WO2022207918A1

WO2022207918A1 - COVID-19 Therapy

Info

Publication number: WO2022207918A1
Application number: PCT/EP2022/058783
Authority: WO
Inventors: Gerald WIRNSBERGER
Original assignee: Apeiron Biologics Ag
Priority date: 2021-04-02
Filing date: 2022-04-01
Publication date: 2022-10-06

Abstract

The present invention provides a method of a treatment or prophylaxis of a SARS-CoV-2 infection in a subject comprising administering an ACE2 polypeptide to the subject, wherein the SARS-CoV-2 infection is with a SARS-CoV-2 variant exhibiting an increase of ACE2 polypeptide binding affinity for SARS-CoV-2 spike protein mutants in comparison to wild type SARS-CoV-2 spike protein and a method of a treatment or prophylaxis of a coronavirus infection in a subject comprising administering an ACE2 polypeptide to the subject, wherein the ACE2 polypeptide is administered by inhalation, wherein a solution of ACE2 polypeptides is aerosolized into aerosol particles with an average particle size of 0.1 µm to 100 µm and at a dose of 100 µg to 600 mg daily.

Description

COVID-19 Therapy

The present invention relates to the field of COVID-19 treatment methods.

Background of the invention

Early in the COVID-19 pandemic, sequencing of SARS-CoV-2 en- abled recognition of the high degree of homology with SARS-CoV-1 and the identification of ACE2 as a receptor for both viruses. Blocking of this interaction thus emerged as an anti-SARS-CoV-2 strategy. Proteins or peptides interacting with either of the binding partners could have therapeutic potential and some very high affinity binders have been reported. Likewise, engineered antibodies can inhibit virus/receptor interaction and two have received Emergency Use Authorization from the FDA as systemic therapeutics. Although these strategies proved effective in the case of antibodies, current approaches targeting viral proteins are in principle vulnerable to viral mutations altering the tar- geted epitopes. This is all the more concerning given the spread of mutant viral strains carryincj substitutions/mutations affect- ing interactions with the host receptor ACE2.

Recombinant ACE2 has been developed to reduce damage to the lung as observed in virus induced ARDS by cleaving Ang II (Imai et al., 2007, Cell. Mol. Life Sci. 64, 2006-2012). In vitro sol- uble recombinant human ACE2 has neutralizing activity against SARS-CoV-2 infection (Monteil, 2020, Cell 181, 905-913). Clini- cal trials for treatment of COVID-19 using intravenously admin- istered ACE2 are ongoing.

The search for improved Covid-19 therapies is ongoing and improved pharmaceutical treatments are needed. It is a goal of the invention to provide such improved therapeutic methods.

Summary of the invention

The present invention provides a method of a treatment or prophylaxis of a SARS-CoV-2 infection in a subject comprising administering an ACE2 polypeptide to the subject, wherein the SARS-CoV-2 infection is with a SARS-CoV-2 variant exhibiting an increase of ACE2 polypeptide binding affinity for SARS-CoV-2 spike protein mutants in comparison to wild type SARS-CoV-2 spike protein. Related thereto, the invention further provides an ACE2 pol- ypeptide for use in the treatment or prophylaxis of COVID-19 caused by SARS-CoV-2 in a subject, wherein the SARS-CoV-2 infec- tion is with a SARS-CoV-2 variant exhibiting an increase of ACE2 polypeptide binding affinity for SARS-CoV-2 spike protein mu- tants.

Also provided is a method for the manufacture of a medica- ment comprising an ACE2 polypeptide for the treatment or prophy- laxis of a SARS-CoV-2 infection in a subject, wherein the SARS- CoV-2 infection is with a SARS-CoV-2 variant exhibiting an in- crease of ACE2 polypeptide binding affinity for SARS-CoV-2 spike protein mutants in comparison to wild type SARS-CoV-2 spike pro- tein.

Further provided is a method of a treatment or prophylaxis of a coronavirus infection in a subject comprising administering an ACE2 polypeptide to the subject, wherein the ACE2 polypeptide is administered by inhalation, wherein a solution of ACE2 poly- peptides is aerosolized into aerosol particles with an average particle size of 0.1 pm to 100 pm and at a dose of 100 pg to 600 mg daily.

Related thereto, the invention provides an ACE2 polypeptide for use in a treatment or prophylaxis of a coronavirus infection in a subject comprising administering an ACE2 polypeptide to the subject, wherein the ACE2 polypeptide is administered by inhala- tion, wherein a solution of ACE2 polypeptides is aerosolized into aerosol particles with an average particle size of 0.1 pm to 100 pm and at a dose of 100 pg to 600 mg daily.

Even further provided is method for the manufacture of a me- dicament comprising an ACE2 polypeptide for use in a treatment or prophylaxis of a coronavirus infection in a subject compris- -ng administering an ACE2 polypeptide to the subject, wherein the ACE2 polypeptide is for administration by inhalation, wherein a solution of ACE2 polypeptides is for aerosolization into aerosol particles with an average particle size of 0.1 pm to 100 pm and at a dose of 100 pg to 600 mg daily.

All aspects and embodiments are related to each other and can be combined. Preferred embodiments are disclosed for all as- pects of the invention. Detailed description of the invention

Provided is a method of a treatment or prophylaxis of a SARS-CoV-2 infection in a subject comprising administering an ACE2 polypeptide to the subject, wherein the SARS-CoV-2 infec- tion is with a SARS-CoV-2 variant exhibiting an increase of ACE2 polypeptide binding affinity for SARS-CoV-2 spike protein mu- tants in comparison to wild type SARS-CoV-2 spike protein. Fur- ther provided is a method of a treatment or prophylaxis of a coronavirus infection in a subject comprising administering an ACE2 poly-peptide to the subject, wherein the ACE2 polypeptide is administered by inhalation, wherein a solution of ACE2 poly- peptides is aerosolized into aerosol particles with an average particle size of 0.1 pm to 100 pm and at a dose of 100 pg to 600 mg daily. These methods can of course be combined and any par- ticular embodiments described further herein relate two both methods. Generally, the invention relates to improvements of ACE2-based therapies by the identification of therapeutic tar- gets and modes with improved efficacy, such as selecting partic- ular SARS-CoV-2 variant as therapy targets, or using inhalation of aerosols as improved delivery system, which works with the SARS-CoV-2 variants and also wild type SARS-CoV-2.

A "method of treatment" or just "treatment" as used herein refers to a therapy of a subject that is infected with SARS-CoV- 2. The infection may be symptomatic or asymptomatic. Neverthe- less SARS-CoV-2 should be detectable in the subject.

A "method of prophylaxis" or just "prophylaxis" refers to a pre-emptive treatment to reduce the risk of gaining a disease of SARS-CoV-2, i.e. COVID-19. A prophylaxis may be administered to a subject that is at risk of getting an infection with SARS-CoV- 2, such as by exposure with the virus SARS-CoV-2 or to a subject who is vulnerable to SARS-CoV-2 such as in case of a higher sus- ceptibility to a viral infection, e.g. by immunosuppression and/or age.

The present invention includes providing an ACE2 polypeptide for administration to a subject or the method step of adminis- tering an ACE2 polypeptide to the subject.

ACE2 is a key metalloprotease of the Renin Angiotensin Sys- tem (RAS), primarily existing as a membrane anchored zinc metal- loprotease (WO 2004/000367). ACE2 is expressed in the vascular system as well as in most organs, but predominantly in the lungs, kidneys, liver, heart, intestine and testis. In a normal adult human lung, ACE2 is expressed primarily in alveolar epi- thelial type II cells, which can serve as a viral reservoir. These cells produce surfactant which reduces surface tension, thus preventing alveoli from collapsing, and hence are critical to the gas exchange function of the lung.

Many variants of ACE2 have been generated for therapeutic uses, including recombinant soluble ACE2 and shorter fragments suitable for glomerular filtration (WO 2008/151347, US 10,443,049 B2). In particular the C-terminus is suitable for large deletions while still maintaining activity of the enzyme.

The ACE2 polypeptide of the invention binds to the Spike glycoprotein of SARS-CoV-2 as was investigated in several refer- ences (Walls et al., Cell (2020) 181(2): 281-292; Wan et al., J Virol (2020) doi:10.1128/JVI.00127-20; Wrapp et al., Science (2020) doi:10.1126/science.abb2507), so that it can act as mo- lecular decoy to block virus entry.

The amino acid sequence of human recombinant ACE2 amino ac- ids 1 to 740 is provided in SEQ ID NO: 1. SEQ ID NO: 2 provides the full-length amino acid sequence of human ACE2 with 805 amino acids in length. The full-length amino acid sequence is also provided in database UniProtKB, database entry Q9BYF1 as of 17 June 2020 (human ACE2). Amino acids 1-17 are the signal se- quence, amino acids 18-740 form the extracellular domain, amino acids 741-761 are the transmembrane domain and amino acids 762- 805 are the cytoplasmic domain. Amino acids 1 to 740 of SEQ ID NO:2 are identical to SEQ ID NO: 1. Corresponding amino acids and domains exist in other ACE2 polypeptides, including ACE2 from other mammals.

ACE2 polypeptides of the invention can be variants of natu- rally occurring ACE2 proteins. Such ACE2 variants may be used in methods and products of the invention. Changes which result in production of a chemically equivalent or chemically similar amino acid sequence are included within the scope of the inven- tion. Variants of ACE2 may occur naturally, for example, by mu- tation, or may be made, for example, with polypeptide engineer- ing techniques such as site directed mutagenesis, which are well known in the art for substitution of amino acids. For example, a hydrophobic residue, such as glycine can be substituted for an- other hydrophobic residue such as alanine. An alanine residue may be substituted with a more hydrophobic residue such as leu- cine, valine or isoleucine. A negatively charged amino acid such as aspartic acid may be substituted for glutamic acid. A posi- tively charged amino acid such as lysine may be substituted for another positively charged amino acid such as arginine.

