WO2022167947A1

WO2022167947A1 - Mutated recombinant ace2-fc fusion proteins for the treatment of covid-19 infections

Info

Publication number: WO2022167947A1
Application number: PCT/IB2022/050901
Authority: WO
Inventors: Dóra KARANCSINÉ MENYHÁRD; András PERCZEL; Imre Kacskovics; Zoltán URBÁNYI
Original assignee: Richter Gedeon Nyrt.; Immunogenes Kft.; Pécsi Tudományegyetem; Eötvös Loránd Tudományegyetem
Priority date: 2021-02-03
Filing date: 2022-02-02
Publication date: 2022-08-11
Also published as: HUP2100038A1

Abstract

The invention relates to a mutated recombinant fusion protein comprising a human ACE2 domain and a human IgG-Fc domain, wherein in comparison to the ACE2 domain of SEQ ID NO: 1, the ACE2 domain of the mutated recombinant fusion protein comprises mutation (i) at positions R275, T373, H507; or (ii) at positions R275, H347, T373, H507, I515; or (iii) at positions R275, H347, H507, I515; or (iv) at position I515; or (v) at positions T373, I515; or (vi) at position H507; or (vii) at positions H507, I515; or (viii) at position T373. The invention also relates to said mutated recombinant fusion protein, further comprising a signal peptide. The invention further relates to said mutated recombinant fusion protein for use in a method of treating an infection disease caused by a coronavirus capable of binding ACE2. The invention also relates to a polynucleotide encoding said mutated recombinant fusion protein, and to a vector comprising said polynucleotide, as well as to a host cell comprising said vector.

Description

MUTATED RECOMBINANT ACE2-FC FUSION PROTEINS

FOR THE TREATMENT OF COVID-19 INFECTIONS

FIELD OF THE INVENTION

The present invention relates to mutated recombinant fusion proteins comprising mutated ACE2 domain(s) and/or fragments thereof and Fc domain(s) and their use for the treatment of COVID-19.

BACKGROUND OF THE INVENTION

In 2019, a new type of corona virus called SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) spread from China to the rest of the world and was declared a pandemic by the WHO (World Health Organization) in March, 2020. This virus is the third of its kind causing severe respiratory illness, following the pathogen SARS- CoV (severe acute respiratory syndrome coronavirus) and MERS-CoV (Middle East Respiratory Syndrome coronavirus), spreading in 2003 and 2012, respectively.

Currently, there is no specific therapy available for the treatment of the SARS-CoV- 2-related disease, which received the name COVID-19 (referring to coronavirus disease 2019). First approvals for vaccines are currently being granted in a fast-track process and will be produced for worldwide vaccination coverage in 2021 . Given the extremely rapid spreading of the disease, whose progression is however mild in most cases, there is an immense global urgency in the development of effective treatment strategies. Especially for severe courses of the disease, which occur in about 20% of patients and which cause fever and serious pneumonia, therapeutic and preventive options are of high importance (European Centre for Disease Prevention and Control, 2020). Risk factors are today considered to include male sex, smoking, hypertension, diabetes, obesity and preexisting cardiovascular disease; all of which are associated with an unfavorable prognosis for COVID-19 (Zou et al, N. Engl. J. Med.; 2020, 382, 1177-1179, Section “Findings”). Symptoms of the newly emerged disease COVID-19 include, among others, dry cough and fever and general malaise. The most severe symptoms triggered by SARS-CoV-2 may also include cytokine release syndrome (CRS), which can be associated with various secondary diseases (e.g. pulmonary edema, acute respiratory distress syndrome (ARDS) or secondary hemophagocytic lym phohistiocytosis (sHLH)). According thereto, patients suffering COVID-19 may develop systemic hyper-inflammation, cause by increased levels of IL-2, II-7, II-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma- inducible protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1 ), macrophage inflammatory protein 1-a (MIP-1a) and the tumor necrosis factor-alpha (TNFa) indicating CRS, as it was shown for example by Huang et al. (The Lancet; 2020, 395, 497-506)

All known variants of the newly appearing coronavirus allows itself to access the human body in a special way: SARS-CoV-2 binds to the cell surface receptor Angiotensin Converting Enzyme-2 (ACE2) in the body, in order to gain access to the attacked body cells allowing its reproduction. This receptor is particularly common in cardiopulmonary tissues, but it is also expressed in hematopoietic cells, such as monocytes and macrophages. The transmembrane protein natively cleaves angiotensin II within an enzymatic reaction. ACE2 thus plays an extremely important role in the renin-angiotensin-aldosterone system (RAAS) and thus in the regulation of fluid balance and blood pressure.

To date, there are no clearly effective drugs against COVID-19, and most patients are treated only on a symptom-specific basis. Accordingly, there is a strong medical need for the therapeutic approaches which allow the prevention, amelioration and the treatment of the disease and symptoms that are associated with inflammation of the respiratory tract and/or immunologically induced injury of the respiratory tract in a human subject. So far, various therapeutic approaches have been considered:

One therapeutic option is the development of receptor-binding domains (RBDs), in which a fragment of the viral protein blocks the cell surface receptor and thus prevents the virus from entering the cell. In 2004, Wong et al. (J. Biol. Chem.; 2004, 279, 3197-3201 ) showed that the RBD approach can be successfully used to block the ACE2 cell surface receptor and thus limit the access of SARS-CoV. Experiments with monoclonal antibodies also showed a similar effect (Desmyter et al., Proc. Natl. Acad. Sci.; 2013, 110, E1371-E1379; Koch et al., Sci. Rep.; 2017, 7, 8390). However, the approach to the use of RBDs also has its limitations: this is shown by the fact that in most cases the body produces neutralizing antibodies against RBDs. In addition, the risks of using RBDs have not yet been described adequately. It is not yet known, whether there is a ratio of ACE2 receptors that are needed to be blocked in order to the viral infection to slow down or even halt. It is also questionable, what the density of ACE2 receptors is in certain organs and in the whole body. In addition, it is also possible that blocking ACE2 could ultimately worsen the clinical symptoms of infection by altering the normal physiological functions of ACE2.

Another strategy deals with the extracellular binding of viral proteins (Li et al., Nature; 2003, 426, 450-454) which are responsible for access to the cell even before the virus docks to the cell. In this case, soluble forms of the membrane-bound proteins are used to bind and block the virus in advance. It takes advantage of the strong affinity of the spike protein (S protein) to ACE2 to effectively prevent it from binding to the receptor. This technique is used in the present invention.

The generation of Fc-fusion proteins in order to improve half-life extension is commonly used in biomedical science, as long-circulating plasma proteins can result in reduced degradation.

ACE2-Fc fusion proteins were designed and tested by Liu et al. (Int. J. Biol. Macromol.; 2020, 165: 1626-1633). Nine mutants of ACE2-Fc (E145A, R273A, H345A, P346A, D368A, H374A, H378A, E402A, H505A) were studied, among them R273A, H378A and E402A were completely lack of ACE2 peptidase activity but maintained their binding capacity toward SARS-CoV-2 spike protein and inhibited the transduction of a pseudotyped reporter virus. According to the conclusions of the article, H378A and E402A mutations may potentially suffer protein instability problems.

Abolishment of peptidase activity together with high affinity binding to SARS-CoV-2 S protein is mentioned in connection with the H374 and H378 mutations of the ACE2 extracellular domain by Kruse (F1000Research; 2020, 9:72) citing Moore et al. (J Virol.; 2004, 10628-10635). ACE-lg proteins containing the same mutations (H374, H378) neutralized virus pseudotyped with SARS-CoV or SARS-CoV-2 spike proteins in vitro (Lei et al., Nat. Comm.; 2020, 11 :2070).

A stepwise engineering approach to generate affinity optimized, enzymatically inactivated ACE2 variants able to tightly bind the RBD of the viral spike protein is described by Glasgow et al. (PNAS, 2020, 117, 45, 28046-55). The study came to the conclusion that H374N/H378N double mutation is unstable, therefore H345L mutation should be incorporated, too. The highest affinity clones contained N33D and H34S mutations and were derived from the K31 F/H34I/E35Q ACE2(614) variant, which bound to the RBD 170-fold more tightly than wild-type ACE2.

ACE2-Fc lgG1 fusion proteins are described in a preprint article of Iwanaga et al. (bioRxiv; 2020, Version 2.). Three mutants were engineered such as MDR503, MDR504 and MDR505. Among them MDR504 appeared to be the best candidate, which had greater binding to the SARS-CoV-2 RBD and spike protein and showed enhanced neutralization of virus in a Vero E6 cell plaque assay. Furthermore, MDR504 had similar serum stability as wild-type ACE2-Fc. A later version of the preprint article was published on 21^st January 2022 (iScience 25, 103670), in which mutations are named: MDR503 has a R273A mutation, MDR504 has a H345A mutation and MDR505 has both R273A and H345A mutation.

