Isolated polypeptides comprising an ace2 moiety and a stability-increasing polypeptide moiety

Abstract

The invention relates to an isolated polypeptide comprising an ACE2 moiety and a stability-increasing polypeptide moiety, wherein the ACE2 moiety consists of amino acid residues 18 to 728 of the ACE2 protein ( SEQ ID NO : 1; UniProt Q9BYF1, ACE2_HUMAN) or a biologically active ACE2 mutant or ACE2 variant polypeptide thereof; and wherein the stability-increasing polypeptide moiety increases the stability of the ACE2 moiety in the course of administration to a patient. The polypeptide according to the present invention shows an increased biological activity and stability compared to the ACE2 polypeptides available in the prior art.

Description

[0001] I solated polypeptides comprising an ACE2 moiety and a stabilityincreasing polypeptide moiety

[0002] The present invention relates to isolated polypeptides comprising an ACE2 moiety and a stability-increasing polypeptide moiety, nucleic acids encoding such polypeptides and methods for producing and using such polypeptides .

[0003] ACE2 (Angiotensin-Converting Enzyme 2 ; UniProt : Q9BYF1 ACE2_HUMAN) is a protein found on the surface of cells in various tissues throughout the human body . It plays a crucial role in the regulation of the renin-angiotensin-aldosterone system (RAAS ) , which controls blood pressure , fluid balance , and electrolyte homeostasis . ACE2 is primarily known for its role in counterbalancing the ef fects of another enzyme called ACE (Angiotensin-Converting Enzyme ) . ACE2 is an 805-amino acid carboxypeptidase that removes a single amino acid from the C terminus of its substrates .

[0004] ACE2 has a central role in the regulation of blood pressure : ACE2 is involved in the conversion of angiotensin I I , a potent vasoconstrictor, into angiotensin- ( 1-7 ) , which has vasodilatory and anti-inflammatory properties . This action helps to regulate blood pressure . ACE2 counteracts ACE (Angiotensin-Converting Enzyme ) which converts angiotensin I to angiotensin I I , which can raise blood pressure . ACE2 counterbalances this by breaking down angiotensin I I into its less harmful form . Other physiological roles of ACE2 include the involvement in the regulation of fluid and electrolyte balance , as well as in protecting various organs and tissues from damage during pathological conditions . Plasma ACE2 activity is usually low in healthy individuals but patients with cardiovascular risk factors and / or disease have increased plasma ACE2 activity, suggesting that it may be an important biomarker of endothelial dys function and atherosclerosis .

[0005] ACE2 is a zinc-containing metalloenzyme located on the surface of intestinal enterocytes , renal tubular cells and other epithelial cells . It contains an N-terminal metallopeptidase domain that contains the HEXXH zinc-binding moti f at the catalytic site and shares homology with ACE and a C-terminal collectrin domain that shows homology to renal amino acid transporters . ACE2 is a single-pass type I membrane protein, with its enzymatically active luminal ectodomain being exposed on the surface of cells in the intestines and other tissues. The extracellular domain of ACE2 can be cleaved from the transmembrane domain by sheddases, and the resulting soluble protein is released into the bloodstream.

[0006] ACE2 is also used by SARS-CoV(-l) and SARS-CoV-2 for host cell entry. Both the membrane-bound and soluble forms of ACE2 harbour the binding site for the SARS-CoV-2 spike protein which resides in the extracellular domain (Jackson et al., Nat Rev Mol Cell Biol. 2022; 23:3-20. doi: 10.1038 / s41580-021-00418-x .2022 ) .

[0007] The repeated emergence of new pathogenic viruses in recent years highlights the need for the development of novel antiviral strategies to foster pandemic preparedness. This is best exemplified by the sudden appearance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) , the etiologic agent of COVID- 19. SARS-CoV-2 is an RNA virus that has its reservoirs in animals, most likely bats, which eventually spread to other animals and humans. Being an RNA virus, SARS-CoV-2 is prone to accumulate mutations in its genome, which resulted in the emergence of several virus variants that show increased infectivity and were driving recent infection waves.

[0008] Since 2019, the COVID-19 pandemic has claimed more than 6 million deaths worldwide, despite the rapid development of vaccines and immunity as consequence of natural infection. SARS- CoV-2 infects the nasal cavity where it replicates in epithelial cells, while in the cells of the lower respiratory tract, including the lungs, the ability to infect and replicate is lower. However, eventually also cells of the lower respiratory tract can get infected (e.g., in patients with chronic diseases) , which may result in the development of severe pneumonia and systemic disease. The results from several studies show that the virus usually takes a firm foothold first in the nasal cavity, but in some instances, the virus may infect the lungs and can cause severe disease including fatal pneumonia.

[0009] Symptoms of COVID-19 can range from mild illness to pneumonia, renal dysfunction, respiratory and multi-organ failure. The major socio-economic threat of the COVID-19 pandemic is caused by cases with severe illness requiring hospitalization and eventually intensive care treatment. Severe COVID-19 results from progression of local infection in the upper respiratory tract to pneumonia and further systemic effects. Progression to pneumonia leads to aggravation of symptoms , which makes treatment in hospitals necessary .

[0010] In view of the continuing threat of SARS-CoV-2 to human health, there is an urgent need for preventive and therapeutic antiviral therapies . In particular, there is a need for medicines that prevent virus replication and thus disease progression from mild local disease in the upper respiratory tract to severe disease with pneumonia requiring hospitali zation and intensive care .

[0011] SARS-CoV-2 is a Beta-coronavirus which has four maj or structural proteins , speci fically the surface-anchored trimeric spike ( S ) , envelope (E ) , membrane (M) and nucleocapsid (N) proteins . The N protein compacts the RNA genome , while the other three structural proteins are components of the viral envelope . The S protein is responsible for allowing the virus to attach and fuse to the membrane of a host cell .

[0012] SARS-CoV-2 enters a host cell by interacting with the ACE2 receptor using its spike glycoprotein . The binding results in the activation of several proteases and to the eventual cleavage of the spike protein, which finally leads to fusion of virus envelope and cell membrane leading to cell infection . The SARS-CoV-2 spike protein is a 1273 amino acid glycoprotein, which can be further subdivided into the S I domain, containing the receptor binding domain (RBD) , and the S2 domain . Viral attachment to the ACE2 receptor is implemented through binding of the RBD to speci fic regions of the extracellular peptidase domain of ACE2 with an equilibrium dissociation constant (KD) of ~ 15nM . Upon interaction, the S2 domain is exposed to proteolytic cleavage which is crucial for subsequent fusion of the viral envelope with the cell membrane allowing entry of the virus and infection of the cell ( s . Figure 3 of Jackson et al . , 2022 ) . Alternatively, SARS-CoV-2 might also infect cells by endocytotic uptake after binding to the ACE2 receptor on the cell surface .

[0013] This is why ACE2 or fragments and derivatives thereof have been used or suggested for the treatment or prevention of diseases , especially for prevention of COVID- 19 and for preventing SARS-CoV- 2 from entering human cells or from causing adverse symptoms or diseases in humans . For example , WO 2021 / 183717 Al , WO 2021 / 207207 A2 , and WO 2022 / 165246 Al disclose de novo proteins , termed "ACE2 protein decoys" that bind to the coronavirus spike protein of SARS- CoV and SARS-CoV-2 . US 2023 / 183668 Al discloses non-naturally occurring ACE2 proteins and their use in coronavirus infection. Svilenov et al. (Research Square (2021) : 4, 6-7, 11-15) report the picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein. US 2024 / 043821 Al discloses an ACE2 polypeptide lacking an N-linked glycosylation or comprising a truncated N-linked glycosylation, e.g., at an amino acid corresponding to Asn90 and / or Asn322. US 2023 / 257726 Al discloses ACE2 polypeptides having improved binding affinity for the SARS-CoV-2 spike protein receptor binding domain relative to wild-type ACE2. Ferrari et. al. (J. Virol. 95 (19) (2021) ) report the characterization of a novel ACE2-based therapeutic with enhanced rather than reduced activity against SARS-CoV-2 variants. WO 2024 / 068777 Al relates to modified ACE2 proteins having increased binding affinity for the SARS-CoV-2 spike protein. WO 2021 / 236957 A2 discloses non-naturally occurring ACE2 muteins. WO 2017 / 180587 A2 relates to regulatable biocircuit systems. WO 2022 / 090469 A2 , WO 2022 / 184854 A2 and WO 2023 / 006935 A2 disclose ACE2 Fc fusion proteins, e.g. with IgG or IgM Fc .

[0014] The proteins disclosed in the prior art have been described as being useful for inhibiting or neutralizing the activity of the virus, e.g. by blocking binding of the virus to its host cell receptor and for preventing the entry of the coronavirus into host cells. These ACE2 variants or ACE2 mutants function by inhibiting the cell-to-cell transmission of the virus. Such polypeptides are therefore useful in preventing, treating or ameliorating at least one symptom of coronavirus infection in a subject.

[0015] The ACE2 decoy polypeptides disclosed are usually difficult to be produced in expression platforms of interest due to many reasons. For example, the polypeptides can undergo post- translational modifications which may cause adverse reactions in humans, e.g. due to inappropriate glycosylation.

[0016] It is therefore an object of the present invention to provide such ACE2 decoy polypeptides with improved features concerning producibility in various expression platforms but without being impeded with respect to their ability to block coronavirus infections .

[0017] Therefore, the present invention provides an isolated polypeptide comprising an ACE2 moiety and a stability-increasing polypeptide moiety, wherein the ACE2 moiety comprises or consists of amino acid (aa) residues 18 to 728 of the ACE2 protein (SEQ ID N0:2; aa 18 to 728 of UniProt Q9BYF1, ACE2_HUMAN (SEQ ID NO : 1 ) or a biologically active ACE2 mutant or ACE2 variant polypeptide thereof; and wherein the stability-increasing polypeptide moiety increases the stability of the ACE2 moiety in the course of administration to a patient, especially in the course of mucosal administration to such patient. Preferably, the stabilityincreasing polypeptide has also the capability to increase the stability of the ACE2 moiety during storage. It is further preferred that the stability-increasing polypeptide has also the capability to extend the half-life of ACE2 in human plasma. The polypeptide according to the present invention therefore shows an increased biological activity and stability compared to the ACE2 polypeptides available in the prior art.

[0018] The ACE2 moiety may either comprise the wild type sequence of human ACE2 (SEQ ID NO : 1 ; UniProt Q9BYF1, ACE2_HUMAN) or an appropriate ACE2 variant or ACE2 mutant polypeptide which is able to inhibit coronavirus infections to at least the same extent as the native ACE2 sequence (SEQ ID NO : 1 ; UniProt Q9BYF1, ACE2_HUMAN) .