Therefore, the invention includes polypeptides having con- servative changes or substitutions in amino acid sequences. Con- servative amino acid substitutions insert one or more amino ac- ids, which have similar chemical properties as the replaced amino acids. The invention includes sequences where conservative amino acid substitutions are made that do not destroy enzymatic activity and/or binding to Spike glycoprotein of SARS-CoV-2. Amino acids 147-555 of SEQ ID NO: 1 or 2 are considered im- portant for catalytic activity and should preferably be retained with a high degree in the ACE2 polypeptide of the invention.

Such as with the ACE2 polypeptide of the invention comprising a sequence with a sequence identity of at least 90%, preferably at least 95& or at least 98% or at least 99%, to amino acids 147- 555 of SEQ ID NO: 1 or 2. Other amino acid changes may lead to a loss of enzymatic activity, however maintaining or even increas- ing enzymatic activity and/or binding to Spike glycoprotein of SARS-CoV-2 is preferred.

Identity is calculated according to methods known in the art. Sequence identity is most preferably assessed by the BLAST version 2.1 program advanced search (parameters as above). BLAST is a series of programs that are available online at blast.ncbi.nlm.nih.gov/. The BLAST search may be set to default parameters (i.e. Matrix BLOSUM62; Gap existence cost 11; Per residue gap cost 1; Lambda ratio 0.85 default). References to BLAST searches are: Altschul et al., J. Mol. Biol. (1990) 215: 403-410; Gish & States, Nature Genet. (1993) 3: 266-272; Madden et al., Meth. Enzymol. (1996) 266: 131-141; Altschul et al. Nu- cleic Acids Res. (1997) 25: 3389-3402. "Conservative amino acid substitutions" are those substitu- tions that are predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference protein. The following Table A pro- vides a list of exemplary conservative amino acid substitutions:

Original Conservative

Residue Substitution

Ala Gly, Ser

Arg His, Lys

Asn Asp, Gin, His

Asp Asn, Glu

Cys Ala, Ser

Gin Asn, Glu, His

Glu Asp, Gin, His

Gly Ala

His Asn, Arg, Gln, Glu ILe Leu, Val

Leu He, Val

Lys Arg, Gln, Glu

Met Leu, lie

Phe His, Met, Leu, Trp, Tyr

Ser Cys, Thr

Thr Ser, Val

Trp Phe, Tyr

Tyr His, Phe, Trp

Val He, Leu, Thr

Conservative amino acid substitutions generally maintain one or more of: (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.

Preferably about 1, 2, 3, 4, 5, 6 to 10, 11 to 25, 26 to 50 or 51 to 100, or 101 to 250 amino acids of SEQ ID NO: 1 or SEQ ID NO: 2 are modified or deleted. The invention includes poly- peptides with mutations that cause an amino acid change in a portion of the polypeptide not involved in providing activity of SARS-Cov-2 binding or an amino acid change in a portion of the polypeptide involved in providing activity or SARS-Cov-2 binding so that the mutation increases or decreases the activity or SARS-Cov-2 binding of the polypeptide. For example, it is possi- ble to increase SARS-Cov-2 spike protein binding of the ACE2 polypeptide according to the known interactions (Walls et al., Cell (2020) 181(2): 281-292; Wan et al., J Virol (2020) doi:10.1128/JVI .00127-20; Wrapp et al., Science (2020) doi:10.1126/science .abb2507). Improvement can be in comparison to an unmodified ACE2 of SEQ ID NO: 1. Amino acid changes to im- prove SARS-Cov-2 spike protein binding of ACE2 polypeptide can e.g. be to the receptor binding domain of the spike protein.

Amin acids of ACE2 polypeptide are e.g. K31, E35, D38, M82 and K353 of SEQ ID NO: 1 or 2. These amino acids and adjacent amino acids or regions including amino acids 25 to 45, 75 to 90 or 345 to 360 corresponding to SEQ ID NO: 1 or 2 may be changed as com- pared to SEQ ID NO: 1 or 2 in an ACE2 polypeptide of the inven- tion to alter spike protein binding.

Polypeptides comprising one or more d-amino acids are con- templated within the invention. Also contemplated are polypep- tides where one or more amino acids are acetylated at the N-ter- minus. Those with skill in the art recognize that a variety of techniques are available for constructing polypeptide mimetics with the same or similar desired compound activity as the corre- sponding polypeptide compound of the invention but with more fa- vourable activity than the polypeptide with respect to solubil- ity, stability, and/or susceptibility to hydrolysis and proteol- ysis.

The invention also includes hybrids and polypeptides, for example where an amino acid sequence is combined with a second sequence. A possibility is a fusion with an antibody portion, such as a Fc fragment or a CH3 domain of a Fc fragment (US 10,443,049 B2).

Preferably, the ACE2 polypeptide is soluble ACE2. "Soluble" refers to solubility in water, especially under physiological conditions, in the sense that the ACE2 polypeptide is not ad- hered to a cellular surface. Soluble ACE2 polypeptides thus lack an anchor region that would bind it to a cell membrane, such as a functional transmembrane domain as found in native ACE2. In particular, the transmembrane domain corresponding to amino ac- ids 741-761 of SEQ ID NO: 2 should be missing in a soluble ACE2 polypeptide.

Besides the amino acid sequence, the solubility of a protein is also influenced by its folding as well as post-translational modifications. Glycosylation structures are the main cause of an increase in the solubility of a protein and have a major influ- ence on its pharmacological profile.

The ACE2 polypeptide of the; invention can be glycosylated, e.g. through expression in suitable expression systems, which further increases solubility. Due to its solubility, ACE2 can be administered intravenous as a bolus. For the same reasons the bioavailability is guaranteed immediately after administration.

Preferably the ACE2 polypeptide is glycosylated with high, highly branched and complex proportion of the glycosylation structures. The glycosylation structures preferably contain si- alic acid, preferably the molar amount of glycosylation struc- tures containing at least one sialic acid is at least 50%. Such glycosylation structures increase the half-life of the ACE2 pol- ypeptide in vivo.

Preferably, the ACE2 polypeptide is glycosylated on at least 70 % of the possible N-glycosylation sites and/or has a propor- tion of glycosylation structure of more than 10 % (w/w of total ACE2). N-glycosylation sites of SEQ ID Nos: 1 and 2 are Asn53, Asn90, Asnl03, Asn322, Asn432, Asn546, Asn690. Corresponding N- glycosylation sites are usually found in other ACE2 polypep- tides, such as from other mammals. ACE2 polypeptides that are fragments of ACE2 may miss one or more N-glycosylation sites.

Preferably, the ACE2 polypeptide has a molecular weight of at least 90 kDa, preferably at least 92 kDa, particularly pref- erably at least 94 kDa, in particular at least 96 kDa, and highly preferably at least 98 kDa, most preferably at least 100 kDa, 100.5 kDa, 101 kDa, 101.5 kDa or at least 102 kDa. An abso- lute molecular mass - i.e. of the peptide per se without the hy- drate sheath - can be determined by peptide mapping. More highly glycosylated forms may also have molecular masses of at least 103 kDa, 104 kDa, 105 kDa, 106 kDa, 107 kDa or at least 108 kDa. ACE2 polypeptides have been expressed with a molecular weight of up to about 120 kDa. Higher molecular weights are possible by modification of the ACE2 polypeptide, for example PEGylation.

PEGylation is one of the preferred modifications of the ACE2 polypeptide but any fusion or modification as known in the art for pharmaceutical proteins can be used according to the inven- tion. Such fusions or modifications are disclosed in Strohl et al., BioDrugs (2015) 29:215-239 (incorporated herein by refer- ence) and include Fc fusion proteins, scFva fusion, fusion to human serum albumin, fusion to human transferrin, fusion to car- boxy-terminal peptide, and other polypeptide fusions, XTENyla- tion, rPEG, PASylation, ELPylation, HAPylation, GLK fusion, CTP fusion. Such fusions and modifications can adjust the ACE2 poly- peptide to a desirable pharmacokinetic profile, in particular increase of the half-life. An Fc fusion is preferably to an Fc of IgG, IgM, IgD, or IgA or a part thereof, such as a CHI, CH2 or CH3 domain, or FcRn. A CH3 domain is preferred. It may or may not include the C-terminus of the Fc part. IgG is preferably hu- man IgGi, IgG2, and IgG₄. Modifications, amino acid changes, se- lected glycosylation patters and fusions can protect the ACE2 polypeptide from proteolytic degradation, e.g. reduce proteo- lytic degradation as compared to unmodified ACE2, such as ac- cording to SEQ ID NO: 1 without modifications or fusions. Prote- olytic degradation may from or in human serum.

In preferred embodiment, the ACE2 polypeptide comprises amino acids 19 to 600 of SEQ ID NO: 1. In particular prefer- ments, the ACE2 polypeptide consists of or comprises amino acids 19 to 605 or amino acids 19 to 619, each of SEQ ID NO: 1, in- cluding embodiments of the ACE2 polypeptide comprising amino ac- ids 1 to 605 or amino acids 1 to 619, each of SEQ ID NO: 1. Such ACE2 polypeptides, being fragments of native human ACE2 but re- taining its activity are disclosed in US 10,443,049. In pre- ferred embodiments the ACE2 polypeptide comprises or consists of an amino acid sequence with at least 70%, preferably at least 80%, at least 90%, at least 95%, at least 98% or at least 99%, sequence identity to amino acids 19 to 619 of SEQ ID NO: 1 or with amino acids 19 to 605 of SEQ ID NO: 1.

