WO2023094571A1

WO2023094571A1 - Stabilization of ace2 fusion proteins

Info

Publication number: WO2023094571A1
Application number: PCT/EP2022/083221
Authority: WO
Inventors: Johann Sebastian BUCHNER; Hristo Lyubomirov SVILENOV
Original assignee: Formycon Ag
Priority date: 2021-11-25
Filing date: 2022-11-25
Publication date: 2023-06-01

Abstract

The present invention relates a combination product comprising a fusion protein of ACE2 with an Fc part of human Ig and an ACE2 inhibitor as well as to the medical use of this combination product, in particular in the prevention or treatment of infections with coronaviruses such as SARS-CoV-2. The present invention also relates to the stabilization of comprising a fusion protein of ACE2 with an Fc part of human Ig with an ACE2 inhibitor..

Description

Stabilization of ACE2 fusion proteins

FIELD OF THE INVENTION

The present invention relates a combination product comprising a fusion protein of ACE2 with an Fc part of human Ig and an ACE2 inhibitor as well as to the medical use of this combination product, in particular in the prevention or treatment of infections with coronaviruses such as SARS-CoV-2. The present invention also relates to the stabilization of a fusion protein of ACE2 with an Fc part of human Ig using an ACE2 inhibitor.

BACKGROUND OF THE INVENTION

ACE2 (angiotensin converting enzyme 2) is a key metalloprotease of the renin-angiotensin system with a catalytic zinc atom in the centre (Donoghue et al. (2002) Circ. Res. 87: e1-e9). Full-length ACE2 consists of an N-terminal extracellular peptidase domain, a collectrin-like domain, a single transmembrane helix and a short intracellular segment. It acts to cleave Angiotensin II to produce Angiotensin (1-7) and Angiotensin I to produce Angiotensin (1-9) which is then processed by other enzymes to become Angiotensin (1-7). ACE2 acts to lower the blood pressure and counters the activity of ACE in order to maintain a balance in the Ras/MAS system. Accordingly, it is a promising target for the treatment of cardiovascular diseases.

Recently, ACE2 has gained much attention as a receptor for coronaviruses and in particular the novel coronavirus SARS-CoV-2. SARS-CoV-2 is a coronavirus which was first discovered in December 2019 in Wuhan, China, but spread rapidly all over the world, leading to a world-wide pandemic. On 21 September 2021, the Johns Hopkins University counted almost 230 millions of confirmed infections worldwide, causing the death of more than 4.7 million people. The pandemic led to several lock-downs in many countries with very significant economic and social effects. It was shown that ACE2 functions as a receptor for SARS-CoV (Li et al. (2003) Nature 426: 450-454; Prakabaran et al. (2004) Biochem. Biophys. Res. Comm. 314: 235-241) and for SARS-CoV-2 (Yan et al. (2020) Science 367: 1444-1485). Further, entry of SARS-CoV-2 into the respiratory cells depends on ACE2 and the serine protease TMPRSS2 (Hoffmann et al. (2020) Cell 181 : 1-10).

In view of the important role of ACE2 for virus entry into the cell, it was proposed to use soluble ACE2 for blocking SARS binding to the cell (WO 2005/032487; WO 2006/122819). The same approach was also suggested for the treatment of infections with SARS-CoV-2 (Kruse (2020) F1000Res. 9:72). Clinical trials with a soluble form of ACE2 in the treatment of infections with SARS-CoV-2 have been initiated by the company Apeiron (Pharmazeutische Zeitung, 10 April 2020) and the first results showed that one patient with severe Covid-19 caused by SARS-CoV-2 recovered fast upon treatment with soluble ACE2 (Zoufaly et al. (2020) The Lancet Respiratory Medicine 8: 1154-1158).

However, isolated receptor domains are typically characterized by a low stability and plasma half-life. For the soluble form of ACE2 a dose-dependent terminal half-life of 10 hours was shown (Haschke et al. (2013) Clin. Pharmacokinet. 52: 783-792). In view of these results, it was decided to administer the soluble form of ACE2 as twice-daily infusion in a later study (Khan et al. (2017) Critical Care 21 : 234). However, an administration of more than one time per day is inconvenient both for the patient and the medical personnel.

A fusion protein of ACE2 consisting of the extracellular domain of either enzymatically active or enzymatically inactive ACE2 linked to the Fc domain of human IgG 1 was constructed and tested. It was shown that both constructs potently neutralized both SARS-CoV and SARS- CoV-2 and inhibited S (Spike) protein-mediated fusion (Lei et al. (2020) Nature Communications 11 : 2070). Further, a fusion protein called COVIDTRAP™ or STI-4398 was developed for clinical testing by the company Sorrento Therapeutics (see https ://www.globenewswire.com/news-release/2020/03/20/2003957/0/en/SORRENTQ- DEVELOPS-STI-4398-COVIDTRAP-PROTEIN-FOR-POTENTIAL-PREVENTION-AND- TREATMENT-OF-SARS-COV-2-CORONAVIRUS-DISEASE-COVID-19.html). An ACE2 fusion protein with the Fc domain of lgG1 called HLX71 is also tested by the company Shanghai Henlius Biotech (see Henlius ACE2-Fc Fusion Protein HLX71 Received IND Approval from US FDA-Media). Liu et al. (2020) Int. J. Biol. Macromol. 165: 1626-1633 describe fusion proteins of wild-type ACE2 and nine ACE2 mutants affecting catalytic activity of ACE2 with the Fc region of human IgG 1. Similarly, Ferrari et al. (2021 ), available at Characterisation of a novel ACE2-based therapeutic with enhanced rather than reduced activity against SARS-CoV2 variants I bioRxiv, describe a fusion protein of enzymatically inactive ACE2 with the Fc domain of human IgG 1. Castilho et al. (2021 ) Biotechnology Journal 16: 2000566 describe the production of ACE2-Fc in glycoengineered Nicotiana benthamiana. However, the interaction of the Fc domain of human IgG 1 with Fc gamma receptors on immune cells may enhance the virus infection (Perlman and Dandekar (2005) Nat. Rev. Immunol. 5(12): 917-927; Chen et al. (2020) Current Tropical Medicine Reports 3:1-4).

Tada et al. (2020), available at https://www.biorxiv.Org/content/10.1101/2020.09.16.300319v1. full, disclose an "ACE2 microbody" in which the extracellular domain of catalytically inactive ACE2 is fused to the CH3 domain of an Fc part of the immunoglobulin heavy chain.

Iwanaga et al. (2020), available at Novel ACE2-lgG1 fusions with improved in vitro and in vivo activity against SARS-C0V2 I bioRxiv discloses ACE2- human IgG 1 fusion proteins with mutations in the catalytic domain of ACE2 and a LALA mutation in the Fc part which abrogates Fey receptor binding, but retains FcRn binding.

Svilenov et al. (2020), available at Efficient inhibition of SARS-CoV-2 strains by a novel ACE2-lgG4-Fc fusion protein with a stabilized hinge region I bioRxiv and PCT/EP2021/063692 describe ACE2 fusion proteins with the Fc domain of lgG4.

European patent application EP21188832.6 discloses ACE2 fusion proteins with the Fc domain of IgM and lgG2.

WO 2021/170113, WO 2021/183404, US 2021/0284716 and WO 2021/189772 also disclose fusion proteins of ACE2 with the Fc part of different immunoglobulins.

Nevertheless, there is still a need for agents which can be used to stabilize fusion proteins of ACE2 with the Fc part of a human antibody to further increase the therapeutic efficacy of the fusion protein and/or to enable cost-effective production of the fusion protein.

SUMMARY OF THE INVENTION

The present invention provides a combination product comprising: (a) a fusion protein comprising a first part comprising a fragment of human ACE2 or a variant of said fragment, said human ACE2 having the amino acid sequence according to SEQ ID NO: 1 , and a second part comprising an Fc portion of a human antibody or a fragment or variant of the Fc portion; and

(b) an inhibitor of ACE2.

In one embodiment, the Fc portion of a human antibody is the Fc portion of a human IgG 1 , lgG2, lgG3, lgG4, or IgM antibody. In one embodiment, the Fc portion of a human antibody may be selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.

In one embodiment, the amino acid sequence of the Fc portion of of the variant of the Fc portion of a human antibody is selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36.

The fragment of human ACE2 may consist of the amino acid sequence according to SEQ ID NO: 2 or may be the extracellular domain of ACE2 consisting of the amino acid sequence according to SEQ ID NO: 3.

The fusion protein may have an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46.

In one embodiment, the variant of the human ACE2 fragment is an enzymatically inactive variant of human ACE2, in particular an enzymatically inactive variant of human ACE2 comprising a H374N and a H378N mutation, the numbering referring to SEQ ID NO: 1. Preferably, the enzymatically inactive variant of human ACE2 has the amino acid sequence according to SEQ ID NO: 37 or SEQ ID NO: 38.

The fusion protein may have an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54. In another embodiment, the enzymatically inactive variant of human ACE2 comprises a R273A mutation, the numbering referring to SEQ ID NO: 1 , Preferably, the enzymatically inactive variant of human ACE2 has the amino acid sequence according to SEQ ID NO: 55 or 56.

The fusion protein may have an amino acid sequence selected from the group consisting of SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 , SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68.

In one embodiment, the variant of the human ACE2 fragment comprises a S645C mutation, the numbering referring to SEQ ID NO: 1 . Preferably, the variant of human ACE2 has the amino acid sequence according to SEQ ID NO: 69 or 70.

The fusion protein may have an amino acid sequence selected from the group consisting of SEQ ID NO: 71 , SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81 and SEQ ID NO: 82.

In one embodiment, the variant of the human ACE2 fragment comprises a S645C mutation, a H374N mutation and a H378N mutation, the numbering referring to SEQ ID NO: 1. Preferably, the variant of human ACE2 has the amino acid sequence according to SEQ ID NO: 83 or SEQ ID NO: 84.

The fusion protein may have an amino acid sequence selected from the group consisting of SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96.

In one embodiment, the variant of the human ACE2 fragment comprises a S645C mutation and a R273A mutation, the numbering referring to SEQ ID NO: 1 . Preferably, the variant of human ACE2 has the amino acid sequence according to SEQ ID NO: 97 or SEQ ID NO: 98.

The fusion protein may have an amino acid sequence selected from the group consisting of SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 , SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 110.

In one embodiment, the inhibitor of ACE2 is a small molecule or a peptide, preferably the inhibitor of ACE2 is selected from MLN-4760 and DX600.

In one embodiment, the inhibitor of ACE2 and the fusion protein are present in the composition in a molar ratio of 100:1 to 2:1 .

The present invention also relates to said combination product for medical use, in particular for use in preventing and/or treating an infection with a coronavirus binding to ACE2, preferably wherein the coronavirus binding to ACE2 is selected from the group consisting of SARS, SARS-CoV-2 and NL63, preferably it is SARS-CoV-2.

In one embodiment, the combination product is to be administered in combination with an anti-viral agent which may be selected from the group consisting of remdesivir, arbidol HCI, ritonavir, lopinavir, darunavir, ribavirin, chloroquin and derivatives thereof, nitazoxanide, camostat mesilate, tocilizumab, siltuximab, sarilumab and baricitinib phosphate.

In one embodiment, the combination product is to be administered in combination with an antibody which may be selected from the group consisting of bamlanivimab, etesevimab, casirivimab, imdevimab, sotrovimab and mixtures thereof.

The present invention also relates to said combination product for use in treating hypertension (including high blood pressure), congestive heart failure, chronic heart failure, acute heart failure, contractile heart failure, myocardial infarction, arteriosclerosis, kidney failure, renal failure, Acute Respiratory Distress Syndrome (ARDS), Acute Lung Injury (ALI), chronic obstructive pulmonary disease (COPD), pulmonary hypertension, renal fibrosis, chronic renal failure, acute renal failure, acute kidney injury, inflammatory bowel disease and multi-organ dysfunction syndrome.

In one embodiment, the combination product further comprises a pharmaceutically acceptable carrier or excipient and/or an anti-viral agent.

The present invention also relates to a method for stabilizing said fusion protein, comprising adding an inhibitor of ACE2 to a cell culture medium comprising host cells producing said fusion protein. The present invention also relates to a method for stabilizing said fusion protein, comprising mixing the fusion protein with an inhibitor of ACE2.

The fusion protein and the inhibitor of ACE2 may be mixed before, during and/or after purification of the fusion protein.

In one embodiment, the fusion protein is stabilized against thermal unfolding.

The inhibitor of ACE2 may be a small molecule or a peptide, preferably it may be selected from MLN-4760 and DX600.

In one embodiment, the fusion protein and the inhibitor of ACE2 are mixed in a molar ratio of 1 :2 to 1 :100.

In one embodiment, the fusion protein is stabilized against aggregation.

The present invention also relates to the use of an inhibitor of ACE2 for stabilizing said ACE2 fusion protein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 A and 1 B: Effect of the ACE2 inhibitor MLN-4760 on the thermal stability of a fusion protein of ACE2 with the Fc part of human lgG2 (A) and with the Fc part of human IgM (B).

Figures 2A and 2B: Effect of the ACE2 inhibitors MLN-4760 and DX600 on the thermal stability of a fusion protein of ACE2 with the Fc part of human lgG4. Figure 2A shows an exemplary measurement. Figure 2B shows the mean melting temperature obtained by three independent measurements as well as the standard deviation.

Figures 3A and 3B: Concentration-dependent effect of the ACE2 inhibitors DX600 (A) and MLN-4760 (B) on the thermal stability of a fusion protein of ACE2 with the Fc part of human igG4.

Figure 4: Effect of the combination of the ACE2 inhibitors MLN-4760 and DX600 on the thermal stability of a fusion protein of ACE2 with the Fc part of human lgG4. Figure 5: Effect of the ACE2 inhibitor MLN-4760 on the aggregation of a fusion protein of ACE2 with the Fc part of human lgG2 after incubation for two hours at 45°C as measured by size exclusion chromatography.

Figure 6: Binding of a fusion protein of ACE2 with the Fc part of human lgG2 to the spike protein of SARS-CoV2 in the presence and absence of the ACE2 inhibitor MLN-4760.

Figures 7A and 7B: Binding of a fusion protein of ACE2 with the Fc part of human lgG4 to the spike protein of SARS-CoV2 in the presence and absence of the ACE2 inhibitors MLN- 4760 and DX600. Figure 7A shows an exemplary measurement. Figure 7B shows the mean KD obtained by three independent measurements as well as the standard deviation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention as illustratively described in the following may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

The present invention will be described with respect to particular embodiments, but the invention is not limited thereto, but only by the claims.

Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting of” is considered to be a preferred embodiment of the term “comprising". If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments.

For the purposes of the present invention, the term “obtained” is considered to be a preferred embodiment of the term “obtainable”. If hereinafter e.g. a cell or organism is defined to be obtainable by a specific method, this is also to be understood to disclose a cell or organism which is obtained by this method.

Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated. As discussed above, the present invention provides a combination product comprising (a) a fusion protein comprising a first part comprising a fragment of human ACE2 or a variant of said fragment, said human ACE2 having the amino acid sequence according to SEQ ID NO: 1 , and a second part comprising an Fc portion of a human antibody or a fragment or variant of the Fc portion; and (b) an inhibitor of ACE2. Surprisingly it has been found that the presence of the inhibitor of ACE2 in the combination product stabilizes said fusion protein against thermal unfolding and aggregation.

The term “combination product” as used herein can refer to (i) a product comprising two or more components that are physically, chemically, or otherwise combined or mixed and produced as a single entity; (ii) two or more separate products packaged together in a single package or as a unit; (iii) a drug, device, or biological product packaged separately that according to its investigational plan or proposed labeling is intended for use with another product; or (iv) any drug, device, or biological product packaged separately that according to its proposed labeling is for use with another product.

Applied to the present case, the term “combination product” may mean that the fusion protein and the inhibitor of ACE2 are (i) physically, chemically, or otherwise combined or mixed and produced as a single entity; or (ii) two separate products packaged together in a single package or as a unit. Alternatively, the term “combination product” may mean that the fusion protein and the inhibitor of ACE2 are packaged separately and according to its investigational plan or proposed labeling the fusion protein is intended for use with the ACE2 inhibitor. Finally, the term “combination product” may mean that the fusion protein and the inhibitor of ACE2 are packaged separately and according to its proposed labeling the fusion protein is for use with the ACE2 inhibitor

An "inhibitor of ACE2" or "ACE2 inhibitor" refers to a molecule which reduces the enzymatic activity of ACE2. In one embodiment, the inhibitor of ACE2 is a selective ACE2 inhibitor. "Selective ACE2 inhibitor" is intended to mean that the inhibitor inhibits ACE2, but not other proteins. In particular, a selective ACE2 inhibitor does not inhibit ACE. The enzymatic activity of ACE2 can be determined by incubating ACE2 with a suitable substrate such as 7-Mca- YVADAPK(Dnp) and measuring fluorescence. Preferably, the inhibitor of ACE2 inhibits the enzymatic activity of ACE2 with an IC50 in the nanomolar or picomolar range.

Suitable inhibitors of ACE2 may be small molecule drugs or peptide drugs. "Small molecule drugs" are molecules of a low molecular weight, preferably a molecular weight of less than 900 daltons. This size allows the small molecule drugs to rapidly diffuse across cell membranes and to reach their intracellular sites of action. "Peptide drugs" are drugs which comprise three to 50 amino acids, preferably 10 to 40 amino acids, more preferably 15 to 35 amino acids and most preferably 20 to 30 amino acids which are linked by peptide bonds. The peptide drugs may comprise chemical modifications at the N-terminal or C-terminal end.