[0019] Native human ACE2 sequence (UniProt Q9BYF1; SEQ ID NO: 1) : MSSSSWLLLS LVAVTAAQST IEEQAKTFLD KFNHEAEDLF YQSSLASWNY NTNITEENVQ NMNNAGDKWS AFLKEQSTLA QMYPLQEIQN LTVKLQLQAL QQNGSSVLSE DKSKRLNTIL NTMSTIYSTG KVCNPDNPQE CLLLEPGLNE IMANSLDYNE RLWAWESWRS EVGKQLRPLY EEYWLKNEM ARANHYEDYG DYWRGDYEVN GVDGYDYSRG QLIEDVEHTF EEIKPLYEHL HAYVRAKLMN AYPSYISPIG CLPAHLLGDM WGRFWTNLYS LTVPFGQKPN IDVTDAMVDQ AWDAQRIFKE AEKFFVSVGL PNMTQGFWEN SMLTDPGNVQ KAVCHPTAWD LGKGDFRILM CTKVTMDDFL TAHHEMGHIQ YDMAYAAQPF LLRNGANEGF HEAVGEIMSL SAATPKHLKS IGLLSPDFQE DNETEINFLL KQALTIVGTL PFTYMLEKWR WMVFKGEIPK DQWMKKWWEM KREIVGWEP VPHDETYCDP ASLFHVSNDY SFIRYYTRTL YQFQFQEALC QAAKHEGPLH KCDISNSTEA GQKLFNMLRL GKSEPWTLAL ENWGAKNMN VRPLLNYFEP LFTWLKDQNK NSFVGWSTDW SPYADQSIKV RISLKSALGD KAYEWNDNEM YLFRSSVAYA MRQYFLKVKN QMILFGEEDV RVANLKPRIS FNFFVTAPKN VSDIIPRTEV EKAIRMSRSR INDAFRLNDN SLEFLGIQPT LGPPNQPPVS IWLIVFGWM GVIWGIVIL IFTGIRDRKK KNKARSGENP YASIDISKGE NNPGFQNTDD VQTSF

[0020] AA 18 to 728 of the native human ACE2 sequence (SEQ ID N0:2) are :

[0021] QST IEEQAKTFLD KFNHEAEDLF YQSSLASWNY NTNITEENVQ NMNNAGDKWS AFLKEQSTLA QMYPLQEIQN LTVKLQLQAL QQNGSSVLSE DKSKRLNTIL NTMSTIYSTG KVCNPDNPQE CLLLEPGLNE IMANSLDYNE RLWAWESWRS EVGKQLRPLY EEYWLKNEM ARANHYEDYG DYWRGDYEVN GVDGYDYSRG QLIEDVEHTF EEIKPLYEHL HAYVRAKLMN AYPSYISPIG CLPAHLLGDM WGRFWTNLYS LTVPFGQKPN IDVTDAMVDQ

[0022] AWDAQRIFKE AEKFFVSVGL PNMTQGFWEN SMLTDPGNVQ KAVCHPTAWD LGKGDFRILM

[0023] CTKVTMDDFL TAHHEMGHIQ YDMAYAAQPF LLRNGANEGF HEAVGEIMSL SAATPKHLKS

[0024] IGLLSPDFQE DNETEINFLL KQALTIVGTL PFTYMLEKWR WMVFKGEIPK DQWMKKWWEM

[0025] KREIVGWEP VPHDETYCDP ASLFHVSNDY SFIRYYTRTL YQFQFQEALC QAAKHEGPLH

[0026] KCDISNSTEA GQKLFNMLRL GKSEPWTLAL ENWGAKNMN VRPLLNYFEP LFTWLKDQNK

[0027] NSFVGWSTDW SPYADQSIKV RISLKSALGD KAYEWNDNEM YLFRSSVAYA MRQYFLKVKN

[0028] QMILFGEEDV RVANLKPRIS FNFFVTAPKN VSDIIPRTEV EKAIRMSRSR INDAFRLNDN

[0029] SLEFLGIQ

[0030] Examples of such ACE2 variants or ACE2 mutants are known in the prior art. An "ACE2 variant" according to the present invention is a naturally occurring variant of the canonical sequence UniProt Q9BYF1, ACE2_HUMAN (SEQ ID NO : 1 ) . An "ACE2 mutant" according to the present invention is an artificial (non-naturally occurring) mutant of the canonical sequence UniProt Q9BYF1, ACE2_HUMAN (SEQ ID NO:1) which differs from the primary sequence of the native ACE2 sequence by exchanges of one or more amino acid residues, deletions of one or more amino acid residues; insertions of one or more amino acid residues; or combinations thereof. Suitable ACE2 variants or ACE2 mutants are specifically disclosed in WO 2021 / 183717 Al, WO 2021 / 207207 A2 , WO 2022 / 165246 Al, US 2023 / 183668 Al, Svilenov et al. (Research Square (2021) : 4, 6-7, 11-15) , US 2024 / 043821 Al, US 2023 / 257726 Al, Ferrari et. al. (J. Virol. 95 (19) (2021) ) , WO 2024 / 068777 Al, WO 2021 / 236957 A2 , WO 2017 / 180587 A2 , WO 2022 / 090469 A2 , WO 2022 / 184854 A2 and WO 2023 / 006935 A2 (some of which already disclose the combination of such an ACE2 moiety and a stability-increasing polypeptide moiety, such as in immunoglobulin) to the extent that these ACE2 variants or ACE2 mutants exhibit the functionality as required by the present invention, especially that the ACE2 variant or ACE2 mutant binds to the spike protein. Specifically preferred embodiments of

[0031] For both, the ACE2 variant and the ACE2 mutant according to the present invention, it is therefore essential that their sequence within the part defined by aa 18 to 728 is functional with respect to binding to the SARS-CoV-2 spike protein which means that the ACE2 variant binds to this spike protein with an affinity comparable to the wild type, i.e. corresponding to a KDof about 10 to 20 nM as measured by surface plasmon resonance (SPR; exemplified in the example section below) . In case of doubt, an ACE2 variant or an ACE2 mutant according to the present invention has a KDof 50 nM or less as measured by SPR (with the details according to the example section, below) . Preferred ACE2 variants or ACE2 mutants according to the present invention have a higher binding affinity than the natural human ACE2 protein. Preferably, the KDof the ACE2 variant or the ACE2 mutant according to the present invention is 20 nM or less, more preferred 10 nM or less, especially 5 nM or less, as measured by SPR. In specific embodiments of the present invention, the ACE2 variant or the ACE2 mutant according to the present invention have a KDof 1 nM or less, preferably of 800 pM or less, as measured by SPR. Examples for such high-affinity ACE2 mutants are disclosed e.g. in Chan et al. (2020) , Science 369:1261-1265.

[0032] In a preferred embodiment of the present invention, residues 729 to 732 (or 729 to 730) of the ACE2 moiety / mutant / variant are deleted, since this provides an additional and significant advantage for the peptide according to the present invention. ACE2 residue Thr730 is O-glycosylated when ACE2 is synthesized in human cells (s. Shajahan et al., Glycobiol. 31 (2021) , 410-424) . However, this modification of Thr730 does not occur when ACE2 is expressed in plants or plant cells. Hence, deletion of residues 729 to 732 (or 729 to 730) eliminates a sequence that leads to immunological complications when a plant-derived ACE2 moiety / mutant / variant is used as treatment of viral infections in humans. Whereas the prior art worked on the improvement of the exact glycosylation on Thr730 in mammalian host cells in the course of optimisation of recombinant ACE2 expression (because specifically O-glycosylation was known to be important for protein stability and function, especially in the process in mediating pathogens binding with human receptors and ACE2 shedding (Mayr et al., Sci. Rep. 8 (2018) , 16382) ) , the question of "plant cell-type glycosylation" and the immunogenic consequences thereof only occurs in connection with the recombinant ACE2 expression in plant host cells. It was also surprising that the deletion of residues 729 to 732 (or 729 to 730) in the ACE2 moiety leads nevertheless to an active, stable and fully working ACE2 component in the fusion polypeptide according to the present invention. In a speci fically preferred embodiment , the ACE2 moiety of the polypeptides according to the present invention consists o f the aa 18 to 728 fragment of native human ACE2 or a fragment corresponding to the 18 to 728 fragment of an ACE2 variant or ACE2 mutant . In the course of the present invention, the ACE2 fragments including the amino acids up to the aa residue 728 have even shown to be preferred in recombinant production compared to polypeptides which include a longer fragment extending up to aa 740 with respect to propensity to form higher oligomers . Surprisingly ( and in addition to the advantages of such ACE2 moieties in plant cells , as described above ) , the ACE2 moiety fragments being shorter than the fragments up to aa 740 ( especially those with a deletion of residues 729 to 732 ( or 729 to 730 ) ) show a far lower propensity to form higher oligomers than those which included aa 740 and further amino acid residues in C-terminal direction, such as aa 741 , etc . . Moreover, speci fically in the production in N. benthamiana, the shorter fragments were still suitable , because the resistance to endogenous N. benthamiana proteases was not af fected, as long as the fragments ( still ) included aa 728 . Moreover, also the neutrali zing ef ficiency of SARS-CoV-2 in tissue culture was not af fected in these shorter versions .

[0033] The present polypeptides act as a "decoy" to block viral interaction and cellular entry of coronaviruses to host cells .

[0034] The polypeptides according to the present invention are fusion polypeptides of ACE2 with the stability-increasing moiety wherein the ACE2 moiety may be based on or derived from native , modi fied or engineered versions of the ACE2 protein . These fusion proteins are created by combining ACE2 with other proteins or molecules , and they serve various purposes in scienti fic research, drug development , and therapeutic applications . Preferred applications of the polypeptides according to the present invention (may also be referred to as " fusion proteins of ACE2" ) include the use of the polypeptides for viral entry inhibition to block the entry of viruses , such as coronaviruses , especially SARS-CoV-2 , into host cells .

[0035] The polypeptides according to the present invention may also be used as therapeutic agents , e . g . for conditions such as acute respiratory distress syndrome (ARDS ) and pulmonary hypertension . With the modi fications of the native ACE2 polypeptide according to the present invention it is possible to enhance the protective ef fects of ACE2 in speci fic tissues .

[0036] The polypeptides according to the present invention may also be used as research tools to better understand the binding mechanisms between ACE2 and other molecules , including viral spike proteins , which can aid in the development of treatments and vaccines .

[0037] The polypeptides according to the present invention may also be used for drug screening in drug screening assays to identi fy compounds that interact with ACE2 or that modulate the reninangiotensin system . This is valuable in drug development and pharmacological research .

[0038] Since ACE2 is a crucial enzyme in the regulation of blood pressure and various physiological processes , the polypeptides according to the present invention as engineered combinations of ACE2 with other molecules , can be used in research and therapeutic applications , particularly in the context of viral infections and cardiovascular diseases .

