) and “including but not consisting of” (i.e., additional require the presence of the other), the former being preferred.

As used herein, the abbreviation "aa" preferably refers to the term "amino acid" and is particularly used in reference to a particular amino acid position in a given amino acid sequence. For example, "aa19 to 103 of SEQ ID NO:3" refers to the amino acid sequence starting with the 19th amino acid residue and ending with the 103rd amino acid residue in the amino acid sequence shown in SEQ ID NO:3.

A first object of the present invention envisages providing a soluble receptor fragment (SRF) of the ACE-2 receptor comprising the peptidase domain (PD) of ACE-2. Optionally, the SRF according to the invention comprises ACE-2 PD fragments and/or derivatives, in particular one, two or more ACE-2 PD fragments. When an SRF according to the present invention contains two or more fragments, the term "fragment combination of two or more fragments" or simply "fragment combination" is used in this disclosure to describe this embodiment. can do. In a further preferred embodiment of the invention, the SRF comprises a derivative of PD of ACE-2 or a fragment or combination of fragments of PD of ACE-2. In a preferred embodiment of the invention, the SRF may be composed of ACE-2 PD, two or more fragments thereof, or derivatives of ACE-2 PD or fragments thereof. In a further preferred embodiment, the SRF comprises or consists of PD of ACE-2 or one or more fragments thereof, or derivatives of PD of ACE-2 or fragments thereof, wherein one or more mutations render the active site inactive. activated.

The SRF ACE-2 PD fragment, fragment combination and/or derivative according to the present invention has a binding affinity or specificity for the viral spike protein, particularly the receptor binding cleft of the viral spike protein S. , essentially as high as or higher than wild-type full-length PD of ACE-2. Here, essentially the same viral spike protein has a binding affinity or binding characteristic for the receptor-binding cleft that is about 70% to about 150%, more preferably from about 80% to about 130%, or from about 90% to about 120%. In a preferred embodiment of the invention, ACE- that effectively binds to the receptor-binding cleft of the viral spike protein S of SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2 or variants thereof. Any fragment, combination of fragments and/or derivative of PD of 2 is a fragment, combination of fragments and/or derivative of SRF according to the present invention. The binding affinity is such that the binding affinity of the tested derivative, fragment, or combination of fragments is at least 50% of that of (9)_ACE2_19-615 according to SEQ ID NO: 12 tested in the Examples herein, 60 %, 70%, 80%, 90%, 100%, 110% or 120% can be considered effective.

The inventors of the present invention have surprisingly found that the SRF according to the invention binds to the receptor-binding cleft of viral spike proteins, in particular to the receptor-binding cleft of viral spike protein S belonging to the family Coronaviridae. . In preferred embodiments, the SRF binds to the receptor binding cleft of the viral spike protein S of SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2. In a particularly preferred embodiment, SRF binds to the receptor binding cleft of viral spike protein S of SARS-CoV-2. Such binding prevents the virus from entering host cells, especially human cells. This results in the inability to initiate viral replication and thus the inability to produce further numbers of viral particles by host cells, such as human cells.

Preferably, the SRF ACE-2 PD of the present invention is a soluble expressed isolated PD of ACE-2 or a soluble expressed isolated fragment, combination of fragments and/or derivative thereof. Fragments or combinations of fragments of full-length PD of ACE-2 can enhance the solubility of SRF. The above fragment or combination of fragments is not essential for the binding activity of PD to the receptor-binding cleft of the viral spike protein, in particular the receptor-binding cleft of the viral spike protein S, of PD of ACE-2 one, two, It can preferably be designed by deleting 3, 4, 5 or more structural elements. Components of the PD of ACE-2 are, for example, those required to form alpha-helices, beta-sheets and beta-turns. Alternatively or additionally, the solubility of PD of ACE-2 or a derivative or fragment thereof is increased by 1, 2, 3, 4, 5 or more mutations such as insertions, substitutions, deletions or additions. be able to. Alternatively or additionally, the solubility of PD of ACE-2 or a derivative or fragment thereof can be increased by removing glycosylation sites.

In a particularly preferred embodiment of the invention, the SRF is any fragment of PD of ACE-2 that binds to the receptor-binding cleft of the viral spike protein, particularly viral spike protein S, and inhibits the binding of spike protein S to host cells. can include

Preferably, SRF 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 acids It has a fragment or a first fragment of ACE-2 PD with a length of residual residues. Preferably, the SRF is 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. A PD fragment of ACE-2 with length. In a preferred embodiment, the SRF comprises an ACE-2 PD fragment or first fragment having a length of about 80 to about 600 amino acids, more preferably about 84 to about 597 amino acids.

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

When the SRF of the present invention comprises a second fragment of PD of ACE-2, said second fragment is preferably about 50 to about 150 amino acid residues, about 60 to about 130 amino acid residues, or has a length of about 64 to about 125 amino acid residues. Particularly preferred fragments are the It has a length of amino acid residues.

In a preferred embodiment of the invention, the SRF comprises Sars-CoV-2 contact residues Q24, T27, F28, D30, K31, H34, E35, E37, D38, Y41, Q42, L79, M82, Y83 of SEQ ID NO:3. of which may include at least 5, 6, 7, 8, 9, 10, 11, 12, 13 or all 14. 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 of Sars-CoV contact residues Q24, T27, F28, K31, H34, E37, D38, Y41, Q42, L45, L79, M82, Y83 of SEQ ID NO:3: It can include at least 5, 6, 7, 8, 9, 10, 11, 12 or all 13.

In a further preferred embodiment of the invention, the SRF comprises 3, 4, 5, 6, 7, 8, 9 or all 10 of the alpha helical structures of ACE-2 PD shown in SEQ ID NOS: 34-43. or a derivative thereof. Any derivative of the amino acid sequences represented by SEQ ID NOS: 34-43 preferably has 1, 2, 3, 4 or 5 deletions or mutations, provided that they are capable of forming an alpha-helical structure. have Deletions may occur within and/or at one or both ends of a given amino acid sequence. The derivative of SEQ ID NO:34 preferably comprises at least 5, 6, of Sars-CoV-2 contact residues Q24, T27, F28, D30, K31, H34, E35, E37, D38, Y41 and Q42 of SEQ ID NO:3. 7, 8, 9, 10 or all 11 can be included. Derivatives of SEQ ID NO:40 are preferably Sars-CoV-2 contact residue N330 of SEQ ID NO:3 and/or one, two of Sars-CoV contact residues Q325, E329 and N330 of SEQ ID NO:3, Or you can have three.

In a preferred embodiment of the invention, the SRF is at least
- aa 21-53 of SEQ ID NO: 3, as set forth in SEQ ID NO: 34, or derivatives thereof as defined above - aa 56-80 of SEQ ID NO: 3, as set forth in SEQ ID NO: 35, or derivatives thereof as defined above and It has the alpha helical structure of aa 91-100 of SEQ ID NO:3, as shown in SEQ ID NO:36, or a derivative thereof as defined above.
Examples of such SRFs include amino acid sequences shown in SEQ ID NOs: 4-14, 17-22, 25-27 and 29-31, as well as SRFs containing the amino acid sequences shown in SEQ ID NOs: 8 or 13. There is SFT. In a further preferred embodiment of the invention the SRF further has an additional alpha helical structure, i.e. comprising aa 110-129 of SEQ ID NO: 3, as shown in SEQ ID NO: 37, or a derivative thereof as defined above. . Examples of such SRFs include SRFs comprising the amino acid sequences shown in SEQ ID NOs:9 or 14, as well as SFTs comprising the amino acid sequences shown in SEQ ID NOs:4-6, 10, 12 and 17-19.

When the SRF of the invention comprises a second fragment of ACE-2 PD, said second fragment preferably comprises
- aa 304-318 of SEQ ID NO: 3 as set forth in SEQ ID NO: 39, or derivatives thereof as defined above and/or - aa 325-330 of SEQ ID NO: 3 as set forth in SEQ ID NO: 40, or as defined above derivatives,
has an alpha-helical structure of
An example of such a second fragment is shown in SEQ ID NO:15. Examples of SRFs containing such second fragments are shown in SEQ ID NOS: 4-7, 10-12, 17-22 and 25-27.
In a further preferred embodiment of the invention the second fragment of SRF comprises an alpha helical structure, i.e. aa 366-385 of SEQ ID NO: 3, as shown in SEQ ID NO: 41, or a derivative thereof as defined above. .
An example of such a second fragment is shown in SEQ ID NO:23. Examples of SRFs containing 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 is
- aa 400-412 of SEQ ID NO: 3, as set forth in SEQ ID NO: 42, or derivatives thereof as defined above, and/or - aa 415-421 of SEQ ID NO: 3, as set forth in SEQ ID NO: 43, or as defined above Derivatives thereof, including the alpha-helical structure of An example of such a second fragment is the amino acid sequence shown in SEQ ID NO:28. Examples of SRFs containing such second fragments are shown in SEQ ID NOS: 4-6, 10, 12, 17-19, 25-27 and 29-31.