Even more preferred, the ACE2 polypeptide comprises or con- sists of amino acids corresponding to amino acids 19 to 700 of SEQ ID NO: 1 or comprises or consists of amino acids correspond- ing to amino acids 18 to 740 of SEQ ID NO: 1. The ACE2 polypep- tide preferably comprises the peptidase domain, or comprises the peptidase domain plus the collectrin-like domain. The ACE2 poly- peptide may comprise the full ACE2 ectodomain.

Preferably, the ACE2 polypeptide consists of or comprises amino acids 18 to 740 of SEQ ID NO: 1. Such ACE2 polypeptides are disclosed in WO 2008/151347 and in WO 2014/108530 (both in- corporated herein by reference) and are preferred embodiments of the ACE2 polypeptide used according to the invention. In preferred embodiments the ACE2 polypeptide comprises or consists of an amino acid sequence with at least 70%, preferably at least 80%, at least 90%, at least 95%, at least 98% or at least 99%, sequence identity to SEQ ID NO: 1 or with amino acids 18 to 740 of SEQ ID NO: 1. The ACE2 polypeptide can be APN01 (a soluble recombinant human ACE2 - "srhACE2") or GSK2586881 (a recombinant human angiotensin converting enzyme type 2 - "rhACE2").

Preferably, a serine (or C-terminal amino acid) of the ACE2 polypeptide corresponding to Ser740 of SEQ ID NO: 1 (for example the C-terminal end) is O-glycosylated.

The ACE2 polypeptide may be a monomer or a dimer, as de- scribed in WO 2008/151347, or a multimer.

The ACE2 polypeptide can be catalytically inactive or active and is preferably catalytically active in hydrolysing angioten- sin II to angiotensin-(1-7) and/or in hydrolysing angiotensin I to angiotensin-(1-9) (Vickers et al., J Biol Chem (2002)

277(17): 14838-14843). Preferably, the catalytic activity of the ACE2 polypeptide or preparation, ccat, is at least 4 s^_1, prefer- ably at least 5 s^_1, particularly preferably at least 6 s^_1, highly preferably at least 7 s^_1, and most preferably at least 7.6 s^_1 with respect to the Ang 1-7 (angiotensin 1-7) conversion. Ang 1-7 is formed from Ang II (angiotensin II) by means of ACE2. The conversion can be tested in a simple manner, as described in WO 2008/151347. This conversion or the catalytic activity of the ACE2 polypeptide can also be extrapolated from other assay data. The activity can, for example, be measured as described in WO 2008/046125 A.

Although human ACE2 (SEQ ID NOs 1 and 2) is preferred for most therapeutic applications, ACE2 from other mammals, for ex- ample mouse, rat, hamster, pig, primates or cattle, can also be used. ACE2 is a universal enzyme in all mammals with the Ang II substrate which is identical in the various species. Hence, in principle it can also be used in other organisms. Thus, the ACE2 polypeptide according to the invention can be used regardless of the origin of the ACE2, for example from humans, mice, rats, hamsters, pigs, primates or cattle. However, in preferred embod- iments, the origin of the ACE2 and the organism (subject or pa- tient) to be treated is the same. Preferably the subject (or pa- tient) is a human.

The ACE2 polypeptide can be administered at a daily dose of 0.01 μg/kg to 10 mg/kg, such as 0.1 μg/kg to 5 mg/kg. The ACE2 polypeptide is preferably for administration at a dose of 10 μg/kg to 1500 μg/kg daily. In particular preferred embodiments, the daily dose is about 400 μg/kg, in other embodiments, the daily dose is about 200 μg/kg. Given that recombinant soluble ACE2 is well tolerated, even at high doses and no adverse ef- fects have been shown by the present invention, a broader dose range is possible. Accordingly, the invention provides for a daily dose of 10 μg/kg to 100 μg/kg, 100 μg/kg to 200 μg/kg, 200 μg/kg to 300 μg/kg, 300 μg/kg to 400 μg/kg, 400 μg/kg to 500 μg/kg, 500 μg/kg to 600 μg/kg, 700 μg/kg to 800 μg/kg, 800 μg/kg to 1000 μg/kg, 1000 μg/kg to 1500 μg/kg and any combination of these ranges, such as 200 μg/kg to 600 μg/kg, or 10 μg/kg to 300 μg/kg. μg/kg refers to the amount of ACE2 polypeptide in pg per kg of the patient's body weight. A skilled artisan (e.g. a phy- sician or veterinarian) may reduce or increase dosage in accord- ance with these or other conditions or requirements.

Variants of ACE2 have been described as ACE2 polypeptides suitable for the present invention. Despite molecular weight differences of different ACE2 polypeptides, the above amounts refer to any ACE2 polypeptide, given that differences in molar concentration for a given mass amount are minor.

A pharmaceutical composition comprising an ACE2 polypeptide preferably contain dosage forms for these daily doses in a con- tainer for a 70 kg subject. E.g. the pharmaceutical composition may comprise the ACE2 polypeptide in an amount of 700 μg to 105 mg per container, more preferably 700 μg to 1 mg, 1 mg to 5 mg,

5 mg to 10 mg, 10 mg to 20 mg, 20 mg to 30 mg, 30 mg to 40 mg,

40 mg to 50 mg, 60 mg to 70 mg, 80 mg to 90 mg, 90 mg to 105 mg. Any combination of these ranges is possible, such as 10 mg to 40 mg, or 1 mg to 20 mg for lower doses than 400 μg/kg as mentioned above, given the efficacy of the inventive combination therapy. Such doses may be provided in a container, such as a syringe, vial, flask, bottle, etc., containing the pharmaceutical compo- sition.

Preferably, the ACE2 polypeptide is administered systemi- cally, preferably intravenously; topically; orally; intranasally or by inhalation, or a combination thereof. Particularly pre- ferred are an intravenous (i.v.) administration and an admin- istration by inhalation. Both can be combined. E.g. an initial i.v. administration that is followed by an administration by in- halation at later doses, such as on other days. An initial i.v. administration provides a strong initial counter to a SARS-CoV-2 infection; later inhalation administration(s) provide a continu- ous counter while being more agreeable to a patient than an i.v. administration. An i.v. administration and the inhalation can also be combined for the same dose, i.e. a parallel administra- tion. Accordingly, a dose may be provided in part as i.v. dose and in part as an inhalation dose.

The daily dose may be administered once per day, or as split doses more than once per day, wherein the daily dose is divided by the number of administrations to the patient on a day. The administration may be 2x per day, 3x per day or more often. It is also possible to administer with intermittent administration- free days, such as every 2^nd day (with a dose twice the daily dose). In case of non-daily administrations, the daily dose is adjusted so that the daily dose is achieved on average per day over the treatment time span. The treatment time span may be 1, day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days or more, such as up to 30 days or up to 40 days or even more. Any ranges in between these treatment times are possible. E.g. ad- ministered can be at least 3, 4, 5 or more consecutive days. In a preferred embodiment and example, administration is for at least 3 days by inhalation treatment twice daily.

In some embodiments, the ACE2 polypeptide is administered to the subject at about 0.4 mg/kg through intravenous (i.v.) injec- tion twice a day up to 7 days. However, in preferred embodi- ments, i.v. administrations are limited to an initial treatment such as up to 3 or up to 2 days and/or doses, followed by inha- lation as following doses and/or on following days. As mentioned above, also parallel i.v. and inhalation administrations are possible. In fact, it is preferred to administer the ACE2 poly- peptide systemically, preferably intravenously, in parallel or followed by administration by inhalation.

In further alternative or combinable embodiments, the ACE2 polypeptide is administered systemically, preferably intrave- nously, at least twice, followed by administration by inhala- tion. In particular preferred embodiments, the systemic admin- istration is at a dose of 100 μg/kg to 4 mg/kg daily and/or preferably wherein administration by inhalation is at a dose of 100 μg to 600 mg, preferably 10 mg to 100 mg, daily. Any partic- ular doses within these ranges, as mentioned above, fall within this preferred embodiment.

In preferred embodiments of the invention the ACE2 polypep- tide is administered at least twice, 3 times, 4 times, 5 times,

6 times, 7 times or more. As mentioned before, the administra- tion, can be daily, twice daily or every other day.

In a preferred embodiment of the invention, the ACE2 poly- peptide is administered systemically, preferably intravenously, at least twice.

The systemically administration of ACE2 polypeptide is pref- ereably in parallel or followed by administration by inhalation of aerosolized ACE2 polypeptide.

In some embodiments, up to 4mg/kg ACE2 polypeptide could be administered systemically to a patient with mild or moderate SARS-CoV-2 infection once, twice or every other day. The treat- ment of the patient can be followed by 5 mg to 40 mg, preferably about 20 mg, aerosolized ACE2 polypeptide once daily, e.g. for up to 28 days.

In an alternative embodiment of the invention ACE2 polypep- tide is administered by inhalation, wherein a solution of ACE2 polypeptides is aerosolized into aerosol particles with an aver- age particle size of 0.1 pm to 100 pm and at a dose of 100 pg to 600 mg daily, wherein preferably the concentration is 1 mg/ml to 50 mg/ml. Especially, a volume of 1 ml to 5 ml is aerosolized per administered dose, preferably in a PARI Vios® PRO Nebulizer.

In particular preferred embodiments of the invention, the SARS-CoV-2 infection is with a SARS-CoV-2 variant exhibiting an increase of ACE2 polypeptide binding affinity for SARS-CoV-2 spike protein mutants in comparison to wild type SARS-CoV-2 spike protein. Such SARS-CoV-2 variants with a SARS-CoV-2 spike protein mutant that has an increased binding affinity with an ACE2 polypeptide are particularly effective and are thus a pre- ferred embodiment of the invention.