[0039] Preferably, the polypeptides according to the present invention contain an ACE2 moiety and a stability-increasing polypeptide moiety which are linked by a linker peptide and / or a hinge region of a human immunoglobulin, preferably a hinge region of human IgGl or human IgA or human IgM .

[0040] According to a preferred embodiment , the polypeptide according to the present invention comprises an ACE2 moiety which lacks at least proline residues corresponding to Pro733 , Pro734 or Pro738 of the ACE2 protein ( SEQ ID NO : 1 ; UniProt Q9BYF1 , ACE2_HUMAN) , preferably wherein the ACE2 moiety lacks proline residues corresponding to Pro733 , Pro734 and Pro738 of the ACE2 protein ( SEQ ID NO : 1 ; UniProt Q9BYF1 , ACE2_HUMAN) .

[0041] These preferred polypeptides according to the present invention are superior with respect to the recombinant production in plant cells , because Pro-hydroxylation and Pro-arabinosylation can be signi ficantly reduced with such ACE2 moieties or even essentially prevented ( in case all three Pro residues are eliminated) .

[0042] Preferably, the ACE2 moiety in the present polypeptide lacks at least the amino acids corresponding to amino acid residues 733 to 738 of the ACE2 protein ( SEQ ID NO : 1 ) , preferably wherein the ACE2 moiety lacks at least the amino acids corresponding to amino acid residues 731 to 740 of the ACE2 protein ( SEQ ID NO : 1 ) , especial ly wherein the ACE2 moiety lacks at least the amino acids corresponding to amino acid residues 729 to 805 of the ACE2 protein (SEQ ID NO:1) . The term "lack" in this connection can either mean that the respective amino acid residue is substituted at the specific position in the ACE2 sequence by another amino acid residue or that the respective amino acid residue is deleted at the specific position in the ACE2 sequence.

[0043] A preferred stability-increasing polypeptide is an Fc moiety, preferably an Fc moiety selected from a human IgGl or a human IgA or a human IgM.

[0044] Passive delivery of antibodies to mucosal sites may be a valuable adjunct to antiviral vaccination to prevent infection, treat viral carriage, or block transmission. Neutralizing monoclonal IgG antibodies are already approved for systemic delivery, and several clinical trials have been reported for delivery to mucosal sites where respiratory viruses, such as SARS-CoV-2, resides and replicates in early infection. In certain instances, IgA, especially secretory IgA, may be preferred because the polymeric complex is adapted for the harsh, unstable external mucosal environment (Gdritzer et al., Mol. Ther. 32 (2024) , 689-703) . Further results show that intranasal administration of an engineered IgM can improve efficacy, reduce resistance and simplify the prophylactic and therapeutic treatment of respiratory viruses, especially COVID-19 (Ku et al., Nature 595 (2021) , 718-723) . Moreover, pentameric IgMs exhibited increased antigen binding and virus neutralization potency (up to 390-fold) , compared to the parental IgGl (Kallolimath et al., Front. Immunol. 14 (2023) , 1147960) .

[0045] According to a preferred embodiment, the stability-increasing polypeptide of the polypeptide according to the present invention is therefore an Fc moiety selected from SEQ ID NO: 3 (UniProt P01857, IGHG1_HUMAN) , SEQ ID NO:4 (UniProt P01876-1, IGHA1_HUMAN) , SEQ ID NO: 5 (UniProt P01877-1, IgHA2_HUMAN) or SEQ ID NO : 6 (UniProt P01871-1, IgHM_HUMAN) or a fragment or variant thereof capable of extending half-life of ACE2, preferably a fragment consisting of amino acid residues 106 to 330 of SEQ ID NO: 3, with the exchange of P01857-1 (329 and 330 being GK instead of EL) ; or a suitable IgAl fragment or a suitable IgA2 fragment or a suitable IgM fragment .

[0046] SEQ ID NO: 3: UniProt P01857, IGHG1_HUMAN, canonical: ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV 50

[0047] HTFPAVLQSS GLYSLSSWT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP 100

[0048] KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS 150

[0049] HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRWSVLT VLHQDWLNGK 200

[0050] EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC 250

[0051] LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 300

[0052] QQGNVFSCSV MHEALHNHYT QKSLSLSPEL QLEESCAEAQ DGELDGLWTT 350

[0053] ITIFITLFLL SVCYSATVTF FKVKWI FSSV VDLKQTI IPD YRNMIGQGA 399

[0054] SEQ ID NO : 4 : UniProt P01876- 1 , IGHA1_HUMAN :

[0055] ASPTSPKVFP LSLCSTQPDG NWIACLVQG FFPQEPLSVT WSESGQGVTA 50 RNFPPSQDAS GDLYTTSSQL TLPATQCLAG KSVTCHVKHY TNPSQDVTVP 100 CPVPSTPPTP SPSTPPTPSP SCCHPRLSLH RPALEDLLLG SEANLTCTLT 150 GLRDASGVTF TWTPSSGKSA VQGPPERDLC GCYSVSSVLP GCAEPWNHGK 200 TFTCTAAYPE SKTPLTATLS KSGNTFRPEV HLLPPPSEEL ALNELVTLTC 250 LARGFSPKDV LVRWLQGSQE LPREKYLTWA SRQEPSQGTT TFAVTS ILRV 300 AAEDWKKGDT FSCMVGHEAL PLAFTQKTID RLAGKPTHVN VSWMAEVDG 350 TOY 353

[0056] SEQ ID NO : 5 : UniProt P01877- 1 , IgHA2_HUMAN :

[0057] ASPTSPKVFP LSLDSTPQDG NVWACLVQG FFPQEPLSVT WSESGQNVTA 50

[0058] RNFPPSQDAS GDLYTTSSQL TLPATQCPDG KSVTCHVKHY TNSSQDVTVP 100

[0059] CRVPPPPPCC HPRLSLHRPA LEDLLLGSEA NLTCTLTGLR DASGATFTWT 150

[0060] PSSGKSAVQG PPERDLCGCY SVSSVLPGCA QPWNHGETFT CTAAHPELKT 200

[0061] PLTANITKSG NTFRPEVHLL PPPSEELALN ELVTLTCLAR GFSPKDVLVR 250

[0062] WLQGSQELPR EKYLTWASRQ EPSQGTTTYA VTS ILRVAAE DWKKGETFSC 300

[0063] MVGHEALPLA FTQKTIDRMA GKPTHINVSV VMAEADGTCY 340

[0064] SEQ ID NO : 6 : UniProt P01871- 1 , IgHM_HUMAN :

[0065] GSASAPTLFP LVSCENSPSD TSSVAVGCLA QDFLPDS ITF SWKYKNNSDI 50 SSTRGFPSVL RGGKYAATSQ VLLPSKDVMQ GTDEHWCKV QHPNGNKEKN 100 VPLPVIAELP PKVSVFVPPR DGFFGNPRKS KLICQATGES PRQIQVSWLR 150 EGKQVGSGVT TDQVQAEAKE SGPTTYKVTS TLTIKESDWL GQSMFTCRVD 200 HRGLTFQQNA SSMCVPDQDT AIRVFAIPPS FAS I FLTKST KLTCLVTDLT 250 TYDSVTISWT RQNGEAVKTH TNISESHPNA TFSAVGEASI CEDDWNSGER 300

[0066] FTCTVTHTDL PSPLKQTISR PKGVALHRPD VYLLPPAREQ LNLRESATIT 350 CLVTGFSPAD VFVQWMQRGQ PLSPEKYVTS APMPEPQAPG RYFAHS ILTV 400 SEEEWNTGET YTCWAHEAL PNRVTERTVD KSTGKPTLYN VSLVMSDTAG 450

[0067] TCY 453

[0068] Preferably, the ACE2 moiety comprises an exchange at an amino acid corresponding to amino acid position 19 (S) and / or at amino acid position 92 (T) of SEQ ID NO:1, preferably wherein the ACE2 moiety comprises an S19P and / or a T92I exchange.

[0069] Further preferred embodiments of the ACE2 moiety may be selected from the ACE2 polypeptides disclosed in the prior art, e.g. in WO 2021 / 183717 Al, WO 2021 / 207207 A2 , WO 2022 / 165246 Al, or Chan et al. (2020) .

[0070] Exemplary heterologous polypeptides can increase the stability of the resultant chimeric polypeptide in the course of administration in vivo, and may, therefore, further enhance the properties of the proteins of the present invention, especially by increasing the stability in the course of mucosal administration. In various embodiments, the polypeptide that increases the stability in the course of administration may be a serum albumin, such as human serum albumin, or the Fc region of immunoglobulins, preferably of the IgG, IgA or IgM subclasses of antibodies that lack the heavy chain variable region. Exemplary Fc regions can include a mutation (e.g., a mutation that inhibits complement fixation and / or Fc receptor binding) or it may be lytic, i.e., able to bind complement or to lyse cells via another mechanism, such as antibody-dependent complement lysis. The "Fc region" can be a naturally occurring or synthetic polypeptide that is homologous to the IgG C-terminal domain produced by digestion of immunoglobulins, especially IgG, IgA or IgM with papain. The fusion proteins can include the entire Fc region, or a smaller portion that retains the ability to increase the stability of a chimeric polypeptide of which it is a part in the course of administration, especially in the course of mucosal administration. In addition, full-length or fragmented Fc regions can be wild-type or variants of the wildtype molecule. That is, they can contain mutations that may or may not affect the function of the polypeptides. For example, they may have effector function or may be modified as to have one or more activities associated with effector function reduced or completely eliminated. Effector function refers to those biological activities attributable to the Fc region of an immunoglobulin, which vary with the immunoglobulin isotype. Examples of effector function include, Clq binding and complement dependent cytotoxicity (CDC) , Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC) , antibody-dependent cellular phagocytosis (ADCP) , cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors, and B cell activation. In some exemplary embodiments, the de novo proteins of the present invention include an IgM, IgA, IgGl, IgG2, IgG3, or IgG4 Fc region. In some exemplary embodiments, the de novo proteins include a variant IgM, IgA, IgGl, IgG2, IgG3, or IgG4 Fc region. In some aspects, the variant Fc region lacks effector function. In some aspects, a de novo protein of the present invention is fused to the C-terminus of an Fc region (e.g., a native or variant IgA, IgM, IgGl, IgG2, IgG3, or IgG4 Fc region) . In some aspects, two de novo proteins of the present invention are fused to a Fc region (e.g., at the N and C terminus) .

[0071] According to a further aspect the present invention also relates to a nucleic acid molecule encoding a polypeptide according to the present invention.

[0072] The nucleic acid may be included in suitable vector systems to enable recombinant production of the polypeptide in host cells or for administration of the polypeptide to patients, especially human patients.