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

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

In a particularly preferred embodiment, the SRF of the invention has at least aa19-103 (relative to the amino acid sequence shown in SEQ ID NO:3) of the PD of ACE-2. Therefore, SRF according to the present invention includes at least the amino acid sequence shown in SEQ ID NO: 8 or derivatives thereof. Said derivatives preferably include at least 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, and preferably consisting of: SEQ ID NO: 34 or derivatives thereof; SEQ ID NO: 35 or derivatives thereof; and SEQ ID NO: 36 or derivatives thereof have at least a structure.

The SRF, ACE-2 PD or fragment, combination of fragments and/or derivatives thereof according to the present invention can be expressed in any suitable expression system such as baculovirus cells, HEK293 cells, CHO or other eukaryotic cells. can be made In some cases, SRF proteins can be expressed in E. coli. Affinity tags such as His-tags or Strep-tags or other standard purification techniques can be used to purify the expressed SRF protein.

Thus, in a preferred embodiment of the invention, the SRF according to the invention may comprise a tag, preferably a His-tag or Strep-tag, MBP-tag or other tags used in standard purification procedures. , and the like.

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

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

An SRF consisting of or comprising the amino acid sequence according to SEQ ID NO: 6 is a further preferred example of an SRF consisting of or comprising a fragment of the amino acid sequence according to SEQ ID NO: 3, which is SEQ ID NO: 3, amino acid 1 of SEQ ID NO: 3 is deleted, ie, it lacks methionine as a translation initiation signal.

An SRF consisting of or comprising the amino acid sequence according to SEQ ID NO: 8 is a further preferred example of an SRF consisting of or comprising a fragment of the amino acid sequence according to SEQ ID NO: 3, which is SEQ ID NO: 8 is composed of amino acids 19-103 of SEQ ID NO:3, and amino acids 1-18 and 104-615 of SEQ ID NO:3 are deleted. An example of an SRF comprising the sequence shown in SEQ ID NO:8 is an SRF consisting of or comprising the sequence shown in SEQ ID NO:13. The SRF having the sequence shown in SEQ ID NO: 13 has, in addition to the sequence shown in SEQ ID NO: 8, methionine as a translation initiation signal and a tag, ie, a His tag.

An SRF consisting of or comprising the amino acid sequence according to SEQ ID NO:9 is a further preferred example of an SRF consisting of or comprising a fragment of the amino acid sequence according to SEQ ID NO:3, which is the sequence This is because number 9 consists of amino acids 19-132 of SEQ ID NO:3 and lacks amino acids 1-18 and 133-615 of SEQ ID NO:3. An example of an SRF comprising the sequence shown in SEQ ID NO:9 is an SRF consisting of or comprising the sequence shown in SEQ ID NO:14. The SRF having the sequence shown in SEQ ID NO: 14 contains, in addition to the sequence shown in SEQ ID NO: 9, methionine as a translation initiation signal and a tag, namely a His tag.

An SRF consisting of or comprising the amino acid sequence according to SEQ ID NO: 17 is a further preferred example of an SRF consisting of or comprising a fragment of the amino acid sequence according to SEQ ID NO: 3, which is SEQ ID NO: 17 is composed of amino acids 19-602 of SEQ ID NO:3, thus deleting amino acids 1-18 and 603-615 of SEQ ID NO:3. An example of an SRF comprising a derivative of the fragment shown in SEQ ID NO:17 is an SRF consisting of or comprising the sequence shown in SEQ ID NO:18 or SEQ ID NO:19. The SRF having the sequence shown in SEQ ID NO: 18 contains the substitution Glu375Gl, i.e. a substitution of glutamic acid to glutamine at position 375 relative to the sequence shown in SEQ ID NO: 3, i.e. at position 375 of SEQ ID NO: 18. . The SRF having the sequence shown in SEQ ID NO: 19 contains, in addition to the sequence shown in SEQ ID NO: 18, methionine as a translation initiation signal and a tag, ie, a His tag.

In a further preferred embodiment of the invention the SRF has more than one fragment of the amino acid sequence according to SEQ ID NO:3, i.e. fragments consisting of 2, 3 or more fragments of the amino acid sequence according to SEQ ID NO:3 including combinations of 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 fragment combinations can be directly linked to each other or linked via linkers. The linker should be flexible enough to be able to bind to a trimer spike subunit or different spike trimer subunits. This linker can be a linker already contained in the link structural module of the ACE-2 receptor, such as the alpha-helix of the full-length protein. In some cases, designed to be stable, flexible enough to allow the variant to fold into a native-like structure, and resistant to protease clefts to increase the half-life of the variant. can be a linked linker. Such linkers preferably have a length of about 5 to about 50 amino acid residues, about 10 to about 30 amino acid residues, or about 15 to about 25 amino acid residues.

In a preferred embodiment, the SRF according to the present invention includes a combination of fragments consisting of the first fragment and the second fragment of SEQ ID NO: 3, the first fragment being selected from the amino acid sequence shown in SEQ ID NO: 8 or a derivative thereof. and the second fragment is selected from the amino acid sequences set forth in SEQ ID NOS: 15, 23, 28 and 32 or derivatives of each. In a preferred embodiment, the SRF according to the present invention includes a combination of fragments consisting of the first fragment and the second fragment of SEQ ID NO:3, and 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 set forth in SEQ ID NOs: 15, 23, 28 and 32 or derivatives of each. In a further preferred embodiment, the second fragment according to SEQ ID NOs:23, 28 and 32 comprises the substitution Glu375Gln, ie a glutamic acid to glutamine substitution at position 375 relative to the sequence shown in SEQ ID NO:3.

Preferably, the first and second fragments of the fragment combination are linked by a linker selected from the amino acid sequences set forth in SEQ ID NOs:16, 24 and 33.

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

In a further preferred embodiment, the SRF of the present invention comprises a combination of fragments set forth in SEQ ID NOS: 20, 21, 22, 25, 26, 27, 29, 30, 31 or derivatives of each of these.

Derivatives with respect to SEQ ID NOS: 3-14, 17-22, 25-27, 29-31 are preferably those with at least 60, 70, 80, 90, 95 or 98% sequence identity to the given sequence. wherein said derivative is preferably at least 5, 6, 7 of Q24, T27, F28, D30, K31, H34, E35, E37, D38, Y41, Q42, L79, M82, Y83 of SEQ ID NO:3 , 8, 9, 10, 11, 12, 13 or 14, and at least an alpha helical structure consisting of: SEQ ID NO: 34 or derivatives thereof; SEQ ID NO: 35 or derivatives thereof; have.

Derivatives with respect to SEQ ID NOs: 34-43 preferably have 1, 2, 3, 4 or 5 deletions, or especially conservative amino acid substitutions, etc., provided that the derivatives are capable of forming an alpha-helical structure. It refers to a derivative containing a mutation such as a substitution of

Preferably, the SRF of the present invention comprises no more domains or sequences of ACE2 in addition to any of the PD fragments or derivatives of ACE-2 described herein, or at least 10 thereof, at least contains no more than 15, at least 20, at least 30, at least 40 at least 50, at least 60 or at least 70 conservative amino acid moieties. In particular the SRF according to the invention preferably does not comprise further domains or sequences according to aa 620-805 of ACE2 shown in SEQ ID NO: 1, or at least 10, at least 15, at least 20, at least 30, at least 40 at least 50 thereof, Does not include portions of at least 60 or at least 70 conservative amino acids.

Thus, the term "comprising" in relation to the definition of SRF according to the present invention means that the SRF is any of the fragments or derivatives of PD of ACE-2 described herein, in particular the following components: "translation initiation methionine as a signal, one or more tags, one or more linkers, proteins, antibodies, or fragments thereof, compounds or molecules further defined below, in addition to one or more components of the above domains of ACE2 or Says it doesn't contain more arrays.

In a preferred embodiment of the invention, the SRF according to the invention does not comprise more than 14, 18 amino acids, more preferably 31 , 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600 amino acids. Preferably, none of these additional amino acids comprise the domains or sequences of ACE2 described above.

The inventors of the present invention have surprisingly found that viruses of the Coronaviridae family, such as the Sars-CoV-2 virus, can be inactivated or neutralized using the soluble receptor fragment (SRF) of the ACE-2 receptor. I found that it is possible. These SRFs consist solely of or include the soluble expressed isolated peptidase domain (PD) of ACE-2 or fragments thereof, combinations of fragments and/or derivatives thereof, herein Yan et al. (2020) The neck domain described in has been truncated. Therefore, the SRF according to the present invention does not include the amino acid sequence shown in SEQ ID NO:2. The active site of PD is preferably inactivated to avoid effects on blood pressure and renal function, as the purpose of the present invention is to suppress Sars-CoV-2 without significantly affecting blood pressure or renal function. This is because it blocks receptor-binding clefts of coronavirus family viruses such as spike protein S. To further prevent viral uptake, or at least to significantly reduce the amount of virus that enters the human body, the inventors have used soluble fragments of host cell receptors, particularly the SRFs of the present invention, in the present invention. It has been found that adaptation to the washing solution can neutralize the virus receptor and preferably wash the neutralized virus away from the point of entry.