Inhibitors of ACE2 are described in Xiu et al. (2020) J. Med. Chem. 63: 12256-12274 and include, but are not limited to, the small molecules NAAE (N-(2-amino-ethyl)-1 aziridine- ethanamine), chloroquine, hydroxychloroquine, GW280264X, TAPI-0, TAPI-2, MLN-4760, HTCC (N-(2- hydroxypropyl)-3-trimethylammonium chitosan chloride), and hydrophobically modified HTCC (HM-HTCC) as well as the peptides SP-4, SP-8, SP-10, P-4, P-5, S471-503, RBD-11B, DX600, HR2-8, HR1-A, GST-removed-HR2, HR2, HR2P, HR2P-M2, EK1 , 229E- HR1 P and 229E-HR2P. Further inhibitors include, but are not limited to, rutin, quercetin-3-O- glucoside, tamarixetin, 3,4-dihydroxyphenylacetic acid (Liu et al. (2020) J. Agric. Food Chem. 68: 13982-13989), saikosaponin A, baicalin, glycyrrhizin, umifenovir (Shakshi-Niaei et al. (2021) Iran J. Public Health 50(5): 1028-1036), quercetin, galangin, quisinostat, fluprofylline, spirofylline, RS 504393, TNP, GNF-5 (Tung et al. (2020) VNU Journal of Science: Medical and Pharmaceutical Sciences 36(4): 1-11), glyasperin A, broussoflavonol F, sulabiroins A, (2S)-5,7-dihydroxy-40-methoxy-8-prenylflavanone and isorhamnetin (Khayrani et al. (2021) Journal of King Saud University - Science 33: 101297).

Further suitable ACE2 inhibitors can be identified by using a commercial ACE2 inhibitor screening kit which is available from companies such as PromoCell and abeam.

In a preferred embodiment, the inhibitor of ACE2 is MLN-4760. MLN-4760 is a small molecule with the chemical formula:

A synonym thereof is (S,S)-2-(1-Carboxy-2-(3-(3,5-dichlorobenzyl)-3H-imidazol-4-yl)- ethylamino)-4-methylpentanoic acid, 2(S)-(1 (S)-Carboxy-2-(3-(3,5-dichlorobenzyl)-3H- imidazol-4-yl)-ethylamino)-4-methylpentanoic acid. It exists as the S- and the R-isomer and the racemate of these two isomers (Joshi et al. (2016) Eur. J. Pharmacol. 774: 25-33). Preferably, the S-isomer is used. MLN-4760 binds ACE2 with an IC50 of about 440 nM (see, e.g., Xiu et al. (2020) J. Med. Chem. 63: 12256-12274). It was shown that MLN-4760 binds ACE2 at the enzymatic active site with high affinity and significantly alters the ACE2 protein conformation to a closed conformation, but has no major effect on the binding between the SARS-CoV2 spike receptor-binding domain and ACE2 (Nami B, Ghanaeian A, Ghanaeian K, Nami N. The Effect of ACE2 Inhibitor MLN-4760 on the Interaction of SARS-CoV-2 Spike Protein with Human ACE2: A Molecular Dynamics Study. ChemRxiv. Cambridge: Cambridge Open Engage; 2020 (preprint).

In another preferred embodiment, the inhibitor of ACE2 is DX600. DX600 is a peptide inhibitor with the sequence GDYSHCSPLRYYPWWKCTYPDPEGGG (SEQ ID NO: 32). It may be acetylated at its N-terminus and/or amidated at its C-terminus. In a preferred embodiment, it is acetylated at its N-terminus and amidated at its C-terminus. DX600 binds ACE2 with an IC50 of 10 nM (see e.g., Xiu et al. (2020) J. Med. Chem. 63: 12256-12274).

A "fusion protein" is a protein which is formed by at least two polypeptide parts which are not naturally linked with each other. The two polypeptide parts are linked by a peptide bond and optionally a linker molecule is inserted between the two polypeptide parts. The two polypeptide parts are transcribed and translated as a single molecule. The fusion protein typically has functionalities derived from both polypeptide parts. In the context of the present invention, the fusion protein retains the binding properties of ACE2, in particular the binding of viruses such as coronaviruses, and the increased half-life and Fc receptor binding conferred by the Fc portion of a human antibody.

The term "human ACE2" refers to angiotensin converting enzyme 2 derived from a human subject. The full-length sequence of human ACE2 has 805 amino acids. It comprises a signal peptide, an N-terminal extracellular peptidase domain followed by a collectrin-like domain, a single transmembrane helix and a short intracellular segment. The full-length sequence of human ACE2 is depicted in SEQ ID NO:1. Unless indicated otherwise, the amino acid numbering used herein refers to the numbering of the full-length sequence of human ACE2 according to SEQ ID NO: 1. The extracellular domain of human ACE2 consists of amino acids 18 to 740 of SEQ ID NO: 1 and is shown in SEQ ID NO: 3.

The term "fragment of human ACE2" refers to a polypeptide which lacks one or more amino acids compared to the full-length sequence of human ACE2 according to SEQ ID NO:1. The fragment of human ACE2 is capable of binding to the S protein of at least one coronavirus, in particular to the S protein of SARS-CoV-2. The binding of a fragment of human ACE2 to the S protein of at least one coronavirus, in particular to the S protein of SARS-CoV-2, can be determined in an ELISA assay in which the S protein is immobilized on a substrate and contacted with the fragment of human ACE2 and the interaction between the S protein and the fragment of human ACE2 is detected. Alternatively, the binding of a fragment of human ACE2 to the S protein of at least one coronavirus, in particular to the S protein of SARS- CoV-2, can be determined by surface plasmon resonance, e.g. as described in Shang et al. (2020) Nature doi: 10.1038/s41586-020-2179-y; Wrapp et al. (2020) Science 367(6483): 1260-1263; Lei et al. (2020) Nature Communications 11(1): 2070. In a further alternative, the binding of a fragment of human ACE2 to the S protein of at least one coronavirus, in particular to the S protein of SARS-CoV-2, can be determined by biolayer interferometry, e.g. as described in Seydoux et al. (2020) https://doi.org/10.1101/2020.05.12.091298.

In one embodiment, the fragment of human ACE2 consists of 360 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 . Preferably, the fragment of human ACE2 consists of 380 to 723, 400 to 723, 420 to 723, 440 to 723, 460 to 723, 480 to 723 or 500 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1. Preferably, the fragment of human ACE2 consists of 520 to 723, 540 to 723, 560 to 723, 580 to 723 or 600 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 . More preferably, the fragment of human ACE2 consists of 620 to 723, 640 to 723, 660 to 723, 680 to 723, 700 to 723 or 720 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises the amino acid residues K31 and K353, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 comprises the amino acid residues Q24, D30, E35 and Q42, the numbering referring to SEQ ID NO:1. In one embodiment, the fragment of human ACE2 comprises the amino acid residues Q24, D30, K31, E35, Q42 and K353, the numbering referring to SEQ ID NO:1.

In one embodiment, the fragment of human ACE2 consists of 360 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues K31 and K353, the numbering referring to SEQ ID NO: 1 . Preferably, the fragment of human ACE2 consists of 380 to 723, 400 to 723, 420 to 723, 440 to 723, 460 to 723, 480 to 723 or 500 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues K31 and K353, the numbering referring to SEQ ID NO: 1 . Preferably, the fragment of human ACE2 consists of 520 to 723, 540 to 723, 560 to 723, 580 to 723 or 600 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues K31 and K353, the numbering referring to SEQ ID NO: 1 . More preferably, the fragment of human ACE2 consists of 620 to 723, 640 to 723, 660 to 723, 680 to 723, 700 to 723 or 720 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues K31 and K353, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of 360 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues Q24, D30, E35 and Q42, the numbering referring to SEQ ID NO: 1 . Preferably, the fragment of human ACE2 consists of 380 to 723, 400 to 723, 420 to 723, 440 to 723, 460 to 723, 480 to 723 or 500 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues Q24, D30, E35 and Q42, the numbering referring to SEQ ID NO: 1 . Preferably, the fragment of human ACE2 consists of 520 to 723, 540 to 723, 560 to 723, 580 to 723 or 600 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues Q24, D30, E35 and Q42, the numbering referring to SEQ ID NO: 1 . More preferably, the fragment of human ACE2 consists of 620 to 723, 640 to 723, 660 to 723, 680 to 723, 700 to 723 or 720 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues Q24, D30, E35 and Q42, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of 360 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues Q24, D30, K31, E35, Q42 and K353, the numbering referring to SEQ ID NO: 1. Preferably, the fragment of human ACE2 consists of 380 to 723, 400 to 723, 420 to 723, 440 to 723, 460 to 723, 480 to 723 or 500 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues Q24, D30, K31 , E35, Q42 and K353, the numbering referring to SEQ ID NO: 1 . Preferably, the fragment of human ACE2 consists of 520 to 723, 540 to 723, 560 to 723, 580 to 723 or 600 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues Q24, D30, K31, E35, Q42 and K353, the numbering referring to SEQ ID NO: 1. More preferably, the fragment of human ACE2 consists of 620 to 723, 640 to 723, 660 to 723, 680 to 723, 700 to 723 or 720 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues Q24, D30, K31, E35, Q42 and K353, the numbering referring to SEQ ID NO: 1 .

In one embodiment, the fragment of human ACE2 consists of amino acids 18 to 380, 18 to 400, 18 to 420, 18 to 440, 18 to 460, 18 to 480 or 18 to 500 of the sequence according to SEQ ID NO: 1 . Preferably, the fragment of human ACE2 consists of amino acids 18 to 520, 18 to 540, 18 to 560, 18 to 580 or 18 to 600 of the sequence according to SEQ ID NO: 1. More preferably, the fragment of human ACE2 consists of amino acids 18 to 605, 18 to 615, 18 to 620, 18 to 640, 18 to 660, 18 to 680 or 18 to 700 of the sequence according to SEQ ID NO: 1 . Even more preferably, the fragment of human ACE2 consists of amino acids 18 to 710, 18 to 720 or 18 to 730 of the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of the amino acid sequence according to SEQ ID NO:2. The amino acid sequence according to SEQ ID NO: 2 starts at amino acid Q18 and ends with amino acid G732 in the sequence according to SEQ ID NO: 1 . The amino acid glycine at the C-terminal end of this fragment provides a high rotational freedom which favours the fusion of the two protein parts and increases the stability of the fusion protein. Additionally, the use of an ACE2 fragment comprising the amino acid sequence which starts at amino acid Q18 and ends with amino acid G732 in the sequence according to SEQ ID NO: 1 provides a better yield than a longer ACE2 fragment.

In one embodiment, the fragment of human ACE2 consists of the complete extracellular domain of human ACE2 which has the amino acid sequence according to SEQ ID NO:3.

In one embodiment, the fragment of human ACE2 consists of the amino acid sequence according to SEQ ID NO:22. The amino acid sequence according to SEQ ID NO: 22 starts at amino acid Q18 and ends with amino acid G605 in the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 is N-glycosylated at at least one amino acid residue selected from N53, N90, N103, N322, N432, N546 and N690, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 is N- glycosylated at amino acid residues N53, N90 and, N322, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 is N-glycosylated at amino acid residues N53, N90, N103, N322, N432, N546 and N690, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 consists of the amino acid sequence according to SEQ ID NO:2 and is N-glycosylated at at least one amino acid residue selected from N53, N90, N103, N322, N432, N546 and N690, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 consists of the amino acid sequence according to SEQ ID NO:2 and is N-glycosylated at amino acid residues N53, N90 and N322, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 consists of the amino acid sequence according to SEQ ID NO:2 and is N-glycosylated at amino acid residues N53, N90, N103, N322, N432, N546 and N690, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of the amino acid sequence according to SEQ ID NO:3 and is N-glycosylated at at least one amino acid residue selected from N53, N90, N103, N322, N432, N546 and N690, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 consists of the amino acid sequence according to SEQ ID NO:3 and is N-glycosylated at amino acid residues N53, N90 and N322, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 consists of the amino acid sequence according to SEQ ID NO:3 and is N-glycosylated at amino acid residues N53, N90, N103, N322, N432, N546 and N690, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by 360 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 . Preferably, the fragment of human ACE2 comprises or is identified by 380 to 723, 400 to 723, 420 to 723, 440 to 723, 460 to 723, 480 to 723 or 500 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1. Preferably, the fragment of human ACE2 comprises or is identified by 520 to 723, 540 to 723, 560 to 723, 580 to 723 or 600 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 . More preferably, the fragment of human ACE2 comprises or is identified by 620 to 723, 640 to 723, 660 to 723, 680 to 723, 700 to 723 or 720 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises the amino acid residues K31 and K353, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 comprises the amino acid residues Q24, D30, E35 and Q42, the numbering referring to SEQ ID NO:1 . In one embodiment, the fragment of human ACE2 comprises the amino acid residues Q24, D30, K31, E35, Q42 and K353, the numbering referring to SEQ ID NO:1.

In one embodiment, the fragment of human ACE2 comprises or is identified by 360 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues K31 and K353, the numbering referring to SEQ ID NO: 1 . Preferably, the fragment of human ACE2 comprises or is identified by 380 to 723, 400 to 723, 420 to 723, 440 to 723, 460 to 723, 480 to 723 or 500 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues K31 and K353, the numbering referring to SEQ ID NO: 1 . Preferably, the fragment of human ACE2 comprises or is identified by 520 to 723, 540 to 723, 560 to 723, 580 to 723 or 600 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues K31 and K353, the numbering referring to SEQ ID NO: 1. More preferably, the fragment of human ACE2 comprises or is identified by 620 to 723, 640 to 723, 660 to 723, 680 to 723, 700 to 723 or 720 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues K31 and K353, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by 360 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues Q24, D30, E35 and Q42, the numbering referring to SEQ ID NO: 1. Preferably, the fragment of human ACE2 comprises or is identified by 380 to 723, 400 to 723, 420 to 723, 440 to 723, 460 to 723, 480 to 723 or 500 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues Q24, D30, E35 and Q42, the numbering referring to SEQ ID NO: 1 . Preferably, the fragment of human ACE2 comprises or is identified by 520 to 723, 540 to 723, 560 to 723, 580 to 723 or 600 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues Q24, D30, E35 and Q42, the numbering referring to SEQ ID NO: 1 . More preferably, the fragment of human ACE2 comprises or is identified by 620 to 723, 640 to 723, 660 to 723, 680 to 723, 700 to 723 or 720 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues Q24, D30, E35 and Q42, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by 360 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues Q24, D30, K31, E35, Q42 and K353, the numbering referring to SEQ ID NO: 1 . Preferably, the fragment of human ACE2 comprises or is identified by 380 to 723, 400 to 723, 420 to 723, 440 to 723, 460 to 723, 480 to 723 or 500 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues Q24, D30, K31, E35, Q42 and K353, the numbering referring to SEQ ID NO: 1 . Preferably, the fragment of human ACE2 comprises or is identified by 520 to 723, 540 to 723, 560 to 723, 580 to 723 or 600 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues Q24, D30, K31, E35, Q42 and K353, the numbering referring to SEQ ID NO: 1 . More preferably, the fragment of human ACE2 comprises or is identified by 620 to 723, 640 to 723, 660 to 723, 680 to 723, 700 to 723 or 720 to 723 contiguous amino acids within the sequence according to SEQ ID NO: 1 and comprises the amino acid residues Q24, D30, K31, E35, Q42 and K353, the numbering referring to SEQ ID NO: 1 .

In one embodiment, the fragment of human ACE2 comprises or is identified by amino acids 18 to 380, 18 to 400, 18 to 420, 18 to 440, 18 to 460, 18 to 480 or 18 to 500 of the sequence according to SEQ ID NO: 1 . Preferably, the fragment of human ACE2 comprises or is identified by amino acids 18 to 520, 18 to 540, 18 to 560, 18 to 580 or 18 to 600 of the sequence according to SEQ ID NO: 1 . More preferably, the fragment of human ACE2 comprises or is identified by amino acids 18 to 605, 18 to 615, 18 to 620, 18 to 640, 18 to 660, 18 to 680 or 18 to 700 of the sequence according to SEQ ID NO: 1. Even more preferably, the fragment of human ACE2 comprises or is identified by amino acids 18 to 710, 18 to 720 or 18 to 730 of the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the amino acid sequence according to SEQ ID NO:2. The amino acid sequence according to SEQ ID NO: 2 starts at amino acid Q18 and ends with amino acid G732 in the sequence according to SEQ ID NO: 1. The amino acid glycine at the C-terminal end of this fragment provides a high rotational freedom which favours the fusion of the two protein parts and increases the stability of the fusion protein. Additionally, the use of an ACE2 fragment comprising or which is identified by the amino acid sequence which starts at amino acid Q18 and ends with amino acid G732 in the sequence according to SEQ ID NO: 1 provides a better yield than a longer ACE2 fragment.

In one embodiment, the fragment of human ACE2 comprises or is identified by the complete extracellular domain of human ACE2 which has the amino acid sequence according to SEQ ID NO:3. In one embodiment, the fragment of human ACE2 comprises or is identified by the amino acid sequence according to SEQ ID NO:22. The amino acid sequence according to SEQ ID NO: 22 starts at amino acid Q18 and ends with amino acid G605 in the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 is N-glycosylated at at least one amino acid residue selected from N53, N90, N103, N322, N432, N546 and N690, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 is N- glycosylated at amino acid residues N53, N90 and, N322, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 is N-glycosylated at amino acid residues N53, N90, N103, N322, N432, N546 and N690, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the amino acid sequence according to SEQ ID NO:2 and is N-glycosylated at at least one amino acid residue selected from N53, N90, N103, N322, N432, N546 and N690, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 comprises or is identified by of the amino acid sequence according to SEQ ID NO:2 and is N-glycosylated at amino acid residues N53, N90 and N322, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 comprises or is identified by the amino acid sequence according to SEQ ID NO:2 and is N-glycosylated at amino acid residues N53, N90, N103, N322, N432, N546 and N690, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the amino acid sequence according to SEQ ID NO:3 and is N-glycosylated at at least one amino acid residue selected from N53, N90, N103, N322, N432, N546 and N690, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 comprises or is identified by the amino acid sequence according to SEQ ID NO:3 and is N-glycosylated at amino acid residues N53, N90 and N322, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 comprises or is identified by the amino acid sequence according to SEQ ID NO:3 and is N-glycosylated at amino acid residues N53, N90, N103, N322, N432, N546 and N690, the numbering referring to SEQ ID NO: 1.