Like other coronaviruses, SARS-CoV-2 has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins. The spike protein is the protein responsible for allowing the SARS-CoV-2 virus to attach to the membrane of a host cell, the receptor binding domain ("RBD") of the spike protein of SARS-CoV-2 recognizes and e.g. attaches to the angiotensin-converting enzyme 2 ("ACE2") of host cells to use them as a mechanism of cell entry. Binding and entry into the cell is further mediated by TMPRSS2 (Hoffmann et al., Cell (2020) 181(2): 271-280.e8). As such, ACE2 is a receptor for the Spike glycoprotein of SARS-CoV-2. As a pharmaceutical, the ACE2 polypeptide acts as competitive inhibitor to SARS-CoV-2 spike protein.

SARS-CoV-2 variants with mutations in the spike protein that increase binding to ACE2 thus have increased affinity to both, the natural ACE2 on host cell surfaces and the competitive ACE2 polypeptide that acts as inhibitor. Surprisingly, the ACE2 poly- peptide of the invention acts in such situations as a strong in- hibitor when compared to wild-type SARS-CoV-2.

Wild-type SARS-CoV-2 spike protein is used as reference for comparison with the stronger targets of the invention. Wild type SARS-CoV-2 spike protein is for example provided in database UniProtKB, database entry P0DTC2 as of entry version 6 of 10 February 2021 (spike_SARS2) (www.uniprot.org/uniprot/P0DTC2) and provided as SEQ ID NO: 3 herein. The stronger binding affinity can e.g. be determined in a binding affinity assay, as shown in the examples, such as by surface plasmon resonance. A soluble ACE2 polypeptide, such as of amino acids 18-740 of SEQ ID NO: 1 (e.g. as of the examples of WO 2008/151347 Al, incorporated herein by reference), can be used as reference in the assay.

In particular preferred, the SARS-CoV-2 mutation comprises a mutation of the SARS-CoV-2 spike protein selected from a group comprising V367F, N354D, W436R, R408I, K417N, E484K, N501Y,

G476S, V483A, A475V, N501Y, D614G, K417T, L452R, T478K, E484Q, R682Q, del69-70, dell44, A570D, P681H, T716I, S982A, D1118H,

L18F, D80A, D215G, del242-244, Q677H, R682W, A701V, S13I, W152C, D138Y, R189S, H655Y, T1027I, T19R, K77T, G142D, E156G, P681R, D950N, A67V, T95I, dell43-145, N211I, del212, ins EPE 214-216, G339D, S371L, S373P, S375F, N440K, G446S, S477N, E484A, Q493R, G496S, Q498R, Y505H, T547K, N679K, N764K, D796Y, N856K, Q954H or combinations thereof. SARS-CoV-2 with any one of these mutations in the spike protein is a particular preferred target for the inventive treatment. One or more mutations in comparisons to wild-type SARS-CoV-2 spike protein may be in a mutant SARS-CoV-2 spike protein of the virus to be treated according to the invention, such as 2, 3, 4, 5, 6 or more mutations. Preferably, the mutation of the SARS-CoV-2 spike protein comprises K417N, E484K and N501Y, or K417T, E484K and N501Y, or L452R and E484Q or L452R and T478K. These mutations significantly increase af- finity to ACE2 and are found in several highly virulent variants of SARS-CoV-2.

In preferments, the SARS-CoV-2 infection is with a SARS-CoV- 2 variant B.l.1.7 (Alpha), B.1.351 (Beta), or B.1.28/P1 also termed B.1.1.28.1/Pl (Gamma), B.1.617.2 (Delta), B.1.526 (Iota), B.1.427 (Epsilon), B.1.429 (Epsilon), B.1.617.1 (Kappa),

B.1.617.3, or B.1.525 (Eta), C.37 (Lambda), P.2 (Zeta), P.3 (Theta), B.1.1.529 (Omicron), A.23.1, A.27, B.1.1.318, B.1.620,

C.36.3 or C.1.2. These variants contain significant mutations in the spike protein that increase binding affinity to ACE2 and are therefore preferred therapeutic targets of the invention.

Preferably, the infection is by SARS-CoV-2 wherein at least 30% of the SARS-CoV-2 infecting the subject comprise any one of said mutations or variants.

As mentioned above, in preferred embodiments, the treatment or prophylaxis comprises administration of the ACE2 polypeptide by inhalation, wherein a solution of ACE2 polypeptides is aero- solized into aerosol particles with an average particle size of 0.1 μm to 100 μm, preferably 0.2 pm to 10 pm, and/or at a dose of 100 μg to 600 mg daily. An ACE2 polypeptide may be formulated for such an administration, such as in a nebulizer or spray. The therapy by inhalation may be therapy or prophylaxis of any SARS- CoV-2 infection, including SARS-CoV-2 with wild-type spike pro- tein or any of the above mutations. The inhalation may be com- bined with other therapies, such as a systemically administered ACE2 therapy.

In preferred embodiments, the ACE2 polypeptide in the solu- tion is at a concentration of 0.5 μg/ml to 125 mg/ml, preferably 10 μg/ml to 100 mg/ml, 0.1 mg/ml to 75 mg/ml, 0.5 mg/ml to 50 mg/ml, 1 mg/ml to 40 mg/ml, 1.5 mg/ml to 30 mg/ml, e.g. about 5 mg/ml or about 10 mg/ml or any range in between these values. In preferred embodiments, the concentration of the ACE2 polypeptide in solution for aerosolization is 0.5 mg/ml and 5 mg/ml.

For inhalation, preferably a volume of 250 μl to 8 ml, pref- erably 500 μl to 6 ml, even more preferred 1 ml to 5 ml, of the solution is aerosolized per administered dose. An example inhalation dose is an inhalation of 4 ml that comprises or con- tains 20 mg ACE2 polypeptide.

As mentioned above, a preferred administration by inhalation is combined with a systemic administration. The administration by inhalation may be parallel or after a systemic administration to the subject. The systemic administration is preferably an in- travenous administration, especially preferred a systemic admin- istration at a dose of 0.01 μg/kg to 10 mg/kg daily or any dose mentioned above, in particular those within this range.

According to the method of therapy, the ACE2 polypeptide can be administered to subjects with detected virus RNA but without symptoms (asymptomatic subjects). The administration is prefera- bly within the first seven days after detection of virus RNA.

Such patients with light cases of COVID-19 are particularly suited for therapy by inhalation, but of course any other ther- apy does, mode of administration, such as i.v. alone or in com- bination with inhalation, is also possible.

According to a method of prophylaxis (or therapy of unde- tected or undetectable virus), the ACE2 polypeptide is adminis- tered to high-risk subjects without detected virus RNA. This serves as a post-exposure prophylaxis but may also be adminis- tered to subjects without known expose who are at risk or may face exposure after the prophylactic administration. A prophy- lactic administration may be only one administration, e.g. by inhalation and/or systemic, like i.v, but other administration routes as mentioned above are also possible.

The treatment can be of subjects with symptomatic mild or moderate SARS-CoV-2 infection. The mild or moderate SARS-CoV-2 infected subjects can be with or with no limitation of activi- ties; they can be hospitalized without oxygen therapy, or they can have oxygen therapy by mask or by nasal prongs.

Mild or moderate SARS-CoV-2 infection can be according to the WHO clinical progression scale as denied in- doi.org/10.1016/S1473-3099 (20)30483-7 of June 12, 2020 by the WHO Working Group on the Clinical Characterisation and Manage- ment of COVID-19 infection. The WHO clinical progression scale is reproduced in the following table B.

Table B: WHO clinical progression scale. ECMO=extracorporeal membrane oxygenation. FiO₂=fraction of inspired oxygen. NIV=non- invasive ventilation. pO₂=partial pressure of oxygen. SpO₂=oxygen saturation. *If hospitalised for isolation only, record status as for ambulatory patient.

Preferably the mild or moderate SARS-CoV-2 infection is with a score of 1, 2, 3, 4 or 5 of the WHO clinical progression scale.

The invention further provides an ACE2 polypeptide for use in a method of treatment or prophylaxis according to the inven- tion, in all its aspects and embodiments. Furthermore, the in- vention provides the manufacture of a pharmaceutical composition comprising an ACE2 polypeptide for the treatment or prophylaxis according to the invention, in all its aspects and embodiments.

In accordance with the invention, a pharmaceutical composi- tion or medicine comprising the ACE2 polypeptide can be pro- vided. The pharmaceutical composition may be in a container, such as a vial, flask or bag, and/or in kit. Such compositions may be pharmaceutically acceptable salts themselves, with addi- tional buffers, tonicity components or pharmaceutically accepta- ble carriers. Pharmaceutical carrier substances serve to improve the compatibility of the composition and provide better solubility as well as better bioavailability of the active in- gredients. Examples are emulsifiers, thickeners, redox compo- nents, starches, alcoholic solutions, polyethylene glycol and lipids. Selection of a suitable pharmaceutical carrier is highly dependent on the administration route. For oral administration, liquid or solid carriers may be used; for injections, liquid fi- nal compositions are required.

Preferably, the ACE2 polypeptide is provided in a composi- tion comprising buffers or tonic substances. The buffer can ad- just the pH of the medicine to the physiological conditions and further, can reduce or buffer variations in pH. An example is a phosphate buffer. Tonic substances can adjust the osmolarity and may include ionic substances, such as inorganic salts, for exam- ple NaCl or KC1, or non-ionic substances such as glycerin or carbohydrates .