Thus, in a highly preferred embodiment of the present invention, PD derivatives, fragments and/or combinations of fragments are preferably 1, 2, 3, 4 active sites of PD such as insertions, additions, deletions or substitutions. , 5, 6, 7, 8, 9 or 10 or more mutations that are inactivated, such as a PD, a fragment or combination of fragments of said PD. In particular, the activity of PD to hydrolyze Angiotensin II (a vasoconstrictor) to Angiotensin (1-7) (a vasodilator) can be inactivated using one or more of the above mutations. Inactivated PD, inactivated fragments of PD or combinations of inactivated fragments have the activity of hydrolyzing Angiotensin II (vasoconstrictor) to Angiotensin (1-7) (vasodilators) is about 60% to about 100%, more preferably about 70% to about 100%, about 80% compared to activated PD, activated fragments of PD or combinations of activated fragments reduced by to about 100% or from about 90% to about 100%.

In a preferred embodiment, the PD of the SRF of the invention comprises 1, 2, 3, 4, 5 or more amino acid insertions, additions, deletions or substitutions, such as those listed in the table below. It is inactivated by the above suitable mutations. A preferred mutation is one that favors inactivation of PD. Suitable mutations can be readily determined by those skilled in the art, eg, by using routine cloning techniques.

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

The SRFs having the sequences shown in SEQ ID NOS: 21, 26 and 30 all have the substitution Glu375Gln, which is a glutamic acid to glutamine substitution at position 375 relative to the sequence shown in SEQ ID NO:3, thus the sequence Substitutions at position 175 of No. 21 and SEQ ID No. 26, and at position 144 of SEQ ID No. 30.

In a further preferred embodiment the PD of the SRF according to the invention comprises, besides the mutation at position 375, in particular the substitution Glu375Gln, 1, 2, 3, 4 or 5 further mutations, which are For example, insertion, addition, deletion or substitution of amino acids listed in the table above.

Depending on the use of SRF according to the invention, SRF and PD of SRF, fragments thereof, combinations of fragments, or derivatives, respectively, can preferably be dimerized. Dimerization can increase the stability of SRF and/or increase the binding affinity of the S protein. Optionally, the PD, fragment thereof, combination of fragments, or derivative of SRF may include insertions, additions, deletions, or substitutions, such as 1, 2, 3, 4, 5, to monomerize the PD. , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more mutations. Monomerization can increase the rate of dispersion in solution.

By applying a solution containing the SRF according to the invention, for example, as an oral rinse or nasal rinse, spikes of free viruses of the coronavirus family, such as Sars-CoV-2, present in these body parts. The binding pocket of a protein (eg, S protein) becomes tethered with SRF, rendering it incapable of infecting the cells of interest. These solutions can also be applied prophylactically to forestall infection. The SRF according to the present invention can also be applied curatively because it inactivates viral particles at sites where it infects the human body without significantly affecting blood pressure or renal function.

In some cases, SRF can be immobilized on beads or other surfaces to trap virus particles, e.g., to create an avidity effect to improve cleaning efficiency, thereby removing virus from infected sites. Particle washout can be facilitated.

In some cases, immobilization of SRF to beads or other surfaces can improve diagnostic quality, or cleaning efficiency, for example. This includes, for example, use in dialysis applications with SRF bound to dialysis membranes or beads for removal of virus particles.

Optionally, the SRF can be anchored or fused to a protein such as the Fc region of IgG1 for application inside the human body.

In some cases, the SRF can be anchored or fused to a protein for application inside the human body and dimerize the PD to increase affinity.

Thus, in a preferred embodiment of the invention, SRF can be immobilized, tethered, bound or linked to beads, membranes such as dialysis membranes, or other surfaces. In further preferred embodiments, the SRF can be immobilized, tethered, linked, bound, or fused to a protein, antibody, antibody fragment such as the Fc region of IgG1, or other compound or molecule.

For example, the SRFs of the present invention can additionally include tags, linkers, proteins, compounds, or molecules that can enhance stability, solubility, dispersibility, half-life in the human body, Or other features of the SRF needed to address the respective application can be facilitated. SRFs in general can be modified to solve the application challenges addressed and as such are known as therapeutic proteins (reviewed for example by Dellas et al. (2021) .) For example, the SRF can additionally comprise a maltose binding protein (MBP) to enhance the stability and/or solubility of the SRF, or an Fc fusion protein for dimerization.

The invention is therefore also used in methods of surgically or therapeutically treating the human or animal body, or as diagnostic methods or vaccines administered to the human or animal body, or with body fluids or other materials from the human or animal body. The SRF according to all the above embodiments of the invention used in the method for

More specifically, the present invention provides an SRF according to all the above embodiments of the present invention for use in a method of treating and/or preventing infection in a subject, particularly by a virus of the Coronaviridae family; Specifically, the SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, which is a variant from the UK, B. 1.1.7 lineage, a mutant strain B. from South Africa. 1.351, B. . 1.617 mutant, or the B. 1.1.28.1 The SRF is provided for use in methods of treating and/or preventing infection by a virus, including any variant, including the 1.1.28.1 variant.

In particular, the SRF according to the present invention is particularly effective in treating and/or preventing viral infections in a subject as long as host cell binding of the viral mutants or variants described above is permitted, and in particular the mutated virus or It is useful in cases where the viral variant still binds to the ACE-2 receptor of potential host cells. To test whether a potential mutation or variant allows binding of host cell receptors, in particular binding of the ACE-2 receptor, a Biacore chip was prepared as described in the Examples below. or by microscale thermophoresis can be used. If such a test results in binding stronger than the standard deviation of the negative control, then the tested virus is a member of the Coronaviridae family, SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS. - Mutants or variants of CoV-2, which can be effectively treated or prevented from viral infections using the SRF of the present invention.

Prevention of viral infection using SRF according to the present invention includes inactivating the virus, particularly by blocking the binding pocket of the viral spike protein, more particularly by binding SRF to the viral binding protein. or neutralizing. Preferably, the prevention of viral infection using SRF according to the present invention includes, in particular, binding the SRF to the binding protein S of the virus, particularly by blocking the binding pocket of the spike protein (S protein) of the virus. This may include inactivating or neutralizing the virus.

It is known that viral replication causes variants or mutations. These mutations may or may not affect viral proteins. Mutations in the viral receptor protein can also occur, but only those mutations that allow host cell receptor binding are replicated. This means that the binding pocket of the viral receptor protein is relatively stable to mutations that affect binding to host cells, which could otherwise impair binding to host cells. and viruses with such mutations will not replicate. Humans are made up of 100 trillion cells. Naturally, the cell does not mutate the biofunctional receptor to avoid viral infection. For this reason, the docking receptors of host cells, especially human cells, are highly conserved. As a result, there is high pressure for coronaviruses not to mutate host cell receptors, or for spike proteins to fail to recognize host cell docking receptors with significant affinity. British lineage mutant B. 1.1.7 (spike mutation (which has high affinity for ACE-2) plus other mutations in the spike) or the mutant strain B. from South Africa. 1.351 (with multiple mutations in the spike protein, including K417N, E484K, and N501Y) shows spike protein binding to the host cell docking receptor ACE-2, although the spike protein is mutated. and therefore the SRF according to the invention is also effective against these mutants.

The SRF of the present invention provides a blocking tool that blocks the viral spike protein to neutralize the virus' ability to infect. The SRF masquerades as a human cell for which the viral docking key seeks. Once docked, the virus cannot replicate because it cannot infect human cells. Inactivated viruses, ie viruses docked to the SRF according to the invention, are destroyed by the immune system. The SRF according to the invention, in particular the SRF according to the invention, which has no enzymatic activity and does not affect the blood pressure system, does not interact with living cells and therefore protects the native human microbiome.

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

In a preferred embodiment, the SRF for use according to the invention is administered in an amount sufficient to reduce the viral load of a virus capable of infecting cells of the subject and/or It is administered in an amount sufficient to inactivate the virus particles at the site of infection. The effective amount of compound to be administered can be readily determined by one of ordinary skill in the art during clinical or preclinical trials using methods well known to physicians or clinicians.

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

According to all embodiments of the present invention, the effective amount of SRF used in the methods of treating and/or preventing viral infections is as required using any of the many known means of administering pharmaceutical agents. can be administered to subjects who are Although the compounds can be administered locally or systemically, local administration is preferred. The route of administration can be nasal, oral, intraocular, topical, systemic, intravenous, inhalation, injection, or topical, or any other suitable route of administration. Accordingly, the SRFs used in the present invention are preferably formulated for nasal, oral, intraocular, topical, systemic, intravenous, or wound cleansing administration.