The term "N-glycosylated" or "N-glycosylation" means that a glycan structure is attached to the amide nitrogen of an asparagine residue of a protein. A glycan is a branched, flexible chain of carbohydrates and the exact structure of the glycan attached to the asparagine residue of a protein depends on the expression system used for glycoprotein production.

A "variant" of the fragment of human ACE2 refers to a fragment as defined above, wherein compared to the corresponding sequence in the amino acid sequence of wild-type, full- length human ACE2 according to SEQ ID NO: 1 at least one amino acid residue is different or at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or at least thirteen amino acids are different. The "variant" of the fragment of human ACE2 comprises one or more amino acid substitutions in the sequence of the fragment of human ACE2. A "variant" of the fragment of human ACE2 does not comprise any amino acid additions or deletions compared to the sequence from which the variant is derived. In one embodiment, the variant the fragment of human ACE2 is a variant of the fragment of human ACE2 according to SEQ ID NO: 2 or 3 and does not comprise any amino acid additions or deletions compared to the sequence according to SEQ ID NO: 2 or 3, i.e. it has the same length as the sequence according to SEQ ID NO: 2 or 3. Within the scope of the present invention a variant of the fragment of human ACE2 is capable of binding to the S protein of at least one coronavirus, in particular to the S protein of SARS-CoV-2. The binding of a variant of the fragment of human ACE2 to the S protein of at least one coronavirus, in particular to the S protein of SARS-CoV-2, can be determined as described above for fragments of human ACE2.

In one embodiment, the variant of the fragment of human ACE2 differs from the corresponding amino acid sequence in the sequence according to SEQ ID NO: 1 by one amino acid. In another embodiment, the variant of the fragment of human ACE2 differs from the corresponding amino acid sequence in the sequence according to SEQ ID NO: 1 by two amino acids. In still another embodiment, the variant of the fragment of human ACE2 differs from the corresponding amino acid sequence in the sequence according to SEQ ID NO: 1 by three amino acids. In still another embodiment, the variant of the fragment of human ACE2 differs from the corresponding amino acid sequence in the sequence according to SEQ ID NO: 1 by four amino acids. In still another embodiment, the variant of the fragment of human ACE2 differs from the corresponding amino acid sequence in the sequence according to SEQ ID NO: 1 by five amino acids. In still another embodiment, the variant of the fragment of human ACE2 differs from the corresponding amino acid sequence in the sequence according to SEQ ID NO: 1 by six amino acids. In still another embodiment, the variant of the fragment of human ACE2 differs from the corresponding amino acid sequence in the sequence according to SEQ ID NO: 1 by seven amino acids. In still another embodiment, the variant of the fragment of human ACE2 differs from the corresponding amino acid sequence in the sequence according to SEQ ID NO: 1 by eight amino acids. In still another embodiment, the variant of the fragment of human ACE2 differs from the corresponding amino acid sequence in the sequence according to SEQ ID NO: 1 by nine amino acids. In still another embodiment, the variant of the fragment of human ACE2 differs from the corresponding amino acid sequence in the sequence according to SEQ ID NO: 1 by ten amino acids. In still another embodiment, the variant of the fragment of human ACE2 differs from the corresponding amino acid sequence in the sequence according to SEQ ID NO: 1 by eleven amino acids. In still another embodiment, the variant of the fragment of human ACE2 differs from the corresponding amino acid sequence in the sequence according to SEQ ID NO: 1 by twelve amino acids. In still another embodiment, the variant of the fragment of human ACE2 differs from the corresponding amino acid sequence in the sequence according to SEQ ID NO: 1 by thirteen amino acids.

One variant of the fragment of human ACE2 may be an enzymatically inactive variant. "The enzymatically inactive variant of the fragment of human ACE2" lacks the ability to cleave angiotensin II to Ang1-7. The enzymatic activity of human ACE2 can be determined by methods known to the skilled person. Suitable kits for determining the enzymatic activity of human ACE2 are commercially available, for example from the companies BioVision or Anaspec. By using an enzymatically inactive ACE2 variant any side effects associated with the enzymatic activity of ACE2 such as effects on the cardiovascular system or the regulation of blood pressure are eliminated. Further, the risk of counterbalancing the RAS - MAS equilibrium is reduced.

The enzymatically inactive variant of the fragment of human ACE2 may comprise one or more mutations of amino acids within the catalytic centre of ACE2. In particular, the enzymatically inactive variant of the fragment of human ACE2 comprises a mutation of the wildtype histidine at residue 374 of the sequence according to SEQ ID NO: 1 and/or a mutation of the wildtype histidine at residue 378 of the sequence according to SEQ ID NO: 1 . The wild-type histidine may be mutated to any amino acid other than histidine and particularly, the wild-type histidine is mutated to asparagine. Preferably, the enzymatically inactive variant of the fragment of human ACE2 comprises a H374N and a H378N mutation, the numbering referring to the sequence according to SEQ ID NO: 1.

In another embodiment, the enzymatically inactive variant of the fragment of human ACE2 comprises a mutation at one or more of the following amino acid residues, the numbering referring to the sequence according to SEQ ID NO: 1 : residue 345 (histidine in wild-type), 273 (arginine in wild-type), 402 (glutamic acid in wild-type) and 505 (histidine in wild-type). In one embodiment, the enzymatically inactive variant of the fragment of human ACE2 comprises a mutation of histidine at residue 345 to alanine or leucine, a mutation of arginine at residue 273 to alanine, glutamine or lysine, a mutation of glutamic acid at residue 402 to alanine and/or a mutation of histidine at residue 505 to alanine or leucine, the numbering referring to the sequence according to SEQ ID NO: 1. In a particular embodiment, the enzymatically inactive variant of the fragment of human ACE2 comprises a mutation of arginine at residue 273 to alanine (also called R273A mutation). It was found that arginine 273 is critical for substrate binding and that its substitution abolishes enzymatic activity (Guy et al. (2005) FEBS J. 272(14): 3512-3520).

In one embodiment, the fragment of human ACE2 consists of amino acids 18 to 380, 18 to 400, 18 to 420, 18 to 440, 18 to 460, 18 to 480 or 18 to 500 of the sequence according to SEQ ID NO: 1 and comprises a H374N and a H378N mutation, the numbering referring to the sequence according to SEQ ID NO: 1 . Preferably, the fragment of human ACE2 consists of amino acids 18 to 520, 18 to 540, 18 to 560, 18 to 580 or 18 to 600 of the sequence according to SEQ ID NO: 1 and comprises a H374N and a H378N mutation, the numbering referring to the sequence according to SEQ ID NO: 1 . More preferably, the fragment of human ACE2 consists of amino acids 18 to 615, 18 to 620, 18 to 640, 18 to 660, 18 to 680 or 18 to 700 of the sequence according to SEQ ID NO: 1 and comprises a H374N and a H378N mutation, the numbering referring to the sequence according to SEQ ID NO: 1. Even more preferably, the fragment of human ACE2 consists of amino acids 18 to 710, 18 to 720 or 18 to 730 of the sequence according to SEQ ID NO: 1 and comprises a H374N and a H378N mutation, the numbering referring to the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 2 and comprises a H374N and a H378N mutation, the numbering referring to the sequence according to SEQ ID NO: 1 In one embodiment, the variant of the fragment of human ACE2 consists of a protein having the amino acid sequence according to SEQ ID No. 37.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 3 and comprises a H374N and a H378N mutation, the numbering referring to the sequence according to SEQ ID NO: 1. In one embodiment, the variant of the fragment of human ACE2 consists of a protein having the amino acid sequence according to SEQ ID No. 38.

In one embodiment, the fragment of human ACE2 consists of amino acids 18 to 380, 18 to 400, 18 to 420, 18 to 440, 18 to 460, 18 to 480 or 18 to 500 of the sequence according to SEQ ID No. 1 and comprises a R273A mutation, the numbering referring to the sequence according to SEQ ID No. 1. Preferably, the fragment of human ACE2 consists of amino acids 18 to 520, 18 to 540, 18 to 560, 18 to 580 or 18 to 600 of the sequence according to SEQ ID No. 1 and comprises a R273A mutation, the numbering referring to the sequence according to SEQ ID No. 1. More preferably, the fragment of human ACE2 consists of amino acids 18 to 615, 18 to 620, 18 to 640, 18 to 660, 18 to 680 or 18 to 700 of the sequence according to SEQ ID No. 1 and comprises a R273A mutation, the numbering referring to the sequence according to SEQ ID No. 1 . Even more preferably, the fragment of human ACE2 consists of amino acids 18 to 710, 18 to 720 or 18 to 730 of the sequence according to SEQ ID No. 1 and comprises a R273A mutation, the numbering referring to the sequence according to SEQ ID No. 1.

In one embodiment, the fragment of human ACE2 consists of a protein having the amino acid sequence according to SEQ ID No. 2 and comprises a R273A mutation. In one embodiment, the fragment of human ACE2 consists of a protein having the amino acid sequence according to SEQ ID No. 3 and comprises a R273A mutation. In one embodiment, the variant of the fragment of human ACE2 consists of a protein having the amino acid sequence according to SEQ ID No. 55. In one embodiment, the variant of the fragment of human ACE2 consists of a protein having the amino acid sequence according to SEQ ID No. 56.

In one embodiment, the fragment of human ACE2 comprises or is identified by amino acids 18 to 380, 18 to 400, 18 to 420, 18 to 440, 18 to 460, 18 to 480 or 18 to 500 of the sequence according to SEQ ID NO: 1 and comprises a H374N and a H378N mutation, the numbering referring to the sequence according to SEQ ID NO: 1 . Preferably, the fragment of human ACE2 comprises or is identified by amino acids 18 to 520, 18 to 540, 18 to 560, 18 to 580 or 18 to 600 of the sequence according to SEQ ID NO: 1 and comprises a H374N and a H378N mutation, the numbering referring to the sequence according to SEQ ID NO: 1 . More preferably, the fragment of human ACE2 comprises or is identified by amino acids 18 to 615, 18 to 620, 18 to 640, 18 to 660, 18 to 680 or 18 to 700 of the sequence according to SEQ ID NO: 1 and comprises a H374N and a H378N mutation, the numbering referring to the sequence according to SEQ ID NO: 1 . Even more preferably, the fragment of human ACE2 comprises or is identified by amino acids 18 to 710, 18 to 720 or 18 to 730 of the sequence according to SEQ ID NO: 1 and comprises a H374N and a H378N mutation, the numbering referring to the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 2 and comprises a H374N and a H378N mutation, the numbering referring to the sequence according to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 3 and comprises a H374N and a H378N mutation, the numbering referring to the sequence according to SEQ ID NO: 1.

Another variant of the fragment of human ACE2 may be a variant which inhibits shedding of ACE2. It was shown that ACE2 is shed from human airway epithelia by cleavage of the ACE2 ectodomain and that ADAM17 regulates ACE2 cleavage. Further, a point mutation at leucine 584 of full-length ACE2 which is located in the ectodomain of ACE2 abolished shedding (Jia et al. (2009) Am. J. Physiol. Lung Cell. Mol. Physiol. 297(1): L84-96). Hence, in one embodiment the variant of the fragment of human ACE2 comprises a mutation at leucine 584, the numbering referring to the sequence according to SEQ ID NO:1. In one embodiment the mutation at leucine 584 is a L584A mutation.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 2 and comprises a L584A mutation, the numbering referring to the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 3 and comprises a L584A mutation, the numbering referring to the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 2 and comprises a L584A mutation, the numbering referring to the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 3 and comprises a L584A mutation, the numbering referring to the sequence according to SEQ ID NO: 1. In one embodiment, the variant of the fragment of human ACE2 comprises a H374N mutation, a H378N mutation and a L584A mutation, the numbering referring to the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 2 and comprises a H374N mutation, a H378N mutation and a L584A mutation, the numbering referring to the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 3 and comprises a H374N mutation, a H378N mutation and a L584A mutation, the numbering referring to the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 2 and comprises a H374N mutation, a H378N mutation and a L584A mutation, the numbering referring to the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 3 and comprises a H374N mutation, a H378N mutation and a L584A mutation, the numbering referring to the sequence according to SEQ ID NO: 1.

Another variant of the fragment of human ACE2 may be a variant which inhibits cleavage of ACE2 by the protease TMPRSS2. It was shown that ACE2 proteolysis by TMPRSS2 augments entry of SARS-CoV (Heurich et al. (2014) J. Virol. 88(2): 1293-1307). TMPRSS2 also plays a role in the entry of SARS-CoV-2 into the cells (Hoffmann et al. (2020) Cell 181 : 1-10). To abolish cleavage of ACE2 by TMPRSS2, the amino acid residues essential for the cleavage may be mutated. It was shown that arginine and lysine residues within the amino acid region spanning amino acids 697 to 716 of ACE2 are essential for ACE2 cleavage by TMPRSS2 (Heurich et al. (2014) J. Virol. 88(2): 1293-1307). Hence, in one embodiment the variant of the fragment of human ACE2 comprises a mutation at at least one residue selected from amino acids 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1. Preferably, the variant of the fragment of human ACE2 comprises a mutation at at least two or three residues selected from amino acids 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1. More preferably, the variant of the fragment of human ACE2 comprises a mutation at at least four or five residues selected from amino acids 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1. Most preferably, the variant of the fragment of human ACE2 comprises a mutation at residues 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1. The wild-type amino acid residue at any of these residues may be mutated to any other amino acid and particularly, the wild-type amino acid residue is mutated to alanine.

In one embodiment, the variant of the fragment of human ACE2 comprises at least one of the following mutations: R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1. Preferably, the variant of the fragment of human ACE2 comprises at least two or three of the following mutations: R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1. More preferably, the variant of the fragment of human ACE2 comprises at least four or five of the following mutations: R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1. Most preferably, the variant of the fragment of human ACE2 comprises the following mutations: R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1.

The variant of the fragment of human ACE2 may further comprise mutations at residues 619, 621 and/or 625, the numbering referring to SEQ ID NO: 1. In particular, the variant of the fragment of human ACE2 may further comprise the following mutations: K619A, R621 A and/or K625A, the numbering referring to SEQ ID NO: 1.

Hence, in one embodiment, the variant of the fragment of human ACE2 comprises the following mutations: K619A, R621A, K625A, R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 2 and comprises a mutation at at least one residue selected from amino acids 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 2 and comprises a mutation at residues 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 2 and comprises at least one of the following mutations: R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 2 and comprises the following mutations: R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 3 and comprises a mutation at at least one residue selected from amino acids 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 3 and comprises a mutation at residues 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 3 and comprises at least one of the following mutations: R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 3 and comprises the following mutations: R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 2 and comprises a H374N mutation, a H378N mutation, a L584A mutation and the following mutations: R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 3 and comprises a H374N mutation, a H378N mutation, a L584A mutation and the following mutations: R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 2 and comprises a mutation at at least one residue selected from amino acids 619, 621, 625, 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 2 and comprises a mutation at residues 619, 621, 625, 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 2 and comprises at least one of the following mutations: K619A, R621A, K625A, R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 2 and comprises the following mutations: K619A, R621A, K625A, R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 3 and comprises a mutation at at least one residue selected from amino acids 619, 621, 625, 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 3 and comprises a mutation at residues 619, 621, 625, 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 3 and comprises at least one of the following mutations: K619A, R621A, K625A, R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 3 and comprises the following mutations: K619A, R621A, K625A, R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 2 and comprises a H374N mutation, a H378N mutation, a L584A mutation and the following mutations: K619A, R621A, K625A, R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 3 and comprises a H374N mutation, a H378N mutation, a L584A mutation and the following mutations: K619A, R621A, K625A, R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 2 and comprises a mutation at at least one residue selected from amino acids 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 2 and comprises a mutation at residues 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 2 and comprises at least one of the following mutations: R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 2 and comprises the following mutations: R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 3 and comprises a mutation at at least one residue selected from amino acids 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 3 and comprises a mutation at residues 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 3 and comprises at least one of the following mutations: R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 3 and comprises the following mutations: R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 2 and comprises a H374N mutation, a H378N mutation, a L584A mutation and the following mutations: R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 3 and comprises a H374N mutation, a H378N mutation, a L584A mutation and the following mutations: R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 2 and comprises a mutation at at least one residue selected from amino acids 619, 621 , 625, 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 2 and comprises a mutation at residues 619, 621, 625, 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 2 and comprises at least one of the following mutations: K619A, R621A, K625A, R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 2 and comprises the following mutations: K619A, R621A, K625A, R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 3 and comprises a mutation at at least one residue selected from amino acids 619, 621 , 625, 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 3 and comprises a mutation at residues 619, 621, 625, 697, 702, 705, 708, 710 and 716, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 3 and comprises at least one of the following mutations: K619A, R621A, K625A, R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 3 and comprises the following mutations: K619A, R621A, K625A, R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 2 and comprises a H374N mutation, a H378N mutation, a L584A mutation and the following mutations: K619A, R621A, K625A, R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to the sequence according to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 3 and comprises a H374N mutation, a H378N mutation, a L584A mutation and the following mutations: K619A, R621A, K625A, R697A, K702A, R705A, R708A, R710A and R716A, the numbering referring to the sequence according to SEQ ID NO: 1.