H354A mutation was introduced in ACE2 microbody proteins, in which the ACE2 ectodomain is fused to Fc domain 3 of the Ig heavy chain. This protein inhibits entry of SARS-CoV-2 spike protein pseudotyped virus and replication of live SARS-CoV-2 both in vitro and in mouse model (Tada et al., Cell Rep.; 2020, 33, 108528).

Mutations at N824 in an Fc domain or fragments thereof are well known in the art (Wang et al., Protein & Cell; 2018, 9, 63-73). They are routinely used in immunologic science, as this specific mutation enables to switch off all effector functions of the Fc domain by depleting the ability to bind a receptor. Wang et al (Protein & Cell; 2018, 9, 63-73) summarize all known modifications concerning human IgG-s, which modifications influence the effector functions of a given antibody. As for the N297A, N297Q and N297G mutations described by Wang et al., the reduction of effector function is achieved by the mutation of asparagine in position 297 of Fc region. WO 2021/217120 A2 discloses ACE2-Fc fusion proteins with mutations H345A, H345V, H345I or H345L, and R273A in the ACE2 domain; and with eliminated FcRy binding. Also disclosed is the use of a product comprising such a fusion protein for the prevention, or the reduction of severity of an infection by a coronavirus that binds to human ACE2 receptor.

WO 2021/203098 A2 discloses ACE2-Fc fusion proteins with mutation H345L in the ACE2 domain, and with an Fc domain having one or more amino acid substitution(s). Said proteins are useful against ACE2-targeted viruses.

WO 2021/183717 A1 discloses triple mutant ACE2-Fc fusion proteins, and discloses mutations H505L, H345A and R273L, separately. The proteins are used for the prevention and treatment of COVID-19 and other such virally induced diseases.

However, there is still a need in the art for improved ACE2-Fc fusion proteins for the prevention and treatment of an infection disease caused by a coronavirus capable of binding ACE2, especially, caused by any variants of SARS-CoV-2.

SUMMARY OF THE INVENTION

The present invention provides a mutated recombinant fusion protein comprising a human ACE2 domain and an Fc domain of human IgG, wherein the human ACE2 domain in comparison to SEQ ID NO: 1 is mutated at one or more position(s), selected from the group consisting of H347, H507, R275, T373 and/or 1515, furthermore, the invention provides said mutated recombinant fusion protein for use in a method of treating infection diseases caused by a coronavirus.

The invention further relates to a mutated recombinant fusion protein comprising a human ACE2 domain and a human IgG-Fc domain, wherein in comparison to the ACE2 domain of SEQ ID NO: 1 , the ACE2 domain of the mutated recombinant fusion protein comprises mutation (i) at positions R275, T373, H507; or (ii) at positions R275, H347, T373, H507, 1515; or (iii) at positions R275, H347, H507, 1515; or (iv) at position 1515; or (v) at positions T373, 1515; or (vi) at position H507; or (vii) at positions H507, 1515; or (viii) at position T373. The invention further relates to said mutated recombinant fusion protein, wherein a mutation (i) at position R275 is an exchange of R for L; (ii) at position H347 is an exchange of H for G, V, A, L or I; (iii) at position T373 is an exchange of T for F, Y or W; (iv) at position H507 is an exchange of H for G, V, A, L or I; and/or (v) at position 1515 is an exchange of I for T or S. The invention further relates to said mutated recombinant fusion protein, wherein a mutation (i) at position R275 is an exchange of R for L; (ii) at position H347 is an exchange of H for A; (iii) at position T373 is an exchange of T for F; (iv) at position H507 is an exchange of H for L; and/or (v) at position 1515 is an exchange of I for T. The invention further relates to any one of said mutated recombinant fusion proteins, wherein the human IgG-Fc domain of the mutated recombinant fusion protein is a human lgG1 -Fc domain. The invention further relates to said mutated recombinant fusion protein, wherein in comparison to the lgG1 -Fc region of SEQ ID NO: 1 , the lgG1-Fc domain of the mutated recombinant fusion protein comprises mutation at position N824. The invention further relates to said mutated recombinant fusion protein, wherein the mutation at position N824 is an exchange of N for G. The invention further relates to a mutated recombinant fusion protein, wherein the mutated recombinant fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 21 , 22, 23 and 24. The invention further relates to a mutated recombinant fusion protein, wherein the mutated recombinant fusion protein has a sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 21 , 22, 23 and 24. The invention further relates to a mutated recombinant fusion protein, wherein the mutated recombinant fusion protein has a sequence as shown by SEQ ID NO: 15.

In another aspect of the invention, the mutated recombinant fusion protein further comprises a signal peptide. The invention also relates to a mutated recombinant fusion protein comprising a human ACE2 domain and a human IgG-Fc domain, wherein in comparison to the ACE2 domain of SEQ ID NO: 1 , the ACE2 domain of the mutated recombinant fusion protein comprises mutation (i) at positions R275, T373, H507; or (ii) at positions R275, H347, T373, H507, 1515; or (iii) at positions R275, H347, H507, 1515; or (iv) at position 1515; or (v) at positions T373, 1515; or (vi) at position H507; or (vii) at positions H507, 1515; or (viii) at position T373, wherein the mutated recombinant fusion protein further comprises a signal peptide. The invention also relates to said mutated recombinant fusion protein, wherein a mutation (i) at position R275 is an exchange of R for L; (ii) at position H347 is an exchange of H for G, V, A, L or I; (iii) at position T373 is an exchange of T for F, Y or W; (iv) at position H507 is an exchange of H for G, V, A, L or I; and/or (v) at position 1515 is an exchange of I for T or S, wherein the mutated recombinant fusion protein further comprises a signal peptide. The invention also relates to said mutated recombinant fusion protein, wherein a mutation (i) at position R275 is an exchange of R for L; (ii) at position H347 is an exchange of H for A; (iii) at position T373 is an exchange of T for F; (iv) at position H507 is an exchange of H for L; and/or (v) at position 1515 is an exchange of I for T, wherein the mutated recombinant fusion protein further comprises a signal peptide. The invention further relates to any one of said mutated recombinant fusion proteins, wherein the human IgG-Fc domain of the mutated recombinant fusion protein is a human lgG1 -Fc domain, wherein the mutated recombinant fusion protein further comprises a signal peptide. The invention further relates to said mutated recombinant fusion protein, wherein in comparison to the lgG1 -Fc region of SEQ ID NO: 1 , the lgG1 -Fc domain of the mutated recombinant fusion protein comprises mutation at position N824, wherein the mutated recombinant fusion protein further comprises a signal peptide. The invention further relates to said mutated recombinant fusion protein, wherein the mutation at position N824 is an exchange of N for G, wherein the mutated recombinant fusion protein further comprises a signal peptide. The invention further relates to a mutated recombinant fusion protein, wherein the mutated recombinant fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 21 , 22, 23 and 24, and wherein the mutated recombinant fusion protein further comprises a signal peptide. The invention further relates to a mutated recombinant fusion protein, wherein the mutated recombinant fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9, 11 , 17, 18, 19 and 20. The invention further relates to a mutated recombinant fusion protein, wherein the mutated recombinant fusion protein has a sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9, 11 , 17, 18, 19 and 20. The invention further relates to a mutated recombinant fusion protein, wherein the mutated recombinant fusion protein has a sequence as shown by SEQ ID NO: 9.

In a further aspect, the invention relates to the mutated recombinant fusion protein as disclosed in the previous paragraphs, for use in a method of treating an infection disease caused by a coronavirus capable of binding ACE2, and optionally, wherein the infection disease is COVID-19. In a further aspect, the invention relates to the mutated recombinant fusion protein as disclosed in the previous paragraphs, for use in a method of treating an infection disease caused by a coronavirus capable of binding ACE2, wherein the coronavirus capable of binding ACE2 is SARS-CoV-2, and optionally, wherein the infection disease is COVID-19.

The invention also relates to a polynucleotide encoding a mutated recombinant fusion protein, wherein the polynucleotide comprises a sequence as shown in any one of SEQ ID NOs: 6, 8, 10, 12. The invention also relates to a polynucleotide encoding a mutated recombinant fusion protein, wherein the polynucleotide has a sequence as shown in any one of SEQ ID NOs: 6, 8, 10, 12. The invention further relates to a vector comprising said polynucleotide. The invention further relates to a host cell comprising said vector. The invention further relates to a CHO host cell comprising said vector.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Proposed structure of the ACE2-Fc fusion protein: the extracellular domain of ACE2 is fused onto human immunoglobulin Fc domain. (Figure 1 is from Kruse, F1000Research; 2020, 9:72.)

Figure 2: Virus neutralization experiment of ACE2-Fc fusion protein against SARS- CoV-2 in Vero E6 cells. Cytopathic effects are only apparent at 1 :160 dilution of ACE2-Fc, and are marked at 1 :640. The micrographs are representative images of the micrographs taken of the wells during the virus neutralization experiment under phase contrast microscope.