A further aspect of the invention relates to pharmaceutical compositions or medicaments comprising SRFs according to all embodiments of the invention together with pharmaceutically or physiologically acceptable excipients and/or carriers.

The pharmaceutical composition according to the invention can also additionally contain one or more conventional excipients. Some examples of such additives include solubilizers such as glycerol, antioxidants such as benzalkonium chloride, benzyl alcohol, chlordane or chlorobutanol, and/or isotonic agents.

The above pharmaceutical compositions or medicaments include tablets, troches, candies, drops, chewing gums, lollipops, sprays (especially nasal, oral, mouth, throat or wound sprays), washes (especially nasal, oral, wound or eye washes) , injectables, balms, ointments, eye drops, or oral or throat washes.

The administration of the SRF according to the invention and/or the pharmaceutical composition according to the invention as described above may be specially formulated or designed to prevent viral uptake or at least reduce the amount of virus entering the human body. can.
Administration is therefore preferably carried out to allow for a washing effect or washing solution for neutralization of virus receptors and preferably for allowing neutralized virus to be washed away from the point of entry. is performed as

Furthermore, the present invention also provides a pharmaceutical pack comprising one or more compartments, wherein at least one compartment comprises an SRF of the invention or a pharmaceutical composition of the 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 viral infections, in particular of the coronavirus family. A viral infection caused by a virus, more preferably a viral infection caused by SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, most preferably a variant B from the UK . 1.1.7 lineage, a mutant strain B. from South Africa. 1.351, B. . 1.617 mutant, or the B. 1.1.28.1 for the manufacture of a medicament for the treatment or prevention of viral infections caused by SARS-CoV-2, including the 1.1.28.1 variant.

In a specific embodiment of the invention, the SRF according to the invention and/or the pharmaceutical composition according to the invention is used as a medicament for the treatment or prevention of viral infections, in particular caused by viruses of the Coronavirus family. A viral infection, more preferably a viral infection caused by SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, most preferably a variant strain from the UK, B. 1.1.7 lineage, a mutant strain B. from South Africa. 1.351, B. . 1.617 mutant, or the B. It is used as a drug for the treatment or prevention of viral infections caused by SARS-CoV-2, including the 1.1.28.1 variant.

A further aspect of the present invention is the administration or application of the SRF according to the present invention or the pharmaceutical composition according to the present invention to a subject, particularly a human being or an animal, to treat a viral infection, particularly a viral infection caused by a virus of the coronavirus family. A method of treating or preventing a disease, more particularly a viral infection caused by SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2, most preferably from the United Kingdom. The mutant B. 1.1.7 lineage, a mutant strain B. from South Africa. 1.351, B. . 1.617 mutant, or the B. 1. A method of treating or preventing a viral infection caused by SARS-CoV-2, including the 1.1.28.1 variant.

In a further aspect, the invention provides a method for capturing viral particles, the method comprising: a) immobilizing, tethering, binding or tethering to a bead, column or column material, membrane, or other surface; and b) contacting the liquid sample or fluid with the SRF of step a) under the condition that the SRF is enabled to bind to virus particles. .

Preferably, the viral particle is a viral particle or whole virus, preferably of the Coronaviridae family, more preferably from the group consisting of SARS coronavirus, SARS coronavirus-2, human coronavirus NL63, or SARS-CoV-2. The SARS-CoV-2 strain was selected for SARS-CoV-2, a mutant strain from the UK, B. 1.1.7 lineage, a mutant strain B. from South Africa. 1.351, B. . 1.617 mutant, or the B. All variants are included, such as the 1.1.28.1 variant.

In a preferred embodiment, the method of trapping virus particles is a method of detecting trapped virus particles. The detection method additionally includes detecting the trapped virus particles. Detection can be performed by detecting trapped virus particles. Optionally, the method can include eluting the viral particles and detecting the eluted viral particles. Additionally, methods of capturing viral particles and/or detecting captured viral particles can include additional 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, characterized in that the amount of virus in the liquid sample or fluid has been reduced by capturing the virus particles. do. The method of washing may be suitable to neutralize the virus receptor and preferably allow washing out of the neutralized virus. This method can be used, for example, in dialysis to reduce the viral load in a subject's bodily fluids. In particular, lavage can result in neutralization of viral receptors, and preferably the neutralized virus can be washed out of the subject's bodily fluids. Alternatively or additionally, this may prevent uptake of the virus by the subject's cells, or at least significantly reduce the amount of virus entering the human body. To this end, the SRF according to the invention is preferably immobilized, tethered, bound or linked to a dialysis membrane and the fluid is a bodily fluid such as whole blood, plasma or a blood fraction.