Another variant of the fragment of human ACE2 may be a variant which provides an additional cysteine for the formation of disulfide bridges between two ACE2 molecules. The disulfide bridge increases the intrinsic stability of the fusion protein and may also have an effect on the binding of the fusion protein to its target. The additional cysteine may be provided by a substitution of serine 645 in the numbering of SEQ ID NO: 1 with cysteine.

Hence, in one embodiment, the variant of the fragment of human ACE2 comprises a S645C mutation, the numbering referring to SEQ ID NO: 1 . In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 2 and comprises a S645C mutation, the numbering referring to SEQ ID NO: 1. In one embodiment, the variant of the fragment of human ACE2 consists of a protein having the amino acid sequence according to SEQ ID No. 69.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 3 and comprises a S645C mutation, the numbering referring to SEQ ID NO: 1. In one embodiment, the variant of the fragment of human ACE2 consists of a protein having the amino acid sequence according to SEQ ID No. 70.

In one embodiment, the fragment of human ACE2 consists of a protein having the amino acid sequence according to SEQ ID No. 2 and comprises a H374N mutation, a H378N mutation, and a S645C mutation, the numbering referring to SEQ ID No. 1 . In one embodiment, the variant of the fragment of human ACE2 consists of a protein having the amino acid sequence according to SEQ ID No. 83.

In one embodiment, the fragment of human ACE2 consists of a protein having the amino acid sequence according to SEQ ID No. 3 and comprises a H374N mutation, a H378N mutation, and a S645C mutation, the numbering referring to SEQ ID No. 1 . In one embodiment, the variant of the fragment of human ACE2 consists of a protein having the amino acid sequence according to SEQ ID No. 84.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 2 and comprises a H374N mutation, a H378N mutation, a L584A mutation and a S645C mutation, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 3 and comprises a H374N mutation, a H378N mutation, a L584A mutation and a S645C mutation, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of a protein having the amino acid sequence according to SEQ ID No. 2 and comprises a R273A mutation and a S645C mutation, the numbering referring to SEQ ID No. 1 . In one embodiment, the variant of the fragment of human ACE2 consists of a protein having the amino acid sequence according to SEQ ID No. 97. In one embodiment, the fragment of human ACE2 consists of a protein having the amino acid sequence according to SEQ ID No. 3 and comprises a R273A mutation and a S645C mutation, the numbering referring to SEQ ID No. 1. In one embodiment, the variant of the fragment of human ACE2 consists of a protein having the amino acid sequence according to SEQ ID No. 98.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 2 and comprises a S645C mutation, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 3 and comprises a S645C mutation, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 2 and comprises a H374N mutation, a H378N mutation, a L584A mutation and a S645C mutation, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 3 and comprises a H374N mutation, a H378N mutation, a L584A mutation and a S645C mutation, the numbering referring to SEQ ID NO: 1.

Another variant of the fragment of human ACE2 may be a variant which inhibits dimerization. Hence, in one embodiment the variant of the fragment of human ACE2 comprises a mutation at amino acid Q139, the numbering referring to SEQ ID NO: 1 . In one embodiment the variant of the fragment of human ACE2 comprises a Q139A mutation, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 2 and comprises a Q139A mutation, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 3 and comprises a Q139A mutation, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 2 and comprises a H374N mutation, a H378N mutation, a L584A mutation and a Q139A mutation, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 3 and comprises a H374N mutation, a H378N mutation, a L584A mutation and a Q139A mutation, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 22 and comprises a Q139A mutation, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 consists of the sequence according to SEQ ID NO: 22 and comprises a H374N mutation, a H378N mutation, a L584A mutation and a Q139A mutation, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 2 and comprises a Q139A mutation, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 3 and comprises a Q139A mutation, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 2 and comprises a H374N mutation, a H378N mutation, a L584A mutation and a Q139A mutation, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 3 and comprises a H374N mutation, a H378N mutation, a L584A mutation and a Q139A mutation, the numbering referring to SEQ ID NO: 1.

In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 22 and comprises a Q139A mutation, the numbering referring to SEQ ID NO: 1. In one embodiment, the fragment of human ACE2 comprises or is identified by the sequence according to SEQ ID NO: 22 and comprises a H374N mutation, a H378N mutation, a L584A mutation and a Q139A mutation, the numbering referring to SEQ ID NO: 1.

The fusion protein present in the combination product of the present invention comprises a second part comprising an Fc portion of a human antibody or a fragment or variant of the Fc portion. Preferably, the Fc portion of a human antibody is the Fc portion of a human IgG 1 , lgG2, lgG3, lgG4, or IgM antibody.

In one embodiment, the second part of the fusion protein comprises the Fc portion of human lgG4. The Fc portion of human lgG4 comprises the CH2 and CH3 domains of human lgG4 linked together to form the Fc portion. In a full-length human lgG4 antibody the Fc portion is connected to the Fab fragment through a hinge region. The Fab fragment comprises the heavy chain variable region and the CH1 domain. Preferably, the Fc portion of human lgG4 used in the fusion protein has the sequence according to SEQ ID NO: 6.

Since the lgG4 subclass of antibodies has only a partial affinity for Fc gamma receptor and does not activate complement (see Muhammed (2020) Immunome Res. 16(1): 173), it does not activate the immune system to the same extent as the IgG 1 subclass of antibodies. Consequently, the cytokine expression is stimulated to a lower extent and the risk for a cytokine storm is reduced. The lgG4 subclass of antibodies is able to bind to FcRn.

"A variant of the Fc portion of human lgG4" refers to the Fc portion of human lgG4 which has one or more amino acid substitutions compared to the wild-type Fc portion of human lgG4 according to SEQ ID NO: 6. In one embodiment, the variant of the Fc portion of human lgG4 has one to twelve, one to eleven, one to ten, one to nine, one to eight, one to seven, one to six, one to five, one to four, one to three, one or two amino acid substitutions compared to the wild-type Fc portion of human lgG4 according to SEQ ID NO: 6. In one embodiment, the variant of the Fc portion of human lgG4 has one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve amino acid substitutions compared to the wild-type Fc portion of human lgG4 according to SEQ ID NO: 6. In one embodiment, the one or more amino acid substitutions lead to decreased effector functions compared to the wild-type Fc portion of human lgG4 according to SEQ ID NO: 6. In one embodiment, the one or more amino acid substitutions lead to an increased half-life compared to the wild-type Fc portion of human lgG4 according to SEQ ID NO: 6. In one embodiment, the one or more amino acid substitutions lead to decreased effector functions compared to the wild-type Fc portion of human lgG4 according to SEQ ID NO: 6 and to an increased half-life compared to the wildtype Fc portion of human lgG4 according to SEQ ID NO: 6.

In one embodiment, the one or more amino acid substitutions compared to the wild-type Fc portion of human lgG4 do not produce the wild-type Fc portion of IgG 1 according to SEQ ID NO: 4. In one embodiment, the one or more amino acid substitutions do not impart upon the altered lgG4 Fc portion the effector functions of wild-type IgG 1.

Preferably, the decreased effector functions comprise a decreased complement-dependent cytotoxicity (CDC). More preferably, the CDC is decreased by at least two-fold, at least three-fold, at least four-fold or at least five-fold compared to the CDC of the wild-type Fc portion of human lgG4 according to SEQ ID NO: 6. Methods to determine and quantify CDC are well-known to the skilled person. In general, CDC can be determined by incubating the Fc portion fused to an antigen-binding portion with suitable target cells and complement and detecting the cell death of the target cells. Complement recruitment can be analyzed with a C1q binding assay using ELISA plates (see, e.g., Schlothauer et al. (2016) Protein Eng. Des. Sei. 29(10): 457-466).

In one embodiment, the variant of the Fc portion of human lgG4 comprises at least one amino acid substitution at an amino acid residue selected from F3, L4, G6, P7, F12, V33, N66 and P98 of the sequence according to SEQ ID NO: 6. These amino acid residues correspond to amino acid residues F234, L235, G237, P238, F243, V264, N297 and P329 of full-length human lgG4. It was shown that amino acid substitutions at these residues lead to a reduced effector function (WO 94/28027; WO 94/29351 ; WO 95/26403; WO 2011/066501 ; WO 2011/149999; WO 2012/130831; Wang et al. (2018) Protein Cell. 9(1): 63-73).

In one embodiment, the variant of the Fc portion of human lgG4 may comprise the amino acid substitutions M21 Y, S23T and T25E in the sequence according to SEQ ID NO: 6 which corresponds to the amino acid substitutions M252Y, S254T and T256E in the amino acid sequence of full-length human lgG4. In one embodiment, the variant of the Fc portion of human lgG4 has the amino acid sequence according to SEQ ID NO: 33. This variant has an increased half-life.

In one embodiment, the variant of the Fc portion of human lgG4 comprises the amino acid substitutions T25D and T76Q in the sequence according to SEQ ID NO: 6 which corresponds to the amino acid substitutions T256D and T307Q in the amino acid sequence of full-length human lgG4. In one embodiment, the variant of the Fc portion of human lgG4 has the amino acid sequence according to SEQ ID NO: 34. This variant has an increased half-life and an enhanced binding to FcRn (Mackness et al. (2019) MABS 11(7): 1276-1288).

In one embodiment, the variant of the Fc portion of human lgG4 comprises the amino acid substitution L4E/A in the sequence according to SEQ ID NO: 6 which corresponds to the amino acid substitution L235E/A in the amino acid sequence of full-length human lgG4. This variant has a reduced effector function, in particular reduced CDC.

In one embodiment, the variant of the Fc portion of human lgG4 comprises the amino acid substitutions F3A and L4A in the sequence according to SEQ ID NO: 6 which correspond to the amino acid substitutions F234A and L235A in the amino acid sequence of full-length human lgG4. This variant has a reduced effector function, in particular reduced CDC.

In one embodiment, the variant of the Fc portion of human lgG4 comprises the amino acid substitutions F3A, L4E, G6A and P7S in the sequence according to SEQ ID NO: 6 which correspond to the amino acid substitutions F234A, L235E, G237A and P238S in the amino acid sequence of full-length human lgG4. This variant has a reduced effector function, in particular reduced CDC.

In one embodiment, the variant of the Fc portion of human lgG4 comprises the amino acid substitutions F12A and V33A in the sequence according to SEQ ID NO: 6 which correspond to the amino acid substitutions F243A and V264A in the amino acid sequence of full-length human lgG4. This variant has a reduced effector function, in particular reduced CDC.

In one embodiment, the variant of the Fc portion of human lgG4 comprises the amino acid substitutions L4E and P98G in the sequence according to SEQ ID NO: 6 which correspond to the amino acid substitutions L235E and P329G in the amino acid sequence of full-length human lgG4. This variant has a reduced effector function, in particular reduced CDC.

In one embodiment, the variant of the Fc portion of human lgG4 comprises the amino acid substitution N66A/Q/G in the sequence according to SEQ ID NO: 6 which corresponds to the amino acid substitution N297A/Q/G in the amino acid sequence of full-length human lgG4. This variant has a reduced effector function, in particular reduced CDC. In one embodiment, the variant of the Fc portion of human lgG4 comprises at least one amino acid substitution at an amino acid residue selected from T250, M252, S254, T256, E258, K288, T307, V308, Q311, V427, M428, H433, N434 and H435 of full-length human lgG4. These amino acid residues correspond to amino acid residues T19, M21 , S23, T25, E27, K57, T76, V77, Q80, V196, M197, H202, N203 and H204 of the sequence according to SEQ ID NO: 6. It was shown that these amino acid substitutions lead to an increased half-life of the Fc-containing protein (WO 00/42072; WO 02/060919; WO 2004/035752;

WO 2006/053301; WO 2009/058492; WO 2009/086320; US 2010/0204454; GB 2013/02878; WO 2013/163630; US 2019/0010243). The half-life of an antibody or Fc fusion protein can be determined by measuring the antibody or Fc fusion protein concentration in the serum at different time-points after administration of the antibody or Fc fusion protein and calculating the half-life therefrom.

In one embodiment, the variant of the Fc portion of human lgG4 comprises the amino acid substitutions M21Y, S23T and T25E in the sequence according to SEQ ID NO: 6 which corresponds to the amino acid substitutions M252Y, S254T and T256E in the amino acid sequence of full-length human lgG4. This variant has an increased half-life.

In one embodiment, the variant of the Fc portion of human lgG4 comprises the amino acid substitutions T19Q/E and M197L/F in the sequence according to SEQ ID NO: 6 which corresponds to the amino acid substitutions T250Q/E and M428L/F in the amino acid sequence of full-length human lgG4. This variant has an increased half-life.

In one embodiment, the variant of the Fc portion of human lgG4 comprises the amino acid substitutions N203S and V77W/Y/F in the sequence according to SEQ ID NO: 6 which corresponds to the amino acid substitutions N434S and V308W/Y/F in the amino acid sequence of full-length human lgG4. This variant has an increased half-life.

In one embodiment, the variant of the Fc portion of human lgG4 comprises the amino acid substitutions M21Y and M197L in the sequence according to SEQ ID NO: 6 which corresponds to the amino acid substitutions M252Y and M428L in the amino acid sequence of full-length human lgG4. This variant has an increased half-life.

In one embodiment, the variant of the Fc portion of human lgG4 comprises the amino acid substitutions T76Q and N203S in the sequence according to SEQ ID NO: 6 which corresponds to the amino acid substitutions T307Q and N434S in the amino acid sequence of full-length human lgG4. This variant has an increased half-life.

In one embodiment, the variant of the Fc portion of human lgG4 comprises the amino acid substitutions M197L and V77F in the sequence according to SEQ ID NO: 6 which corresponds to the amino acid substitutions M428L and V308F in the amino acid sequence of full-length human lgG4. This variant has an increased half-life.

In one embodiment, the variant of the Fc portion of human lgG4 comprises the amino acid substitutions Q80V and N203S in the sequence according to SEQ ID NO: 6 which corresponds to the amino acid substitutions Q311 V and N434S in the amino acid sequence of full-length human lgG4. This variant has an increased half-life.

In one embodiment, the variant of the Fc portion of human lgG4 comprises the amino acid substitutions H202K and N203F in the sequence according to SEQ ID NO: 6 which corresponds to the amino acid substitutions H433K and N434F in the amino acid sequence of full-length human lgG4. This variant has an increased half-life.

In one embodiment, the variant of the Fc portion of human lgG4 comprises the amino acid substitutions E27F and V196T in the sequence according to SEQ ID NO: 6 which corresponds to the amino acid substitutions E258F and V427T in the amino acid sequence of full-length human lgG4. This variant has an increased half-life.

In one embodiment, the variant of the Fc portion of human lgG4 comprises the amino acid substitutions K57E and H204K in the sequence according to SEQ ID NO: 6 which corresponds to the amino acid substitutions K288E and H435K in the amino acid sequence of full-length human lgG4. This variant has an increased half-life.

In one embodiment, the variant of the Fc portion of human lgG4 comprises the amino acid substitution R178K in the sequence according to SEQ ID NO: 6 which corresponds to the amino acid substitution R409K in the amino acid sequence of full-length human lgG4. This variant prevents acid-induced aggregation of human lgG4 (see Namisaki et al. (2020) PloS ONE 15(3): e0229027).

In one embodiment, the variant of the Fc portion of human lgG4 does not comprise an amino acid substitution at one or more of positions 37, 43, 65, 96, 99, 100, 124, 125, 127, 187 and 214 in the sequence according to SEQ ID NO: 6. In one embodiment, the variant of the Fc portion of human lgG4 does not comprise one or more of the amino acid substitutions Q37H, Q43K, F65Y, G96A, S99A, SWOP, Q124R, E125D, M127L, R178K, E187Q and L214P. In one embodiment, the variant of the Fc portion of human lgG4 does not comprise any amino acid substitution at any of the positions 37, 43, 65, 96, 99, 100, 124, 125, 127, 187 and 214 in the sequence according to SEQ ID NO: 6. In one embodiment, the variant of the Fc portion of human lgG4 does not comprise any of the amino acid substitutions Q37H, Q43K, F65Y, G96A, S99A, SWOP, Q124R, E125D, M127L, R178K, E187Q and L214P.

In one embodiment, the second part of the fusion protein of the present invention comprises the Fc portion of human IgG 1 or a variant thereof. The Fc portion of human IgG 1 comprises the CH2 and CH3 domains of human IgG 1 linked together to form the Fc portion. In a full- length human IgG 1 antibody the Fc portion is connected to the Fab fragment through a hinge region. The Fab fragment comprises the heavy chain variable region and the CH1 domain. Preferably, the Fc portion of human IgG 1 used in the fusion protein has the sequence according to SEQ ID NO: 4.

"A variant of the Fc portion of human IgGI" refers to the Fc portion of human lgG1 which has one or more amino acid substitutions compared to the wild-type Fc portion of human lgG1 according to SEQ ID NO: 4. In one embodiment, the one or more amino acid substitutions lead to decreased effector functions compared to the wild-type Fc portion of human lgG1 according to SEQ ID NO: 4.

Preferably, the decreased effector functions comprise a decreased complement-dependent cytotoxicity (CDC). More preferably, the CDC is decreased by at least two-fold, at least three-fold, at least four-fold or at least five-fold compared to the CDC of the wild-type Fc portion of human IgG 1 according to SEQ ID NO: 4. Methods to determine and quantify CDC are well-known to the skilled person and have been described above.