Figure 3: Peptide sequence of ACE2 (native)-Fc fusion protein according to SEQ ID NO: 1 ; functional domains of the protein (signal peptide, ACE2 extracellular domain, lgG1 Hinge and Fc region) are indicated.

Figure 4: Peptide sequence of ACE2 (native)-Fc(N824G) fusion protein according to SEQ ID NO: 3; functional domains of the protein (signal peptide, ACE2 extracellular domain, lgG1 Hinge and Fc region) are indicated.

Figure 5: Peptide sequence of ACE2 (H347A, H507L, R275L, T373F, I515T)- Fc(N824G) fusion protein according to SEQ ID NO: 5; functional domains of the protein (signal peptide, ACE2 extracellular domain, lgG1 Hinge and Fc region) are indicated.

Figure 6: Peptide sequence of ACE2 (l515T)-Fc(N824G) fusion protein according to SEQ ID NO: 7; functional domains of the protein (signal peptide, ACE2 extracellular domain, lgG1 Hinge and Fc region) are indicated.

Figure 7: Peptide sequence of ACE2 (H507L, R275L, T373F)-Fc(N824G) fusion protein according to SEQ ID NO: 9; functional domains of the protein (signal peptide, ACE2 extracellular domain, lgG1 Hinge and Fc region) are indicated.

Figure 8: Peptide sequence of ACE2 (1515T, H507L, R275L, H347A)-Fc(N824G) fusion protein according to SEQ ID NO: 11 ; functional domains of the protein (signal peptide, ACE2 extracellular domain, lgG1 Hinge and Fc region) are indicated.

Figure 9: Peptide sequence of ACE2 (H507L, R275L, T373F)-Fc(N824G) fusion protein according to SEQ ID NO: 15; functional domains of the protein (ACE2 extracellular domain, lgG1 Hinge and Fc region) are indicated.

Figure 10: A bar graph based on data of column "Specific activity [AU/s/ml] Average" of Table 1 showing the results of the measurement of ACE2 activity of proteins of the present invention (Example 1 ). Legend: "Native" is ACE2(native)-Fc fusion protein; "Fc" is mutant variant with mutation N824G; "Fc+1x" is mutant variant with mutations 1515T, N824G; "Fc+3x" is mutant variant with mutations R275L, T373F, H507L, N824G; "Fc+4x" is mutant variant with mutations R275L, H347A, H507L, 1515T, N824G; "Fc+5x" is mutant variant with mutations R275L, H347A, T373F, H507L, 1515T, N824G.

Figure 11 : Representative sensorgram of the native ACE2-Fc fusion protein, BLI assay.

Figure 12: Representative sensorgram of the 3x mutant variant (mutant variant with mutations R275L, T373F, H507L and N824G in comparison to the ACE2 domain and lgG1 -Fc region of SEQ ID NO: 1 , respectively), BLI assay.

Figure 13: Equilibrium binding (KD), native ACE2-Fc fusion protein and 3x mutant variant (mutant variant with mutations R275L, T373F, H507L, N824G), BLI assay. DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, “recombinant protein” defines a protein that is produced artificially with the help of for example genetically modified microorganisms or cell cultures. Such organisms and cell cultures include, but are not limited to those, cells that express the desired mutated recombinant fusion protein.

“Mutated” as used herein is related to biological mutations in the nucleotide sequence, including for example point mutations, insertions or deletions at specific positions. At mentioned positions, mutations include point mutations, insertions and deletions. The term "mutated" is also used in connection with peptides, referring to an alteration of an amino acid sequence, for example by deletion, insertion and/or substitution of one or more amino acids.

In the context of the present invention, the terms “neutralizing” and “blocking” are used in an exchangeable way. The term "neutralization" as used here refers to the complete or partial blocking of viruses or viral particles by binding the fusion protein to the viral S-protein. Coronaviruses in general use S-proteins for the attachment to the cell to be infected in order to gain access. S-proteins are spiky envelope proteins that are located on the outer surface of the virus and give it its typical shape. The spikes consist of a glycoprotein with which the virus is coupled to the host cell via the ACE2 receptor. SARS-CoV-2 has a very strong binding affinity to ACE2.

A signal peptide is hereby determined as a short, 3 to 60 amino acid long peptide, which after translation determines the transport target of a protein within the cell.

As used herein, fragment crystallizable (Fc) region domains are regions of antibodies that allow interaction with cell surface receptors. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains. IgM and IgE Fc regions contain three heavy chain constant domains in each polypeptide chain. IgG Fc domain interacts with the neonatal Fc receptor (FcRn) that protects this immunoglobulin isotype from intracellular degradation in capillary endothelial cells, or macrophages, dendritic cells, among other cell types and thus this mechanism provides long half-life for IgG molecules. As used herein, antibody effector functions or effector functions, allowing antibodies to act in a large number of mechanisms, can be for example the recruitment of effector cells of the immune system to bind viral particles or binding to a receptor, for example FcyR (IgG), FcsRI (IgE), FcaRI (IgA), FcpR (IgM) and FcbR (IgD).

As used herein, the term “treat,” “treating,” and “treatment” refer to therapeutic or preventative measures described herein. The methods and uses of “treatment” employ administration of a mutated recombinant fusion peptide to a subject having an infection disease caused by a coronavirus.

As used herein, infection diseases caused by a coronavirus include, but are not limited to those: respiratory tract infections such as common cold diseases, characterized by fever, sore throat, runny nose, cough and headaches, Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). Coronaviruses, which are sought to be treated by the present invention, include all 4 genera of coronaviruses, namely: Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus. Included are especially Alphacoronavirus 1, Human coronavirus 229E, Human coronavirus NL63, Miniopterus bat coronavirus 1, Miniopterus bat coronavirus HKU8, Rhinolophus bat coronavirus HKU2, Scotophilus bat coronavirus 512, Betacoronavirus 1 (Bovine Coronavirus, Human coronavirus OC43), Hedgehog coronavirus 1, Human coronavirus HKU1, Middle East respiratory syndrome-related coronavirus, Murine coronavirus, Pipistrel I us bat coronavirus HKU5, Rousettus bat coronavirus HKU9, Severe acute respiratory syndrome-related coronavirus (SARS-CoV, SARS-CoV-2), Tylonycteris bat coronavirus HKU4, Avian coronavirus, Beluga whale coronavirus SW1, Bulbul coronavirus HKU11 and Porcine coronavirus HKU15. Also included herein are secondary diseases, appearing following a coronavirus infection, such as acute respiratory distress syndrome (ARDS), secondary hemophagocytic lym phohistiocytosis (sHLH) or bacterial infections such as pneumonia. This applies in particular to diseases that follow a CRS. ACE2-Fc fusion protein according to the invention

The present invention comprises a mutated recombinant fusion peptide of ACE2 and/or fragments thereof and an Fc domain of human IgG (i.e. human IgG-Fc domain) and/or fragments thereof for binding viral proteins to prevent infection of a cell with said virus. Physiological ACE2 has an enzymatic activity, which, by hydrolysis of peptide bonds at the C-terminal end of angiotensin II, carried out by the peptidase domain (PD) of the enzyme, serves to generate angiotensin-(1-7), an important substance in the regulation of the renin-angiotensin-aldosterone system (RAAS). Mutations can reduce or completely eliminate this enzymatic activity of the peptidase domain (PD). While enzymatic activity of ACE2 is diminished, capability to bind viral spike-proteins (S-protein) is maintained, making it a powerful neutralizing agent against COVID-19 infections. Although these mutations influence the enzymatic activity of the protein, they do not affect the binding to the S-protein of the virus. For this reason, enzymatically inactive proteins can also be used for extracellular neutralization of the virus. The present invention relates to a mutated recombinant ACE2-Fc fusion peptide for effective neutralization of viral particles in infection diseases caused by a coronavirus. The invention disclosed herein offers effective therapy option(s) against corona viruses using enzymatically inactive ACE2 constructs for virus neutralization, wherein the mutated ACE2 domain is fused to an Fc domain, thereby increasing stability and half-life of the fusion protein in order to improve medical application.

Accordingly, in a first aspect, the present invention relates to a mutated recombinant fusion protein comprising a human ACE2 domain and an Fc domain of human IgG, wherein the human ACE2 domain in comparison to SEQ ID NO: 1 is mutated at one or more position(s), selected from the group consisting of H347, H507, R275, T373 and/or 1515.

Consequently, the fusion protein of the invention comprises, apart from the mutations identified above, the ACE2 domain of SEQ ID NO:1 , but may comprise another IgG Fc domain.

As it is shown in Example 1 , mutations in the ACE2 domain as disclosed in the present invention can reduce ACE2 activity as it is illustrated in Table 1 . ACE2 activity was determined by performing an assay, which uses a specific quenched fluorescent substrate, as it was previously shown by Uri et al. in 2016 (J. Renin-Angiotensin- Aldosterone Syst.; 2016; 17(4): 1470320316668435). For the detection of ACE2 activity, purified recombinant ACE2 was used and the activity level was measured in a fluorescent microplate reader.