[0073] Preferably, the nucleic acid molecule according to the present invention further comprises a signal peptide encoding region, wherein the signal coding region is not an ACE2 signal peptide or an IgGl, IgAl, IgA2 or IgM signal peptide, wherein the signal peptide is preferably a plant signal peptide, more preferred a signal peptide of barley, especially barley a-amylase signal peptide of barley.

[0074] According to a further aspect, the present invention also relates to a method for producing a polypeptide according to the present invention, wherein a nucleic acid according to the present invention is expressed in a host cell, preferably in a plant host cell, especially in a Nicotiana benthamiana cell.

[0075] According to a further aspect, the present invention also relates to a polypeptide according to the present invention for use in neutralising SARS-CoV-1 and SARS-CoV-2.

[0076] Preferably, the polypeptide for use according to the present invention has an ACE2 moiety which does not comprise amino acid residues 729 to 740 of the ACE2 protein. According to a further aspect , the present invention also relates to a pharmaceutical preparation comprising a polypeptide or a nucleic acid according to the present invention and a pharmaceutically acceptable carrier, preferably provided and finished for inhalation or nasal delivery, especially in an aerosol formulation .

[0077] An aerosol formulation usually consists of two components : the product to be delivered as an aerosol ( i . e . the polypeptide according to the present invention) and a propellant . The polypeptide according to the present invention may be provided in a formulation combined with additional ingredients or co-solvents required to make a stable and ef ficacious product ( e . g . as a solution, suspension, emulsion, semisolid, or powder ) ; the propellant provides the force that expels the product concentrate from the container and additionally is responsible for the delivery of the formulation in the proper form ( i . e . , spray, foam, semisolid) . When the propellant is a liquefied gas or a mixture of liquefied gases , it can also serve as the solvent or vehicle for the product concentrate . I f the product characteristics are to change on dispensing, additional energy in the form of a mechanical breakup system may be required . A propellant may be a chemical with a vapor pressure greater than atmospheric pressure at 40 ° C ( 105 ° F) . Types of propellants commonly used in pharmaceutical aerosols include chlorofluorocarbons , hydrocarbons , hydrochlorofluorocarbons and hydrofluorocarbons , and compressed gases ( including compressed air, e . g . air compressed immediately before administration ( e . g . by the patients themselves ) . The ef fectiveness of a pharmaceutical aerosol depends on achieving the proper combination of product concentrate formulation, container, and valve assembly . The valve mechanism is the part of the product package through which the contents of the container are emitted . The valve must withstand the pressure required by the product concentrate and the container, be corrosive resistant , and must contribute to the form of the emitted product concentrate . The primary purpose of the valve is to regulate the flow of product concentrate from the container . But the valve must also be multi functional and regulate the amount of emitted material (metered valves ) , be capable of delivering the product concentrate in the desired form, and be easy to turn on and of f . Among the materials used in the manufacture of the various valve parts are plastic, rubber, aluminium, and stainless steel . Aerosols are used to mucosally deliver active drugs to the pulmonary airways, the nasal passages, or the oral cavity. They are also used to administer drugs via mucosal administration topically and into body cavities such the vagina and rectum. Pulmonary, nasal, and oral administration is intended to achieve either local or systemic therapeutic effect, while topical, vaginal, and rectal administration is only intended for local effect. Inhalation therapy (i.e., drug delivery to the pulmonary airways and nasal passages) as a preferred mucosal administration according to the present invention may also be performed using nebulizers or. Preferably, metered dose inhalers may be used for accurate dosing. Preferred inhalation therapy according to the present invention is performed under formulation of droplets or particles that are the optimum size (e.g. for bronchial administration in the 1 to 10 pm range; smaller particles (0.1 to 1 pm) penetrate to the alveolar sacs) . Preferred particle sizes may therefore be from 0.1 to 20 pm, preferably from 1 to 10 pm, especially from 3 and 6 pm. Nasal aerosols deliver the drug directly to the nasal mucosa. Aerosols used to deliver drugs to the oral cavity generally administer the product sublingually.

[0078] According to a preferred embodiment, the present polypeptide is administered together with further effective agents, preferably in combination with further antiviral drugs. Antiviral drugs are well available to the skilled person targeting several fundamental steps in virus entry and replication (s. e.g. Meganck et al., Nat. Med. 27 (2021) , 401-410; Goncalves et al., Fund. Clin. Pharmacol. 35 (2021) 305-320) ; various functional classes of antivirals are known, targeting either directly the virus or act indirectly via host factors, for example convalescent plasma (direct-acting; derived from recovered individuals with high-titer neutralizing antibodies) , fusion inhibitors (direct-acting; peptide mimics that block interactions required for fusion of some enveloped viruses) , monoclonal or polyclonal antibodies (direct-acting; produced bio- technologically or as purified antibodies often isolated from patients; both can be neutralizing or block receptor interactions) , nucleoside analogs (direct-acting; analogs are incorporated into DNA or RNA chains, terminating the chain or leading to mutations) , polymerase inhibitors (direct-acting; directly block viral polymerases to inhibit replication) , receptor decoys (direct-acting; overexpression or delivery of free cellular proteins or attachment factors as a decoy to bind virus) , protease inhibitors (direct- acting and host-factor; inhibit proteases necessary for infection; can be viral proteases or host proteases required for cleavage of viral peptides) , translation inhibitors (direct-acting and hostfactor; block translation of viral mRNAs) , endocytosis inhibitors (host-factor; target cellular endocytosis pathways used for viral entry) , interferons (host-factor; upregulate host immune responses; often used in combination with other drugs) , kinase inhibitors (host-factor; target host kinases and modulate cellular environment) , and lipidomic drugs (host-factor; target lipid biosynthesis pathways required for some enveloped viruses) . Both direct-acting and host-factor therapeutics can be broadly applicable if the antiviral targets conserved genes / motifs within a virus family, or if diverse virus family members co-opt the same host pathways to promote virus replication and / or pathogenesis (such as the furin protease) . Antiviral therapeutic treatment windows are typically limited to the viral phase of infection, after which they are not as effective anymore. In the inflammatory phase, treatment options include anti-inflammatory and immunomodulatory drugs, such as corticosteroids, which dampen the host immune response in an attempt to control inflammatory damage.

[0079] Preferably, the pharmaceutical preparation according to the present invention therefore further comprises an antiviral agent, preferably an antiviral agent selected from the group convalescent plasma, fusion inhibitors, monoclonal or polyclonal antibodies, nucleoside analogs, polymerase inhibitors, receptor decoys, protease inhibitors, translation inhibitors, endocytosis inhibitors, interferons, kinase inhibitors, and lipidomic drugs, especially 3, 3 ' , 4, 4 ' , 5, 5 ' -hexahydroxy-trans-stilbene, nirmatrelvir , ritonavir, remdesivir, dexamethasone or combinations thereof, either as a physical combination or as a set of at least two separate pharmaceutical preparations.

[0080] Therefore, the present invention relates to the following preferred embodiments:

[0081] 1. An isolated polypeptide comprising an ACE2 moiety and a stability-increasing polypeptide moiety, wherein the ACE2 moiety comprises or consists of amino acid residues 18 to 728 of the ACE2 protein (SEQ ID NO: 2) or a biologically active ACE2 mutant or ACE2 variant polypeptide thereof; and wherein the stability-increasing polypeptide moiety increases the stability of the ACE2 moiety in the course of administration to a patient.

[0082] 2. A polypeptide according to embodiment 1, wherein the ACE2 moiety and the stability-increasing polypeptide moiety are linked by a linker peptide and / or a hinge region of a human immunoglobulin, preferably a hinge region of human IgGl, human IgA or human I gM .

[0083] 3. A polypeptide according to embodiment 1 or 2, wherein the ACE2 moiety lacks at least proline residues corresponding to Pro733, Pro734 or Pro738 of the ACE2 protein (SEQ ID NO:1; UniProt Q9BYF1, ACE 2 _HUMAN ) , preferably wherein the ACE2 moiety lacks pro- line residues corresponding to Pro733, Pro734 and Pro738 of the ACE2 protein (SEQ ID NO:1) ; and / or wherein the ACE2 protein (SEQ ID NO:1) lacks residues 729 to 732 or 729 to 730.

[0084] 4. A polypeptide according to any one of embodiments 1 to

[0085] 3, wherein the ACE2 moiety lacks at least the amino acids corresponding to amino acid residues 733 to 738 of the ACE2 protein (SEQ ID NO:1) , preferably wherein the ACE2 moiety lacks at least the amino acids corresponding to amino acid residues 731 to 740 of the ACE2 protein (SEQ ID NO:1) , especially wherein the ACE2 moiety lacks at least the amino acids corresponding to amino acid residues 729 to 805 of the ACE2 protein (SEQ ID NO:1) .

[0086] 5. A polypeptide according to any one of embodiments 1 to

[0087] 4, wherein the stability-increasing polypeptide is an Fc moiety, preferably an Fc moiety selected from a human IgGl or a human IgA or a human IgM.

[0088] 6. A polypeptide according to any one of embodiments 1 to

[0089] 5, wherein the stability-increasing polypeptide is a Fc moiety selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 or a fragment or variant thereof capable of extending half-life of ACE2, preferably a fragment consisting of amino acid residues 106 to 330, with the exchange of P01857-1 (329 and 330 being GK instead of EL) ; or a suitable IgAl fragment or a suitable IgA2 fragment or a suitable IgM fragment.

[0090] 7. A polypeptide according to any one of embodiments 1 to

[0091] 6, wherein the ACE2 moiety comprises an exchange at an amino acid corresponding to amino acid position 19 (S) and / or at amino acid position 92 (T) of SEQ ID NO:1, preferably wherein the ACE2 moiety comprises an S19P and / or a T92I exchange.

[0092] 8. A polypeptide according to any one of embodiments 1 to

[0093] 7, wherein the ACE2 moiety consists of amino acid residues 18 to 728 of the ACE2 protein ( SEQ ID NO : 2 ) or a biologically active ACE2 mutant or ACE2 variant polypeptide thereof .

[0094] 9 . A nucleic acid molecule encoding a polypeptide according to any one of embodiments 1 to 8 .

[0095] 10 . A nucleic acid molecule according to embodiment 9 further comprising a signal peptide encoding region, wherein the signal coding region is not an ACE2 signal peptide , or an IgGl , IgAl , IgA2 or IgM signal peptide , wherein the signal peptide is preferably a plant signal peptide , more preferred a signal peptide of barley, especially barley a-amylase signal peptide of barley .

[0096] 11 . A method for producing a polypeptide according to any one of embodiments 1 to 8 , wherein a nucleic acid according to any one of embodiments 9 or 10 is expressed in a host cell , preferably in a plant host , especially in Ni cotiana benthamiana .

[0097] 12 . A polypeptide according to any one of embodiments 1 to 8 for use in neutralising SARS-CoV- 1 and SARS-CoV-2 .