In one embodiment, the variant of the Fc portion of human lgG1 may comprise the amino acid substitutions M21 Y, S23T and T25E in the sequence according to SEQ ID NO: 4 which corresponds to the amino acid substitutions M252Y, S254T and T256E in the amino acid sequence of full-length human lgG1. In one embodiment, the variant of the Fc portion of human IgG 1 has the amino acid sequence according to SEQ ID NO: 35. This variant has an increased half-life. In one embodiment, the variant of the Fc portion of human lgG1 comprises the amino acid substitutions T25D and T76Q in the sequence according to SEQ ID NO: 4 which corresponds to the amino acid substitutions T256D and T307Q in the amino acid sequence of full-length human IgG 1. In one embodiment, the variant of the Fc portion of human lgG1 has the amino acid sequence according to SEQ ID NO: 36. This variant has an increased half-life and an enhanced binding to FcRn (Mackness et al. (2019) MABS 11(7): 1276-1288).

In one embodiment, the variant of the Fc portion of human lgG1 comprises at least one amino acid substitution at an amino acid residue selected from L3, L4 and P98 of the sequence according to SEQ ID NO: 4. These amino acid residues correspond to amino acid residues L234, L235, and P329 of full-length human lgG1.

In one embodiment, the variant of the Fc portion of human lgG1 comprises the amino acid substitution L4E/A in the sequence according to SEQ ID NO: 4 which corresponds to the amino acid substitution L235E/A in the amino acid sequence of full-length human lgG1 . This variant has a reduced effector function, in particular reduced CDC.

In one embodiment, the variant of the Fc portion of human lgG1 comprises the amino acid substitutions L3A and L4A in the sequence according to SEQ ID NO: 4 which correspond to the amino acid substitutions L234A and L235A in the amino acid sequence of full-length human IgG 1. This variant has a reduced effector function, in particular reduced CDC.

In one embodiment, the variant of the Fc portion of human lgG1 comprises the amino acid substitutions L3A, L4A, P98G in the sequence according to SEQ ID NO: 4 which correspond to the amino acid substitutions L234A, L235A and P329G in the amino acid sequence of full- length human IgG 1. This variant has a reduced effector function, in particular reduced CDC.

In one embodiment, the variant of the Fc portion of human lgG1 comprises the amino acid substitutions L4A and P98G in the sequence according to SEQ ID NO: 4 which correspond to the amino acid substitutions L235A and P329G in the amino acid sequence of full-length human IgG 1. This variant has a reduced effector function, in particular reduced CDC.

In one embodiment, the second part of the fusion protein used in the present invention comprises the Fc portion of human lgG3 or a variant of the Fc portion of human lgG3 and has reduced binding to FcyRHIa compared to a fusion protein comprising the same first part and a second part comprising the Fc portion of wild-type human IgG 1. The binding to FcyRllla is decreased by at least two-fold, at least three-fold, at least four-fold, at least fivefold or at least 10-fold compared to the binding of a fusion protein comprising the same first part and a second part comprising the Fc portion of wild-type human lgG1 according to SEQ ID NO: 4.

In one embodiment, the second part of the fusion protein used in the present invention comprises the Fc portion of human lgG3 or a variant of the Fc portion of human lgG3 and has reduced binding to FcyRllla and essentially the same binding to FcRn compared to a fusion protein comprising the same first part and a second part comprising the Fc portion of wild-type human IgG 1. The term "essentially the same binding to FcRn" means that the binding of the fusion protein comprising the Fc portion of human lgG3 or a variant of the Fc portion of human lgG3 to FcRn differs by not more than 20% or not more than 15%, preferably not more than 10% or not more than 5%, more preferably not more than 3% or not more than 2% and most preferably not more than 1% from the binding of a fusion protein comprising the same first part and a second part comprising the Fc portion of wild-type human lgG1 according to SEQ ID NO: 4.

The binding of fusion proteins to FcyRllla or FcRn can be determined by surface plasmon resonance as described in the examples herein.

In one embodiment, the second part of the fusion protein used in the present invention comprises the Fc portion of human IgM or human lgG2 or a fragment thereof.

In one embodiment, the second part of the fusion protein used in the present invention comprises the Fc portion of human IgM. The full-length Fc portion of human IgM comprises the CH2, CH3, CH4 domains and the tailpiece of human IgM linked together to form the Fc portion. A full-length human IgM antibody additionally comprises the Fab fragment which comprises the heavy chain variable region and the CH1 domain. Preferably, the Fc portion of human IgM used in the fusion protein has the sequence according to SEQ ID NO: 7.

It is known that immunoglobulins of the IgM subclass exist as pentamers and hexamers of homodimers due to disulfide bonds between the Fc domain of the homodimers (Muller et al. (2013) Proc. Natl. Acad. Sci USA 110(25): 10183-10188), resulting in ten or twelve antigenbinding sites that allow them to potently neutralize antigens. Further, immunoglobulins of the IgM subclass do not bind to Fey receptors. Since it has been discussed that Fey receptors play a role in antibody-dependent enhancement of viral pathogenesis (see, e.g., Bournazos et al. (2020) Nature Reviews Immunology 20: 633-643), the antibody-dependent enhancement can be avoided by using a fusion protein comprising the Fc portion of human IgM or a variant or fragment of the Fc portion of human IgM.

"A variant of the Fc portion of human IgM" refers to the Fc portion of human IgM which has one or more amino acid substitutions compared to the wild-type Fc portion of human IgM according to SEQ ID NO: 7. In one embodiment, the variant of the Fc portion of human IgM has one to twelve, one to eleven, one to ten, one to nine, one to eight, one to seven, one to six, one to five, one to four, one to three, one or two amino acid substitutions compared to the wild-type Fc portion of human IgM according to SEQ ID NO: 7. In one embodiment, the variant of the Fc portion of human IgM has one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve amino acid substitutions compared to the wild-type Fc portion of human IgM according to SEQ ID NO: 7. The variant of the Fc region of human IgM is capable of forming multimers, preferably hexamers. The formation of multimers can be determined by size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) as described in the examples herein.

In one embodiment, the variant of the Fc portion of human IgM comprises a mutation at position 221 and/or 222, the numbering referring to SEQ ID NO: 7. In one embodiment the variant of the fragment of human ACE2 comprises a L221C mutation and/or a H222L mutation, the numbering referring to SEQ ID NO: 7. It has been shown that a cysteine at position 221 is important for the formation of polymeric antibodies. The histidine at position 222 may be mutated to leucine, since a histidine at this position may interfere with disulfide bond formation and thereby with polymeric assembly (Mekhaiel et al. (2011) Scientific Reports 1, Article Number 124).

"A fragment of the Fc portion of human IgM" refers to a polypeptide which lacks one or more amino acids compared to the full-length sequence of the Fc region of human IgM according to SEQ ID NO: 7. The fragment of the Fc region of human IgM is capable of forming multimers, preferably hexamers. The formation of multimers can be determined by size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) as described in the examples herein.

In one embodiment, the fragment of the Fc portion of human IgM consists of SEQ ID NO: 27. In one embodiment, the fragment of the Fc portion of human IgM consists of SEQ ID NO: 28. In one embodiment, the fragment of the Fc portion of human IgM consists of SEQ ID NO: 29. In one embodiment, the fragment of the Fc portion of human IgM consists of SEQ ID NO: 30. In one embodiment, the fragment of the Fc portion of human IgM consists of the sequence according to SEQ ID NO: 20. In one embodiment, the fragment of the Fc portion of human IgM consists of SEQ ID NO: 31.

In one embodiment, the second part of the fusion protein used in the present invention comprises the Fc portion of human lgG2 or a variant thereof. The Fc portion of human lgG2 comprises the CH2 and CH3 domains of human lgG2 linked together to form the Fc portion. In a full-length human lgG2 antibody the Fc portion is connected to the Fab fragment through a hinge region. The Fab fragment comprises the heavy chain variable region and the CH1 domain. Preferably, the Fc portion of human lgG2 used in the fusion protein has the sequence according to SEQ ID NO: 5.

The Fc portion of human lgG2 has lower ability to bind to Fc receptors and recruit the immune system (Stewart et al. (2014) Journal of Immunotherapy of Cancer 1-10, available at https://doi.orQ/10.1186/s40425-014-0029-x). Hence, using the Fc portion of human lgG2 instead of the Fc portion of human IgG 1 could be beneficial in the development of a medicament.

"A variant of the Fc portion of human lgG2" refers to the Fc portion of human lgG2 which has one or more amino acid substitutions compared to the wild-type Fc portion of human lgG2 according to SEQ ID NO: 5. In one embodiment, the one or more amino acid substitutions lead to decreased effector functions compared to the wild-type Fc portion of human lgG2 according to SEQ ID NO: 5. In one embodiment, the variant of the Fc portion of human lgG2 has one to twelve, one to eleven, one to ten, one to nine, one to eight, one to seven, one to six, one to five, one to four, one to three, one or two amino acid substitutions compared to the wild-type Fc portion of human lgG2 according to SEQ ID NO: 5. In one embodiment, the variant of the Fc portion of human lgG2 has one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve amino acid substitutions compared to the wild-type Fc portion of human lgG2 according to SEQ ID NO: 5.

In one embodiment, the variant of the Fc portion of human lgG2 comprises a mutation at at least one position selected from the positions 16, 18, 19, 49, 90, 111 and 112, the numbering referring to SEQ ID NO: 5. In one embodiment, the variant of the Fc portion of human lgG2 comprises a mutation at positions 16, 18, 19, 49, 90, 111 and 112, the numbering referring to SEQ ID NO: 5. In one embodiment, the variant of the Fc portion of human lgG2 comprises at least one mutation selected from the group consisting of V16A, G18A, P19S, H49A, V90L, A111S and P112S. In one embodiment, the variant of the Fc portion of human lgG2 comprises the mutations V16A, G18A, P19S, H49A, V90L, A111S and P112S. It has been shown that these mutations eliminate all effector functions of human lgG2 (Vafa et al. (2014) Methods 65: 114-126. In one embodiment, the variant of the Fc portion of human lgG2 comprises the tailpiece of human IgM or human IgA. Such variants are described in Sorensen et al. (1996) J. Immunol. 156(8): 2858-2865.

In one embodiment, the first and the second part of the fusion protein used in the present invention are linked by a linker sequence. The linker sequence is a short amino acid sequence which does not have a function on its own and which does not affect the folding of the fusion protein. In one embodiment, the linker sequence comprises five to twenty amino acids, preferably six to 18 amino acids, more preferably seven to 17 amino acids and most preferably eight or 13 amino acids.

In one embodiment, the linker sequence consists of small amino acids selected from glycine and serine. An overview of linker sequences is provided in Chen et al. (2013) Adv. Drug Deliv. Rev. 65(10): 1357-1369. In one embodiment, the linker sequence comprises five to twenty amino acids and consists of small amino acids selected from glycine and serine. In one embodiment, the linker sequence comprises six to 18 amino acids and consists of small amino acids selected from glycine and serine. In one embodiment, the linker sequence comprises seven to 17 amino acids and consists of small amino acids selected from glycine and serine. In one embodiment, the linker sequence comprises eight amino acids and consists of small amino acids selected from glycine and serine. In one embodiment, the linker sequence has the amino acid sequence according to SEQ ID NO: 18.

In another embodiment, the linker sequence is the hinge region of a human IgG antibody. In one embodiment, the linker sequence is the hinge region of a human IgG 1 antibody, a human lgG4 antibody or a human lgG2 antibody. In one embodiment, the hinge region is the hinge region of a human lgG4 antibody and has the amino acid sequence according to SEQ ID NO: 24. In one embodiment, the hinge region is the hinge region of a human lgG1 antibody and has the amino acid sequence according to SEQ ID NO: 25. In one embodiment, the hinge region is the hinge region of a human lgG2 antibody and has the amino acid sequence according to SEQ ID NO: 26. An overview of antibody structure and the role of the hinge region is provided in the Chapter "Antibody structure-function relationships" in Therapeutic Antibody Engineering - Current and Future Advances Driving the Strongest Growth Area in the Pharmaceutical Industry, Woodhead Publishing Series in Biomedicine 2012, pages 37-56, 459-595 (available at Antibody structure-function relationships - ScienceDirect).

In a fusion protein with the Fc part of IgG 1 or a variant or fragment thereof preferably the hinge region of a human IgG 1 antibody is used. More preferably, the hinge region of a human IgG 1 antibody according to SEQ ID No: 25 is used in a fusion protein with the Fc part of IgG 1 or a variant or fragment thereof.

In a fusion protein with the Fc part of lgG4 or a variant or fragment thereof preferably the hinge region of a human lgG4 antibody is used. More preferably, the hinge region of a human lgG4 antibody according to SEQ ID No: 24 is used in a fusion protein with the Fc part of lgG4 or a variant or fragment thereof.

In a fusion protein with the Fc part of lgG2 or a variant or fragment thereof preferably the hinge region of a human lgG2 antibody is used. More preferably, the hinge region of a human lgG2 antibody according to SEQ ID No: 26 is used in a fusion protein with the Fc part of lgG2 or a variant or fragment thereof.

In one embodiment, the fusion protein used in the present invention comprises a signal peptide which is located N-terminal of the ACE2 part of the fusion protein. The signal peptide functions to target the protein to the endoplasmic reticulum and ultimately to secretion from the cell. The skilled person knows suitable signal peptides. In one embodiment, the signal peptide is selected from a human albumin signal peptide, a human chymotrypsinogen signal peptide, a human interleukin-2 signal peptide, a human trypsinogen-2 signal peptide or a heavy or light chain signal peptide. In a preferred embodiment, the signal peptide is a human albumin signal peptide. In a particularly preferred embodiment, the signal peptide is a human albumin signal peptide according to SEQ ID NO: 19.

In a particular embodiment, the fusion protein used in the present invention has the amino acid sequence according to SEQ ID NO: 8 which comprises amino acids 18 to 732 of human ACE2 (SEQ ID NO: 2), the hinge region according to SEQ ID NO: 24 and the Fc portion of human lgG4 according to SEQ ID NO: 6. In a particular embodiment, the fusion protein used in the present invention comprises amino acids 18 to 732 of human ACE2 (SEQ ID NO: 2), a linker sequence and the Fc portion of human lgG4 according to SEQ ID NO: 6.

In a particular embodiment, the fusion protein used in the present invention has the amino acid sequence according to SEQ ID NO: 9 which comprises amino acids 18 to 732 of human ACE2 (SEQ ID NO: 2), the hinge region according to SEQ ID NO: 25 and the Fc portion of human IgG 1 according to SEQ ID NO: 4.

In a particular embodiment, the fusion protein used in the present invention comprises amino acids 18 to 732 of human ACE2 (SEQ ID NO: 2), a linker sequence and the Fc portion of human IgG 1 according to SEQ ID NO: 4.

In a particular embodiment, the fusion protein used in the present invention has the amino acid sequence according to SEQ ID NO: 10 which comprises amino acids 18 to 740 of human ACE2 (SEQ ID NO: 3), the hinge region according to SEQ ID NO: 24 and the Fc portion of human lgG4 according to SEQ ID NO: 6.

In a particular embodiment, the fusion protein used in the present invention comprises amino acids 18 to 740 of human ACE2 (SEQ ID NO: 3), a linker sequence and the Fc portion of human lgG4 according to SEQ ID NO: 6.

In a particular embodiment, the fusion protein used in the present invention has the amino acid sequence according to SEQ ID NO: 11 which comprises amino acids 18 to 740 of human ACE2 (SEQ ID NO: 3), the hinge region according to SEQ ID NO: 25 and the Fc portion of human lgG1 according to SEQ ID NO: 4.

In a particular embodiment, the fusion protein used in the present invention comprises amino acids 18 to 740 of human ACE2 (SEQ ID NO: 3), a linker sequence and the Fc portion of human IgG 1 according to SEQ ID NO: 4.

In a particular embodiment, the fusion protein used in the present invention has the amino acid sequence according to SEQ ID NO: 14 which comprises amino acids 18 to 732 of human ACE2 (SEQ ID NO: 2) comprising a H374N and a H378N mutation, the hinge region according to SEQ ID NO: 24 and the Fc portion of human lgG4 according to SEQ ID NO: 6. In a particular embodiment, the fusion protein used in the present invention comprises amino acids 18 to 732 of human ACE2 (SEQ ID NO: 2) comprising a H374N and a H378N mutation, a linker sequence and the Fc portion of human lgG4 according to SEQ ID NO: 6.

In a particular embodiment, the fusion protein used in the present invention has the amino acid sequence according to SEQ ID NO: 15 which comprises amino acids 18 to 732 of human ACE2 (SEQ ID NO: 2) comprising a H374N and a H378N mutation, the hinge region according to SEQ ID NO: 25 and the Fc portion of human lgG1 according to SEQ ID NO: 4.

In a particular embodiment, the fusion protein used in the present invention comprises amino acids 18 to 732 of human ACE2 (SEQ ID NO: 2) comprising a H374N and a H378N mutation, a linker sequence and the Fc portion of human IgG 1 according to SEQ ID NO: 4.

In a particular embodiment, the fusion protein used in the present invention has the amino acid sequence according to SEQ ID NO: 16 which comprises amino acids 18 to 740 of human ACE2 (SEQ ID NO: 3) comprising a H374N and a H378N mutation, the hinge region according to SEQ ID NO: 24 and the Fc portion of human lgG4 according to SEQ ID NO: 6.

In a particular embodiment, the fusion protein used in the present invention comprises amino acids 18 to 740 of human ACE2 (SEQ ID NO: 3) comprising a H374N and a H378N mutation, a linker sequence and the Fc portion of human lgG4 according to SEQ ID NO: 6.

In a particular embodiment, the fusion protein used in the present invention has the amino acid sequence according to SEQ ID NO: 17 which comprises amino acids 18 to 740 of human ACE2 (SEQ ID NO: 3) comprising a H374N and a H378N mutation, the hinge region according to SEQ ID NO: 25 and the Fc portion of human lgG1 according to SEQ ID NO: 4.

In a particular embodiment, the fusion protein used in the present invention comprises amino acids 18 to 740 of human ACE2 (SEQ ID NO: 3) comprising a H374N and a H378N mutation, a linker sequence and the Fc portion of human lgG1 according to SEQ ID NO: 4.