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

のうち一以上の位置での、挿入、付加、欠失または置換などの変異、が含まれていることを特徴とする上記項目の何れか一項に記載のＳＲＦ。
＜１３＞
前記ＳＲＦに、配列番号３、配列番号４、配列番号５、または配列番号６に係るアミノ酸配列、またはその誘導体および／またはフラグメントが含まれ、
特に前記ＳＲＦには、配列番号７、配列番号８、配列番号９、配列番号１０、配列番号１１、配列番号１２、配列番号１３、配列番号１４、配列番号１７、配列番号１８、配列番号１９、配列番号２０、配列番号２１、配列番号２２、配列番号２５、配列番号２６、配列番号２７、配列番号２９、配列番号３０、配列番号３１に係るアミノ酸配列、または配列番号３～１４、配列番号１７～２２、配列番号２５～２７、または配列番号２９～３１と、少なくとも６０％、少なくとも７０％、少なくとも８０％、少なくとも９０％、少なくとも９５％または少なくとも９８％の、配列同一性を有する誘導体が含まれ、
ただし前記誘導体には、
配列番号３のＱ２４、Ｔ２７、Ｆ２８、Ｄ３０、Ｋ３１、Ｈ３４、Ｅ３５、Ｅ３７、Ｄ３８、Ｙ４１、Ｑ４２、Ｌ７９、Ｍ８２、Ｙ８３のうち、少なくとも５、少なくとも６、少なくとも７、少なくとも８、少なくとも９、少なくとも１０、少なくとも１１、少なくとも１２、少なくとも１３、または少なくとも１４、が含まれ、また、
・配列番号３４もしくはその誘導体、
・配列番号３５もしくはその誘導体、および
・配列番号３６もしくはその誘導体
から構成されるアルファヘリックス構造体を少なくとも含む、
という条件が課される、
ことを特徴とする上記何れかの項目に記載のＳＲＦ。
＜１４＞
前記ＳＲＦが、タンパク質、抗体、ＩｇＧ１のＦｃ領域などの抗体フラグメント、または他の化合物もしくは分子に、固定化、束縛、連結、結合、または融合している、ことを特徴とする上記項目の何れか一項に記載のＳＲＦ。
＜１５＞
前記ＳＲＦがビーズ、透析膜等の膜、カラムもしくはカラム材料、または他の表面に、固定化、束縛、結合または連結されていることを特徴とする上記項目の何れか一項に記載のＳＲＦ。
＜１６＞
人体もしくは動物体を手術または治療により処置する方法に使用するための、人体もしくは動物体に施されるワクチンとしてもしくは診断方法で使用するための、または人体もしくは動物体からの体液その他の試料と共に使用するための、上記項目の何れか一項に記載のＳＲＦ。
＜１７＞
ウイルス感染症を治療および／または予防する方法に使用するためのSRFであって、特にコロナウイルス科のウイルスにより生じたウイルス感染症、より詳細にはＳＡＲＳコロナウイルス、ＳＡＲＳコロナウイルス－２、ヒトコロナウイルスＮＬ６３、またはＳＡＲＳ－ＣｏＶ－２により生じたウイルス感染症であって、前記ＳＡＲＳ－ＣｏＶ－２には、英国由来の変異株であるＢ．１．１．７系統、南アフリカ由来の変異株Ｂ．１．３５１、インド由来のＢ．１．６１７変異株、もしくはブラジル由来のＢ．１．１．２８．１変異株などのあらゆる変異体が含まれる、ことを特徴とする項目１～１４の何れか一項に記載のＳＲＦ。
＜１８＞
ウイルス感染症の予防にウイルスの不活性化または中和が含まれ、特にスパイクタンパク質の結合ポケットをブロックすることによる不活性化または中和、より詳細にはウイルスの結合タンパク質に前記ＳＲＦを結合させることによる不活性化または中和、さらに詳細にはコロナウイルス科のウイルスのスパイクタンパク質（Ｓタンパク質）の結合ポケットをブロックすることによる不活性化または中和、より詳細にはウイルスの結合タンパク質Ｓに前記ＳＲＦを結合させることによる不活性化または中和、が含まれていることを特徴とする項目１７に記載の、使用するためのＳＲＦ。
＜１９＞
前記ＳＲＦが、対象者の細胞を感染させることのできるウイルスのウイルス量を低減させるのに十分な量で投与され、および／または、対象者の体内における感染箇所においてウイルス粒子を不活性化させるのに十分な量で投与されることを特徴とする項目１７または１８に記載の、使用するためのＳＲＦ。
＜２０＞
前記ＳＲＦが、経鼻、経口、眼内、局所、全身、静脈内、もしくは創傷洗浄投与のために調合されているか、または吸入もしくは注射による投与のために調合されていることを特徴とする項目１７～１９の何れか一項に記載の、使用のためのＳＲＦ。
＜２１＞
項目１～１４の何れか一項に記載のＳＲＦと、
薬学的または生理的に許容される賦形剤および／またはキャリアと、
を含む医薬組成物または医薬品であって、
特に前記医薬組成物または医薬品が、錠剤、トローチ、キャンディー、ドロップ、チューインガム、ロリポップ、スプレー、特に鼻腔、口腔、口、喉もしくは傷のスプレーとして、洗浄液、特に鼻腔、口腔、傷口もしくは目の洗浄液として、注射液、バーム、軟膏として、目薬として、または口腔もしくは咽頭の洗浄液として、調合されていることを特徴とする医薬組成物または医薬品。
＜２２＞
ａ）項目１４または１５に記載のSRFを提供するステップと、
ｂ）ＳＲＦをウイルス粒子に結合可能にさせる条件下で、液体サンプルまたは流体を、ステップａ）のＳＲＦに接触させるステップと、
を含むことを特徴とするウイルス粒子を捕捉する方法。
＜２３＞
前記方法が捕捉されたウイルス粒子を検知する方法であって、前記方法には捕捉されたウイルス粒子を検知するステップが追加で含まれることを特徴とする項目２２に記載の方法。
＜２４＞
前記方法が液体サンプルまたは流体を洗浄する方法であって、前記液体サンプルまたは流体のウイルスの量が、ウイルス粒子の捕捉によって低減され、特に前記SFRが透析膜に固定化、束縛、結合、または連結されており、また前記流体は全血、血漿、または血液分画などの体液であることを特徴とする項目２２に記載の方法。 <1>
A soluble receptor fragment (SRF) of the ACE-2 receptor, characterized in that said SRF comprises the peptidase domain (PD) of ACE-2 or fragments and/or derivatives thereof (SRF ).
<2>
Said SRF binds to the receptor-binding cleft of the viral spike protein, in particular the viral spike of viruses of the Coronaviridae family, more particularly SARS coronavirus, SARS coronavirus-2, human coronavirus NL63, or SARS-CoV-2. SARS-CoV-2, which binds to the receptor-binding cleft of protein S, contains a mutant strain from the UK, B. 1.1.7 lineage, a mutant strain B. from South Africa. 1.351, B. . 1.617 mutant, or the B. 1. The SRF according to item <1>, which includes all mutants such as the 1.28.1 mutant.
<3>
said fragments and/or derivatives include one, two or more fragments of PD of ACE-2 and/or derivatives of one, two or more fragments of PD of ACE-2; The SRF according to item <1> or <2>, characterized by comprising:
<4>
Said fragments and/or derivatives include 3, 4, 5, 6, 7 of the alpha helical structures composed of PDs of ACE-2 shown in SEQ ID NOS: 34-43 or derivatives thereof , 8, 9 or 10, and in particular said derivative includes 1, 2, 3, 4 or 5, provided that said derivative is capable of forming an alpha-helical structure. The SRF of any one of the preceding items, characterized in that it contains a deletion or mutation.
<5>
wherein said fragment and/or derivative comprises among Sars-CoV-2 contact residues Q24, T27, F28, D30, K31, H34, E35, E37, D38, Y41, Q42, L79, M82, Y83 of SEQ ID NO:3 The SRF of any one of the preceding items, comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.
<6>
said fragment and/or derivative is at least 5 of Sars-CoV-2 contact residues Q24, T27, F28, K31, H34, E37, D38, Y41, Q42, L45, L79, M82, Y83 of SEQ ID NO:3 , 6, 7, 8, 9, 10, 11, 12 or 13.
<7>
said fragment and/or derivative is 1, 2, 3, 4, 5 or 6 of Sars-CoV-2 contact residues N330, K353, G354, D355, R357, R393 of SEQ ID NO: 3 The SRF of any one of the preceding items, comprising:
<8>
said fragment and/or derivative is 1, 2, 3, 4, 5 of Sars-CoV-2 contact residues Q325, E329, N330, K353, G354, D355, R357 of SEQ ID NO: 3 , six or seven.
<9>
said fragments and/or derivatives are
- SEQ ID NO: 34 or a derivative thereof,
- SEQ ID NO: 35 or a derivative thereof; and - SEQ ID NO: 36 or a derivative thereof.
<10>
said fragment and/or derivative has at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence according to SEQ ID NO:8 or with SEQ ID NO:8 comprising at least a derivative,
In particular, said derivatives include
at least 5, 6, 7, 8, 9, 10, 11 of residues Q6, T9, F10, D12, K13, H16, E17, E19, D20, Y23, Q24, L61, M64, Y65 of SEQ ID NO:8; including at least 12, 13 or 14;
Also preferably,
- SEQ ID NO: 34 or a derivative thereof,
- SEQ ID NO: 35 or a derivative thereof, and - an alpha helical structure composed of SEQ ID NO: 36 or a derivative thereof,
The SRF according to any one of the above items, characterized in that:
<11>
said SRF comprises a combination of fragments consisting of two fragments of PD of ACE-2 or a derivative thereof;
In particular, the first fragment is selected from the amino acid sequences set forth in SEQ ID NO: 8 and SEQ ID NO: 9 or derivatives thereof, and the second fragment is selected from the amino acid sequences set forth in SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 28 and SEQ ID NO: 32 or these selected from any derivative,
The SRF according to any one of the above items, characterized in that:
<12>
said SRF comprises an inactivated PD of ACE-2 or a derivative thereof, or an inactivated fragment or combination of fragments of ACE-2 PD;
In particular, said inactivated PD, derivative, fragment or combination of fragments may include the following positions:

A SRF according to any one of the preceding items, characterized in that it contains mutations such as insertions, additions, deletions or substitutions at one or more positions of
<13>
said 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, said SRFs include: 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, amino acid sequence according to SEQ ID NO: 31, or SEQ ID NO: 3-14, SEQ ID NO: 17 -22, SEQ ID NOs:25-27, or SEQ ID NOs:29-31 at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity with be,
However, the derivative
at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14, and
- SEQ ID NO: 34 or a derivative thereof,
- SEQ ID NO: 35 or a derivative thereof; and - SEQ ID NO: 36 or a derivative thereof.
is subject to the condition that
The SRF according to any one of the above items, characterized in that:
<14>
Any of the above items characterized in that the SRF is immobilized, tethered, linked, bound, or fused to a protein, antibody, antibody fragment such as the Fc region of IgG1, or other compound or molecule. The SRF of item 1.
<15>
SRF according to any one of the preceding items, characterized in that said SRF is immobilized, tethered, bound or linked to beads, membranes such as dialysis membranes, columns or column materials, or other surfaces.
<16>
for use in methods of surgically or therapeutically treating the human or animal body, as vaccines administered to the human or animal body, or for use in diagnostic methods, or with body fluids or other samples from the human or animal body The SRF according to any one of the preceding items, for
<17>
SRF for use in a method of treating and/or preventing viral infections, particularly viral infections caused by viruses of the coronavirus family, more particularly SARS coronavirus, SARS coronavirus-2, human coronavirus A viral infection caused by the virus NL63, or SARS-CoV-2, which includes a variant strain from the UK, B. 1.1.7 lineage, a mutant strain B. from South Africa. 1.351, B. . 1.617 mutant, or the B. SRF according to any one of items 1 to 14, characterized in that all variants are included, such as the 1.1.28.1 variant.
<18>
Prevention of viral infection includes inactivation or neutralization of the virus, particularly by blocking the binding pocket of the spike protein, more particularly by binding said SRF to the binding protein of the virus. more particularly by blocking the binding pocket of the spike protein (S protein) of the Coronaviridae virus, more particularly to the binding protein S of the virus. 18. SRF for use according to item 17, comprising inactivating or neutralizing by binding said SRF.
<19>
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 to inactivate viral particles at the site of infection within the subject's body. 19. SRF for use according to item 17 or 18, characterized in that it is administered in an amount sufficient for
<20>
wherein said SRF is formulated for nasal, oral, intraocular, topical, systemic, intravenous, or wound wash administration, or is formulated for administration by inhalation or injection. SRF for use according to any one of clauses 17-19.
<21>
the SRF according to any one of items 1 to 14;
a pharmaceutically or physiologically acceptable excipient and/or carrier;
A pharmaceutical composition or medicament comprising
In particular said pharmaceutical composition or medicament is used as a tablet, troche, candy, lozenge, chewing gum, lollipop, spray, especially nasal, oral cavity, mouth, throat or wound spray, as a washing solution, especially a nasal, oral cavity, wound or eye washing solution. , an injection, a balm, an ointment, an eye drop, or an oral or pharyngeal wash.
<22>
a) providing an SRF according to item 14 or 15;
b) contacting the liquid sample or fluid with the SRF of step a) under conditions that allow the SRF to bind to viral particles;
A method of trapping virus particles, comprising:
<23>
23. The method of item 22, wherein said method is a method of detecting trapped virus particles, said method additionally comprising the step of detecting trapped virus particles.
<24>
Said method is a method of washing a liquid sample or fluid, wherein the amount of virus in said liquid sample or fluid is reduced by capture of virus particles, in particular said SFR is immobilized, bound, bound or linked to a dialysis membrane. and said fluid is a bodily fluid such as whole blood, plasma or a blood fraction.