Furthermore, the disclosed mutated recombinant fusion protein as disclosed herein can be used to neutralize SARS-CoV-2. For testing the virus neutralization, SARS- CoV-2 from an anonymous patient sample was propagated using Vero E6 cells. The cytopathic effect (CPE) of the coronavirus was demonstrated by the destruction of the cell monolayer. For demonstrating the neutralization effect of the fusion protein, Vero E6 cells were incubated with purified recombinant ACE2 fusion protein and diluted virus. Afterwards, the virus neutralizing effect of the ACE2-Fc fusion protein was evaluated by light microscopy. See Example 2 and Figure 2. Vero E6 cells are derived from an African green monkey (Chlorocebus sp.) and are routinely used in vaccine production for virus-related diseases. These experiments show a clear virus neutralization effect of the recombinant proteins of the invention.

As for the native ACE2-Fc fusion construct, its amino acid sequence is known in the art (Kruse: Therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in Wuhan, China. F1000Research; 2020, 9:72), in addition, native ACE2-Fc fusion protein is commercially available. Also known in the art are preparation methods of mutant ACE2-Fc fusion proteins (see e.g. Liu et al.: Designed variants of ACE2-Fc that decouple anti-SARS-CoV-2 activities from unwanted cardiovascular effects. Int. J. Biol. Macromol.; 2020, 165: 1626-1633; Iwanaga et al.: Novel ACE2-lgG1 fusions with improved in vitro and in vivo activity against SARS- CoV-2. bioRxiv; 2020, Version 2.). In example 3, preparation method of the recombinant ACE2-Fc fusion proteins of the present invention is disclosed. SEQ ID NOs: 2, 4, 6, 8, 10 and 12 show nucleotide sequences. Representative nucleotide sequences and codons for ACE2-Fc fusion proteins of the present invention are shown in SEQ ID NOs: 6, 8, 10, 12, which in this order correspond to ACE2-Fc fusion proteins according to SEQ ID NOs: 5, 7, 9, 11. For the preparation of a mutated recombinant ACE2-Fc fusion protein of the invention a polynucleotide encoding the protein according to the invention and a respective ACE2-Fc expression vector, such as a plasmid, is constructed, then host cells, such as CHO (Chinese hamster ovary) cells, are transfected with the nucleic acid, such as DNA, construct coding ACE2-Fc. After selection the transfected pools are cultivated. Expressed ACE2-Fc fusion proteins are characterized, and at last, fusion proteins of the invention are purified from the supernatant of the cell cultures. SARS-CoV-2 has a very strong binding affinity to ACE2, which is exploited in the present invention. Therefore, in one embodiment, the mutated recombinant fusion peptide of the present invention is capable of binding S-proteins of coronaviruses, in order to allow proper neutralization. The interaction between an ACE2-Fc fusion protein and S-protein has been tested by BLI assay (Example 4, Figures 11 -13).

In a further embodiment, the mutated recombinant fusion protein additionally further comprises a signal peptide. In an embodiment, the signal peptide is a 3 to 60 amino acid long peptide. In a preferred embodiment, the signal peptide is between 10 and 30 amino acids, and in a more preferred embodiment, the signal peptide is between 10 and 25 amino acids. In one embodiment the signal peptide can be located at the N terminus of the protein, and in another embodiment, the signal peptide can be positioned at the C terminus of the protein.

In another embodiment of the present invention, the mutated recombinant fusion protein does not encompass a signal peptide. In yet another embodiment, the mutated recombinant fusion protein encompasses a signal peptide that is different from the one disclosed in SEQ ID NO: 1 .

In yet another embodiment, the mutated recombinant fusion protein is modified. Modifications include, but are not limited to those, e.g. protein tags and posttranslational modifications. Examples for tags may be tagged fluorescent proteins, His-tags, Myc-tags, HA-tag, FLAG-tag, T7-tag and any other modification allowing the detection by standard biological methods.

As discussed above, the recombinant polypeptide of the invention comprises an ACE2-domain. ACE2 mutations on mentioned positions impair enzymatic peptidase activity of ACE2 but, of importance, maintain all necessary binding affinities to Spikeproteins (S-proteins) of coronaviruses. By mutating ACE2 domains, the enzymatic activity of ACE2 can be impaired by 10%, 20%, 30%, 40% or about 50%, compared to the peptide variant of SEQ ID NO: 1. Included are also variants, where the complete enzymatic peptidase activity of ACE2 is abolished. Therefore, in one embodiment, the mutated recombinant fusion protein comprises an ACE2 domain, which is mutated to exhibit diminished enzymatic activity. In particular, the activity may be inhibited by any possible reduction. This includes activity reductions of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90%. In another embodiment, the activity is completely absent.

In still another embodiment the mutated recombinant fusion peptide comprises an ACE2 domain, wherein the ACE2 domain is mutated in the peptidase domain, comprising amino acid 20 to amino acid 615, respectively.

The recombinant polypeptide further comprises an Fc domain of human IgG. As stated above, the present invention relates to a mutated recombinant fusion protein, comprising an ACE2 domain and an Fc domain.

In the context of the present invention, the Fc domain of the mutated recombinant fusion protein may be from any IgG, including lgG1 , lgG2, lgG3 and lgG4.

In a preferred embodiment of the present invention, the Fc domain of the mutated recombinant fusion protein is an lgG1 -Fc domain.

Depending on the structure of the Fc domain, the construct can either have antibody effector functions or not.

In one embodiment, the Fc domain can be non-mutated. In one embodiment, the Fc domain has the sequence of the Fc domain of SEQ ID NO: 1 . In another embodiment, the Fc domain can differ from the sequence of the Fc domain of SEQ ID NO: 1. Preferably, the sequence identity of the Fc domain is at least 70% homologue to the sequence of the Fc domain of SEQ ID NO:1. Other embodiments include Fc domains, wherein at least 80, 90 or 99% of the Fc domain is equal to the Fc domain of SEQ ID NO: 1 . In an embodiment, the Fc domain of the fusion protein has a sequence identity of at least 80%, 90% or 99% with the Fc domain of SEQ ID NO: 1 .

Furthermore, in another embodiment of the present invention, the Fc domain can be modified by non-mutation modifications, to adjust its activity. Accordingly, in one embodiment the Fc domain can be glycosylated thereby maintaining all the effector functions of an antibody Fc region, such as recruiting the immune system to bound viral particles, or activating the complement cascade contributing to their elimination. In another embodiment the Fc domain is unglycosylated, resulting in a complete loss of immune activating functions. In those embodiments, the fused Fc domain only serves to increase the fusion protein’s half-life.

In still another embodiment, the Fc domain can be mutated. Preferably, the mutation is located at N824. More preferably, the mutation comprises an amino acid change from N to G, resulting in N824G, compared to SEQ ID NO: 1. Therefore, in one embodiment, the Fc domain comprises the sequence of the Fc domain of SEQ ID NO: 3. N824 is usually referred as N297 in literature and commonly used to adapt effector functions of an Fc domain.

In yet another embodiment, the Fc domain exhibits reduced effector functions, in particular reduced complement and Fc gamma receptor mediated (ADCC, ADCP) activation.

In preferred embodiments, the Fc domain exhibits 10%, 20%, 30%, 40% or 50% reduced effector functions, compared to the wildtype Fc domain. Included are also variants, wherein the complete effector function of the Fc domain is abolished.

Positions of the mutations according to the present invention as stated above refer to the following sequence representing a reference sequence (comprising the ACE2 extracellular domain, an lgG1 hinge and an lgG1 Fc region without mutations). Important sections (signal peptide, ACE2 extracellular domain and lgG1 hinge and lgG1 Fc region) are highlighted as described below the sequence.

SEQ ID NO: 1

MDWIWRILFLVGAATGAHSQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNI TEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVL SEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWA WESWRSEVGKQLRPLYEEYWLKNEMARANHYEDYGDYWRGDYEVNGVDGYD YSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWG RFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQG FWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQ YDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEI NFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGWEP VPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDIS NSTEAGQKLFNMLRLGKSEPWTLALENWGAKNMNVRPLLNYFEPLFTWLKDQN KNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQ YFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRI NDAFRLNDNSLEFLGIQPTLGPPNQPPVSEP SCD THTCPPCB4PELLGGPSVE LFPPKPKDTLMISRTPE VTC VVVD VSHEDPEVKFNWYVDG VE VHNAKTKPREEQ Y NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQ VSLTCL VKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Signal peptide

ACE2 extracellular domain lgG1 Hinge and lgG1 Fc region

In a preferred embodiment, the mutated recombinant fusion protein comprises a human ACE2 domain and an Fc domain of human IgG, wherein the human ACE2 domain in comparison to SEQ ID NO: 1 is mutated at one or more position(s), selected from the group consisting of H347, H507, R275, T373 and/or 1515. In a further preferred embodiment, in said mutated recombinant fusion protein the mutation at one or more position(s) is selected from the group consisting of H347A, H507L, R275L, T373F and/or 1515T.