In a particular embodiment, the fusion protein used in the present invention has the amino acid sequence according to SEQ ID NO: 13 which comprises the signal peptide according to SEQ ID NO: 19, amino acids 18 to 740 of human ACE2 (SEQ ID NO: 3), the linker sequence according to SEQ ID NO: 18 and the Fc portion of human IgM according to SEQ ID NO: 7. In a particular embodiment, the fusion protein used in the present invention comprises amino acids 18 to 740 of human ACE2 (SEQ ID NO: 3), the linker sequence according to SEQ ID NO: 18 and the Fc portion of human IgM according to SEQ ID NO: 7.

In a particular embodiment, the fusion protein used in the present invention comprises amino acids 18 to 740 of human ACE2 (SEQ ID NO: 3), a linker sequence and the Fc portion of human IgM according to SEQ ID NO: 7.

In another particular embodiment, the fusion protein used in the present invention has the amino acid sequence according to SEQ ID NO: 21 which comprises the signal peptide according to SEQ ID NO: 19, amino acids 18 to 740 of human ACE2 (SEQ ID NO: 3), the linker sequence according to SEQ ID NO: 18 and the fragment of the Fc portion of human IgM according to SEQ ID NO: 20.

In another particular embodiment, the fusion protein used in the present invention comprises amino acids 18 to 740 of human ACE2 (SEQ ID NO: 3), the linker sequence according to SEQ ID NO: 18 and the fragment of the Fc portion of human IgM according to SEQ ID NO: 20.

In another particular embodiment, the fusion protein used in the present invention comprises amino acids 18 to 740 of human ACE2 (SEQ ID NO: 3), a linker sequence and the fragment of the Fc portion of human IgM according to SEQ ID NO: 20.

In a particular embodiment, the fusion protein used in the present invention has the amino acid sequence according to SEQ ID NO: 12 which comprises the signal peptide according to SEQ ID NO: 19, amino acids 18 to 740 of human ACE2 (SEQ ID NO: 3), the linker sequence according to SEQ ID NO: 18 and the Fc portion of human lgG2 according to SEQ ID NO: 5.

In a particular embodiment, the fusion protein used in the present invention comprises amino acids 18 to 740 of human ACE2 (SEQ ID NO: 3), the linker sequence according to SEQ ID NO: 18 and the Fc portion of human lgG2 according to SEQ ID NO: 5.

In a particular embodiment, the fusion protein used in the present invention comprises amino acids 18 to 740 of human ACE2 (SEQ ID NO: 3), a linker sequence and the Fc portion of human lgG2 according to SEQ ID NO: 5. The combination product of the present invention may also comprise multimers of the fusion protein of a fragment of human ACE2 and the Fc portion of human IgM or a fragment of the Fc portion of human IgM. The term "multimer" is intended to mean a structure wherein two to twelve fusion proteins of a fragment of human ACE2 and the Fc portion of human IgM or a fragment of the Fc portion of human IgM are associated by disulfide bonds. In one particular embodiment, the term multimer means hexamer, i.e. six fusion proteins of the present invention are associated by disulfide bonds. It was surprisingly shown that the IgM fusion proteins are able to predominantly form hexamers. By the formation of hexamers the antigen binding can be enhanced.

The fusion protein used in the combination product of the present invention is preferably produced in mammalian host cells. Suitable mammalian host cells for expressing the fusion protein include Chinese Hamster Ovary (CHO) cells (including dhfr negative CHO cells used with a DHFR selectable marker), NSO myeloma cells, COS cells, SP2 cells, monkey kidney CV1 , human embryonic kidney line 293, baby hamster kidney cells (BHK), mouse Sertoli cells (TM4), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDC), buffalo rat liver cells (BRL 3 A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells, MRC 5 cells and FS4 cells. More preferably, the host cells are derived from a rodent. Most preferably, the mammalian cells are Chinese hamster ovary (CHO) or 293 cells. In one embodiment the CHO cells are ExpiCHO cells. In one embodiment the 293 cells are Expi293 cells.

To produce the fusion protein, the host cells are cultured in a suitable culture medium. The terms “medium", "cell culture medium" and "culture medium" are interchangeably used herein and refer to a solution containing nutrients which are required for growing mammalian cells. Typically, a cell culture medium provides essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for minimal growth and/or survival. The cell culture medium may also comprise growth factors. Preferably, the medium is chemically defined in that all its components and their concentration are known. Also preferably, the medium is serum-free and hydrolysate-free and does not contain any components derived from animals. In a more preferred embodiment the medium is serum- free and hydrolysate-free and does not contain any components derived from animals or insulin. In one embodiment the medium used to produce the fusion protein is a commercially available medium such as Freestyle 293 expression medium (Life Technologies), PolCHO P Powder Base CD, ActiPro (both available from GE), PowerCHO-2, ProCHO-5 (both available from Lonza) or EX-CELL® Advanced CHO fed batch medium (available from Sigma). If the cells used for expressing the fusion protein are ExpiCHO cells, the medium is ExpiCHO medium available from ThermoFisher. If the cells used for expressing the fusion protein are Expi293 cells, the medium is Expi293 medium available from ThermoFisher.

For culturing the mammalian cells different strategies are available, including batch culture, perfusion culture, continuous culture and fed-batch culture. Preferably, a fed-batch culture process is used. In fed-batch culture the culturing process is started with a certain volume of the basal medium and one or more feed media comprising one or more nutrients are fed at later time-point(s) of the culture process to prevent nutrient depletion while no product is removed from the cell culture broth. Accordingly, the term "feeding" means that at least one component is added to an existing culture of cells.

The term "basal medium" is intended to refer to the medium which is used from the beginning of the cell culture process. The mammalian cells are inoculated into the basal medium and grown in this medium for a certain period until the feeding is started. The basal medium meets the definition of the culture medium as provided above. If a commercially available medium is used, additional components may be added to the basal medium.

The feed medium is added to the cell culture after the cells have been cultured in the basal medium for a certain period. The feed medium serves to prevent nutrient depletion and therefore may not have the same composition as the basal medium. In particular, the concentration of one or more nutrients may be higher in the feed medium than in the basal medium. In one embodiment, the feed medium has the same composition as the basal medium. In another embodiment, the feed medium has another composition as the basal medium. The feed medium may be added continuously or as a bolus at defined time points.

Suitable feed media are known to the skilled person and include PolCHO Feed-A Powder Base CD, PolCHO Feed-B Powder Base CD, Cell Boost 7a and Cell Boost 7b (all available from GE), BalanCD® CHO Feed 3 Medium (available from Irvine Scientific) and EX-CELL® Advanced CHO feed 1 (available from Sigma). The culturing of the host cell may be performed at a constant temperature, e.g. at a temperature of 37°C± 0.2°C. Alternatively, the culture temperature may be reduced from a first temperature to a second temperature, i.e. the temperature is actively downregulated. Hence, the second temperature is lower than the first temperature. The first temperature may be 37°C ± 0.2°C. The second temperature may be in the range of from 30°C to 36°C.

After the fusion protein has been produced by culturing the host cell in a suitable culture medium, the fusion protein is harvested from the cell culture. Since Fc fusion proteins expressed from mammalian cells are typically secreted into the cell culture fluid during the cultivation process, the product harvest at the end of the cultivation process occurs by separating cell culture fluid comprising the fusion protein from the cells. The cell separation method should be gentle to minimize cell disruption to avoid the increase of cell debris and release of proteases and other molecules that could affect the quality of the fusion protein product. Usually, the harvesting of the cell culture fluid comprising the fusion protein involves centrifugation and/or filtration, whereby the fusion protein is present in the supernatant and the filtrate, respectively. Expanded bed adsorption chromatography is an alternative method to avoid centrifugation/filtration methods.

After harvesting the cell culture fluid comprising the fusion protein the fusion protein has to be purified from the cell culture fluid. The purification of Fc fusion proteins is usually accomplished by a series of standard techniques that can include chromatographic steps such as anion exchange chromatography, cation exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography and size exclusion chromatography. The affinity chromatography may comprise protein A affinity chromatography for lgG1, lgG2, lgG3 or lgG4 fusion proteins and affinity chromatography with IgM affinity ligands such as CaptureSelect IgM affinity resin (ThermoFisher) for IgM fusion proteins and fusion proteins comprising at least the cm4 part of IgM. Further, the purification process may comprise one or more ultra-, nano- or diafiltration as well as tangential flow filtration and/or cross flow filtration steps.

After purifying the fusion protein it can be used to prepare the combination product of the present invention. A combination product is intended to be delivered to a patient for treating or preventing a disease or condition. The components of the combination product, i.e. the fusion protein comprising a first part comprising a fragment of human ACE2 or a variant of said fragment, said human ACE2 having the amino acid sequence according to SEQ ID NO: 1 , and a second part comprising an Fc portion of a human antibody or a fragment or variant of the Fc portion; and the inhibitor of ACE2, can be administered separately or as a composition comprising both components, i.e. the fusion protein comprising a first part comprising a fragment of human ACE2 or a variant of said fragment, said human ACE2 having the amino acid sequence according to SEQ ID NO: 1 , and a second part comprising an Fc portion of a human antibody or a fragment or variant of the Fc portion; and the inhibitor of ACE2.

In the combination product of the present invention, the inhibitor of ACE2 and the fusion protein are present in a molar ratio of 100:1 to 2:1, preferably in a molar ratio of 80:1 to 3:1 or 70:1 to 4:1, more preferably in a molar ratio of 50:1 to 5:1, even more preferably in a molar ratio of 30:1 to 7:1 and most preferably in a molar ratio of 10:1. The molar ratio is calculated using the molarity of the inhibitor of ACE2 and of the fusion protein. For example, a concentration of the inhibitor of ACE2 of 10 mM and a concentration of the fusion protein of 1 mM provides a molar ratio of 10:1.

In one embodiment, the concentration of the fusion protein in the combination product of the invention is 1 to 60 mg/ml, preferably 5-50 mg/mL or 8-40 mg/mL, more preferably 10-30 mg/mL or 15-25 mg/mL, and most preferably 20 mg/mL. In another embodiment the concentration of the ACE2 Fc fusion protein in the combination product of the present invention is 20-60 mg/mL, preferably 30-50 mg/mL, more preferably 40 mg/mL.

In addition to the fusion protein and the inhibitor of ACE2, a pharmaceutical composition typically contains at least one pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients are substances which do not interfere with the physiological activity of the fusion protein and which stabilize the pharmaceutical composition and/or enhance solubility or decrease viscosity of the pharmaceutical composition. Typical pharmaceutically acceptable excipients for recombinant proteins include buffers, salts, sugars or sugar alcohols, amino acids and surface-active agents.

The combination product comprises a therapeutically effective amount of the fusion protein. The term “therapeutically effective amount” refers to an amount of the fusion protein sufficient to treat a specified disorder, condition or disease such as to ameliorate, palliate, lessen, and/or delay one or more of its symptoms. With respect to an infection with a coronavirus and in particular SARS-CoV-2 the therapeutically effective amount of the fusion protein ameliorates, palliates, lessens, and/or delays one or more of symptoms selected from coughing, shortness of breath, difficulty breathing, fever, chills, tiredness, muscle aches, sore throat, headache, chest pain and loss of smell and/or taste. A therapeutically effective amount can be administered in one or more administrations.

The combination product of the present invention is for medical use, i.e. it is intended to be used to prevent and/or treat a disease.

As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, and/or prolonging survival. The use of the present invention contemplates any one or more of these aspects of treatment.

The term “prevent,” and similar words such as “prevented”, “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the recurrence of, a disease or condition. It also refers to delaying the recurrence of a disease or condition or delaying the recurrence of the symptoms of a disease or condition. As used herein, “prevention” and similar terms also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to recurrence of the disease or condition.

In one embodiment, the combination product of the present invention is used to prevent and/or treat an infection with a coronavirus binding to ACE2. Coronaviruses are enveloped viruses with a positive sense, single-stranded RNA genome and an icosahedral protein shell. The Spike protein consisting of the S1 and S2 subunits forms a homotrimer which projects from the envelope and mediates the interaction with the target cells by binding to ACE2. Coronaviruses often cause respiratory diseases in humans and other mammalian as well as bird species. In humans, seven coronavirus strains are known: HCoV-OC43, HCoV-HKU1, HCOV-229E, HCOV-NL63, MERS-CoV, SARS-CoV and SARS-CoV-2. The first four coronavirus strains (HCoV-OC43, HCoV-HKU1, HCoV-229E, HCoV-NL63) cause only mild symptoms, whereas infection with MERS-CoV, SARS-CoV and SARS-CoV-2 may lead to severe, potentially life-threatening disease. It has been shown that SARS-CoV, SARS-CoV-2 and HCoV-NL63 bind to ACE2 and use this binding to enter the target cells (Li et al. (2003) Nature 426(6965): 450-4; Hoffmann et al. (2020) Cell 181 : 1-10; Hofmann et al. (2005) Proc Natl Acad Sci U S A. 102(22):7988-93). Accordingly, the combination product of the present invention can be used to treat and/or prevent infection with a coronavirus binding to ACE2, in particular infection with SARS-CoV, SARS-CoV-2 or HCoV-NL63. Further coronaviruses binding to ACE2 can be identified by inoculating cells expressing ACE2 either transiently or constitutively with pseudotyped VSV (vesicular stomatitis virus) expressing the coronavirus Spike protein and a reporter protein and detecting the activity of the reporter protein after the inoculation period (see protocol in Hoffmann et al. (2020) Cell 181 : 1-10). In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is not SARS-CoV.

In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is SARS-CoV-2 or a variant of SARS-CoV-2 comprising the amino acid substitution D614G and/or the amino acid substitution N439K. The variant of SARS-CoV-2 comprising the amino acid substitution D614G is described in Korber et al. (2020) Cell 182(4): 812-827 and the amino acid substitution N439K is described in Thomson et al. (2021) Cell 184(5): 1171,1187.e20; available at https://doi.Org/10.1101/2020.11.04.355842. In one embodiment, the combination product is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 comprising the amino acid substitution D614G. The amino acid substitution D614G is caused by an A- to-G nucleotide mutation at position 23,403 in the Wuhan reference strain. The numbering of the amino acids in the variants refers to the numbering in the Spike protein of SARS-CoV-2 according to SEQ ID NO: 23. Hence, a SARS-CoV-2 virus with the Spike protein according to SEQ ID NO: 23 is defined to be the wild-type SARS-CoV-2 from which any variants described herein are derived.

In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 comprising at least one amino acid substitution selected from the group consisting of L452R, E484K, K417N and N501Y. In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS- CoV-2 comprising the amino acid substitutions E484K, K417N and N501 Y. In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 comprising the amino acid substitution D614G and at least one additional amino acid substitution. In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 comprising the amino acid substitutions D614G, N501Y, A570D, P681H, T716I, S982A and D1118H and comprising a deletion of amino acids 69, 70 and 145. In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 comprising the amino acid substitutions D614G, Y453F, I692V and M1229I and comprising a deletion of amino acids 69 and 70. In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 comprising the amino acid substitutions D614G, S13I, W152C and L452R. In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS- CoV-2 comprising the amino acid substitutions D614G, E484K and V1176F. In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 comprising the amino acid substitutions D614G, L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I and V1176F. In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 comprising the amino acid substitutions D614G, D80A, D215G, K417N, E484K, N501 Y and A701V and comprising a deletion of amino acids 242, 243 and 244. In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 comprising the amino acid substitutions D614G, L18F, D80A, D215G, K417N, E484K, N501Y and A701V and comprising a deletion of amino acids 242, 243 and 244. In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 comprising the amino acid substitutions D614G, D80A, R246I, K417N, E484K, N501Y and A701V and comprising a deletion of amino acids 242, 243 and 244 In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 comprising the amino acid substitutions E484Q and L452R. In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 comprising the amino acid substitutions E484K and D614G and comprising a deletion of amino acids 145 and 146. In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 comprising the amino acid substitutions D614G, T478K, P681R and L452R and not comprising amino acid substitution E484Q. In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 comprising the amino acid substitutions T19R, D614G, T478K, P681R, D950N and L452R and comprising a deletion of amino acids 157 and 158 and not comprising amino acid substitution E484Q. In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 comprising the amino acid substitutions G75V, T76I, L452Q, F490S, D614G and T859N and comprising a deletion of amino acids 246 to 252.

The numbering of the amino acids in the variants refers to the numbering in the Spike protein of SARS-CoV-2 according to SEQ ID NO: 23. For the purposes of defining variants, the amino acid sequence according to SEQ ID NO: 23 is considered to be the wild-type sequence of the Spike protein of SARS-CoV-2.

In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 comprising one or more amino acid substitutions in the receptor-binding domain of the Spike protein of SARS-CoV-2. The receptor-binding domain of the Spike protein of SARS-CoV-2 comprises amino acids 331 to 524 of SEQ ID NO: 23 (see Tai et al. (2020) Cell. Mol. Immunol. 17: 613-620). In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 comprising the amino acid substitution N501Y. In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 comprising the amino acid substitution E484K. In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 comprising the amino acid substitution K417T/N.

In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 which has a higher binding affinity to ACE2 compared to the SARS-CoV-2 comprising the wild-type Spike protein according to SEQ ID NO: 23. The affinity of a variant of SARS-CoV-2 to ACE2 can for example be determined using a pseudovirus assay. The pseudovirus assay uses a lentivirus which is pseudotyped with the S protein of wild-type SARS-CoV-2 or of a variant thereof and which contains a reporter gene such as the luciferase gene. Such lentiviruses can be obtained for example from BPS Bioscience. The pseudotyped lentivirus is incubated with ACE2 expressing cells to allow the virus to enter the cells and to express the reporter gene. If the expression of the reporter gene from a lentivirus pseudotyped with the S protein of a variant SARS-CoV-2 is higher than the expression of the reporter gene from a lentivirus pseudotyped with the S protein of wild-type SARS-CoV-2, the variant has a higher binding affinity to ACE2. It is assumed that the fusion proteins of the present invention have a higher affinity to those SARS-CoV-2 variants which have a higher binding affinity to ACE2, such as the variant B.1.1.7 or the so- called delta variant.