Preferably, the composition is suitably prepared for sys- temic, topical, oral or intranasal administration or as an inha- lation preparation. Such administration routes are preferred em- bodiments of the inventive methods. These forms of administra- tion for the composition of the present invention allow fast, uncomplicated take-up. When the ACE2 polypeptide is intended for oral administration, it is preferably provided in a formulation which is resistant to stomach acid or it is encapsulated. For oral administration, solid or liquid medicines can be taken di- rectly or dissolved or diluted, for example. The pharmaceutical composition or ACE2 polypeptide for use in accordance with the invention is preferably produced for intravenous, intra-arte- rial, intramuscular, intravascular, intraperitoneal or subcuta- neous administration. Injections or transfusions, for example, are suitable for this purpose. Administration directly into the bloodstream has the advantage that the active ingredient of the medicine can be distributed through the entire body and the tar- get tissue, such as lungs, heart, kidney, intestine or liver, is reached quickly.

The term "pharmaceutically acceptable" indicates that the designated carrier, vehicle, diluent, excipient(s), and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologi- cally compatible with the recipient thereof. In some embodi- ments, compounds, materials, carriers, compositions, and/or dosage forms that are pharmaceutically acceptable refer to those approved by a regulatory agency (such as U.S. Food and Drug Ad- ministration, National Medicine or European Medicines Agency) or listed in generally recognized pharmacopoeia (such as U.S. Phar- macopoeia, China Pharmacopoeia or European Pharmacopoeia) for use in animals, and more particularly in humans.

Pharmaceutical acceptable carriers for use in the pharmaceu- tical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, tonicity-adjusting agents, antioxi- dants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non- toxic auxiliary substances, other components known in the art, or various combinations thereof. Suitable carriers and auxiliary components may include, for example, fillers, binders, disinte- grants, buffers, preservatives, lubricants, flavorings, thicken- ers, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrins. In some embodiments, the suitable buffers may include, for example, a phosphate buffer or a MES (2-(N-morpho- lino)ethane sulfonic acid) buffer.

To further illustrate, pharmaceutical acceptable carriers may include, for example, aqueous vehicles such as sodium chlo- ride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's in- jection, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, an- timicrobial agents at bacteriostatic or fungistatic concentra- tions, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers or MES (2-(N-mor- pholino)ethane sulfonic acid) buffers, antioxidants such as so- dium bisulfate, local anesthetics such as procaine hydrochlo- ride, suspending and dispersing agents such as sodium carbox- ymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyr- rolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80), sequestering or chelating agents such as EDTA (ethylenedia- minetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, so- dium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that in- clude phenols or cresols, mercurials, benzyl alcohol, chlorobu- tanol, methyl and propyl p-hydroxybenzoic acid esters, thimero- sal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

The pharmaceutical compositions for the ACE2 polypeptide can be a liquid solution, suspension, emulsion, pill, capsule, tab- let, sustained release formulation, or powder. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrol- lidone, sodium saccharine, cellulose, magnesium carbonate, etc.

The form of pharmaceutical compositions depends on a number of criteria, including, but not limited to, route of administra- tion, extent of disease, or dose to be administered. The pharma- ceutical compositions can be formulated for intravenous, oral, nasal, rectal, percutaneous, intramuscular or inhalation admin- istration. For example, dosage forms for intravenous administra- tion, may be formulated as lyophilized powder or fluid formula- tion; dosage forms for nasal or inhalation administration may conveniently be formulated as aerosols, solutions, drops, gels or dry powders. In accordance to the desired route of admin- istration, the pharmaceutical compositions can be formulated in the form of tablets, capsule, pill, dragee, powder, granule, sa- chets, cachets, lozenges, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), spray, in- halant, or suppository.

In some embodiments, the pharmaceutical compositions are formulated into an injectable composition. The injectable phar- maceutical compositions may be prepared in any conventional form, such as for example liquid solution, suspension, emulsion, or solid forms suitable for generating liquid solution, suspen- sion, or emulsion. Preparations for injection may include ster- ile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions. The so- lutions may be either aqueous or nonaqueous. Aqueous is pre- ferred.

In preferred embodiments, the ACE2 polypeptide is formulated in a solution comprising the ACE2 polypeptide, a buffer, a sta- bilizer, such as a carbohydrate like sucrose and/or arginine and/or a salt like sodium chloride, an ACE2 cofactor, like zinc, and a surfactant, such as a polysorbate or a polyglycol, like PEG. Such a preparation is e.g. 5 mg/ml soluble ACE2 of SEQ ID NO: 1 with 10 mM sodium phosphate (buffer), 210 mM sucrose (sta- bilizer), 25 mM arginine HC1 (stabilizer), 10 mM sodium chloride (stabilizer), 50 mM zinc chloride (cofactor), 0.02 % (w/v) poly- sorbate 80 (surfactant), in water pH 7.0 (solvent).

In some embodiments, unit-dose i.v. or parenteral prepara- tions are packaged in an ampoule, a vial, bag or a syringe with a needle. All preparations for parenteral administration should be sterile and not pyretic, as is known and practiced in the art.

Depending on the route of administration, the ACE2 polypep- tide can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally af- fect its ability to perform its intended function. It may be ad- ministered alone, or in conjunction with a pharmaceutically ac- ceptable carrier.

Throughout the present disclosure, the articles "a", "an" and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the arti- cle.

As used herein, words of approximation such as, without lim- itation, "about", "substantial" or "substantially" refer to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skill in the art recognize the modi- fied feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as "about" may vary from the stated value by e.g. ±10%.

As used herein, the terms "include" and "including" have the same meaning as the terms "comprise" and "comprising". The terms "comprise" and "comprising" should be interpreted as being "open" transitional terms that permit the inclusion of addi- tional components further to those components that are recited. "Comprising" in connection with a component connected to a range shall mean that further non-recited components are allowed but the recited component linked to that range shall be within said range and not outside said range. The terms "consist" and "con- sisting of" should be interpreted as being "closed" transitional terms that do not permit the inclusion of additional components other than the recited. The term "consisting essentially of" should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamen- tally alter the nature of the recited subject matter, such as not permitting further non-recited active ingredients but allow- ing further non-recited auxiliary substances, like buffer compo- nents, fillers, and the like.

The present invention is further illustrated by the follow- ing figures and examples, without being limited to these embodi- ments of the invention.

Figures:

Figure 1. Serial dilutions of APN01 were prepared in assay me- dium and a suspension of SARS-CoV-2. After one-hour incubation at 37°C, the dilutions were transferred to wells containing Vero E6 target cells (MOI 0.001). Incubation was continued for four days and cell numbers were assessed with a neutral red endpoint. Figure 2. Dose response curve from APN01 samples generated by SARS-CoV-2 Spike Protein-Binding Assay

Figure 3. ACE2 Enzymatic activity assay data for Trial 3 rec- orded for Pre-Nebulization, Un-nebulized volume, and Post-Nebu- lization samples at APN01 starting dilutions of 25 ng/ml, 50 ng/ml, and 100 ng/ml. Data are plotted as ARFU vs. time (min). Figure 4: Sensorgram Overlay of spike protein RBD Variants

Examples: Example 1: In Vitro Anti-SARS-CoV-2 activity of ACE2

While anti-SARS-CoV-2 activity of soluble recombinant human ACE2 ("APN01") has been reported previously (Monteil et al,

2020, Cell 181, 905-913), we evaluated the cGMP produced APN01 used in these studies for neutralizing activity. As shown in Figure 1, one-hour exposure to concentrations of APN01 as low as 25 μg/ml completely neutralized SARS-CoV-2 as assessed in a four day CPE assay on Vero E6 cells. It is reasoned that direct in- troduction of ACE2 polypeptide into the airways could neutralize virus to limit spreading of the infection, and limit damage to the lung by cleaving Ang II and des-Arg(9)-bradykinin, another relevant ACE2 substrate. The aerosol formulation of ACE2 poly- peptides retains virus-binding activity and enzymatic activity for cleaving Ang II.

Example 2:Activity of aerosolized ACE2

A PARI LC PLUS nebulizer was selected for use in preclinical studies. Aerosolized APN01 was collected using a custom fabri- cated condenser and analyzed for virus-binding activity and en- zymatic activity for cleaving a fluorogenic substrate. Clinical grade APN01 is formulated for i.v. use at 5 mg/ml in an isotonic buffer containing 50 μM ZnCl2 (Zn is required for enzymatic ac- tivity of ACE2) and 0.02% Polysorbate 80 (to inhibit aggregation and sticking to glass surfaces). Recognizing that in vitro anti- SARS-CoV-2 activity was observed at concentrations as low as 25 μg/ml, we studied a range of concentrations spanning 100 μg/ml to 5 mg/ml (the maximum feasible concentration). The concentra- tion of recovered APN01 was assessed by HPLC or ELISA measure- ments.

Binding ELISAs were conducted by coating plates with SARS- CoV-2 receptor binding domain and assessing APN01 binding. A representative experiment for APN01 aerosolized at 100 μg/ml is shown in Figure 2.

As shown in Table 1, the SARS-CoV-2 binding activity was not statistically different pre- and post-nebulization.

Table 1: RBD binding analysis of Trial 1-4 samples. Only minor changes in activity (-27.0%, -10.8%, -2.6%, and -0.14% respec- tively) between Pre-Nebulization and Post-Nebulization samples, with Unnebulized Volume (material remaining in nebulizer cup at the end of the trial) also testing similarly. Similar results were obtained with APN01 aerosolized at 5 mg/ml.

Enzymatic activity was assessed using a fluorogenic peptide substrate. Kinetic analysis was performed at multiple concentra- tions of APN01 and expressed as the change in Relative Fluores- cence Units per minute per ng [(dRFU/min)/ng]. Enzymatic results for Trial 3 of Table 1 are illustrated in Figure 3.

Enzymatic activity for cleaving fluorescently labeled pep- tide substrate for APN01 aerosolized at higher concentrations was not affected (Table 2).