In a preferred embodiment of the invention, the mutation at positions H347, H507, and R275 is an exchange of H for G, V, A, L, and I, or is an exchange of R for L, respectively.

In another preferred embodiment, the mutation at position 1515 is an exchange for T or S. In a further embodiment, the exchange at position T373 is for F, Y or W. In a further embodiment of the present invention, the mutated recombinant fusion peptide comprises a human ACE2 domain and an IgG-Fc domain, wherein the human ACE2 domain in comparison to SEQ ID NO: 1 is mutated at one or more position(s) selected from the group consisting of H347A, H507L, R275L, T373F and/or 1515T.

In another embodiment, the mutated recombinant fusion protein has the sequence(s) as shown in any of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11.

In another preferred embodiment, the mutated recombinant fusion protein comprises a human ACE2 domain and an Fc domain of human IgG, wherein the human ACE2 domain in comparison to SEQ ID NO: 1 is mutated at one or more position(s), selected from the group consisting of H347, H507, R275, T373 and/or 1515, and wherein the Fc domain is an Fc domain of the human IgG 1 and is mutated at position N824, preferably wherein the mutation is N824G.

In another preferred embodiment, the mutated recombinant fusion protein comprises a human ACE2 domain and an Fc domain of human IgG, wherein the human ACE2 domain in comparison to SEQ ID NO: 1 is mutated at one or more position(s), selected from the group consisting of H347, H507, R275, T373 and/or 1515, and in said mutated recombinant fusion protein the mutation at one or more position(s) is selected from the group consisting of H347A, H507L, R275L, T373F and/or 1515T, and wherein the Fc domain is an Fc domain of the human lgG1 and is mutated at position N824, preferably wherein the mutation is N824G.

Accordingly, in one embodiment, the mutated recombinant fusion protein can comprise the sequence as provided in SEQ ID NO: 5 and Figure 5. SEQ ID NO: 5 is, in comparison to SEQ ID NO: 1 , mutated at the following positions: 1515, H507, R275, T373, H347 and N824.

Furthermore, in another embodiment, the mutated recombinant fusion protein can comprise the sequence as provided in SEQ ID NO: 7 and Figure 6. SEQ ID NO: 7 is, in comparison to SEQ ID NO: 1 , mutated at the following positions: 1515 and N824.

Moreover, in another embodiment, the mutated recombinant fusion protein can comprise the sequence as provided in SEQ ID NO: 9 and Figure 7. SEQ ID NO: 9 is, in comparison to SEQ ID NO: 1 , mutated at the following positions: H507L, R275L, T373F and N824.

Additionally, in still another embodiment, the mutated recombinant fusion protein can comprise the sequence of SEQ ID NO: 11 and Figure 8. SEQ ID NO: 11 is, in comparison to SEQ ID NO: 1 , mutated at the following positions: 1515, H507, R275, H347 and N824.

In a preferred embodiment, the mutated recombinant fusion protein comprises a human ACE2 domain and an Fc domain of human IgG, wherein the human ACE2 domain in comparison to SEQ ID NO: 1 is mutated at one or more position(s), selected from the group consisting of H347, H507, R275, T373 and/or 1515, wherein the mutated recombinant fusion protein does not comprise a signal peptide.

In a further preferred embodiment, the mutated recombinant fusion protein comprises a human ACE2 domain and an Fc domain of human IgG, wherein the human ACE2 domain in comparison to SEQ ID NO: 1 is mutated at one or more position(s), selected from the group consisting of H347, H507, R275, T373 and/or 1515, wherein in said mutated recombinant fusion protein the mutation at one or more position(s) is selected from the group consisting of H347A, H507L, R275L, T373F and/or 1515T, and wherein the mutated recombinant fusion protein does not comprise a signal peptide.

In another preferred embodiment, the mutated recombinant fusion protein comprises a human ACE2 domain and an Fc domain of human IgG, wherein the human ACE2 domain in comparison to SEQ ID NO: 1 is mutated at one or more position(s), selected from the group consisting of H347, H507, R275, T373 and/or 1515, and wherein the Fc domain is an Fc domain of the human IgG 1 and is mutated at position N824, preferably wherein the mutation is N824G, and wherein the mutated recombinant fusion protein does not comprise a signal peptide.

In another preferred embodiment, the mutated recombinant fusion protein comprises a human ACE2 domain and an Fc domain of human IgG, wherein the human ACE2 domain in comparison to SEQ ID NO: 1 is mutated at one or more position(s), selected from the group consisting of H347, H507, R275, T373 and/or 1515, and in said mutated recombinant fusion protein the mutation at one or more position(s) is selected from the group consisting of H347A, H507L, R275L, T373F and/or 1515T, and wherein the Fc domain is an Fc domain of the human lgG1 and is mutated at position N824, preferably wherein the mutation is N824G, wherein the mutated recombinant fusion protein does not comprise a signal peptide.

In a preferred embodiment, the mutated recombinant fusion protein has a sequence as shown in any of SEQ ID NO: 5, 7, 9, or 11 ; in a more preferred embodiment, said mutated recombinant fusion protein does not comprise a signal peptide (SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16). In another embodiment, the mutated recombinant fusion protein can comprise the sequence of SEQ ID NO: 17. SEQ ID NO: 17 is, in comparison to SEQ ID NO: 1 , mutated at the following positions: H507, N824. In another embodiment, the mutated recombinant fusion protein can comprise the sequence of SEQ ID NO: 18. SEQ ID NO: 18 is, in comparison to SEQ ID NO: 1 , mutated at the following positions: T373, N824. In another embodiment, the mutated recombinant fusion protein can comprise the sequence of SEQ ID NO: 19. SEQ ID NO: 19 is, in comparison to SEQ ID NO: 1 , mutated at the following positions: T373, 1515, N824. In another embodiment, the mutated recombinant fusion protein can comprise the sequence of SEQ ID NO: 20. SEQ ID NO: 20 is, in comparison to SEQ ID NO: 1 , mutated at the following positions: H507, 1515, N824. In another embodiment, the mutated recombinant ACE2-Fc fusion protein has a sequence as shown in any one of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 20. In a further embodiment, said mutated recombinant fusion protein does not comprise a signal peptide (SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23; SEQ ID NO: 24).

The present invention also relates to a mutated recombinant fusion protein for use in a method of treating infection diseases caused by a coronavirus.

Furthermore, the present invention relates to a mutated recombinant fusion protein for use in a method of treating infection diseases caused by a virus capable of binding ACE2.

In a preferred embodiment, the coronavirus causing an infection disease is SARS- CoV-2 and the infection disease is COVID-19. In a further preferred embodiment, the coronavirus causing an infection disease is SARS-CoV-2 including any variants thereof.

In a preferred embodiment, the mutated recombinant fusion protein comprising a human ACE2 domain and an Fc domain of human IgG, wherein the human ACE2 domain in comparison to SEQ ID NO: 1 is mutated at one or more position(s), selected from the group consisting of H347, H507, R275, T373 and/or 1515, is for use in a method of treating infection diseases caused by a coronavirus, optionally, wherein the coronavirus is SARS-CoV-2 and wherein the infection disease is COVID- In a preferred embodiment, the mutated recombinant fusion protein comprising a human ACE2 domain and an Fc domain of human IgG, wherein the human ACE2 domain in comparison to SEQ ID NO: 1 is mutated at one or more position(s), selected from the group consisting of H347, H507, R275, T373 and/or 1515, wherein the mutation at one or more position(s) is selected from the group consisting of H347A, H507L, R275L, T373F and/or 1515T, is for use in a method of treating infection diseases caused by a coronavirus, optionally, wherein the coronavirus is SARS-CoV-2 and wherein the infection disease is COVID-19.

In a preferred embodiment, the mutated recombinant fusion protein comprising a human ACE2 domain and an Fc domain of human IgG, wherein the human ACE2 domain in comparison to SEQ ID NO: 1 is mutated at one or more position(s), selected from the group consisting of H347, H507, R275, T373 and/or 1515, and wherein the Fc domain is an Fc domain of the human IgG 1 and is mutated at position N824, preferably wherein the mutation is N824G, is for use in a method of treating infection diseases caused by a coronavirus, optionally, wherein the coronavirus is SARS-CoV-2 and wherein the infection disease is COVID-19.