In one embodiment, the combination product of the present invention is used to treat and/or prevent infection with a coronavirus binding to ACE2, wherein the coronavirus binding to ACE2 is a variant of SARS-CoV-2 which has a higher transmissibility compared to the SARS-CoV-2 comprising the wild-type Spike protein according to SEQ ID NO: 23. The viral transmissibility can be determined using the basic reproduction number Ro which is the average number of people who will catch a disease from one contagious person.

The route of administration of the combination product is in accordance with known and accepted methods, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intra-arterial, intralesional or intraarticular routes. In another embodiment the combination product of the present invention is to be administered intranasally, e.g. by means of a nasal spray, a nasal ointment or nasal drops. In another embodiment, the combination product of the present invention is administered by topical administration or by inhalation. Preferably, the combination product of the present invention is administered by intravenous injection or infusion.

Dosages and desired drug concentrations of the combination product of the present invention may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W.“The Use of Interspecies Scaling in Toxicokinetics,” In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp.42-46.

In one embodiment, the fusion protein within the combination product is administered intravenously at a dosage of 0.1 mg/kg body weight to 4 mg/kg body weight, such as a dosage of 0.1 mg/kg body weight, 0.2 mg/kg body weight, 0.3 mg/kg body weight, 0.4 mg/kg body weight, 0.5 mg/kg body weight, 0.6 mg/kg body weight, 0.7 mg/kg body weight, 0.8 mg/kg body weight, 0.9 mg/kg body weight, 1.0 mg/kg body weight, 1.1 mg/kg body weight, 1 .2 mg/kg body weight, 1 .3 mg/kg body weight, 1 .4 mg/kg body weight, 1 .5 mg/kg body weight, 1.6 mg/kg body weight, 1.7 mg/kg body weight, 1.8 mg/kg body weight, 1.9 mg/kg body weight, 2.0 mg/kg body weight, 2.1 mg/kg body weight, 2.2 mg/kg body weight, 2.3 mg/kg body weight, 2.4 mg/kg body weight, 2.5 mg/kg body weight, 2.6 mg/kg body weight, 2.7 mg/kg body weight, 2.8 mg/kg body weight, 2.9 mg/kg body weight, 3.0 mg/kg body weight, 3.1 mg/kg body weight, 3.2 mg/kg body weight, 3.3 mg/kg body weight, 3.4 mg/kg body weight, 3.5 mg/kg body weight, 3.6 mg/kg body weight, 3.7 mg/kg body weight, 3.8 mg/kg body weight, 3.9 mg/kg body weight or 4.0 mg/kg body weight.

In one embodiment, the fusion protein within the combination product is administered intravenously at a dosage of 10 mg/kg body weight to 150 mg/kg body weight, such as a dosage of 10 mg/kg body weight, 15 mg/kg body weight, 20 mg/kg body weight, 25 mg/kg body weight, 30 mg/kg body weight, 35 mg/kg body weight, 40 mg/kg body weight, 45 mg/kg body weight, 50 mg/kg body weight, 55 mg/kg body weight, 60 mg/kg body weight, 65 mg/kg body weight, 70 mg/kg body weight, 75 mg/kg body weight, 80 mg/kg body weight, 85 mg/kg body weight, 90 mg/kg body weight, 95 mg/kg body weight, 100 mg/kg body weight, 105 mg/kg body weight, 110 mg/kg body weight, 115 mg/kg body weight, 120 mg/kg body weight, 125 mg/kg body weight, 130 mg/kg body weight, 135 mg/kg body weight, 140 mg/kg body weight, 145 mg/kg body weight or 150 mg/kg body weight. The combination product may be administered once per day, twice per day, three times per day, every other day, once per week or once every two weeks.

The combination product may be administered for a period of three days, four days, five days, six days, seven days, eight days, nine days or ten days.

By administering the combination product of the present invention, the infection with a coronavirus and in particular with SARS-CoV-2 is treated, i.e. at least one of the symptoms of an infection with SARS-CoV-2 is reduced or abolished. Symptoms of an infection with SARS-CoV-2 include coughing, shortness of breath, difficulty breathing, fever, chills, tiredness, muscle aches, sore throat, headache, chest pain and loss of smell and/or taste. In one embodiment, by the administration of the combination product of the present invention the fever caused by infection with SARS-CoV-2 is reduced. In one embodiment, the administration of the combination product of the present invention to a subject reduces the risk that the subject experiences a severe course of infection with SARS-CoV-2. In one embodiment, the administration of the combination product of the present invention to a subject reduces the risk that the subject experiences multi-organ failure, acute respiratory distress syndrome (ARDS) or pneumonia. In one embodiment, the administration of the combination product of the present invention to a subject reduces the risk that the subject experiences long-term effects of the infection with SARS-CoV-2 such as lung damage, neurological disorders, dermatological disorders and cardiovascular disease. In one embodiment, the administration of the combination product of the present invention to a subject reduces the concentration of the cytokines IL6 and/or IL8 in the blood. In one embodiment, the administration of the combination product of the present invention to a subject reduces the concentration of SARS-CoV-2 virus particles in the blood. In one embodiment, the administration of the combination product of the present invention to a subject stimulates the production of antiviral antibodies. In one embodiment, the administration of the combination product of the present invention to a subject stimulates the production of antiviral IgA and/or IgG antibodies.

In one embodiment, the combination product of the present invention is administered to a subject suffering from a severe infection with SARS-CoV-2. In one embodiment, the combination product of the present invention is administered to a subject infected with SARS-CoV-2 and requiring artificial ventilation. In one embodiment, the combination product of the present invention is administered to a subject infected with SARS-CoV-2 and requiring extracorporeal membrane oxygenation (ECMO).

By administering the combination product of the present invention to a healthy subject, the infection with a coronavirus and in particular with SARS-CoV-2 is prevented, i.e. the treated subject does not develop symptoms of an infection with SARS-CoV-2.

In one embodiment, the combination product of the present invention is administered to a subject which has been in contact with a subject infected with SARS-CoV-2. Subjects which have been in contact with a subject infected with SARS-CoV-2 can be identified by use of a "Corona warning app" installed on the smartphone.

In one embodiment, the combination product of the present invention is administered to a subject for which a test with a throat or nasal swab of said subject indicates that it is infected with SARS-CoV-2, but which has not developed any symptoms of an infection with SARS- CoV-2.

In the treatment or prevention of an infection with a coronavirus binding to ACE2 and in particular SARS-CoV-2 the combination product of the present invention may be combined with a known anti-viral agent. Anti-viral agents are medicaments used to treat viral infections and include both specific anti-viral agents and broad-spectrum viral agents. Suitable antiviral agents include, but are not limited to, nucleoside analogs, inhibitors of viral protease, inhibitors of viral polymerase, blockers of virus entry into the cell, Janus kinase inhibitors, but also inhibitors of inflammatory mediators.

In specific embodiments, the anti-viral agent is selected from the group consisting of remdesivir, arbidol HCI, ritonavir, lopinavir, darunavir, ribavirin, chloroquin and derivatives thereof such as hydroxychloroquin, nitazoxanide, camostat mesilate, anti-l L6 and anti-l L6 receptor antibodies such as tocilizumab, siltuximab and sarilumab and baricitinib phosphate.

In the treatment or prevention of an infection with SARS-CoV-2 the combination product of the present invention may be combined with an anti-SARS-CoV-2 monoclonal antibody. Anti- SARS-CoV2 monoclonal antibodies include, but are not limited to, LY-CoV555 (LY3819253) developed by Eli Lilly and Company, REGN-COV2 which is a cocktail of REGN10933 and REGN10987 and which is developed by Regeneron, VIR-7831 (GSK4182136) developed by Vir Biotechnology and GlaxoSmithKline, CT-P59 developed by Celltrion, AZD 7442 which is a combination of antibodies AZD8895 and AZD1061 and which is developed by Astra Zeneca, JS016 developed by Junshi Biosciences, TY027 developed by Tychan Pte Ltd, BRII-96 and BRII-98 developed by Brii Biosciences, SCTA01 developed by Sinocelltech Ltd, ADM03820 developed by Ology Bioservices, BI767551 developed by Boehringer Ingelheim and others and COR-101 developed by Corat Therapeutics.

Apart from its function in binding coronaviruses, ACE2 has also been implicated in several disorders and diseases such as hypertension (including high blood pressure), congestive heart failure, chronic heart failure, acute heart failure, contractile heart failure, myocardial infarction, arteriosclerosis, kidney failure, renal failure, Acute Respiratory Distress Syndrome (ARDS), Acute Lung Injury (ALI), chronic obstructive pulmonary disease (COPD), pulmonary hypertension, renal fibrosis, chronic renal failure, acute renal failure, acute kidney injury, inflammatory bowel disease and multi-organ dysfunction syndrome. Hence, the combination product of the present invention can also be used in the treatment of these disorders and diseases.

The present invention also relates to a method for stabilizing a fusion protein as described herein by adding an inhibitor of ACE2 to a cell culture medium comprising host cells producing said fusion protein.

The term "stabilizing" means that the degradation and/or aggregation of the fusion protein is prevented.

In one embodiment, the fusion protein is stabilized against thermal unfolding. Thermal unfolding means that the fusion protein changes its conformation when exposed to increasing temperatures. The fusion protein is stabilized against thermal unfolding, if in the presence of the inhibitor of ACE2 a change of the protein conformation does not occur or occurs at a higher temperature than in the absence of the inhibitor of ACE2. The degree of conformational changes upon temperature increase, i.e. the thermal unfolding, can be detected by circular dichroism spectroscopy as described in the examples herein.

In one embodiment, the fusion protein is stabilized against aggregation. Aggregation means the formation of aggregates. Aggregates form by association of two or more fusion protein molecules. The fusion protein is stabilized against aggregation, if the amount of aggregates detected in the presence of the inhibitor of ACE2 is lower than in the absence of the inhibitor of ACE2. The amount of aggregates can be detected by methods known to the skilled person, e.g. by size exclusion chromatography as described in the examples herein.

The method of stabilizing the fusion protein may involve the mixing of the fusion protein and the inhibitor of ACE2 before, during and/or after purification of the fusion protein. The molar ratio of the inhibitor of ACE2 and the fusion protein may be 100:1 to 2:1. In one embodiment, the harvested cell culture fluid obtained by centrifuging or filtering the cell culture medium is mixed with the inhibitor of ACE2. In one embodiment, the purification method comprises at least one chromatography step on a chromatography medium and the solution applied to the chromatography medium is mixed with the inhibitor of ACE2. In one embodiment, the purification comprises at least one chromatography step on a chromatography medium and the eluate from the chromatography medium is mixed with the inhibitor of ACE2.

In one embodiment, the present invention relates to a combination product comprising:

(a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 2, and a second part comprising an Fc portion of a human IgG 1 antibody; and

(b) MLN-4760.

(a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 2, and a second part comprising an Fc portion of a human lgG2 antibody; and

(b) MLN-4760.

(a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 2, and a second part comprising an Fc portion of a human lgG3 antibody; and

(b) MLN-4760.

(a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 2, and a second part comprising an Fc portion of a human lgG4 antibody; and

(b) MLN-4760. In one embodiment, the present invention relates to a combination product comprising:

(a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 2, and a second part comprising an Fc portion of a human IgM antibody; and

(b) MLN-4760.

(a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 2, and a second part comprising a variant of the Fc portion of a human lgG1 antibody; and

(b) MLN-4760.

(a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 2, and a second part comprising a variant of the Fc portion of a human lgG4 antibody; and

(b) MLN-4760.

(a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 2, and a second part comprising a variant of the Fc portion of a human IgG 1 antibody having an amino acid sequence selected from SEQ ID NO: 35 and SEQ ID NO: 36; and

(b) MLN-4760.

(a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 2, and a second part comprising a variant of the Fc portion of a human lgG4 antibody having an amino acid sequence selected from SEQ ID NO: 33 and SEQ ID NO: 34; and

(b) MLN-4760.

In one embodiment, the present invention relates to a combination product comprising: (a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 3, and a second part comprising an Fc portion of a human lgG1 antibody; and

(b) MLN-4760.

(a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 3, and a second part comprising an Fc portion of a human lgG2 antibody; and

(b) MLN-4760.

(a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 3, and a second part comprising an Fc portion of a human lgG3 antibody; and

(b) MLN-4760.

(a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 3, and a second part comprising an Fc portion of a human lgG4 antibody; and

(b) MLN-4760.

(a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 3, and a second part comprising an Fc portion of a human IgM antibody; and

(b) MLN-4760.

(a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 3, and a second part comprising a variant of the Fc portion of a human IgG 1 antibody; and

(b) MLN-4760.

In one embodiment, the present invention relates to a combination product comprising: (a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 3, and a second part comprising a variant of the Fc portion of a human lgG4 antibody; and

(b) MLN-4760.

(a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 3, and a second part comprising a variant of the Fc portion of a human IgG 1 antibody having an amino acid sequence selected from SEQ ID NO: 35 and SEQ ID NO: 36; and

(b) MLN-4760.

(a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 3, and a second part comprising a variant of the Fc portion of a human lgG4 antibody having an amino acid sequence selected from SEQ ID NO: 33 and SEQ ID NO: 34; and

(b) MLN-4760.

(b) DX600.

(b) DX600. In one embodiment, the present invention relates to a combination product comprising:

(b) DX600.

(a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 2, and a second part comprising a variant of the Fc portion of a human IgG 1 antibody; and

(b) DX600.

In one embodiment, the present invention relates to a combination product comprising: (a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 2, and a second part comprising a variant of the Fc portion of a human lgG4 antibody having an amino acid sequence selected from SEQ ID NO: 33 and SEQ ID NO: 34; and

(b) DX600.

(a) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 3, and a second part comprising an Fc portion of a human IgG 1 antibody; and

(b) DX600.

In one embodiment, the present invention relates to a combination product comprising: (c) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 3, and a second part comprising a variant of the Fc portion of a human IgG 1 antibody; and

(d) DX600.

(c) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 3, and a second part comprising a variant of the Fc portion of a human lgG4 antibody; and

(d) DX600.

(c) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 3, and a second part comprising a variant of the Fc portion of a human IgG 1 antibody having an amino acid sequence selected from SEQ ID NO: 35 and SEQ ID NO: 36; and

(d) DX600.

(c) a fusion protein comprising a first part comprising a fragment of human ACE2 consisting of the amino acid sequence according to SEQ ID NO: 3, and a second part comprising a variant of the Fc portion of a human lgG4 antibody having an amino acid sequence selected from SEQ ID NO: 33 and SEQ ID NO: 34; and

(d) DX600.

(a) a fusion protein according to SEQ ID NO: 8; and

(b) MLN-4760.

(a) a fusion protein according to SEQ ID NO: 9; and

(b) MLN-4760.

(a) a fusion protein according to SEQ ID NO: 10; and

(a) a fusion protein according to SEQ ID NO: 11 ; and

(b) MLN-4760.

(a) a fusion protein according to SEQ ID NO: 12; and

(b) MLN-4760.

(a) a fusion protein according to SEQ ID NO: 13; and

(b) MLN-4760.

(a) a fusion protein according to SEQ ID NO: 14; and

(b) MLN-4760.

(a) a fusion protein according to SEQ ID NO: 15; and

(b) MLN-4760.

(a) a fusion protein according to SEQ ID NO: 16; and

(b) MLN-4760.

(a) a fusion protein according to SEQ ID NO: 17; and

(b) MLN-4760.

(a) a fusion protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO:

48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO:

53, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO:

60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 71 , SEQ ID NO:

72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO:

77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO:

82, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO:

89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO:

94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 110; and

(b) MLN-4760.

(a) a fusion protein according to SEQ ID NO: 8; and

(b) DX600.

(a) a fusion protein according to SEQ ID NO: 9; and

(b) DX600.

(a) a fusion protein according to SEQ ID NO: 10; and

(b) DX600.

(a) a fusion protein according to SEQ ID NO: 11 ; and

(b) DX600.

(a) a fusion protein according to SEQ ID NO: 12; and

(b) DX600.

(a) a fusion protein according to SEQ ID NO: 13; and

(b) DX600.

In one embodiment, the present invention relates to a combination product comprising: (a) a fusion protein according to SEQ ID NO: 14; and

(b) DX600.

(a) a fusion protein according to SEQ ID NO: 15; and

(b) DX600.

(a) a fusion protein according to SEQ ID NO: 16; and

(b) DX600.

(a) a fusion protein according to SEQ ID NO: 17; and

(b) DX600.

48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO:

53, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO:

60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO:

65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO:

72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO:

77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO:

82, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO:

89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO:

(b) DX600.

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. The invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing a claimed invention, from a study of the drawings, the disclosure, and the dependent claims.

The detailed description is merely exemplary in nature and is not intended to limit application and uses. The following examples further illustrate the present invention without, however, limiting the scope of the invention thereto. Various changes and modifications can be made by those skilled in the art on the basis of the description of the invention, and such changes and modifications are also included in the present invention.