[0098] 13 . A polypeptide for use according to embodiment 12 , wherein the ACE2 moiety does not comprise amino acid residues 729 to 740 of the ACE2 protein .

[0099] 14 . A pharmaceutical preparation comprising a polypeptide according to anyone of embodiments 1 to 8 or a nucleic acid according to any one of embodiments 9 or 10 and a pharmaceutically acceptable carrier, preferably provided and finished for inhalation or nasal delivery, especially in an aerosol formulation .

[0100] 15 . A pharmaceutical preparation according to embodiment 14 further comprising an antiviral agent , preferably an antiviral agent selected from the group convalescent plasma, fusion inhibitors , monoclonal or polyclonal antibodies , nucleoside analogs , polymerase inhibitors , receptor decoys , protease inhibitors , translation inhibitors , endocytosis inhibitors , interferons , kinase inhibitors , and lipidomic drugs , especially 3 , 3 ' , 4 , 4 ' , 5 , 5 ' - hexahydroxy-trans-stilbene , nirmatrelvir , ritonavir, remdesivir, dexamethasone or combinations thereof , either as a physical combination or as a set of at least two separate pharmaceutical preparations .

[0101] The invention is further illustrated by way of the following examples and the figures , yet without being restricted thereto . Figure 1 shows the impact of ACE2-615-Fc N-glycosylation on its SARS-CoV-2 virus (Wuhan strain) neutralizing activity in VeroE6 cells (adapted from Capraz et al., eLife 10 (2021) , e73641) .

[0102] Figure 2 shows SARS-CoV-2 (Wuhan strain) neutralization assay on VeroE6 cells with different concentrations (lOpg / ml, 20pg / ml, 50pg / ml) of wild-type ACE2-615-Fc decoys produced in HEK293 cells (wt) and N. benthamiana (N. benth) compared to HEK293-derived ACE2 decoys with modified glycosylation patterns. DeSial, desialylated wild-type ACE2-Fc; DeGlyco, deglycosylated wild-type ACE2-Fc; T92Q, mutant ACE2-Fc lacking the N-glycan attached to N90; N322Q, mutant ACE2-Fc lacking the N-glycan attached to N322; DM, mutant ACE2-Fc lacking the N-glycans attached to N90 and N322.

[0103] Figure 3 shows neutralizing activity of ACE2-615-Fc fusion protein produced in N. benthamiana (black) and HEK293 cells (grey) . Data are represented as mean + / - SD and refer to four independent experimental series, each performed in triplicates (n=12) . The data demonstrate that ACE2-Fc produced in N. benthamiana neutralizes SARS-CoV-2 (Delta variant) more effectively than HEK- derived ACE2-Fc (median Ct values at 4 pg / ml: 27.3 vs. 23.0; at 0.8 pg / ml: 21.0 vs. 16.2) . Ct values >30: complete neutralization; Ct values <15: no neutralization. Positive control: no ACE2-Fc added .

[0104] Figure 4 shows N-linked glycoforms produced in (A) wild-type and (B) glycoengineered plants. The predominant N-glycan species in plants comprise complex glycans containing pl,2-xylose and al,3-fucose residues (GnGnXF) . Elongation of the core glycan gives rise to the Lewis A structure, whereas removal of the terminal GlcNAc residues yields paucimannosidic structures (MMXF) . Elimination of the enzymes responsible for imparting plantspecific glycan moieties has enabled the production of 'humanlike' glycan structures (bisected and branched) , which have been further decorated with galactose and sialic acid residues. Introduction of the appropriate biosynthetic machinery has enabled the production of N-glycan structures in plants with sialic acid extensions. Sialic acid structures are depicted as N- acetylneuraminic acid (Neu5Ac) . Figure based on Margolin et al. (2020) but modified.

[0105] Figure 5 shows that ACE2-740-Fc is less sensitive to proteolysis than ACE2-615-Fc, as evident from the reduction in Fc fragments. The constructs also differ in their linkers between the ACE2 and Fc domains (L, synthetic GGGGSGGGGS linker (SEQ ID NO: 8) ; H, natural IgGl hinge region) . Sample 1, ACE2-615L-Fc; sample 2, ACE2-615H-Fc; sample 3, ACE2-740L-Fc; sample 4, ACE2-740H-Fc .

[0106] Figure 6 shows that ACE2-740-Fc produced in N. benthamiana neutralizes SARS-CoV-2 (Delta variant) more effectively than ACE2- 615-Fc (mean Ct values at 4 pg / ml: 30.3 / 30.1 vs. 28.3 / 24.7; at 0.8 pg / ml: 26.6 / 26.8 vs. 22.1 / 20.5) . The constructs differ in their linkers between the ACE2 and Fc domains (L, synthetic GGGGSGGGGS linker (SEQ ID NO: 8) ; H, IgGl hinge region) . The data are derived from 3 independent experiments performed in triplicates (n=9) . ACE2- (L) -Fc, ACE2-615L-Fc; ACE2- (L) -Fc Sia4, partially sialylated ACE2-615L-Fc (co-expression of sialylation pathway) ; ACE2- (H) -Fc, ACE2-615H-Fc; ACE2-Fc HEK, ACE2-615H-Fc produced in HEK293 cells; positive control, no ACE2-Fc added.

[0107] Figure 7 shows that the tryptic peptide (SEQ ID NO: 13) encompassing the linker sequence of ACE2-740-Fc contains up to three hydroxylated proline residues (hyP) when produced in N. benthamiana .

[0108] Figure 8 shows size-exclusion chromatography of affinity- purified ACE2-728H-Fc (A) and ACE2-740H-Fc (B) produced in N. benthamiana . The oligomers elute in the void volume of the column.

[0109] Figure 9 shows N-glycosylation profiles of ACE2-740H-Fc and ACE2-728H-Fc produced in N. benthamiana as shown here for N- glycosite 6 (peptide CDISNSTEAGQK; SEQ ID NO: 11; ACE2 aa 542-553) . In both proteins, the most abundant N-glycan structure is the complex-type N-glycan GnGn (GlcNAc2Man3GlcNAc2) .

[0110] Figure 10 shows binding of ACE2-740H-Fc and ACE2-728H-Fc produced in N. benthamiana to the RBD of SARS-CoV-2 spike protein (Wuhan strain) as measured by surface plasmon resonance (SPR) . KD, equilibrium dissociation constant.

[0111] Figure 11 shows the neutralization of SARS-CoV-2 (Delta variant) by ACE2-740-Fc and ACE2-728H-Fc produced in N. benthamiana. The virus-neutralizing activity of the two proteins is very similar (mean Ct values at 0.8 pg / ml: 29.0 vs. 27.6) . The data are derived from 3 independent experiments performed in triplicates (n=9) . Ct values >30: complete neutralization; Ct values <20: no neutralization. Positive control: no ACE2-Fc added.

[0112] Figure 12 shows the neutralization of different SARS-CoV-2 variants of concern (VOC) by ACE2-728H-Fc produced in N. benthamiana and nirmatrelvir (Pf332) . ACE2-728H-Fc neutralizes Omicron strains of SARS-CoV-2 (om21, om23) equally well as Delta (de21) and up to 6 times better than the ancestral Wuhan isolate (whl9) . This is not observed for Pf 322. Data are presented as ratios of the IC50 values for the respective VOC and the Wuhan strain .

[0113] Figure 13 shows neutralization of SARS-CoV-2 (Delta variant) by dimeric and oligomeric ACE2-728H-Fc produced in N. benthamiana . The virus-neutralizing activity of the oligomeric fraction (tetramer plus higher oligomers) is higher than of the dimer (mean Ct values at 0.8 pg / ml: 25.7 vs. 15.1) . The data are derived from 2 independent experiments performed in triplicates (n=6) . Ct values >30: complete neutralization; Ct values <13: no neutralization. Positive control (Ctrl) : no ACE2-Fc added. ACE2- 728H-Fc mix: unfractionated mixture of dimer, tetramer and higher oligomers before size-exclusion chromatography.

[0114] Figure 14 shows neutralization of SARS-CoV-2 (Delta variant) by ACE2-728H-Fc produced in N. benthamiana (pTrak728) and hexahydroxystilbene (M08) . The virus-neutralizing activity of ACE2-728H-Fc can be enhanced by the addition of hexahydroxystilbene (mean Ct values (- / + M08) at 0.16 pg / ml: 16.6 vs. 20.3) . Ct values >28: complete neutralization; Ct values <13: no neutralization. Positive control (Ctrl) : no ACE2-Fc or M08 added .

[0115] Figure 15 shows neutralization of SARS-CoV-2 (Delta variant) by ACE2-728H-Fc produced in N. benthamiana (pTrak728) and nirmatrelvir (Pf332) . The virus-neutralizing activity of ACE2- 728H-Fc can be enhanced by the addition of nirmatrelvir (mean Ct values (- / + Pf332) at 0.16 pg / ml: 16.6 vs. 21.8) . Ct values >28: complete neutralization; Ct values <13: no neutralization. Positive control (Ctrl) : no ACE2-Fc or Pf332 added.

[0116] Figure 16 shows neutralization of SARS-CoV-2 (Omicron variant) by unfractionated ACE2-728H-Fc produced in N. benthamiana (pTRAK-728H-Fc mix) and hexahydroxystilbene (M08) in human Calu-3 ALT cultures (RT-qPCR analysis) . Pre-treatment with 20 pg / ml ACE2- 728H-Fc alone or in combination with 60 pM hexahydroxystilbene (M08) leads to minimal release of viral particles into the supernatant during 24 h of incubation. Positive control: no ACE2- Fc or M08 added.

[0117] Figure 17 shows neutralization of SARS-CoV-2 (Omicron variant) by unfractionated ACE2-728H-Fc produced in N. benthamiana and hexahydroxystilbene (M08) in human Calu-3 ALI cultures (immunohistochemical staining) . Pre-treatment with 20 pg / ml ACE2- 728H-Fc alone or in combination with 60 pM hexahydroxystilbene (M08) eliminates the detection of SARS-CoV-2 N protein (dark) in ALI cultures fixed 24 h after addition of SARS-CoV-2. Positive control: no ACE2-Fc or M08 added. Negative control: no SARS-CoV-2 added .

[0118] Figure 18 shows neutralization of SARS-CoV-2 (Omicron variant) by unfractionated ACE2-728H-Fc produced in N. benthamiana in ALI cultures of primary human lung epithelial cells (immunohistochemical staining) . Pre-treatment with 20 pg / ml ACE2- 728H-Fc eliminates the detection of SARS-CoV-2 N protein (dark) in ALI cultures fixed 24 h after addition of SARS-CoV-2. Positive control: no ACE2-Fc added.