In a preferred embodiment, the mutated recombinant fusion protein comprising a human ACE2 domain and an Fc domain of human IgG, wherein the human ACE2 domain in comparison to SEQ ID NO: 1 is mutated at one or more position(s), selected from the group consisting of H347, H507, R275, T373 and/or 1515, wherein the mutation at one or more position(s) is selected from the group consisting of H347A, H507L, R275L, T373F and/or 1515T, and wherein the Fc domain is an Fc domain of the human lgG1 and is mutated at position N824, preferably wherein the mutation is N824G, is for use in a method of treating infection diseases caused by a coronavirus, optionally, wherein the coronavirus is SARS-CoV-2 and wherein the infection disease is COVID-19.

In a preferred embodiment, the mutated recombinant fusion protein, wherein the Fc domain exhibits reduced effector functions, is for use in a method of treating infection diseases caused by a coronavirus, optionally, wherein the coronavirus is SARS-CoV- 2 and wherein the infection disease is COVID-19.

The present invention also relates to a mutated recombinant fusion protein having a sequence as shown in any of SEQ ID NO: 5, 7, 9, or 11 , for use in a method of treating infection diseases caused by a coronavirus, optionally, wherein the coronavirus is SARS-CoV-2 and wherein the infection disease is COVID-19.

In a preferred embodiment, the mutated recombinant fusion protein comprising a human ACE2 domain and an Fc domain of human IgG, wherein the human ACE2 domain in comparison to SEQ ID NO: 1 is mutated at one or more position(s), selected from the group consisting of H347, H507, R275, T373 and/or 1515, and wherein said mutated recombinant fusion protein does not comprise a signal peptide, is for use in a method of treating infection diseases caused by a coronavirus, optionally, wherein the coronavirus is SARS-CoV-2 and wherein the infection disease is COVID-19.

In a preferred embodiment, the mutated recombinant fusion protein comprising a human ACE2 domain and an Fc domain of human IgG, wherein the human ACE2 domain in comparison to SEQ ID NO: 1 is mutated at one or more position(s), selected from the group consisting of H347, H507, R275, T373 and/or 1515, wherein the mutation at one or more position(s) is selected from the group consisting of H347A, H507L, R275L, T373F and/or 1515T, and wherein said mutated recombinant fusion protein does not comprise a signal peptide, is for use in a method of treating infection diseases caused by a coronavirus, optionally, wherein the coronavirus is SARS-CoV-2 and wherein the infection disease is COVID-19.

In a preferred embodiment, the mutated recombinant fusion protein comprising a human ACE2 domain and an Fc domain of human IgG, wherein the human ACE2 domain in comparison to SEQ ID NO: 1 is mutated at one or more position(s), selected from the group consisting of H347, H507, R275, T373 and/or 1515, and wherein the Fc domain is an Fc domain of the human IgG 1 and is mutated at position N824, preferably wherein the mutation is N824G, and wherein said mutated recombinant fusion protein does not comprise a signal peptide, is for use in a method of treating infection diseases caused by a coronavirus, optionally, wherein the coronavirus is SARS-CoV-2 and wherein the infection disease is COVID-19.

In a preferred embodiment, the mutated recombinant fusion protein comprising a human ACE2 domain and an Fc domain of human IgG, wherein the human ACE2 domain in comparison to SEQ ID NO: 1 is mutated at one or more position(s), selected from the group consisting of H347, H507, R275, T373 and/or 1515, wherein the mutation at one or more position(s) is selected from the group consisting of H347A, H507L, R275L, T373F and/or 1515T, and wherein the Fc domain is an Fc domain of the human lgG1 and is mutated at position N824, preferably wherein the mutation is N824G, and wherein said mutated recombinant fusion protein does not comprise a signal peptide, is for use in a method of treating infection diseases caused by a coronavirus, optionally, wherein the coronavirus is SARS-CoV-2 and wherein the infection disease is COVID-19.

In a preferred embodiment, the mutated recombinant fusion protein, wherein the Fc domain exhibits reduced effector functions, and wherein said mutated recombinant fusion protein does not comprise a signal peptide, is for use in a method of treating infection diseases caused by a coronavirus, optionally, wherein the coronavirus is SARS-CoV-2 and wherein the infection disease is COVID-19.

In a preferred embodiment, the mutated recombinant fusion protein having a sequence as shown in any of SEQ ID NO: 5, 7, 9, or 11 , wherein said mutated recombinant fusion protein does not comprise a signal peptide, is for use in a method of treating infection diseases caused by a coronavirus, optionally, wherein the coronavirus is SARS-CoV-2 and wherein the infection disease is COVID-19.

In a preferred embodiment, the mutated recombinant fusion protein having a sequence as shown in any of SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, is for use in a method of treating infection diseases caused by a coronavirus, optionally, wherein the coronavirus is SARS-CoV-2 and wherein the infection disease is COVID-19.

Preferably, the mutated recombinant fusion protein having a sequence as shown in SEQ ID NO: 9 or 15 is used in a method of treating infection diseases caused by a coronavirus, optionally, wherein the coronavirus is SARS-CoV-2 and wherein the infection disease is COVID-19.

The disease Coronavirus-dependent disease 2019 (COVID-19) caused by the emerging virus SARS-CoV-2 is particularly influenced by the binding of the S-protein of SARS-CoV-2 to the membrane bound receptor ACE2. In other forms, the fusion protein described in the present invention can also be used for the treatment of other diseases, in particular in diseases caused by the pathogens SARS-CoV and MERS. The methods and uses of treatment employ administration of a mutated recombinant fusion peptide to a subject having an infection disease caused by a coronavirus. Forms of administration for the infectious disease to be treated caused by coronavirus comprise both, local and systemic administration and include, but are not limited to those: intravenous administration, buccal administration, endobronchial administration, inhalative administration, intranasal administration, intraocular administration, intrapulmonal administration, oral administration.

The invention is further described with the help of the following examples and figures, which are intended to illustrate, but not to limit the invention.

EXAMPLES

EXAMPLE 1

Measurement of recombinantly expressed angiotensin converting enzyme 2 (ACE2) activity

In this example, for the detection of ACE2 activity, purified recombinant enzyme was used and the activity level was measured in a fluorescent microplate reader.

Recombinant enzyme from supernatant of fed-batch harvest broth was diluted with its medium (CD FortiCHO medium, Gibco Cat.No: A1148301 ) by 4-20 fold (preparations with low enzymatic activity: Seq. ID NO: 03, 05, 07, 09, 11 ) or by 1 GO- 72, 900-fold (preparations with high enzymatic activity: Seq. ID No: 01 ).

ACE2 activity measurement was performed using a specific quenched fluorescent substrate as it was previously shown in 2016 by Uri et al. (J. Renin-Angiotensin- Aldosterone Syst.; 2016; 17(4): 1470320316668435) described, performed with some modifications: The reaction mixture (200 pl) contained 10 pl diluted enzyme preparation, 25 pM ACE2-specific fluorescent substrate (7-methoxycoumarin-4- yl)acetyl-Ala-Pro-Lys(2,4-dinitrophenyl)-OH [Mca-APK(Dnp)] (custom synthesized by using Peptide 2.0 Software (Peptide 2.0 Inc. Chantilly, VA, US) in a buffer composed of 500 mM NaCI, 100 pM ZnC , 75 mM TRIS HCI, pH 6.5. Activity was measured in black 96 well plates in a fluorescent microplate reader (Clariostar). ACE2 activity was monitored by measuring the increase in fluorescence (excitation wavelength: 320 nm, emission wavelength: 405 nm) in a kinetic assay. The increase in fluorescence was plotted as a function of reaction time and fitted with a linear regression.

ACE2 activity was calculated by the equation: ACE2 activity=S*D, where S is the rate of increase in fluorescence intensity (Slope of the linear regression) and D is the dilution of the sample. Slope values were accepted when fits resulted in r² >0.95. The unit of the activity is an arbitrary unit (AU), which is proportional with the substrate conversion.

Nonspecific activity was determined by the addition of the ACE2 specific inhibitor MLN-4760 (Merck) at a final concentration of 1 pM. The non-specific activity was less than 10% and generally omitted from the evaluations.

Results

The results of the measurement of enzymatic activity of the ACE2 domain of mutated recombinant fusion proteins of the invention are summarized in Table 1 and in Fig. 10. According to these results, advantageously it has been found that mutations in the ACE2 domain as disclosed in the present invention reduce ACE2 activity in a great extent. As for mutant variant Fc+3x, it can be seen in Table 1 that the specific activity (average) of said ACE2-Fc mutant variant is 0.03% of the native form.

In Table 1 the form of the results is as follows: the values are given in numericals written according to the English grammatical rules (using decimal point). Under the values given in English (e.g. 5.795 or 8155722), the output data in the original language, i.e. in Hungarian, are also given in brackets (i.e. with numericals written according to the Hungarian grammatical rules - using decimal comma, and using point for separating groups of hundreds within a number; e.g.: HU: 5,795 or HU: 8.155.722).