Table 2: ACE2 Enzymatic Assay data from Trials 1-4 recorded as (ARFU/min)/ng for the three starting dilutions of APN01: 25 ng/ml, 50 ng/ml, 100 ng/ml. The difference between Pre-Nebuliza- tion and Post-Nebulization enzymatic activity is also indicated as a % change at each APN01 dilution and the mean change across all 3 dilutions.

Example 3: Tolerability of aerosolized APN01

This study was performed to provide a comprehensive evalua- tion of the toxicity of twice daily inhalation administration of APN01 aerosols to dogs for 14 consecutive days. Goals of this study included: characterization of the toxicity of repeat-dose inhalation exposure to APN01 aerosols in dogs for fourteen days; Identification of sensitive target tissues for the toxicity of inhaled APN01 in dogs; characterization of serum levels and tox- icokinetics (TK) of inhaled APN01 in dogs; and identification of a No Observed Adverse Effect Level [NO(A)EL] for twice daily in- halation administration of APN01 to dogs for fourteen consecu- tive days

The study design is summarized below: Table 6:

The target APN01 concentration of 0.075 mg/L used for the high dose group was demonstrated to be the maximum feasible con- centration (MFC), obtained by aerosolizing the neat i.v. formu- lation, in a preliminary range-finding study.

For administration to experimental animals, multiple PARI LC PLUS nebulizers were multiplexed through a distribution plenum with hoses connected to oronasal masks. Analytical data (ob- tained by HPLC analysis of filters) demonstrated that test aero- sols consistently achieved target exposure concentrations. Par- ticle size distribution data showed that the generated particles were within the respirable size range for dogs (10) and met tar- gets for both MMAD and GSD. Characteristics of the generated aerosol are summarized in Table 3.

Inhaled Dose: Calculated inhaled APN01 dose levels for each group after a single exposure (Study Day 1) and after repeat- dose exposure (after the first exposure on Study Day 11) are provided in Table 4.

Systemic exposure [defined as serum levels of APN01 above the limit of quantitation (LOQ)] was very low in dogs in the low dose and mid dose groups on both Days 1 and 14; in both groups, serum levels of APN01 were below the LOQ (0.5 ng/mL) in most an- imals at most time points. In the high dose group, serum levels of APN01 on Days 1 and 14 were above the LOQ in 5 of 6 dogs at all time points after 0.5 hr. Although interanimal variability was substantial, mean Cmax (pooled across both sexes) in the high dose group was approximately 8 ng/mL on both Day 1 and Day 14.

Dog serum samples were analyzed for levels of APN01 using en- zyme-linked immunosorbent assay (ELISA). Individual animal serum drug concentrations at scheduled (nominal) blood sampling times from animals in Groups 3-5 were used to model toxicokinetic (TK) parameters. Reported TK parameters are Tmax, Cmax, and AUC6hr; terminal phase parameters [e.g., tl/2] could not be calculated reliably for any of the profiles due to a lack of amenable data in the terminal phase of the profiles. Summary TK results are shown in Table 5.

Table 5: Serum levels of APN01 and toxicokinetic parameters.

Toxicology endpoints included mortality/moribundity observa- tions; clinical observations for signs of toxicity; physical ex- aminations; heart rate and blood pressure measurements; body weight measurements; food consumption measurements; ophthalmic examinations; electrocardiographic evaluations; respiratory function evaluations; measurements of blood oxygen saturation and pH; neurotoxicity evaluations (functional observational bat- tery [FOB]); clinical pathology assessments (clinical chemistry, hematology, coagulation, and urinalysis); quantitation of serum drug levels; limited modeling of serum toxicokinetics (TK); gross pathology at necropsy; organ weights; and microscopic evaluation of tissues.

No early deaths occurred during the study, and no gross clinical signs of toxicity were seen in any study animal. Inha- lation administration of APN01 aerosols had no effects on body weight, food consumption, clinical pathology parameters, heart rate, blood pressure, electrocardiography, blood oxygen saturation, blood pH, FOB parameters, or ophthalmology. Respira- tory function evaluations (respiratory rate, tidal volume and minute volume) were inconclusive due to excitement and/or pant- ing exhibited by study animals during measurement periods. Organ weights were comparable in all study groups. No gross or micro- scopic pathology was linked to APN01 administration.

No evidence of systemic or organ-specific toxicity was iden- tified in any dog receiving twice daily 60 minute exposures to APN01 aerosols at target concentrations of 0.019, 0.038, or 0.075 mg/L for fourteen consecutive days. On this basis, the No- Observed-Adverse-Effect Level [NO(A)EL] for twice daily one hour exposures to aerosolized APN01 for fourteen days is 0.075 mg/L.

The results presented here support the feasibility of aero- sol administration of APN01 for treatment of SARS-CoV-2 infec- tion. APN01 retains virus binding and enzymatic activities fol- lowing aerosolization. The aerosol generated using a commercial nebulizer has a particle size distribution consistent with de- livery throughout the respiratory tract and could be delivered repeatedly at high dose to experimental animals without evidence of toxicity. The observation of in vitro SARS-CoV-2 neutraliza- tion at concentrations as low as 25 ug/ml suggests that aerosol administration should deliver effective antiviral therapy to the airways. Recently ACE2-mimetic peptides have been shown to pro- vide effective SARS-CoV-2 treatment when administered intrana- sally in a hamster model of COVID-19 (Linsky et al., 2020, Sci- ence) A novel lipopeptide designed to inhibit virus entry showed efficacy when administered as a nasal spray in a ferret model.

Example 4: ACE2 - SARS-CoV-2 spike protein affinity measurements by surface plasmon resonance

SARS-CoV-2 variants containing mutations within the ACE2 re- ceptor binding region may influence target binding behaviour and affinity and thus efficacy of APN01. Virus mutation regularly accompanies pandemic scenarios which may result in altered in- fectivity and/or pathogenicity. Apart from an early widespread D614G spike protein variant other mutant strains have been iden- tified with amino acid changes within the RBD region, in partic- ular strains B.l.1.7 (British Mutant, Alpha), B.1.351 (South Af- rican Mutant, Beta), PI / B.1.1.28.1 (Brazilian Mutant, Gamma), B.1.617.2 (Delta), B.1.526 (Iota), B.1.427 (Epsilon), B.1.429 (Epsilon), B.1.617.1 (Kappa), B.1.617.3, or B.1.525 (Eta), C.37 (Lambda), P.2 (Zeta), P.3 (Theta), B.1.1.529 (Omicron), A.23.1, A.27, B.1.1.318, B.1.620, C.36.3 or C.1.2 containing a series of mutated amino acids, some of them within the receptor biding mo- tif (RBM) of the RBD region (for example N501Y, E484K/Q,

K417N/T, T478K, L452R). Efficacy and potency of vaccines, immu- notherapeutics and recombinant proteins targeting the viral host cell receptor (ACE2) docking mechanism may be affected by muta- tions at relevant RBD positions. Comparative Sars-CoV-2 variant binding analysis is therefore a valuable tool to generate a data set for potency evaluation of APN01.

Mutant SARS-CoV-2 full length pre-fusion conformation spike proteins and RBD fragments were compared by SPR kinetic analysis with the corresponding wild type variant isolated from an early pandemic virus strain. Mutants with amino acid changes residing within the ACE2 receptor binding motif (RBM) or total RBD frag- ment, including mutations found in the recent epidemiologically relevant strains (B.l.1.7 (Alpha); B.1.351 (Beta); B.1.28/P1 (Gamma), B.1.617.2 (Delta), B.1.617.1 (Kappa), and B.1.1.529 (Omicron) were predominantly analyzed.

For kinetic analysis APN01 was applied as ligand on optical sensor chips thus avoiding affinity/avidity bias of kinetic con- stants due to the dimeric structure of APN01. Since APN01 con- tains no capture tag the protein was immobilized to optical sen- sor chip surfaces by covalent amine coupling. Feasibility and reproducibility of this approach was confirmed by determination of inter assay variation on 9 separate chip surfaces on 3 dif- ferent sensor chips being 4.0% for sensorgram similarity and 7.8% for kinetic constants obtained from sensorgram fitting.

Kinetic constants and affinities were determined by sensor- gram fitting applying a Langmuir 1:1 binding model. 8 out of 11 RBD mutants displayed ~ five-fold increased affinity for APN01, including the "epidemiologically" relevant mutants M6, M12, M13, and M14.

Binding on-rates (association constants, k_a) , off-rates (dissociation constants, k_d) and binding affinities (K_D reported as nM) of SARS-CoV-2 RBD/ACE2 interactions were determined by mathematical sensorgram fitting, applying a monomeric Langmuir 1:1 interaction model (A + B = AB) using BiaEvaluation 4.1 soft- ware. The results are summarized in Table 7.

Table 7: Increased affinity of APN01 interactions with SARS-CoV- 2-RBD variants

Surface Plasmon Resonance analysis to derive kinetic con- stants (k_a, k_d) and affinity values (K_D) of SARS-CoV-2 RBD/APN01 interaction. Table 7 lists both the tested variants and the in- troduced amino acid substitution as well as the designation of the respective Variants of Concern tested. Reference strain RBD sequence corresponds to the Wuhan SARS-CoV-2 isolate.

Comparative kinetic analysis of selected variants was car- ried out by applying a Two State binding algorithm (A + B = AB - > ABx) for sensorgram fitting in addition to the standard Lang- muir 1:1 (A + B = AB) fit. The results indicate structural flex- ibility of the SARS-CoV-2 / ACE2-APN01 binding process (conformational change) to adopt a stabilized binding complex following the initial ligand / analyte contact.