The following examples illustrate the invention, but are not to be considered limiting. Since various modifications and improvements within the spirit and scope of the invention will become apparent to those skilled in the art from this description, the detailed description and specific specifications disclosed herein, which represent specific embodiments of the invention, will become apparent to those skilled in the art. It should be understood that the examples are provided for illustration only.

Example 1 - Expression and Purification of SRF and Spike Protein in Baculovirus Cells SRF (SRF according to SEQ ID NO: 4 with or without Strep-tagging) was incubated with baculovirus according to the manufacturer's (BD Biosciences) protocol. It was expressed in the system (Sf9). Briefly, 2 μg of the SRF-encoding plasmid (according to SEQ ID NO: 4) was mixed with 0.5 μg of BDBaculoGold linearized baculoviral DNA and left at room temperature for 5 minutes before transfection with 1 ml of BDBaculoGold. Buffer B was added. This mixture was added dropwise to Sf9 insect cells (ATCC) precoated with 1 ml of transfection buffer A, after which the cells were incubated at 28°C for 4 hours, and the transfected cells were transfected with fresh SF-900_II_SFN (Invitrogen company) for an additional 4 days. Supernatants were harvested after 4 days and used to infect cells for an additional 3-day culture cycle for viral amplification. Cell culture supernatants from the 4th cycle were collected and subjected to purification of the SRF protein (according to SEQ ID NO:4).

SRF (SRF according to SEQ ID NO: 5 with or without Strep-tagging) was expressed in the baculovirus system (Sf9) according to the manufacturer's protocol (BD Biosciences). Briefly, 2 μg of the SRF-encoding plasmid (according to SEQ ID NO: 5) was mixed with 0.5 μg of BD_BaculoGold linearized baculoviral DNA and left at room temperature for 5 minutes before transfection with 1 ml of BD_BaculoGold. Buffer B was added. This mixture was added dropwise to Sf9 insect cells (ATCC) pre-coated with 1 ml of transfection buffer A, after which the cells were incubated at 28°C for 4 hours and the transfected cells were transfected with fresh SF-900_II_SFM (Invitrogen company) for an additional 4 days. Supernatants were harvested after 4 days and used to infect cells for an additional 3-day culture cycle for viral amplification. Cell culture supernatants from the 4th cycle were collected and subjected to purification of the SRF protein (according to SEQ ID NO:5).

His-tagged spike protein S (40592-V08B-1) was obtained from Sino Biological. Strep-tag mutants were cloned into expression vectors and solublely expressed in baculovirus SF9 cells to allow proper glycosylation. Purification was performed using Strep-tags or other standard purification techniques.

Example 2 - Determination of binding of SRF to spike protein S in an enzyme-linked immunosorbent assay Microtiter plates coated with SRF (according to SEQ ID NO: 4) were incubated with solubilized spike protein S (Strep-tagged) . Unbound spike protein was washed with PBS buffer by three subsequent washing steps. S protein bound to immobilized SRF (according to SEQ ID NO: 4) was then quantified using streptavidin-conjugated horseradish peroxidase. The binding of S protein to immobilized SRF (according to SEQ ID NO:4) was successfully determined.

Microtiter plates coated with S protein were further incubated with SRF (according to SEQ ID NO:5) containing a Strep-tag. Unbound SRF (according to SEQ ID NO:5) was washed with PBS buffer by three subsequent washing steps. SRF (according to SEQ ID NO:5) bound to immobilized S protein was then quantified using streptavidin-conjugated horseradish peroxidase. The binding of SRF (according to SEQ ID NO:5) to immobilized S protein was successfully determined.

Example 3-Measurement of Affinity with Biacore Chip-Part 1
A Sensor Chip SA with pre-immobilized streptavidin was used to capture SRF tagged with the Strep tag. Spike protein S was then injected and binding to immobilized SRF (according to SEQ ID NO: 4) was measured. A blank flow cell was used for correction of binding reactions. The binding affinity of SRF was measured by using surface plasmon resonance (SPR) with a Biacore system. While the binding of S protein to immobilized SRF (according to SEQ ID NO:4) was successfully determined, S protein was incubated with solubilized SRF (according to SEQ ID NO:4) and injected into the Biacore system. However, a distinctly reduced binding was observed for immobilized SRF (according to SEQ ID NO:4). No binding was observed when the S protein (according to SEQ ID NO:4) was incubated with soluble SRF at high concentration and then injected onto a Biacore chip with immobilized SRF (according to SEQ ID NO:4).

Example 4 - Viral Inactivation Assay: Treatment of VeroE6 Cells with SRF VeroE6 cells were seeded in 48-well plates in DMEM containing 10% FBS. Twenty-four hours after seeding, SRF was mixed (1:1) at various virus concentrations in a final volume of 100 ml/well in DMEM (0% FBS) at 37°C. After 30 minutes Vero-E6 was infected with SRF or a mixture containing SRF/SARS-CoV-2 for 1 hour followed by washing or no washing for 15 hours and cells were washed 3 times with PBS. and 500 ml of fresh complete medium supplemented with SRF was added. Fifteen hours post-infection, the supernatant was removed, the cells were washed three times with PBD, lysed with Trizol, and analyzed by qRT-PCR for viral RNA detection. Infection of VeroE6 cells with virus was used as a positive control. Infection of VeroE6 cells with SRF only was used as a negative control. Mixing SRF and SARS-CoV-2 before infection of VeroE6 cells showed a clear decrease in infectivity. No significant difference was obtained between application of SRF (according to SEQ ID NO: 4) and SRF (according to SEQ ID NO: 5).

Example 5 Expression and Purification of SRF and Spike Protein in HEK293 Cells Genes encoding SRF comprising amino acid sequences according to SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:4, SEQ ID NO:8 and SEQ ID NO:9 were transfected into mammalian HEK293 cells. was cloned into the standard expression vector pTZ (Trenzyme) suitable for transient expression in the supernatant of . For translation and purification reasons, the amino acid sequences listed above were complemented with methionine and His-tag as translation signals, respectively. Thus, the encoded sequence actually used for cloning, including methionine as translation initiation signal, secretion signal, and His-tag, is as follows:
- (3) ACE2_19-615_E375Q represented by SEQ ID NO: 10 (including the amino acid sequence represented by SEQ ID NO: 5);
(4) ACE — 2 — 19-103; 301-365 shown in SEQ ID NO: 11 (including the amino acid sequence shown in SEQ ID NO: 7);
(9) ACE2 — 19-615 represented by SEQ ID NO: 12 (including the amino acid sequence represented by SEQ ID NO: 4);
(1) ACE2_19-103 represented by SEQ ID NO: 13 (including the amino acid sequence represented by SEQ ID NO: 8), and (8) ACE2_19 represented by SEQ ID NO: 14 (including the amino acid sequence represented by SEQ ID NO: 9) -132.

Protein expression was performed in shake flasks and the target protein was present in the cell culture supernatant. Supernatants were collected after expression and target proteins were purified using His-tag affinity chromatography.

Constructs (1) and (8) were expressed as MBP fusions by fusing each construct to MBP via a short linker. The encoded sequence contained a methionine as translation initiation signal, a secretion signal, MBP, a short linker and a His-tag. Protein expression was performed in shake flasks and the target protein was present in the cell culture supernatant. Supernatants were collected after expression and target proteins were purified using His-tag affinity chromatography.