Table 1 - Specific Activity and Titer of the Mutants - begins on next page

The average specific activity (AU/s/ml) of mutant variants tested in this measurement are also shown by a bar graph in Fig. 10. Especially, Fc+3x (mutant variant with mutations R275L, T373F, H507L, N824G), Fc+4x (mutant variant with mutations R275L, H347A, H507L, 1515T, N824G) and Fc+5x (mutant variant with mutations R275L, H347A, T373F, H507L, 1515T, N824G) exhibit a greatly reduced ACE2 activity.

EXAMPLE 2

Virus Neutralization Assay

1 . Cells and Virus

Vero E6 (ATCC® CRL-1586™) cells were maintained in DMEM (Lonza) supplemented with 10% heat inactivated fetal bovine serum (FBS; Gibco) and 1 % Penicillin/ Streptomycin (Lonza). Cells were kept in a 37°C, 5% CO2 incubator. hCoV- 19/Hungary/SRC_isolate_2/2020 (Accession ID: EPI_ISL_483637) was used during experiments, originated form anonym human patient.

2. Virus Propagation

Patient sample was inoculated on VeroE6 cells at 37°C, 5% CO2 incubator. The absorption period was 30 minutes with shaking every 10 minutes. Cells were observed for cytopathic effect (CPE) every 24 hours. The virus was passaged three times before collected, clarified, aliquoted and stored at -80°C.

3. TCID50 assay hCoV-19/Hungary/SRC_isolate_2/2020 viral stock was titrated using the TCID50 method. Briefly, serial 10-fold dilutions of hCoV-19/Hungary/SRC_isolate_2/2020 supernatant were inoculated (50 pl) on 80-90% confluent VeroE6 cells (40000 cells/well) in 96-well plates. Viral adsorption was allowed for 1 hour at 37°C. After washing cells with DMEM three times, cells were incubated for 3 days at 37°C in DMEM supplemented with 2% FBS (Gibco). The percentage of infected wells was observed with microscopy and recorded for each virus dilution then results were used to mathematically calculate a TCID50 result with the Spearman-Karber method. From the results obtained, 100TCID50 was calculated and used in further experiments. 4. Neutralization assay

For demonstrating the neutralization effect of the fusion protein, Vero E6 cells were incubated with purified recombinant ACE2 fusion protein and diluted virus.

Vero E6 cells were seeded into 96 well plates at 80-90% confluency (40000 cells/well) in DMEM supplemented with 10% heat-inactivated fetal bovine serum (Gibco US origin). Serial dilutions of the ACE2-Fc fusion protein (Seq-ID: 01 ) in DMEM from 1 :10 to 1 :640 were prepared. Virus stock was diluted to obtain 100 of the fifty-percent tissue culture infective dose (TCID50). For the infection 50 pl of an appropriate dilution of the ACE2-Fc fusion protein together with 50 pl of the 100TCID50 was then added to VeroE6 cells. The plates were then incubated for 1 hour at 37°C and several times agitated.

The supernatant was then gently removed and replaced with 100 pl maintaining medium (DMEM: 2% FBS, 1 % Pen I Strep) and incubated for three days at 37°C. Afterwards, the virus neutralizing effect of the ACE2-Fc fusion protein was evaluated by light microscopy. The 100 pl supernatant was removed for nucleic acid extraction (Monarch, NEB). Droplet Digital PCR (ddPCR) (BIO-RAD) was performed from supernatant’s nucleic acid.

5. Droplet Digital PCR

QX200 Droplet Digital PCR system (Bio-Rad, CA, USA) was used to determine virus copy number decrease triggered by ACE2-Fc fusion protein from supernatants. One- Step RT-ddPCR advanced kit for probes (Bio-Rad, CA, USA) was used in our experiments. The RT-ddPCR reaction mixture consisted of 5 pl of a ddPCR Supermix, 2 pl reverse transcriptase, 1 pl 300 mM DTT, 900 nM CoV specific primers and 250 nM probe, 1 pl of sample nucleic acid solution and nuclease-free H2O in a final volume of 22 pl. The entire reaction mixture was loaded into a disposable plastic cartridge (Bio-Rad, CA, USA) together with 70 pl of droplet generation oil for probes (Bio-Rad, CA, USA) and placed in the QX200 Droplet Generator (Bio-Rad, CA, USA). After processing, the droplets generated from each sample were transferred to a 96- well PCR plate (Bio-Rad CA, USA) and heat-sealed with PX1TM PCR Plate Sealer (Bio-Rad, CA, USA). PCR amplification was carried out on a C1000 TouchTM Thermal Cycler with 96-Deep Well Reaction Module (Bio-Rad, CA, USA) using a thermal profile of beginning at reverse transcription. After amplification, the plate was loaded on the QX200 Droplet Reader (Bio-Rad, CA, USA) and the droplets from each well of the plate were read automatically. Positive droplets, containing amplification products, were partitioned from negative droplets by applying a fluorescence amplitude threshold in QuantaSoftTM analysis software (Bio-Rad, CA, USA). Quantification of the target molecule was presented as the number of copies per pl of the PCR mix.

Practical implementation

1. Using a 96-well plate (VeroE6); VeroE6 cells: 40,000 cells I well. Cells should be 80-90% confluent at the start of treatment.

2. Dilution of ACE2-Fc material, “neutralization”, infection of the cells; Dilution of ACE2-Fc material: half dilution from 1 :10 to 1 : 640 with a platen channel pipette, 5 replicates of each dilution:

■ Concentrated ACE2-Fc material

■ 1 :10 - 495 pl DMEM + 55 pl ACE2-Fc material (measure every 100 pl, of which take 50 pl)

■ 1 :20 - 50 pl DMEM + 50 pl 1 : 10 ACE2-Fc material

■ 1 :40 - 50 pl DMEM + 50 pl 1 :20 ACE2-Fc material

■ 1 :80 - 50 pl DMEM + 50 pl 1 :40 ACE2-Fc material

■ 1 : 160 - 50 pl DMEM + 50 pl 1 :80 ACE2-Fc material

■ 1 :320 - 50 pl DMEM + 50 pl 1 :160 ACE2-Fc material

■ 1 :640 - 50 pl DMEM + 50 pl 1 :320 ACE2-Fc material

3. Add 100 TCID50 viruses (dilute virus from stock):

There are 12,720,000 TCID50s in 1000 pl of virus stock (we know from a preliminary experiment). 100 TCID50 is in ~0.079 pl. For the whole 96-well plate, you need ~ 1.78 pl from the virus stock. Infection requires 50 pl I well diluted in DMEM. For 1 plate: 5000 pl DMEM + £ ~ 1 .78 pl virus.

4. Neutralization

50 pl ACE2-Fc material (at appropriate dilution) I well + 50 pl 100 TCID50 virus I well; Incubation: 1 hour at 37 °C (neutralization). Meanwhile, move every 10 minutes. 5. Infection of cells: Measurement of neutralized virus (100 l I well) incubated for 1 hour on cells.

6. Incubation: 30 min at 37 °C. Meanwhile, move every 10 minutes. After incubation, cells were aspirated and 100 pl of maintainer (DMEM: 2% FBS, 1 % Pen I Strep) was added. Incubation 3 days 37 °C

7. Microscopic reading of the result (whether the cells were infected in the given well). Nucleic acid extraction from supernatant for ddP.

Results

The results are shown by Figure 2. Cytopathic effects of the coronavirus (demonstrated by the destruction of the cell monolayer) are only apparent at 1 :160 dilution of ACE2-Fc, and are marked at 1 :640. The experiments show a clear virus neutralization effect of the recombinant protein.

On the basis of the results of a preliminary virus neutralization experiment carried out on ACE2-Fc mutant variant with mutations R275L, T373F, H507L and N824G in comparison to the ACE2 domain and IgG 1 -Fc region of SEQ ID NO: 1 , respectively, a virus neutralization effect of said ACE2-Fc mutant variant is expected.

EXAMPLE 3

Preparation of the mutated recombinant ACE2-Fc fusion proteins of the invention

Materials and methods

Construction of ACE2-Fc expression plasmids

Structurally, the recombinantly produced ACE2-Fc molecule contains the extracellular domain of ACE2 protein fused with hinge and Fc region of lgG1. Nucleotide sequences of mutant forms disclosed in the present application (including mutants listed in the Sequence Listing) were designed and codon-optimized for CHO cells, where the submitted protein sequence was supplemented with the signal peptides of IgG heavy chain, an undefined 5’ UTR sequence, Kozak sequence, and the restriction recognition sites fit for the conventional T4 ligation based cloning work. Artificial nucleic acid sequence was synthesized by solid phase synthesis through the service of Thermo fisher, GeneArt.

Construction of the expression vector was achieved through digestion both of the vector and the fragments with the same pair of restriction enzymes and then the ligation of the fragments into the correct positions of the expression vector using T4 ligase enzyme.

Sequence verification of the final plasmids was performed using complete restriction analysis and DNA sequencing of the coding subunits to ensure the correctness of the construct. Before transfection, plasmids were linearized with controlled restriction digestion followed by ethanol precipitation providing sterility.