This effect could be impressively demonstrated for mutant M7, carrying a G -> S mutation at RBD position 476 which lies within the RBD / ACE2 contact region in close vicinity to gluta- mine 24 of the ACE2 / APN01 molecule. The stabilizing effect may be caused by insertion of the polar serine-OH group in place of the wild type glycine hydrogen and thus be responsible for the significant affinity increase from KD 16.2 nM to KD 2.25 nM, mainly due to the 15 times lower dissociation rate of the serine mutant.

APN01 binding to recombinant prefusion trimeric SARS-CoV-2 Spike proteins was assessed to test whether the increased affin- ity of variants of concern (VOC) RBD/APN01 interaction is also observed in the context of the full-length Spike protein. APN01 is a dimeric molecule thus allowing for bivalent target interac- tion. Therefore, the VOC trimeric and coated pre-fusion Spike variant proteins were immobilized to an optical sensor chip sur- face by covalent amine coupling. APN01 was passed over the immo- bilized Spike proteins in serial dilution in single binding cy- cles. Using BiaEvaluation 4.1 software, subsequent kinetic anal- ysis was carried out by sensorgram fitting applying a Langmuir binding and a bivalent analyte binding model. Kinetic binding constants derived from the Langmuir model represent apparent af- finity, composed of the affinity of the molecular interaction plus an avidity term contributed by bivalent target binding. The bivalent analyte model calculates separate kinetic constants for the affinity determining first step A + B = AB and the avidity determining second step AB + B == AB2 of the binding process. Sen- sorgram fitting showed enhanced apparent affinity (Langmuir model, Table 10) and avidity (bivalent analyte model, Table 11) of all VOC trimeric Spike proteins, except the Kappa VOI.

Table 10: Increased binding affinity of APN01 to full-length pre-fusion trimeric Spike proteins from SARS-CoV-2 variants of concern

Table 11: Increased binding avidity of APN01 to full-length pre- fusion trimeric Spike proteins from SARS-CoV-2 variants of con- cern

Tables 10 and 11 are listing k_a, k_d, as well as K_D values for the interaction of APN01 and full-length trimeric spike pro- teins. Values are derived from calculations based upon the Lang- muir (Table 10) or Bivalent Analyte sensorgram fitting (Table 11).

These results of Table 10 and 11 demonstrate that, when com- pared to Spike trimers of the reference Wuhan strain, APN01 (di- meric recombinant soluble human ACE2) binds to the pre-fusion Spike trimers from all current VOCs with increased affinity and avidity.

As expected, all presented data confirm an increase of APN01 binding affinity for SARS-CoV-2 mutants which potentially dis- play increased infectivity or pathogenicity caused by more tight binding of the virus to its main cellular receptor ACE2. This is a sound argument in favour of APN01 in contrast to other passive immunotherapies under development, based on anti-SARS-CoV-2 spike protein antibodies or antibody fragments. 4.2. Materials and Methods

4.2.1. Ligand:

APN01 samples originating from different production batches have been obtained from APEIRON Biologic AG. APN01 contains sol- uble recombinant human ACE2 of SEQ ID NO: 1.

4.2.2. Analytes:

A panel of recombinant SARS-CoV-2 Spike Protein SI and RBD variants were purchased from Aero Biosystems Inc. and used as analytes for SPR binding.

Table 8: SARS-CoV-2 Spike Protein SI and RBD Variants Under In- vestigation

Selected SARS-CoV-2 Spike Protein Variants: Supplier Information (Citations):

M1: SARS-CoV-2 (COVID-19) SI protein (D614G)

Citation:

SARS-CoV-2 mutation 614G creates an elastase cleavage site en- hancing its spread in high AAT- deficient regions Bhattacharyya, Das, Ghosh et al InfectGenet Evol (2021)

M2: SARS-CoV-2 (COVID-19) S protein RBD (V367F)

Citation:

Cross-neutralization antibodies against SARS-CoV-2 and RBD muta- tions from convalescent patient antibody libraries. BioRxiv preprint, doi: doi: https://doi.org/10.1101/2020.06.06.137513 Authors: Yan Lou, Wenxiang Zhao, Haitao Wei, et al Journal: BioRxiv 2020

M3: SARS-CoV-2 (COVID-19) S protein RBD (N354D)

Citation:

M4: SARS-CoV-2 (COVID-19) S protein RBD (W436R)

Citations:

High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme Authors: Esparza, Thomas J et al.

Journal: Scientific reports 2020

M5: SARS-CoV-2 (COVID-19) S protein RBD (R408I)

Citation:

M6: SARS-CoV-2 (COVID-19) S protein RBD (K417N, E484K, N501Y) Citation: In Silico Investigation of the New UK (B.l.1.7) and South Afri- can

(501Y.V2) SARS-CoV-2 Variants with a Focus at the ACE2-Spike RBD Interface

Villoutreix, Calvez, Marcelin et al Int J Mol Sci (2021) 22 (4)

Ml2: SARS-CoV-2 (COVID-19) S protein RBD (N501Y)

Citation:

In Silico Investigation of the New UK (B.l.1.7) and South Afri- can (B.1.351) SARS-CoV-2

Variants with a Focus at the ACE2-Spike RBD Interface Villoutreix, Calvez, Marcelin et al Int J Mol Sci (2021) 22 (4)

4.2.3. Equipment and protocols used by NBS-C BioScience:

Biacore™ Instrument 3002 (Serial No.: 33-1150109-3736)

Biacore CM5 sensor chips (Order Code: 29127558)

Biacore HBS-EP buffer, (Order Code: BR-1000-12; GE-Healthcare, Uppsala)

Instrument handling and operation following protocols provided in Biacore® 3000 Instrument Handbook

4.2.4. SPR method used for comparative kinetic analysis:

For kinetic analysis APN01 was applied as ligand on optical sensor chips thus avoiding affinity/avidity bias of kinetic con- stants due to the dimeric structure of APN01. Since APN01 con- tains no capture tag the protein was immobilized on optical sen- sor chip surfaces by covalent amine coupling. Feasibility and reproducibility of this approach has been described in the pre- vious data report NBS-APN01; Appendix 1 of APEIRON / NBSC- BioScience MTA, and confirmed by determination of inter assay variation on 9 separate sensor chip surfaces on 3 different sen- sor chips.

4.2.5. SPR sensorgram running protocol:

Ligand APN01-ID5 CM5 sensor chip coupling: EDC/NHS (9 surfaces, 3 sensor chips; 2672 ± 145 RU immobilized)

Multi cycle sensorgram runs (167nM - 6nM analyte concentration) Running buffer: HBS-EP Flow Cell Temp.: 25°C Chip surface regeneration: 3M Mg-chloride

Table 9: s

4 . 2 . 6 . Kinetic analysis:

Kinetic constants and affinities were determined by sensorgram fitting applying a Langmuir 1:1 binding model using BiaEvalua- tion 4.1 software.

Double referencing: subtraction of amine activated control sur- face (FC1) and zero concentration (HBS-EP buffer) sensorgrams Serial Langmuir 1:1 fitting of multi cycle sensorgrams:

Global and local. Fittings with Chi2 / Residuals < 3% of Rmax

4.3. Determination of Inter Assay Variation

In order to confirm data consistency and comparability inter as- say variation was determined by covalent immobilization of the ligand APN01-ID5 to 9 different sensor chip surfaces on 3 dif- ferent Biacore CMS sensor chips at ligand densities of 2672 ±

145 RU applying the Biacore amine coupling kit protocol. Immobi- lization was performed at pH=4.5.

Sample RBD ref. was passed over the APN01-ID5 surfaces at 42nM concentration. Blank (flow cell 1) and HBS-EP buffer subtracted sensorgrams (double referencing) were used for comparison.

Inter assay variation was determined in two parameters: 1. Similarity plot of double referenced sensorgram overlays (= sensorgram assay variation)

2. Langmuir 1:1 model curve fitting of double referenced sensor- grams and determination of kinetic constants and affinities (= kinetic assay variation)

4.4 Kinetic SARS-CoV-2 Spike Protein Mutant Analysis

SARS-CoV-2 spike protein RBD variants were analyzed by

1. Intra-assay comparison with RBD ref. (= original "wild type" sample) at 42nM analyte concentration in order to control RBD ref. data consistency throughout the whole program.

2. Six concentration multi cycle kinetic analysis followed by determination of kinetic constants and affinities as described in Methods. Kinetic constants presented in the tables were cal- culated from fitted curves with Chi2 values < 3% of Rmax.

As shown in Fig. 4, compared to the wild type RBD ref. sen- sorgram (bold red curve), most mutants display slower associa- tion and significantly slower dissociation curves. The RBD /

APN01 bindingsensorgrams of mutants representative for B.l.1.7 (British mutant) and B.1.351 / B.1.28(=P.l) (South African and Brazilian mutants) are shown as bold brown and blue curves, re- spectively.

Furthermore, comparison of curve shapes point to a more com- plex RBD (mutant) / APN01 (ACE2) interaction for some variants (e.g. M9 and Mil) than simple Langmuir 1:1 binding.

A summary of kinetic constants and affinities calculated from mathematical sensorgram fitting applying a Langmuir 1:1 al- gorithm is shown in the table above at the introduction of this example.

The results show that 8 out of 11 RBD mutants displayed ~ five-fold increased affinity for APN01, including the "epidemio- logically relevant" mutants M6 and M12.

4.5. Conclusions

Mutant SARS-CoV-2 spike protein SI and RBD fragments were compared with the corresponding wild type variants isolated from early pandemic virus strains. Mutants with amino acid changes residing within the ACE2 receptor binding motif (RBM) of the RBD fragment, including mutations found in the recent epidemiologi- cally relevant strains (B.l.1.7; B.1.351; B.1.28/P1) were pre- dominantly analyzed.