[0119] Figure 19 shows proline hydroxylation of ACE2-740-Fc expressed in plants. The chymotryptic / tryptic peptide encompassing the linker sequence of ACE2-740-Fc (L725-K743; SEQ ID NO: 14) contains up to three hydroxylated proline residues (hyP) when produced in N. benthamiana . Quantitation of the peak areas resulted in the following estimates of the abundance of each isoform: unmodified peptide 48%, IhyP 30%, 2hyP 15%, 3hyP 7% (mean of three biological replicates) .

[0120] Figure 20 shows proline hydroxylation of ACE2-740-Fc expressed in plants. MS / MS analysis of the chymotryptic / tryptic peptide L725-K743 (SEQ ID NO: 14) containing three hydroxylated proline residues (Pro733, Pro734 and Pro738) and a single arabinose trisaccharide attached to Pro733 or Pro734.

[0121] Figure 21 shows that the non-neutralizing variant of ACE2- 728H-Fc (mutACE2-728H-Fc (IgG) ) does not neutralize SARS-CoV-2 even at the highest concentration tested (10 pg / ml) ; in contrast, less than 0.15 pg / ml wild-type ACE2-728H-Fc (IgG) are required for 50% inhibition in plaque-reduction neutralization tests; data was normalized to a virus control (no compound, 0% Inhibition, left panel) and a cell control (no virus, 100% inhibition, right panel) ; IC50 values were calculated by nonlinear regression analysis with variable slopes in GraphPad PRISM Version 9.

[0122] Figure 22 shows that intranasal application of 2.5 mg / ml ACE2- 728H-Fc significantly reduced weight loss whereas weight loss could not be prevented by treatment with 2.5 mg / ml of the nonneutralizing variant mutACE2-728H-Fc; furthermore, the median infectious virus levels were significantly lower in the lungs of hamsters treated with ACE2-728H-Fc than in those receiving PBS or mutACE2-728H-Fc instead.

[0123] Figure 23 shows the binding behavior to SARS-CoV-2 receptorbinding domain (RBD) ; ACE2-728H-Fc ( IgG) and ACE2-728H-Fc ( IgA) fusion protein exhibit similar binding properties; binding was assessed by coating 2 pg / ml RBD on ELISA plates; affinity purified proteins were incubated and detected using an anti-ACE2 antibody. Figure 24 shows that the IgA-Fc fusion variant of ACE2-728H-Fc does neutralize SARS-CoV-2 in a similar extent as the IgG-Fc fusion and that less than 0.6 pg / ml ACE2-728H-Fc ( IgA) are required for 50% inhibition in plaque-reduction neutralization tests (calculated values: Ace2-728H-Fc ( IgA) : 0.57 pg / ml; Ace2-728H- Fc(IgG) : 0.25 pg / ml) ; data was normalized to a virus control (no compound, 0% Inhibition, right panel) and a cell control (no virus, 100% inhibition, left panel) ; IC50 values were calculated by nonlinear regression analysis with variable slopes in GraphPad PRISM Version 9.

[0124] Examples :

[0125] ACE2 decoys as therapeutics

[0126] ACE2 decoys such as soluble ACE2 have already been developed and tested in Phase 1 and 2 clinical trials to prevent SARS-CoV-2 binding to ACE2 located on cell surfaces (Haschke et al., Clin Pharmacokinet . 52 (2013) , 783-92; doi: 10.1007 / s40262-013-0072-7 2013; Zoufaly et al., Lancet Respir. Med. 8 (2020) , 1154-1158; doi 10.1016-S2213-2600 (20) 30418-5) . These drugs mimic the membranebound cellular ACE2 receptor and bind to the spike protein thereby preventing the virus to bind to and infect human cells. This approach, however, has not yet demonstrated sufficient efficacy in clinical studies (Zoufaly et al., 2020) .

[0127] The virus-neutralizing activity of ACE2 decoys is strongly influenced by their glycosylation status. In a previous study, we have shown that elimination or reduction of ACE2 glycosylation increases the virus-neutralizing activity of a wild-type ACE2 decoy (ACE2-wt-Fc) produced in human embryonic kidney 293 (HEK293) cells by a factor of up to 10-fold (Capraz et al., 2021; Figure 1) . Figure 1 shows the impact of ACE2-615-Fc N-glycosylation on its SARS-CoV-2 (Wuhan strain) neutralizing activity in VeroE6 cells (from Capraz et al., 2021) .

[0128] Advantage of ACE2 production in glycoengineered N benthamiana

[0129] The enhanced virus-neutralizing activity of deglycosylated ACE2 decoys can be recapitulated by producing human ACE2 in glycoengineered Nicotiana benthamiana plants (Figure 2) . This effect can most likely be attributed to differences in the glycosylation patterns between ACE2 produced in HEK cells and ACE2 produced in glycoengineered N. benthamiana. Figure 2 shows SARS- CoV-2 (Wuhan strain) neutralization assay on VeroE6 cells with different concentrations (lOpg / ml, 20pg / ml, 50pg / ml) of wild-type ACE2-615-Fc decoys produced in HEK293 cells (wt) and N. benthamiana (N. benth) compared to HEK293-derived ACE2 decoys with modified glycosylation patterns. DeSial, desialylated wild-type ACE2-Fc; DeGlyco, deglycosylated wild-type ACE2-Fc; T92Q, mutant ACE2-Fc lacking the N-glycan attached to N90; N322Q, mutant ACE2-Fc lacking the N-glycan attached to N322; DM, mutant ACE2-Fc lacking the N- glycans attached to N90 and N322.

[0130] SARS-CoV-2 variants of concern and their increased inactivation by ACE2 decoys

[0131] Among the various options to block SARS-CoV-2 infections and replication by drugs, the inhibition of the binding of the virus to its receptor on human cells is one of the most promising approaches. This is mainly because the emerging virus variants such as Delta, Omicron and other variants of concern are characterized by increased binding of their spike protein to the ACE2 receptor on human cells due to natural selection. In contrast to other antivirals such as monoclonal antibodies, ACE2 decoys show increased binding to the emerging virus variants, making the decoy more active in neutralizing the virus. This is consistent with our findings that the enhanced virus-neutralizing activity of a human ACE2 decoy produced in N. benthamiana is also seen in virus-neutralization assays performed with the Delta variant of SARS-CoV-2 in the human respiratory cell line Calu3 (Figure 3) . Figure 3 shows neutralizing activity of ACE2-615-Fc fusion protein produced in N. benthamiana (black) and HEK293 cells (grey) . Data are represented as mean + / - SD and refer to four independent experimental series, each performed in triplicates (n=12) . The data demonstrate that ACE2-Fc produced in N. benthamiana neutralizes SARS-CoV-2 (Delta variant) more effectively than HEK- derived ACE2-Fc (median Ct values at 4 pg / ml: 27.3 vs. 23.0; at 0.8 pg / ml: 21.0 vs. 16.2) . Ct values >30: complete neutralization; Ct values <15: no neutralization. Positive control: no ACE2-Fc added .

[0132] These findings show that emerging SARS-CoV-2 variants are naturally selected for better binding to the ACE2 receptor which results in increased infectivity and eventually reduced immune recognition. Therefore, ACE2 decoys that are based on the human wild-type ACE2 sequence are even more potent in neutralizing any newly emerging SARS-CoV-2 variant including the recently identified variant BA.2.86 as compared to the earlier circulating variants of the virus (Wang et al., 2023; Nature 624, 639-644. doi: 10.1038 / s41586- 023- 06750-w) .

[0133] Use of Plant Molecular Pharming for ACE2 production in a glycoengineered Nicotiana benthamiana plant-based expression system

[0134] In recent years, plants have gained increasing attention as manufacturing hosts for recombinant proteins, particularly for glycoprotein production including different human immunoglobulin subtypes or enzymes for treatment of lysosomal storage diseases. While mammalian expression systems like Chinese hamster ovary (CHO) cells or HEK293 cells are still widely used by industry, plants provide an interesting alternative to these established hosts. One major advantage of plants is their comparably simple N- glycan processing pathway and the complete absence of mammaliantype O-glycosylation . While mammalian cells produce quite heterogeneous glycans on recombinant proteins and need extensive genome editing to make glycans more uniform (Yang et al., Nat. Biotechnol. 33 (2015) , 842-844) , the same result can be achieved in plants by very few engineering steps (Strasser et al., Curr. Opin. Biotechnol. 30 (2014) , 95-100) . Importantly, plant cells and whole plants like N. benthamiana tolerate the introduction of novel N- and O-glycan processing steps quite well.

[0135] Elimination of non-human and potentially immunogenic sugar residues

[0136] The majority of the currently used expression systems for glycoprotein therapeutics produce N-glycans carrying non-human structures that can lead to potential side effects of the drugs. These non-human epitopes may elicit unwanted immune responses that neutralize the applied drug or, even worse, cause a hypersensitivity reaction. Glycosylation of recombinant proteins derived from human cell lines such as HEK293 can be highly heterogeneous including different biantennary and branched structures with variable terminal sugar residues and thus do not necessarily resemble N-glycosylation of serum-derived glycoproteins. Comparison of several proteins expressed in CHO and HEK293 cells with their natural counterparts may show significant differences in their N-glycosylation profiles.

[0137] N-glycoengineering in plants

[0138] Plants typically generate rather simple biantennary complex N-glycans containing pl,2-xylose and core al,3-fucose residues that are not found on N-glycans in mammals and therefore represent potentially immunogenic residues (Altmann, Int Arch Allergy Immunol. 142 (2007) , 99-115) . Genetic knockout as well as knockdown approaches have successfully been used to eliminate pl,2-xylose and core al,3-fucose from recombinant glycoprotein therapeutics produced in different plants (Strasser et al., Plant Biotechnol J. 6 (2008) , 392-402) . Compared to conventional CHO-produced recombinant monoclonal antibodies, the N-glycans from plant- derived IgGs are highly homogeneous and display mainly the GnGn glycan (Figure 6) , which is the optimal substrate for further modifications like branching or galactosylation . Notably, the N. benthami ana-based expression platform enables also the production of functionally active recombinant IgM variants with defined N- glycosylation, demonstrating the ability of the N. benthamiana expression system to correctly assemble pentameric and hexameric glycoengineered forms of IgM. Another important example of using the glycoengineered N. benthamiana expression system is the manufacturing of the monoclonal antibody cocktail ZMapp™ protective against Ebola virus (Qiu et al., Nature 514 (2014) , 47- 53) .

[0139] Figure 4 shows N-linked glycoforms produced in (A) wild-type and (B) glycoengineered plants. The predominant N-glycan species in plants comprise complex glycans containing pl,2-xylose and al,3-fucose residues (GnGnXF) . Elongation of the core glycan gives rise to the Lewis A structure, whereas removal of the terminal GlcNAc residues yields paucimannosidic structures (MMXF) . Elimination of the enzymes responsible for imparting plantspecific glycan moieties has enabled the production of "humanlike" glycan structures (bisected and branched) , which have been further decorated with galactose and sialic acid residues. Introduction of the appropriate biosynthetic machinery has enabled the production of N-glycan structures in plants with sialic acid extensions. Sialic acid structures are depicted as N- acetylneuraminic acid (Neu5Ac) . Figure 4 is based on Margolin et al. (Trends Biotechnol. 38 (2020) , 1034-1044) and modified.