Cultivation of Chinese hamster ovary (CHO) cell lines

CHO host cell line was thawed and passaged to recover the viability of the cell line. Cultivation of the host cell line was performed in CD FortiCHO (Thermo Fisher) and in BalanCD CHO Growth A (Irvine Scientific) supplemented with 4 mM L-glutamine (Thermo Fisher) in shake flasks (37°C, 85% humidity, 5% CO2, in a shake flask incubator (Kuhner)). Transfection of ACE2-Fc coding DNA constructs into CHO cells was performed by electroporation applying 4D Nucleofector system (Lonza) and its reagents (SF Cell Line 4D-Nucleofector, Lonza) as described by the manufacturer. Selection of the transfected cells was performed until the viability of the pools reached 90% meanwhile selection medium of the pools were changed in every 3-5 days. Selection medium contained 50 pM methionine-sulfoxide (MSX) without L-glutamine supplementation. Single-cell isolation from the heterogenic pools were performed by limiting dilution technique or using VIPS device (Solentim) onto 96 well plates (Corning).

Batch cultivation of the transfected pools and expanded monoclones was performed in selection medium in shake flasks as detailed before, while fed-batch cultivation of the cell lines was performed in unsupplemented CD FortiCHO or BalanCD CHO Growth A media with 5 VA/% feed (Efficient Feed C+, Thermo Fisher, Feed 4, Irvine Scientific, respectively) addition every two days from day 3 and D-glucose supplementation. Cell free supernatant of the cultures were used for expressed ACE2-Fc protein characterization.

Protein Purification from supernatant

Cell culture was clarified by centrifugation. The supernatant was loaded onto an affinity chromatography column containing protein A beads. The column was washed with 0.1 M sodium citrate pH 6.2 buffer and 1.5 M L-arginine, 0.1 M citric acid pH 4.2 buffer for ACE2-Fc elution.

The elution sample was instantly neutralized with addition of 2 M Tris base. The purified ACE2-Fc fractions were analyzed using size exclusion HPLC and UV spectrometry to continue the purification of fractions with the best monomer ratio. The selected fractions were pooled and concentrated on a LIF/DF system.

As a second purification step the concentrated pool was loaded on size exclusion column and eluted with 50 mM Na2PO4, 100 mM NaCI pH 6.8 to separate aggregates. The separated ACE2-Fc fractions were analyzed using size exclusion HPLC and UV spectrometry.

After pooling of fractions with the best quality, the selected fractions were pooled and concentrated again on UF/DF system.

Final ACE2-Fc concentration was 1 .0 mg/ml in the final composition.

EXAMPLE 4

Analytical characterization

The BLI method

Direct measurement of biomolecular interactions plays an important role in biotherapeutic drug discovery and development. The Octet platform is a label-free analytical technology by which we can obtain accurate information about rate of biomolecular complex formation and complex stability. BLI is an optical analytical technique that measures interference patterns between waves of light. White light is directed down the fiber-optic biosensor towards two interfaces separated by a thin layer at the tip of the fiber: a biocompatible layer on the surface of the tip, and an internal reference layer. Light reflects from each of the two layers, and the reflected beams interfere constructively or destructively at different wavelengths in the spectrum. When the tip of a biosensor is dipped into a sample, target molecules bind to the 2-dimensional coated surface. This binding forms a molecular layer that increases in thickness as more target molecules bind to the surface. As the thickness at the tip increases, creating a shift in the interference patterns of the reflected light. The spectral pattern of the reflected light therefore changes as a function of the optical thickness of the molecular layer. This spectral shift is monitored at the detector and reported on a sensorgram as a change in wavelength (nm shift). Monitoring the interference pattern in real time provides kinetic data on molecular interactions.

In the BLI experiments the molecule which binds to the sensor’s surface called ligand and the analyte is the molecule, which binds to the ligand.

BLI assay

Assay buffer (20 mM HEPES, 150 mM NaCI, 0.02% (v/v) Tween20) was used to dilute the samples and references and to neutralize the biosensors. Glycine pH 1.5 (Cytiva) was used to regenerate the biosensors. We used Protein A biosensors (Sartorius) to attach the ACE2-Fc molecule to the biosensor’s surface. Then the analyte SARS-CoV-2 (COVID-19) S1 protein, His Tag (Aero Biosystems) was bound to the immobilized ACE2-Fc, which was the association phase. After 150 sec of association the next step was the dissociation for additional 150 sec. After evaluation the KD, kdis, k_a values of ACE2-Fc were determined (by methods well-known in the art of enzyme kinetics) - S1 protein interaction, hence the binding strength between these molecules.

Steps of one cycle are shown in Table 2. Each sample means a new cycle. Table 2 - steps of one cycle of a BLI assay

*After reach the appropriate wavelength threshold (0.5 nm) it continues the next step. Results

The results of the BLI assay carried out on native ACE2-Fc fusion protein and on 3x mutant variant (mutant variant having mutations R275L, T373F, H507L and N824G in comparison to the ACE2 domain and lgG1 -Fc region of SEQ ID NO: 1 , respectively) are shown in Table 3: Table 3

Representative sensorgram of the native ACE2-Fc fusion protein is shown in Figure 11 , representative sensorgram of the 3x mutant variant is shown in Figure 12. Furthermore, Figure 13 shows the respective equilibrium binding (KD) values on a diagram. The results clearly show the advantageous characteristics of the interaction between 3x mutant variant and S1 protein in comparison to the interaction between the native ACE2-Fc and S1 protein.

Claims

39

CLAIMS A mutated recombinant fusion protein comprising a human ACE2 domain and a human IgG-Fc domain, wherein in comparison to the ACE2 domain of SEQ ID NO: 1 , the ACE2 domain of the mutated recombinant fusion protein comprises mutation

(i) at positions R275, T373, H507; or

(ii) at positions R275, H347, T373, H507, 1515; or

(iii) at positions R275, H347, H507, 1515; or

(iv) at position 1515; or

(v) at positions T373, 1515; or

(vi) at position H507; or

(vii) at positions H507, 1515; or

(viii) at position T373. The mutated recombinant fusion protein according to claim 1 , wherein a mutation

(i) at position R275 is an exchange of R for L;

(ii) at position H347 is an exchange of H for G, V, A, L or I;

(iii) at position T373 is an exchange of T for F, Y or W;

(iv) at position H507 is an exchange of H for G, V, A, L or I; and/or

(v) at position 1515 is an exchange of I for T or S. The mutated recombinant fusion protein according to claim 2, wherein a mutation

(i) at position R275 is an exchange of R for L;

(ii) at position H347 is an exchange of H for A;

(iii) at position T373 is an exchange of T for F;

(iv) at position H507 is an exchange of H for L; and/or

(v) at position 1515 is an exchange of I for T. The mutated recombinant fusion protein according to any one of claims 1-3, wherein the human IgG-Fc domain of the mutated recombinant fusion protein is a human lgG1-Fc domain. 40 The mutated recombinant fusion protein according to claim 4, wherein in comparison to the lgG1 -Fc region of SEQ ID NO: 1 , the lgG1-Fc domain of the mutated recombinant fusion protein comprises mutation at position N824. The mutated recombinant fusion protein according to claim 5, wherein the mutation at position N824 is an exchange of N for G. The mutated recombinant fusion protein according to claim 6, wherein the mutated recombinant fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 21 , 22, 23 and 24. The mutated recombinant fusion protein according to claim 6, wherein the mutated recombinant fusion protein has a sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 21 , 22, 23 and 24. The mutated recombinant fusion protein according to claim 6, wherein the mutated recombinant fusion protein has a sequence as shown by SEQ ID NO: 15. The mutated recombinant fusion protein according to any one of claims 1 -6, wherein the mutated recombinant fusion protein further comprises a signal peptide. The mutated recombinant fusion protein according to claim 10, wherein the mutated recombinant fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9, 11 , 17, 18, 19 and 20. The mutated recombinant fusion protein according to claim 10, wherein the mutated recombinant fusion protein has a sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9, 11 , 17, 18, 19 and 20. The mutated recombinant fusion protein according to claim 10, wherein the mutated recombinant fusion protein has a sequence as shown by SEQ ID NO: 9. 41 The mutated recombinant fusion protein according to any one of claims 1 -13 for use in a method of treating an infection disease caused by a coronavirus capable of binding ACE2. The mutated recombinant fusion protein for use according to claim 14, wherein the coronavirus capable of binding ACE2 is SARS-CoV-2. The mutated recombinant fusion protein for use according to claim 14 or 15, wherein the infection disease is COVID-19. A polynucleotide encoding a mutated recombinant fusion protein, wherein the polynucleotide comprises a sequence as shown in any one of SEQ ID NOs: 6, 8, 10, 12. A vector comprising the polynucleotide according to claim 17. A host cell comprising the vector according to claim 18.