For kinetic analysis APN01 was applied as ligand on optical sensor chips thus avoiding affinity/avidity bias of kinetic con- stants due to the dimeric structure of APN01. Since APN01 con- tains no capture tag the protein was immobilized to optical sen- sor chip surfaces by covalent amine coupling. Feasibility and reproducibility of this approach was confirmed by determination of inter assay variation on 9 separate chip surfaces on 3 dif- ferent sensor chips being < 5% for sensorgram similarity and <

8% for kinetic constants obtained from sensorgram fitting.

Kinetic constants and affinities were determined by sensorgram fitting applying a Langmuir 1:1 binding model. 8 out of 11 RBD mutants displayed ~ five-fold increased affinity for APN01, in- cluding the "epidemiologically" relevant mutants M6 and M12. Comparative kinetic analysis of selected variants was carried out by applying a Two State binding algorithm (A + B = AB ->

ABx) for sensorgram fitting in addition to the standard Langmuir 1:1 (A + B = AB) fit. The results indicate structural flexibil- ity of the SARS-CoV-2 / ACE2-APN01 binding process (conforma- tional change) to adopt a stabilized binding complex following the initial ligand / analyte contact.

As expected, all presented data confirm an increase of APN01 binding affinity for SARS-CoV-2 mutants which potentially dis- play increased infectivity or pathogenicity caused by more tight binding of the virus to its main cellular receptor ACE2. This is a sound argument in favor of APN01 in contrast to other passive immunotherapies under development, based on anti-SARS-CoV-2 spike protein antibodies or antibody fragments. As a COVID-19 intervention, APN01 offers advantages for treatment of emerging variants. Mutations detected in variants of concern do occur in the virus binding domain, but the vari- ants continue to use ACE2 as primary receptor. Thus, immune es- cape, as can occur with respect to therapeutic MoAbs or natural Abs, should be less of an issue.

A Placebo controlled, double blind, randomized prospective Phase 2 trial (04 - 122020; NCT04335136) in 178 hospitalized Covid-19 patients (aged 18-80 years) demonstrated safety, toler- ability and efficacy of APN01 on top of best standard of care (SOC) in patients with severe COVID-19. Patients were treated for 7 days (follow-up till day 28) with 0,4 mg/kg APN01 compared to sterile 0.9% NaCl.

APN01 was safe and well-tolerated, with no drug-related se- rious Adverse Event and no clinically significant changes in vi- tal signs and ECG were observed.

The primary endpoint of the trial was a composite endpoint of all-cause death or invasive mechanical ventilation up to 28 days or until hospital discharge. Fewer patients treated with APN01 (n=9) died or received invasive ventilation vs. placebo (n=12), although statistical significance was not achieved due to the low total number of events.

Significantly more Ventilator free days (VFD) - mechanical ventilation (subgroup of alive patients) ie 28,2 days in APN01 treated patients vs. 26,9 days in placebo treated patients were observed.

A significant reduction of viral RNA levels compared to pla- cebo could be achieved in APN01 treated patients.

APN01 displayed a positive significant impact on key RAS bi- omarkers like reduction of Angll and increase of Angl-7 and Ang 1-5. The enzymatic ACE2 function of APN01 dials down the RAS and thereby potentially reduces blood pressure, diminishes inflamma- tion and protects many organs from injury.

Additionally, in APN01 arm there was a higher proportion of responders (day 7, 10, 14 and 28), as defined by WHO 11-point score system (2-point improvement) and mSOFA score. Thus, a ten- dency to faster recovery compared to the control group was ob- served.

Therefore, the treatment of APN01 as described above impli- cates lower need for mechanical ventilation in APN01. It reduces the time on mechanical ventilation and lowers the risk of medi- cal complications and comorbidities associated with this inva- sive measure, while also reducing the burden on the Intensive Care Unit and the overall healthcare system.

As a result of these data of the phase II clinical trial it is suggested to combine an i.v. application of APN01 followed by an inhalative therapy of APN01 in patients with mild or moderate symptoms of SARS-CoV-2 infection according to the WHO score shown in this application.

In a preferred embodiment of the invention, APN01 is admin- istered systemically, preferably intravenously, at least twice.

The systemically administration of APN01 could be in paral- lel or followed by administration by inhalation of aerosolized APN01.

Up to 4mg/kg APN01 could be administered systemically to a patient with mild or moderate SARS-CoV-2 infection once, twice or every other day. The treatment of the patient can be followed by 20 mg aerosolized APN01 once daily for up to 28 days.

In an alternative embodiment of the invention APN01 is ad- ministered by inhalation, wherein a solution of ACE2 polypep- tides is aerosolized into aerosol particles with an average par- ticle size of 0.1 ym to 100 ym and at a dose of 100 yg to 600 mg daily, wherein preferably the concentration is 1 mg/ml to 50 mg/ml.

Especially, a volume of 1 ml to 5 ml is aerosolized per ad- ministered dose, preferably in a PARI Vios® PRO Nebulizer.

The aerosolized APN01 could also be administered to subjects with detected virus RNA but without symptoms within the first seven days after detection of virus RNA or to high-risk subjects without detected virus RNA as post-exposure prophylaxis.

Claims

Claims:

1. A method of a treatment or prophylaxis of a SARS-CoV-2 in- fection in a subject comprising administering an ACE2 polypep- tide to the subject, wherein the SARS-CoV-2 infection is with a SARS-CoV-2 variant exhibiting an increase of ACE2 polypeptide binding affinity for SARS-CoV-2 spike protein mutants in compar- ison to wild type SARS-CoV-2 spike protein.

2. The method of claim 1, wherein the ACE2 polypeptide com- prises the peptidase domain and/or collectrin-like domain.

3. The method of claim 1 or 2, wherein the SARS-CoV-2 mutation comprises a mutation of the SARS-CoV-2 spike protein selected from a group comprising V367F, N354D, W436R, R408I, K417N,

E484K, N501Y, G476S, V483A, A475V, N501Y, D614G, K417T, L452R, T478K, E484Q, R682Q, del69-70, dell44, A570D, P681H, T716I,

S982A, D1118H, L18F, D80A, D215G, del242-244, Q677H, R682W,

A701V, S13I, W152C, D138Y, R189S, H655Y, T1027I, T19R, K77T, G142D, E156G, P681R, D950N, A67V, T95I, dell43-145, N211I, del212, ins EPE 214-216, G339D, S371L, S373P, S375F, N440K,

G446S, S477N, E484A, Q493R, G496S, Q498R, Y505H, T547K, N679K, N764K, D796Y, N856K, Q954H or combinations thereof.

4. The method of claim 3 wherein the mutation of the SARS-CoV-2 spike protein comprises K417N, E484K, and N501Y, or K417T,

E484K, and N501Y, or L452R and E484Q, or L452R and T478K.

5. The method of any one of claims 1 to 4, wherein the SARS- CoV-2 infection is with a SARS-CoV-2 variant B.l.1.7 (Alpha),

B.1.351 (Beta), or B.1.1.28.1/Pl (Gamma), B.1.617.2 (Delta),

B.1.526 (Iota), B.1.427 (Epsilon), B.1.429 (Epsilon), B.1.617.1 (Kappa), B.1.617.3, or B.1.525 (Eta), C.37 (Lambda), P.2 (Zeta), P.3 (Theta), B.1.1.529 (Omicron), A.23.1, A.27, B.1.1.318,

B.1.620, C.36.3 or C.1.2.

6. The method of any one of claims 1 to 5, wherein the ACE2 polypeptide is administered at least twice.

7. The method of any one of claims 1 to 6, wherein the ACE2 polypeptide is administered systemically, preferably intrave- nously, in parallel or followed by administration by inhalation.

8. The method of any one of claims 1 to 7, wherein the ACE2 polypeptide is administered systemically, preferably intrave- nously, at least twice, followed by administration by inhala- tion; wherein the systemic administration is at a dose of 100 μg/kg to 4 mg/kg daily and/or preferably wherein administration by inhalation is at a dose of 100 μg to 600 mg, preferably 10 mg to 100 mg, daily.

9. A method of a treatment or prophylaxis of a coronavirus in- fection in a subject comprising administering an ACE2 polypep- tide to the subject, wherein the ACE2 polypeptide is adminis- tered by inhalation, wherein a solution of ACE2 polypeptides is aerosolized into aerosol particles with an average particle size of 0.1 pm to 100 pm and at a dose of 100 pg to 600 mg daily.

10. The method of claim 9, wherein the ACE2 polypeptide in the solution is at a concentration of 0.5 μg/ml to 125 mg/ml, pref- erably 1 mg/ml to 50 mg/ml.

11. The method of claim 9 or 10, wherein the administration by inhalation is parallel or after a systemic administration to the subject, preferably an intravenous administration, preferably a systemic administration at a dose of 0.01 μg/kg to 10 mg/kg daily.

12. The method of any one of claims 8 to 11, wherein a volume of 250 pi to 8 ml, preferably 500 μl to 5 ml, of the solution is aerosolized per administered dose.

13. The method of any one of claims 9 to 12, wherein the ACE2 polypeptide is administered to subjects with detected virus RNA but without symptoms within the first seven days after detection of virus RNA.

14. The method of any one of claims 1 to 13, wherein the ACE2 polypeptide is administered to high-risk subjects without de- tected virus RNA as post-exposure prophylaxis, to asymptomatic subjects with detected virus RNA, or to subjects with sympto- matic mild or moderate SARS-CoV-2 infection, with or with no limitation of activities, hospitalized and no oxygen therapy, or oxygen by mask or nasal prongs.

15. An ACE2 polypeptide for use in a method of any one of claims 1 to 14.