Example 6-Measurement of Affinity with Biacore Chip-Part 2
SARS-CoV-2 spike protein S1 (aa14-683), three SRF variants ((3) _ACE2_19-615_E375Q, 1.24 mg/ml; (4) _ACE_2_19-103; 301-365, 1 mg/ml; (9) _ACE2_19-615, 0.74 mg/ml) of the His-Avi-tagged recombinant protein (Invitrogen, Cat # RP-87681) was measured on a Cytiva Biacore X100 using the Biotin CAPture kit from SensorChip CAP. went. The buffer was PBS, pH 7.4. After equilibration and adjustment of the Biacore system, the CAP chip was coated with biotin CAPture reagent. Immobilization of spike protein S1 on the flow cell yielded ˜100 RU (ligand concentration 0.43 μM; contact time: 420 sec; stabilization time: 300 sec). Relative binding responses were measured at the highest concentrations of ACE-2 variants without flow cell 2 being referenced for non-specific reference binding (contact time: 120 sec; dissociation time: 600 sec; regeneration ).

Three initiation cycles were made with buffer.
Results were as follows:
(3)_ACE2_19-615_E375Q 443.6RU
(4)_ACE_2_19-103; 301-365, 1 mg/ml 352.0 RU
(9)_ACE2_19-615 55.1RU

result:
The binding of the spike protein of Sars-CoV-2 immobilized on Biacore chips was observed for all three proteins tested, with variant (3)_ACE2_19-615_E375Q showing the highest binding response.
Variants (4)_ACE_2_19-103; 301-365 showed less binding and variant (9)_ACE2_19-615 showed the least binding.
(9) _ACE2_19-615 showed the lowest binding compared to other constructs, but binding to SARS-CoV-2 spike protein S1 was clearly confirmed.

Example 7 Measurement of Binding Affinity by Microscale Thermophoresis Microscale thermophoresis was used to measure the affinity of the Sars-CoV-2 spike protein to its respective target protein. In principle, the binding affinity of the two molecules was detected by detecting the change in fluorescent signal caused by the temperature change induced by the IR laser. The extent of change in fluorescent signal correlates with the binding of the ligand to the fluorescent target. This enables quantitative analysis of molecular interactions in solution with high sensitivity on the microliter scale.

Fluorescent labeling of spike proteins:
SARS-CoV-2 spike protein S1 (aa 14-683), His-tag, Avi-tag; Invitrogen, catalog number: RP87681, stock concentration of 10.0 μM in 1×PBS buffer at pH 7.4.

Target protein was diluted in labeling buffer (1×PBS, pH 7.4, 0.005% Tween) to reach a labeling concentration of 9.7 μM. DMSO was introduced into the labeling buffer as dye stock solutions were prepared with DMSO. The dye labeling concentration was 20.1 μM (3:1=stain:protein molar ratio). The fluorescent dye RedNHS650 ^{2nd gen} was used. Labeling was performed at 25°C for 30 minutes. After labeling, unbound dye was removed by a gel filtration step, bringing the labeled protein into the final buffer. Addition of 0.005% Tween 20 reduced protein stickiness/stickiness and increased protein recovery. The concentration of labeled target protein was determined by measuring dye fluorescence. Total protein concentration and protein recovery were measured using the instrument TychoNT. 6 was determined by relative fluorescence measurements. Labeling was performed to introduce approximately two fluorescent labels per protein.

Binding measurements:
(9) Variant: Measurement setup for ACE2_19-615
Target: SARS_CoV2_Spike S1, used at constant 10 nM Ligand: ACE2_19-615, 16 1:1 dilution steps from 4.73 μM Titration instrument: Monolith_NT. 115_Pico
Buffer: 1×PBS pH 7.4, 0.005% Tween 20

(3) Measurement setup for ACE2_19-615 E375Q Target: SARS_CoV2_Spike S1, used at constant 10 nM Ligand: (3) ACE_2_19-615_E375Q, titration instrument: Monolith_NT. 115_Pico
Buffer: 1×PBS pH 7.4, 0.005% Tween 20

(4) Measurement setup for _ACE_2_19-103;301-365 Target: SARS_CoV2_Spike S1, used at constant 10 nM Ligand: (4) _ACE_2_19-19-103;301-365, 1:1 dilution of 16 from 22.9 μM Titration instrument in steps: Monolith_NT. 115_Pico
Buffer: 1×PBS pH 7.4, 0.005% Tween 20

(1) Measurement setup for ACE2_19-103 fused with MBP Target: SARS_CoV2_Spike S1, constant 10 nM used Ligand: (1) ACE2_19-103 fused with MBP, 1:1 dilution of 16 from 1.26 μM Titration instrument in steps: Monolith_NT. 115_Pico
Buffer: 1×PBS pH 7.4, 0.005% Tween 20

(8) Measurement setup for ACE219-132 fused with MBP Target: SARS_CoV2_Spike S1, constant 10 nM used Ligand: (8) ACE2_19-132 fused with MBP, 1:1 dilution of 16 from 1.29 μM Titration instrument in steps: Monolith_NT. 115_Pico
Buffer: 1×PBS pH 7.4, 0.005% Tween 20

（８）ＭＢＰに融合したバリアントＡＣＥ２＿１９－１３２と（１）ＭＢＰと融合したＡＣＥ２＿１９－１０３が最も良好な親和性を示した。他の３つのバリアントはこれより小さな結合であり、
（３）＿ＡＣＥ２＿１９－６１５＿Ｅ３７５Ｑ＿は、＿（４）＿＿ＡＣＥ＿２＿１９－１０３；３０１－３６５＿（ｅｘ ＡＣＥ２＿１９－１０３）よりも結合性が強く、バリアント（９）＿ＡＣＥ２＿１９－６１５が最も小さな結合性を示した。（９）＿ＡＣＥ２＿１９－６１５は他のコンストラクトと比較して最も低い結合性を示したものの、ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質Ｓ１への結合性は明確に確認された。 result:

(8) variant ACE2_19-132 fused to MBP and (1) ACE2_19-103 fused to MBP showed the best affinity. The other three variants are smaller bindings,
(3)_ACE2_19-615_E375Q_ bound more strongly than _(4)__ACE_2_19-103; 301-365_(ex ACE2_19-103), and variant (9)_ACE2_19-615 showed the lowest binding. (9) _ACE2_19-615 showed the lowest binding compared to other constructs, but binding to SARS-CoV-2 spike protein S1 was clearly confirmed.

Example 8 - Neutralizing plaque assay of SARS-CoV-2 (strain B.3) using recombinant SRF variants
sample:
DMEM medium: Thermo Fisher 41965039
FBS: PAN-Biotech #P30-3702
Penicillin/streptomycin: Sigma #P4333
OptiPro-SFM: Thermo Fisher #12309019
DPBS (no Ca/Mg): 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, 1.25×10 ⁵ VeroE6 cells/well were plated in 24-well plates and incubated for 24 hours at 37° C., 5% CO ₂ . On day 1, serial dilutions of recombinant SRF variants were performed as follows.

Stock concentration:
Variant_19-615_E375Q: Mw = 70.6 kDa, c = 1.24 mg/m
→17,563 μM
Variant_19-615: Mw=70.6 kDa, c=0.74 mg/ml
→ 10,481 μM
SRF variants were thawed on ice. Round-bottom 96-well plates were prepared with three 120 μl SRF variant dilutions in OptiPro-SFM (serum-free medium). An additional 12 wells were filled with 120 μl OptiPro-SFM for controls and several 120 μl vehicle control samples were prepared with PBS in OptiPro-SFM. The plate was sealed without air bubbles.

Thaw one aliquot of SARS-CoV-2 (B.3 strain, isolated in February 2020, 1.8×10 ⁶ PFU/ml) grown in vitro in the laboratory in viral dilution BSL-3 and a 1:2250 predilution in OptiPro-SFM (final dilution 1:4500, 80 PFU/200 μl). For each well consisting of protein diluents and wells consisting of positive controls, 120 μl of the diluent is required.

A 1:2250 virus dilution of the SRF variant/virus co-culture was poured into the vessel. Using a multichannel pipette, 120 μl of virus diluent was added to each of the SRF variant diluent and positive control wells. Plates were sealed and incubated for 1 hour at 37° C., 5% CO ₂ .

Using a VeroE6 cell infection vacuum pump or a P1000, media was removed from up to 6 wells at a time of a 24-well plate and 200 μl of SRF variant diluent/virus mixture, virus (positive control), or OptiProSFM (negative control) was added. ) was added. After the first 24 plates were completed, the timer was started for counting and the plates were placed back into the incubator. Continue in this manner until all samples in the 96-well plate have been added to the cells. A timer was recorded each time the plate was completed. Each cell was incubated at 37°C for 1 hour.

Post-infection medium change 1.5% (w/v) carboxymethylcellulose (autoclave sterilized) and 2x MEM (sterile filtration, 2x Pen/Strep, 4% FBS, and _NaHCO3 for pH) 1: 1 mixture was prepared and preheated in a hot water bath. After the timer reached 1 hour after virus addition, the supernatant of each 24-well plate was aspirated (using vacuum or P1000) and replaced with 1 ml carboxymethylcellulose-MEM/well. The plate was returned to the incubator and cultured for 72 hours.