[0140] Production of ACE2 decoys in glycoengineered N. benthamiana plants

[0141] For the ongoing SARS-CoV-2 pandemic as well as for newly emerging viruses it is highly important to establish efficient, versatile and scalable production systems for recombinant protein therapeutics such as ACE2 decoys that are required worldwide in high demands. In addition to COVID-19 treatment and prophylaxis, different ACE2 variants are essential components of diagnostic assays and test kits, for example, to characterize neutralizing antibodies or to screen for novel inhibitors that interfere with the binding of SARS-CoV-2 to ACE2. Therefore, the feasibility of the glycoengineered N. benthamiana expression system for producing a functional recombinant ACE2 decoy was successfully evaluated. It was shown that a functional recombinant ACE2 decoy with high binding affinity for the RBD of the SARS-CoV-2 spike protein can be efficiently produced in the N. benthamiana expression system and may be used as receptor decoy to treat coronavirus infections in humans (Castilho et al., 2021) . Importantly, the N. benthamiana produced ACE2 decoy proved to exhibit significantly higher affinity to the SARS-CoV-2 RBD and higher virus-neutralization activity compared to ACE2 produced in HEK293 cells (see also Figures 4 and 3) .

[0142] In our initial studies (Castilho et al., Biotechnol. J. 2021, 2000566) , we have used a truncated version of the luminal ACE2 ectodomain consisting of amino acids 18-615 fused to the Fc domain of human IgGl. This variant, referred to as ACE2-615-Fc, contains the RBD-binding interface as well as the complete protease domain. Hence, ACE2-615-Fc displays full enzymatic activity and is capable of neutralizing SARS-CoV-2 effectively. However, we have observed substantial fragmentation of ACE2-615-Fc in planta due to the action of endogenous N. benthamiana proteases, which cleave the fusion protein within or in close proximity of the linker peptide connecting its two subdomains. Notably, a variant containing a synthetic linker (termed ACE2-615L-Fc) is less prone to proteolytic cleavage than ACE2-615H-Fc, which contains the natural hinge region of IgGl as a linker between the ACE2 and Fc domains (Castilho et al., 2021) . The susceptibility of ACE2-615-Fc to proteolytic attack could relate to the absence of the collectrin domain responsible for intrinsic ACE2 dimerization. Therefore, we have now also expressed the full-length ectodomain of ACE2 (amino acids 18-740) as an Fc fusion protein (ACE2-740-Fc) in N. benthamiana and compared it to ACE2-615-Fc. We have found that this form is far more resistant to proteolytic fragmentation in planta than ACE2-615-Fc, which is particularly apparent for ACE2- 740H-Fc versus ACE2-615H-Fc (Figure 5) .

[0143] Sequence of ACE2 ectodomain (S19 / T92 isoform; SEQ ID NO: 7)

[0144] Q18STIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQ NLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLW AWESWRSEVGKQLRPLYEEYWLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLY EHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEA EKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAY AAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKW RWMVFKGEIPKDQWMKKWWEMKREIVGWEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAA KHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENLWGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGW STDWSPYAD615QSIKVRISLKSAGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRI SFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVS740

[0145] Sequence of the ACE2 ectodomain. The region missing in ACE2-

[0146] 615 is underlined. In other natural ACE2 isoforms, Serl9 and Thr92 are replaced by Pro and lie respectively.

[0147] Figure 5 shows that ACE2-740-Fc is less sensitive to proteolysis than ACE2-615-Fc, as evident from the reduction in Fc fragments. The constructs also differ in their linkers between the ACE2 and Fc domains (L, synthetic GGGGSGGGGS linker (SEQ ID NO: 8) ; H, natural IgGl hinge region) . Sample 1, ACE2-615L-Fc; sample 2, ACE2-615H-Fc; sample 3, ACE2-740L-Fc; sample 4, ACE2-740H-Fc .

[0148] ACE2-740H-Fc also displays a much higher virus-neutralizing activity than ACE2-615H-Fc and ACE2-615L-Fc (Figure 6) . Figure 6 shows that ACE2-740-Fc produced in N. benthamiana neutralizes SARS-CoV-2 (Delta variant) more effectively than ACE2-615-Fc (mean Ct values at 4 pg / ml: 30.3 / 30.1 vs. 28.3 / 24.7; at 0.8 pg / ml: 26.6 / 26.8 vs. 22.1 / 20.5) . The constructs differ in their linkers between the ACE2 and Fc domains (L, synthetic GGGGSGGGGS linker (SEQ ID NO: 8) ; H, IgGl hinge region) . The data are derived from 3 independent experiments performed in triplicates (n=9) . ACE2- (L) - Fc, ACE2-615L-Fc; ACE2- (L) -Fc Sia4, partially sialylated ACE2- 615L-Fc (co-expression of sialylation pathway) ; ACE2- (H) -Fc, ACE2- 615H-Fc; ACE2-Fc HEK, ACE2-615H-Fc produced in HEK293 cells; positive control, no ACE2-Fc added.

[0149] However, ACE2-740H-Fc made in N. benthamiana is affected by unwanted adventitious non-human post-translational modifications of its linker peptide connecting the ACE2 and Fc domains. Up to three of the six proline residues located in the linker peptide are hydroxylated by endogenous N. benthamiana prolyl hydroxylases (Figure 7) . These modifications are not observed when this protein is expressed in human HEK293 cells. Hence, these modifications could result in adverse immunological reactions when ACE2-740H-Fc produced in N. benthamiana is included in a drug designed to treat coronavirus infections of humans.

[0150] Sequence of ACE2-740H-Fc (S19 / T92 isoform; SEQ ID NO: 9)

[0151] QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLT VKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWES WRSEVGKQLRPLYEEYWLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHA YVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVS VGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLL RNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGE IPKDQWMKKWWEMKREIVGWEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKC DISNSTEAGQKLFNMLRLGKSEPWTLALENWGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQS IKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVS DIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSEPKSCDKTHTCPPCPAPELLGGPS VFI, FPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFL YSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0152] Sequence of ACE2-740-Fc. The linker peptide between the ACE2 domain and the hinge region (bold) of human IgGl Fc (italics) is underlined. In other natural ACE2 isoforms, Serl9 and Thr92 are replaced by Pro and lie respectively. Sequence of ACE2-728H-Fc (S19 / T92 isoform; SEQ ID NO : 10)

[0153] QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNL TVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAW ESWRSEVGKQLRPLYEEYWLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEH LHAYVRAKLMNAYPSYI SPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRI FKEAEK FFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAA QPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRW MVFKGEI PKDQWMKKWWEMKREIVGWEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKH EGPLHKCDI SNSTEAGQKLFNMLRLGKSEPWTLALENWGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTD WSPYADQSIKVRI SLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRI SFNF FVTAPKNVSDI I PRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQTHTCPPCPAPELLGGPS VFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFL YSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0154] Figure 7 shows that the tryptic peptide encompassing the linker sequence of ACE2 -740-Fc contains up to three hydroxylated proline residues (hyP ) when produced in N. benthamiana .

[0155] We have therefore developed a hitherto undescribed ACE2 -Fc variant , ACE2-728H-Fc ( Figure 8 ) . Figure 8 shows si ze-exclusion chromatography of af finity-puri fied ACE2-728H-Fc and ACE2-740H-Fc produced in N. benthamiana . The oligomers elute in the void volume of the column .

[0156] Sequence of ACE2-728H-Fc . The hinge region of human IgGl Fc ( italics ) is highlighted by bold letters . In other natural ACE2 isoforms , Serl 9 and Thr92 are replaced by Pro and l ie respectively .

[0157] ACE2-728H-Fc lacks the structurally flexible 12 C-terminal amino acids of the full-length ACE2 ectodomain, including the 0- glycosylation site Thr730 ( only used in mammals ) as well as five proline residues . ACE2-728H-Fc also lacks the first 7 amino acids (EPKSCDK) of the hinge region, which includes another proline and an unpaired cysteine residue . The absence of the latter could be responsible for the far lower propensity of ACE2 -728H-Fc to form higher oligomers as compared to ACE2-740H-Fc ( Figure 8 ) .

[0158] Importantly, the absence of the proline-rich segment in ACE2- 728H-Fc did not af fect its resistance to endogenous N. benthamiana proteases . We also observed no changes to the N-glycosylation profile of the protein ( Figure 9 ) as well as to its high af finity for the RBD of the SARS-CoV-2 spike protein as measured by surface plasmon resonance (Figure 10) . Figure 9 shows N-glycosylation profiles of ACE2-740H-Fc and ACE2-728H-Fc produced in N. benthamiana as shown here for N-glycosite 6 (peptide CDISNSTEAGQK; SEQ ID NO: 11; ACE2 aa 542-553) . In both proteins, the most abundant N-glycan structure is the complex-type N-glycan GnGn (GlcNAc2Man3GlcNAc2) . Figure 10 shows binding of ACE2-740H-Fc and ACE2-728H-Fc produced in N. benthamiana to the RBD of SARS-CoV-2 spike protein (Wuhan strain) as measured by surface plasmon resonance (SPR) . KD, equilibrium dissociation constant.

[0159] Furthermore, ACE2-728H-Fc was as efficient as ACE2-740H-Fc in neutralizing SARS-CoV-2 in tissue culture (Figure 11) . Figure 11 shows neutralization of SARS-CoV-2 (Delta variant) by ACE2-740-Fc and ACE2-728H-Fc produced in N. benthamiana. The virusneutralizing activity of the two proteins is very similar (mean Ct values at 0.8 pg / ml: 29.0 vs. 27.6) . The data are derived from 3 independent experiments performed in triplicates (n=9) . Ct values >30: complete neutralization; Ct values <20: no neutralization. Positive control: no ACE2-Fc added.

[0160] Figure 12 shows the neutralization of different SARS-CoV-2 variants of concern (VOC) by ACE2-728H-Fc produced in N. benthamiana and nirmatrelvir (Pf332) . ACE2-728H-Fc neutralizes Omicron strains of SARS-CoV-2 (om21, om23) equally well as Delta (de21) and up to 6 times better than the ancestral Wuhan isolate (whl9) . This is not observed for Pf 322. Data are presented as ratios of the IC50 values for the respective VOC and the Wuhan strain .