EXAMPLES

A. Materials and methods

1. Production and purification of the ACE2-Fc fusion proteins a) Fusion proteins with the Fc part of IgM or lgG2

The genes encoding the sequences (ACE2-Fc (IgM) (SEQ ID NO: 13) and ACE2-Fc (lgG2) (SEQ ID NO: 12) were ordered and obtained from a commercial gene synthesis provider. The codons were optimized for Homo sapiens with the Gene optimization tool from GeneArt. Kozak sequence was added before the start codon. A stop codon was added after the codon that encodes the last amino acid in the sequence. The synthesized genes were delivered in a pcDNA3.1(+) plasmid. The genes have Hindi II and Xhol restriction sites. Larger amounts of the delivered plasmids were produced and isolated by standard techniques with XL-1 bacterial cells. The purified plasmids were sent for sequencing with several primers. The entire range of the gene encoding the ACE2-Fc fusion protein was verified.

The ACE2-Fc fusion proteins were produced by transient expression in Expi293 or ExpiCHO cells after transfection with Expifectamine using the manufacturer’s protocol. After 6 days, the supernatant was collected, and the ACE2-Fc fusion proteins were purified by a combination of two standard techniques - affinity chromatography followed by size-exclusion chromatography. For the ACE2-Fc (lgG2) fusion protein a protein A chromatography step was applied as affinity chromatography. For the ACE2-Fc (IgM) fusion protein a chromatography step using CaptureSelect IgM resin (ThermoFisher) was applied as affinity chromatography. The amounts of ACE2-Fc fusion proteins that are obtained from transient transfection of 1 L cell culture with the exemplary sequence are in the range of tens to hundreds of milligrams. b) Fusion proteins with the Fc part of lgG4

The nucleic acid sequence encoding the construct ACE2-Fc (lgG4) according to SEQ ID NO: 8 was inserted into a variant of the expression vector pcDNA3.1 (Invitrogen V860-20) using Hindi ll/Xhol restriction enzymes. The albumin signal sequence according to SEQ ID NO: 19 was attached to the N-terminus of the constructs. The expression vector was then used to transiently transfect 293 cells using the Freestyle expression system (available from ThermoFisher). On day six samples were analyzed for cell viability and cell density and supernatants were harvested by two step centrifugation and were sterile-filtered. The material was pooled and half of it was stored at -80°C until purification. The other half was subjected to Protein A purification. Additionally, small samples (~ 0.5 mL) were taken from the pools to determine expression by bio-layer interferometry (BLI).

Purification of the transient material was performed by protein A column chromatography followed by preparative SEC. For protein A purification, after loading the sample, the column was washed and the ACE2-Fc fusion proteins were eluted using 40 mM NaAc, pH=3.0. Following elution, samples were first neutralized to pH=7.5 using 1 M Tris, pH=9.0, subsequently diluted 1 :1 with 50 mM Tris, pH=7.5, 300 mM NaCI and concentrated to 10 mg/mL using spin filters. Concentrated proteins were further purified with a Superdex 200 increase (GE Healthcare) column equilibrated with 50 mM Tris, pH=7.5, 150 NaCI. The main peak was pooled, the protein concentration was adjusted to 1 mg/mL, passed over a sterilizing filter and stored at 4 °C until further usage.

2. Preparation of the ACE2 inhibitors

MLN4760 (MilliporeSigma, Cat. Nr. 5.30616.0001, Batch Nr. 3585043, 2 mg) was reconstituted in DMSO according to manufacturer’s instructions to obtain a stock solution with a concentration of 4.67 mM.

DX-600 (Selleckchem/Biozol, Cat. Nr. S9666, Batch Nr. 01, 5 mg) was reconstituted in ultrapure water according to the manufacturer's protocol to obtain a 5 mM solution. From the stock solutions, 10 pL aliquots were frozen and stored at - 80 C. Before experiments, an aliquot of the inhibitor stock solution was thawed at room temperature.

3. Effect of ACE2 inhibitors on the thermal stability of ACE2 fusion proteins a) Fusion proteins with the Fc part of IgM or lgG2

All samples were measured by circular dichroism using a Jasco J-1500 spectropolarimeter. The sample buffer was PBS. The Far-UV CD spectra were obtained in a 1 mm quartz cuvette using a protein concentration of 0 1 mg/mL. The sample temperature was increased stepwise (0.5 °C steps) from 20 to 90 °C and the CD signal was collected at each step. The raw CD signal was normalized from 0 to 1. b) Fusion proteins with the Fc part of lgG4

All samples were measured with a Microcal PEAQ-DSC system (Malvern Panalytical). The ACE2 fusion proteins were measured alone or in the presence of MLN-4760 and/or DX-600. The sample buffer was phosphate-buffered saline. The concentration of the ACE2 fusion proteins was 1 mg/mL. The concentration of MLN-4760 and DX-600 was 50 pM in the experiments shown in Figures 1, 2 and 4 and 5 pM and 50 pM in the experiment shown in Figure 3. All measurements were performed with a temperature ramp of 1 °C/min to collect thermograms in the range from 20 °C to 100 °C. The collected data was processed with the PEAQ-DSC software by subtracting buffer measurements from the thermograms of the proteins and using a progress baseline function.

MLN-4760 significantly increases the thermal stability of both an ACE2-Fc (lgG2) fusion protein and an ACE2-Fc (IgM) fusion protein (see Figures 1A and 1B).

Further, both MLN-4760 and DX600 significantly increase the thermal stability of an ACE2- Fc (lgG4) fusion protein (see Figures 2A and 2B).

Figure 3 shows that at a molar ratio of inhibitor to fusion protein of 1 :1 not all protein molecules are stabilized against thermal unfolding, whereas a stabilization of all protein molecules could be observed when a molar ratio of inhibitor to fusion protein of 10:1 was used. No synergistic effect of the two ACE2 inhibitors MLN-4760 and DX600 when used in a concentration of 50 pM each on the stabilization of the ACE2-Fc fusion proteins against thermal unfolding could be observed (see Figure 4).

4. Effect of MLN-4760 on the aggregation of ACE2 fusion proteins

The ACE2-lgG2-Fc fusion protein had a concentration of 0.1 mg/mL in PBS. 330 pL of the sample were placed in a 1 .5 mL Eppendorf tube and incubated for 2 h at 45 °C. The ACE2- lgG2-Fc was incubated either alone or in the presence of 10:1 molar excess of MLN-4760. After incubation, the sample was centrifuged shortly and the amount of aggregates was assessed on a SEC-MALS system consisting of a Shimadzu HPLC, a Heleos II MALS detector (Wyatt technology) and a Superdex 200 Increase 10/300 GL column (Cytiva). The running buffer was PBS and the flow was 0.5 mL/min.

As shown in Figure 5, MLN-4760 also reduces the aggregation propensity of an ACE2-Fc (lgG2) fusion protein during incubation at a temperature of 45°C.

5. Binding of ACE2 fusion proteins to the spike protein of SARS-CoV2 in the presence of MLN-4760

The binding of the ACE2-Fc fusion proteins was measured with a Biacore X-100 system and the Biotin CAPture kit (Cytiva). The running buffer was HBS-EP+ pH 7.4 (Cytiva). The ligand SARS-CoV-2 RBD with an AviTag (Acrobiosystems) was captured on the streptavidin chip to around 100 RD. Increasing concentrations of the analyte ACE2-Fc fusion protein (0.32, 1.6, 8, 40 and 200 nM) were injected over the immobilized ligand in a single-cycle kinetic mode. The ACE2- Fc fusion protein was injected either alone or in the presence of DX-600 or MLN- 4760. The inhibitor: ACE2- Fc fusion protein ratio was 10:1. The obtained sensorgrams were evaluated with the Biacore X-100 software to obtain a binding constant (KD).

As shown in Figure 6, the presence of MLN-4760 had no significant effect on the binding of the ACE2-lgG2 fusion protein to the spike protein of SARS-CoV2, as the KD in the absence of MLN-4760 was 4.8 nM and in the presence of MLN-4760 the KD was 4.4 nM.

Similarly, Figures 7A and 7B show that the presence of either 50 pM MLN-4760 or 50 pM DX-600 had no significant effect on the binding of the ACE2-lgG4 fusion protein to the spike protein of SARS-CoV2. Some embodiments of the present invention relate to:

1. A combination product comprising:

(c) a fusion protein comprising a first part comprising a fragment of human ACE2 or a variant of said fragment, said human ACE2 having the amino acid sequence according to SEQ ID NO: 1 , and a second part comprising an Fc portion of a human antibody or a fragment or variant of the Fc portion; and

(d) an inhibitor of ACE2.

2. Combination product according to item 1 , wherein the Fc portion of a human antibody is the Fc portion of a human IgG 1 , lgG2, lgG3, lgG4, or IgM antibody.

3. Combination product according to item 1 or 2, wherein the amino acid sequence of the Fc portion of a human antibody is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.

4. Combination product according to item 1 , wherein the amino acid sequence of the variant of the Fc portion of a human antibody is selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36.

5. Combination product according to any one of the preceding items, wherein the fragment of human ACE2 consists of the amino acid sequence according to SEQ ID NO: 2.

6. Combination product according to any one of items 1 to 4, wherein the fragment of human ACE2 is the extracellular domain of ACE2 consisting of the amino acid sequence according to SEQ ID NO: 3.

7. Combination product according to any one of items 1 to 3 and 5, wherein the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 9.

8. Combination product according to any one of items 1 to 3 and 6, wherein the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12 and SEQ ID NO: 13. 9. Combination product according to any one of items 1 , 4 and 5, wherein the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42.

10. Combination product according to any one of items 1 , 4 and 6, wherein the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46.

11. Combination product according to any one of items 1 to 6, wherein the variant of the human ACE2 fragment is an enzymatically inactive variant of human ACE2.

12. Combination product according to item 11 , wherein the enzymatically inactive variant of human ACE2 comprises a H374N and a H378N mutation, the numbering referring to SEQ ID NO: 1 .

13. Combination product according to item 11 or 12, wherein the enzymatically inactive variant of human ACE2 has the amino acid sequence according to SEQ ID NO: 37 or SEQ ID NO: 38.

14. Combination product according to any one of items 11 to 13, wherein the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, . SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54.

15. Combination product according to item 11 , wherein the enzymatically inactive variant of human ACE2 comprises a R273A mutation, the numbering referring to SEQ ID NO: 1.

16. Combination product according to item 11 or 15, wherein the enzymatically inactive variant of human ACE2 has the amino acid sequence according to SEQ ID NO: 55 or 56.

17. Combination product according to any one of items 11 , 15 and 16, wherein the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68.

NO:NO:NO:

18. Combination product according to any one of items 1 to 6, wherein the variant of the human ACE2 fragment comprises a S645C mutation, the numbering referring to SEQ ID NO: 1.

19. Combination product according to item 18, wherein the variant of human ACE2 has the amino acid sequence according to SEQ ID NO: 69 or 70.

20. Combination product according to any one of items 18 and 19, wherein the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81 and SEQ ID NO: 82.

21. Combination product according to any one of items 1 to 6, wherein the variant of the human ACE2 fragment comprises a S645C mutation, a H374N mutation and a H378N mutation, the numbering referring to SEQ ID NO: 1.

22. Combination product according to item 21 , wherein the variant of human ACE2 has the amino acid sequence according to SEQ ID NO: 83 or SEQ ID NO: 84.

23. Combination product according to any one of items 21 and 22, wherein the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96.

24. Combination product according to any one of items 1 to 6, wherein the variant of the human ACE2 fragment comprises a S645C mutation and a R273A mutation, the numbering referring to SEQ ID NO: 1.

25. Combination product according to item 24, wherein the variant of human ACE2 has the amino acid sequence according to SEQ ID NO: 97 or SEQ ID NO: 98. 26. Combination product according to any one of items 24 and 25, wherein the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 110.

27. Combination product according to any one of the preceding items, wherein the inhibitor of ACE2 is a small molecule or a peptide.

28. Combination product according to any one of the preceding items, wherein the inhibitor of ACE2 is selected from MLN-4760 and DX600.

29. Combination product according to any one of the preceding items, wherein the inhibitor of ACE2 and the fusion protei n are present in a molar ratio of 100: 1 to 2: 1.

30. Combination product according to any one of the preceding items for medical use.

31. Combination product according to any one of items 1 to 29 for use in preventing and/or treating an infection with a coronavirus binding to ACE2.

32. Combination product for use according to item 31 , wherein the coronavirus binding to ACE2 is selected from the group consisting of SARS, SARS-CoV-2 and NL63, preferably it is SARS-CoV-2.

33. Combination product for use according to item 31 or 32, wherein the combination product is to be administered in combination with an anti-viral agent.

34. Combination product for use according to item 33, wherein the anti-viral agent is selected from the group consisting of remdesivir, arbidol HCI, ritonavir, lopinavir, darunavir, ribavirin, chloroquin and derivatives thereof, nitazoxanide, camostat mesilate, tocilizumab, siltuximab, sarilumab and baricitinib phosphate.

35. Combination product for use according to item 31 or 32, wherein the combination product is to be administered in combination with an antibody. 36. Fusion protein for use according to claim 35, wherein the antibody is selected from the group consisting of bamlanivimab, etesevimab, casirivimab, imdevimab, sotrovimab and mixtures thereof.

37. Combination product according to any one of items 1 to 29 for use in treating hypertension (including high blood pressure), congestive heart failure, chronic heart failure, acute heart failure, contractile heart failure, myocardial infarction, arteriosclerosis, kidney failure, renal failure, Acute Respiratory Distress Syndrome (ARDS), Acute Lung Injury (ALI), chronic obstructive pulmonary disease (COPD), pulmonary hypertension, renal fibrosis, chronic renal failure, acute renal failure, acute kidney injury, inflammatory bowel disease and multi-organ dysfunction syndrome.

38. Combination product according to any one of items 1 to 29, further comprising a pharmaceutically acceptable carrier or excipient.

39. Combination product according to item 38, further comprising an anti-viral agent.

40. Method for stabilizing a fusion protein as characterized in any one of claims 1 to 29, comprising adding an inhibitor of ACE2 to a cell culture medium comprising host cells producing said fusion protein.

41. Method for stabilizing a fusion protein as characterized in any one of items 1 to 29, comprising mixing the fusion protein with an inhibitor of ACE2.

42. Method according to item 41 , wherein the fusion protein and the inhibitor of ACE2 are mixed before, during and/or after purification of the fusion protein.

43. Method according to item 41 or 42, wherein the fusion protein is stabilized against thermal unfolding.

44. Method according to any one of items 40 to 43, wherein the inhibitor of ACE2 is a small molecule or a peptide.

45. Method according to any one of items 40 to 44, wherein the inhibitor of ACE2 is selected from MLN-4760 and DX600. 46. Method according to any one of items 41 to 45, wherein the fusion protein and the inhibitor of ACE2 are mixed in a molar ratio of 1 :2 to 1 :100.

47. Method according to any one of items 40 to 46, wherein the fusion protein is stabilized against aggregation.

48. Use of an inhibitor of ACE2 for stabilizing a fusion protein as characterized in any one of items 1 to 29.

Claims

1 . A combination product comprising:

(a) a fusion protein comprising a first part comprising a fragment of human ACE2 or a variant of said fragment, said human ACE2 having the amino acid sequence according to SEQ ID NO: 1 , and a second part comprising an Fc portion of a human antibody or a fragment or variant of the Fc portion; and

(b) an inhibitor of ACE2.

2. Combination product according to claim 1 , wherein the Fc portion of a human antibody is the Fc portion of a human IgG 1 , lgG2, lgG3, lgG4, or IgM antibody, preferably wherein the amino acid sequence of the Fc portion of a human antibody is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 orwherein the variant of the Fc portion of a human antibody is a variant of the Fc portion of a human lgG1 or lgG4 antibody, preferably wherein the amino acid sequence of the variant of the Fc portion of a human antibody is selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36.

3. Combination product according to any one of the preceding claims, wherein the fragment of human ACE2 consists of the amino acid sequence according to SEQ ID NO: 2 or is the extracellular domain of ACE2 consisting of the amino acid sequence according to SEQ ID NO: 3.

4. Combination product according to any one of claims 1 to 3, wherein the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46.

5. Combination product according to any one of the preceding claims, wherein the variant of the human ACE2 fragment is an enzymatically inactive variant of human ACE2, preferably wherein the enzymatically inactive variant of human ACE2 comprises a H374N and a H378N mutation, the numbering referring to SEQ ID NO: 1.

6. Combination product according to any one of claims 1 to 3 and 5, wherein the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54.

7. Combination product according to any one of the preceding claims, wherein the inhibitor of ACE2 is a small molecule or a peptide, preferably wherein the inhibitor of ACE2 is selected from MLN-4760 and DX600.

8. Combination product according to any one of the preceding claims, wherein the inhibitor of ACE2 and the fusion protein are present in a molar ratio of 100:1 to 2:1 .

9. Combination product according to any one of claims 1 to 8 for medical use.

10. Combination product according to any one of claims 1 to 8 for use in preventing and/or treating an infection with a coronavirus binding to ACE2, preferably wherein the coronavirus binding to ACE2 is selected from the group consisting of SARS, SARS-CoV-2 and NL63, preferably it is SARS-CoV-2.

11 . Method for stabilizing a fusion protein as characterized in any one of claims 1 to 8, comprising adding an inhibitor of ACE2 to a cell culture medium comprising host cells producing said fusion protein.

12. Method for stabilizing a fusion protein as characterized in any one of claims 1 to 8, comprising mixing the fusion protein with an inhibitor of ACE2, preferably wherein the fusion protein and the inhibitor of ACE2 are mixed before, during and/or after purification of the fusion protein.

13. Method according to claim 12, wherein the fusion protein is stabilized against thermal unfolding and/or aggregation.

14. Method according to any one of claims 11 to 13, wherein the inhibitor of ACE2 is a small molecule or a peptide, preferably wherein the inhibitor of ACE2 is selected from MLN-4760 and DX600.

15. Method according to any one of claims 12 to 14, wherein the fusion protein and the inhibitor of ACE2 are mixed in a molar ratio of 1 :2 to 1 :100.