On day 4, cells were fixed and stained. Using a vacuum or a 10 ml serological pipette, a portion of the supernatant was removed from each well without contacting or coming close to the bottom surface of the well. All 24-well plates were submerged in 6% formaldehyde in a fixation/transport container and lids were added to the 24-well plates. Each plate was left at room temperature for 30 minutes. The outside of the container was wiped with a disinfecting Pursept™ wipe and the container was removed from the BSL-3. The container was opened and as much formaldehyde as possible was poured back into the container. Each well was washed 3 times with tap water. A 1% crystal violet staining solution was then added to each well, the lid was closed and the plate was incubated at room temperature for 30 minutes. The crystal violet was funneled back into the bottle, the plate was washed with tap water until the drain was no longer blue, and the plate and lid were allowed to air dry completely. The plaques in each well were counted.

result:

The table above and the results shown in FIGS. 1 and 2 reveal the following. Neutralization studies performed at 80 pfu/ml: Variants 4_ACE_2_19-103; 301-365 did not show significant reduction in pfu at all concentrations tested. Variant 9_ACE2_19-615 showed a small reduction already at 1 nM and a 50% reduction at 250 nM. Adaptation of variant 3_ACE2_19-615_E375Q to the neutralization assay resulted in a large reduction of up to about 40% at all concentrations tested. Neutralization studies performed at 40 pfu/ml: Variants 4_ACE_2_19-103; 301-365 did not show significant reduction in pfu at all concentrations tested. Variant 9_ACE2_19-615 showed 50% inhibition at 250 nM and complete inhibition at 1.5 μM. Application of variant 3_ACE2_19-615_E375Q resulted in a significant reduction in the concentration range from 250 nM to 1 μM. Nearly complete reduction was observed at 1.5 μM and 2 μM.

Neutralizing Plaque Assay of SARS-CoV-2 UK Mutant (B.1.1.7) Using Recombinant SRF Variants Mutants of SARS-CoV-2, namely Rambaut, A., Loman, N., Pybus , O., Barclay, W., Barrett, J., Carabelli, A., Connor, T., Peacock, T., Robertson, DL, and Volz, E. (2020). Neutralizing plaque assays with recombinant SRF variants described in Example 8 were performed using the UK mutant (B.1.1.7). Preliminary genomic evaluation of the new UK SARS-CoV-2 lineage defined by the new spike mutation set.

図４と上記表に認められる通り、バリアント９＿ＡＣＥ２＿１９－６１５＿およびバリアント３＿ＡＣＥ２＿１９－６１５＿Ｅ３７５Ｑは、２５０ｎＭで既に大きな減少が認められ、１μＭで完全な減少が認められた。 result:

As can be seen in FIG. 4 and the table above, variants 9_ACE2_19-615_ and 3_ACE2_19-615_E375Q showed a large reduction already at 250 nM and a complete reduction at 1 μM.

References

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

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

Jiancheng Zhang, Bing Xie and Kenji Hashimoto, Current status of potential therapeutic candidates for the COVID-19 crisis, Brain, Behavior, and Immunity, online available since 22 April 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. 2020 Apr 24;11(1):2070.

Monteil V, Kwon H, Prado P, Hagelkrueys 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. 2020 Apr 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.; 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, Andre NM, Jaimes JA, Millet JK, Whittaker GR. Coronaviruses in cats and other companion animals: Where does SARS-CoV-2/COVID-19 fit Vet Microbiol. 2020 Aug;247:108777.

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

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

Claims (15)

A soluble receptor fragment (SRF) of the ACE-2 receptor, characterized in that said SRF comprises the peptidase domain (PD) of ACE-2 or fragments and/or derivatives thereof (SRF ). Said SRF binds to the receptor-binding cleft of the viral spike protein, in particular the viral spike of viruses of the Coronaviridae family, more particularly SARS coronavirus, SARS coronavirus-2, human coronavirus NL63, or SARS-CoV-2. SARS-CoV-2, which binds to the receptor-binding cleft of protein S, contains a mutant strain from the UK, B. 1.1.7 lineage, a mutant strain B. from South Africa. 1.351, B. . 1.617 mutant, or the B. 2. The SRF of claim 1, including all variants, such as the 1.1.28.1 variant. said fragments and/or derivatives include one, two or more fragments of PD of ACE-2 and/or derivatives of one, two or more fragments of PD of ACE-2; 3. The SRF of claim 1 or 2, comprising: said fragments and/or derivatives are
3, 4, 5, 6, 7, 8, 9 or 10 of the alpha helical structures composed of PDs of ACE-2 shown in SEQ ID NOS: 34-43 or derivatives thereof optionally (i) 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 of which 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 and/or
(ii) 5, 6, 7 of Sars-CoV-2 contact residues Q24, T27, F28, K31, H34, E37, D38, Y41, Q42, L45, L79, M82, Y83 of SEQ ID NO:3 one, eight, nine, ten, eleven, twelve or thirteen,
and optionally,
(iii) 1, 2, 3, 4, 5 or 6 of Sars-CoV-2 contact residues N330, K353, G354, D355, R357, R393 of SEQ ID NO:3, and/or ,
(iv) 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 One
The SRF according to any one of claims 1 to 3, comprising: said 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, said inactivated PD, derivative, fragment or combination of fragments may include the following positions:

SRF according to any one of claims 1 to 4, characterized in that it contains mutations such as insertions, additions, deletions or substitutions at one or more positions of said 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, said SRFs include: 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, amino acid sequence according to SEQ ID NO: 31, or SEQ ID NO: 3-14, SEQ ID NO: 17 -22, SEQ ID NOs:25-27, or SEQ ID NOs:29-31 at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity with be,
However, the derivative
at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14, and
- SEQ ID NO: 34 or a derivative thereof,
- SEQ ID NO: 35 or a derivative thereof; and - SEQ ID NO: 36 or a derivative thereof.
is subject to the condition that
The SRF according to any one of claims 1 to 5, characterized in that: Claims 1-6, characterized in that said SRF is immobilized, tethered, linked, conjugated or fused to a protein, antibody, antibody fragment such as the Fc region of IgG1, or other compound or molecule. The SRF according to any one of . 8. A method according to any one of claims 1 to 7, characterized in that the SRF is immobilized, tethered, bound or linked to a bead, a membrane such as a dialysis membrane, a column or column material or other surface. 's SRF. for use in methods of surgically or therapeutically treating the human or animal body, as vaccines administered to the human or animal body, or for use in diagnostic methods, or with body fluids or other samples from the human or animal body The SRF according to any one of claims 1 to 8, for SRF for use in a method of treating and/or preventing a viral infection, said viral infection in particular caused by a virus of the Coronaviridae family, more particularly SARS coronavirus, SARS coronavirus A viral infection caused by virus-2, human coronavirus NL63, or SARS-CoV-2, in which SARS-CoV-2 includes a variant strain from the United Kingdom, B. 1.1.7 lineage, a mutant strain B. from South Africa. 1.351, B. . 1.617 mutant, or the B. 1.1.28.1 variants, including all variants,
In particular prevention of viral infections includes inactivation or neutralization of the virus, in particular by blocking the binding pocket of the spike protein, more particularly binding said SRF to the binding protein of the virus. more particularly by blocking the binding pocket of the spike protein (S protein) of the Coronaviridae virus, more particularly the viral binding protein S SRF according to any one of claims 1 to 8, characterized in that inactivation or neutralization by binding said SRF to is included. 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 to inactivate viral particles at the site of infection within the subject's body. 11. SRF for use according to claim 10, characterized in that it is administered in an amount sufficient for Claims characterized in that said SRF is formulated for nasal, oral, intraocular, topical, systemic, intravenous, or wound wash administration, or is formulated for administration by inhalation or injection. SRF for use according to any one of clauses 10-11. The SRF according to any one of claims 1 to 7;
a pharmaceutically or physiologically acceptable excipient and/or carrier;
A pharmaceutical composition or medicament comprising
In particular said pharmaceutical composition or medicament is used as a tablet, troche, candy, lozenge, chewing gum, lollipop, spray, especially nasal, oral cavity, mouth, throat or wound spray, as a washing solution, especially a nasal, oral cavity, wound or eye washing solution. , an injection, a balm, an ointment, an eye drop, or an oral or pharyngeal wash. a) providing an SRF according to claim 7 or 8;
b) contacting the liquid sample or fluid with the SRF of step a) under conditions that allow the SRF to bind to viral particles;
A method of trapping virus particles, comprising: said method comprising:
(i) a method of detecting trapped viral particles, said method additionally comprising the step of detecting trapped viral particles, or (ii) washing a liquid sample or fluid A method, wherein the amount of virus in said liquid sample or fluid is reduced by capture of viral particles, in particular said SRF is immobilized, bound, bound or linked to a dialysis membrane, and said fluid is whole blood , plasma, or a body fluid such as a blood fraction,
15. The method of claim 14, wherein:

Images