[0161] ACE2-728H-Fc isolated by affinity chromatography represents a mixture of dimeric, tetrameric and higher oligomeric forms of the protein (see Figure 8) . By means of size-exclusion chromatography, the affinity-purified material can be fractionated into dimer and oligomer fractions. The oligomer fraction consisting of tetrameric and higher oligomeric forms of the protein showed higher virus-neutralizing activity than dimeric ACE2-728H- Fc . Notably, the virus-neutralizing activity of the unfractionated mixture of ACE2-728H-Fc forms was similar to that of the isolated ACE2-728H-Fc oligomers (Figure 13) . Figure 13 shows neutralization of SARS-CoV-2 (Delta variant) by dimeric and oligomeric ACE2-728H- Fc produced in N. benthamiana. The virus-neutralizing activity of the oligomeric fraction (tetramer plus higher oligomers) is higher than of the dimer (mean Ct values at 0.8 pg / ml: 25.7 vs. 15.1) . The data are derived from 2 independent experiments performed in triplicates (n=6) . Ct values >30: complete neutralization; Ct values <13: no neutralization. Positive control (Ctrl) : no ACE2- Fc added. ACE2-728H-Fc mix: unfractionated mixture of dimer, tetramer and higher oligomers before size-exclusion chromatography .

[0162] Taken together, the biophysical properties of ACE2-728H-Fc are superior to those of the canonical variant ACE2-740H-Fc . These features make ACE2-728H-Fc preferable from the manufacturing point of view since they will allow for a less sophisticated downstream process and thus result in higher product yields. ACE2-728H-Fc is also of higher biological quality since it lacks the proline residues present in the linker peptide of ACE2-740H-Fc, which are subjected to undesired post-translational hydroxylation and glycosylation reactions when ACE2-740H-Fc is produced in N. benthamiana . The presence of these unwanted hydroxyproline and arabinose residues could lead to immunological complications when ACE2-740H-Fc is applied to humans. This potential matter of concern does not apply to any drug containing ACE2-728H-Fc .

[0163] Combination of ACE2 decoys with other substances active against SARS-CoV-2

[0164] The virus-neutralizing activity of ACE2 decoys can be further enhanced by combination with other substances active against SARS- CoV-2 which exhibit different modes of action. We have therefore tested the combination of unfractionated ACE2-728H-Fc with a proprietary resveratrol analogue (hexahydroxystilbene; M08) . This combination showed a >10-fold higher virus-neutralizing activity than ACE2-728H-Fc alone (Figure 14) . Figure 14 shows neutralization of SARS-CoV-2 (Delta variant) by ACE2-728H-Fc produced in N. benthamiana (pTrak728) and hexahydroxystilbene (M08) . The virus-neutralizing activity of ACE2-728H-Fc can be enhanced by the addition of hexahydroxystilbene (mean Ct values (- / + M08) at 0.16 pg / ml: 16.6 vs. 20.3) . Ct values >28: complete neutralization; Ct values <13: no neutralization. Positive control (Ctrl) : no ACE2-Fc or M08 added.

[0165] Hexahydroxystilbene (3, 3', 4, 4', 5, 5' -hexahydroxy- transstilbene; M08) is a synthetic analogue of resveratrol, an ingredient of red wine. This compound is an excellent free radical scavenger and inhibits cyclooxygenase 2 more potently than celecoxib, a drug used for the symptomatic relief of rheumatoid arthritis patients. Interestingly, it was also demonstrated that it potently inhibits infection by HIV-1 by targeting a very early step in HIV entry into host cells. Furthermore, we have found that hexahydroxystilbene is highly active in inhibiting SARS-CoV-2 replication (WO 2022 / 200086 Al) .

[0166] We have also tested ACE2-728H-Fc in combination with the SARS- CoV-2 main protease inhibitor nirmaltrevir (Pf332; Abdelnabi et al., Nat Commun. 2022; 13:719. doi : 10.1038 / s41467-022-28354-0) , which is already clinically used for the treatment of COVID-19 patients. The combination of ACE2-728H-Fc and nirmaltrevir showed a >20-fold higher virus-neutralizing activity than ACE2-728H-Fc alone (Figure 15) . Figure 15 shows neutralization of SARS-CoV-2 (Delta variant) by ACE2-728H-Fc produced in N. benthamiana (pTrak728) and nirmatrelvir (Pf332) . The virus-neutralizing activity of ACE2-728H-Fc can be enhanced by the addition of nirmatrelvir (mean Ct values (- / + Pf332) at 0.16 pg / ml: 16.6 vs. 21.8) . Ct values >28: complete neutralization; Ct values <13: no neutralization. Positive control (Ctrl) : no ACE2-Fc or Pf332 added .

[0167] These results show that a combined formulation of human wildtype ACE2-728H-Fc (isoforms Serl9 / Thr92, Serl9 / Ile92, Prol9 / Thr92 or Prol9 / Ile92) produced in N. benthamiana together with hexahydroxystilbene or nirmaltrevir represents a superior treatment for COVID-19.

[0168] Neutralization of SARS-CoV-2 by ACE2-728H-Fc in air liquid interface cultures

[0169] Air liquid interface (ALI) cultures are physiologically relevant models for studying the respiratory epithelium in vitro. We have found that pre-treatment of SARS-CoV-2 with 20 pg / ml unfractionated ACE2-728H-Fc efficiently protects human Calu-3 ALI cultures and ALI cultures of primary human lung epithelial cells from viral infection (Figures 16, 17 and 18) . Figure 16 shows neutralization of SARS-CoV-2 (Omicron variant) by unfractionated ACE2-728H-Fc produced in N. benthamiana (pTRAK-728H-Fc mix) and hexahydroxystilbene (M08) in human Calu-3 ALI cultures (RT-qPCR analysis) . Pre-treatment with 20 pg / ml ACE2-728H-Fc alone or in combination with 60 pM hexahydroxystilbene (M08) leads to minimal release of viral particles into the supernatant during 24 h of incubation. Positive control: no ACE2-Fc or M08 added. Figure 17 shows neutralization of SARS-CoV-2 (Omicron variant) by unfractionated ACE2-728H-Fc produced in N. benthamiana and hexahydroxystilbene (M08) in human Calu-3 ALI cultures (immunohistochemical staining) . Pre-treatment with 20 pg / ml ACE2- 728H-Fc alone or in combination with 60 pM hexahydroxystilbene (M08) eliminates the detection of SARS-CoV-2 N protein (dark) in ALI cultures fixed 24 h after addition of SARS-CoV-2. Positive control: no ACE2-Fc or M08 added. Negative control: no SARS-CoV-2 added. Figure 18 shows neutralization of SARS-CoV-2 (Omicron variant) by unfractionated ACE2-728H-Fc produced in N. benthamiana in ALI cultures of primary human lung epithelial cells (immunohistochemical staining) . Pre-treatment with 20 pg / ml ACE2- 728H-Fc eliminates the detection of SARS-CoV-2 N protein (dark) in ALI cultures fixed 24 h after addition of SARS-CoV-2. Positive control: no ACE2-Fc added.

[0170] Proline hydroxylation and arabinosylation of ACE2-740-Fc in plants The "canonical" ACE2-740-Fc made in N. benthamiana is affected by unwanted adventitious non-human post-translational modifications of its linker peptide connecting the ACE2 and Fc domains. Up to three of the six proline residues located in the linker peptide are hydroxylated by endogenous N. benthamiana prolyl hydroxylases (Figure 7) . These modifications are not observed when this protein is expressed in human HEK293 cells. Hence, these modifications could result in adverse immunological reactions when ACE2-740-Fc produced in N. benthamiana is included in a drug designed to treat coronavirus infections of humans. All three hydroxylated proline residues are located in the chymotryptic / tryptic peptide Leu725-Lys743 (Figure 19) . Figure 19 shows proline hydroxylation of ACE2-740-Fc expressed in plants. The chymotryptic / tryptic peptide encompassing the linker sequence of ACE2-740-Fc (L725-K743) contains up to three hydroxylated proline residues (hyP) when produced in N. benthamiana. Quantitation of the peak areas resulted in the following estimates of the abundance of each isoform: unmodified peptide 48%, IhyP 30%, 2hyP 15%, 3hyP 7% (mean of three biological replicates) . Notably, substantial fractions of the 2hyP and 3hyP isoforms can be further modi fied with a single trisaccharide unit consisting of three arabinose residues ( 2hyP : 16% ; 3hyP : 23% ) .

[0171] MS / MS-based analysis demonstrated that the three modi fied proline residues are Pro733 , Pro734 and Pro738 . These experiments also revealed that the arabinose trisaccharide is attached to Pro733 or Pro734 ( Figure 20 ) . Figure 20 shows proline hydroxylation of ACE2-740-Fc expressed in plants . MS / MS analysis of the chymotryptic / tryptic peptide L725-K743 containing three hydroxylated proline residues ( Pro733 , Pro734 and Pro738 ) and a single arabinose trisaccharide attached to Pro733 or Pro734 .

[0172] The presence of these partially arabinosylated hydroxyproline residues could lead to immunological complications when ACE2-740- Fc is applied to humans . This potential matter of concern does not apply to any drug containing ACE2-728-Fc, since this protein lacks Pro733 , Pro734 and Pro738 .

[0173] Generation of a non-neutralizing ACE2-728H-Fc variant

[0174] In contrast to human ACE2 , mouse ACE2 does not bind to the

[0175] Sequence of non-neutralizing ACE2-728H-Fc (SEQ ID NO : 12 )

[0176] QSTIEENAKTFLNNFNQEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTTAQSFPLQEIQNLT VKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWES WRSEVGKQLRPLYEEYWLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHA YVRAKLMNAYPSYI SPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRI FKEAEKFFVS VGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGHGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLL RNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGE

[0177] I PKDQWMKKWWEMKREIVGWEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKC DISNSTEAGQKLFNMLRLGKSEPWTLALENWGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQS IKVRI SLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRI SFNFFVTAPKNVS DII PRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTC VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDEL TKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKL T VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK spike protein of many SARS-CoV-2 isolates including the original Wuhan strain and the Delta variant ( Li et al . , Cell Discov . 2022 ; 8 : 65 . doi : 10 . 1038 / s41421 - 022- 00431- 0 ) . Human and mouse ACE2 di ffer at 8 positions in the Spike-binding region ( Li et al . , J . Virol . 2020 ; 94 : e01283-20 . doi : 10 . 1128 / JVI . 01283-20 ) . We have developed a hitherto undescribed human ACE2 -Fc variant , non- neutralizing ACE2-728H-Fc, by replacing these 8 amino acids with their mouse ACE2 counterparts (SEQ ID NO: 12) .

[0178] Sequence of non-neutralizing ACE2-728H-Fc . The changed residues (N24, N30, N31, Q34, T79, S82, F83, H353; preproprotein numbering) are underlined. The hinge region of human IgGl Fc (italics) is highlighted by bold letters.

[0179] Figure 21 shows that the non-neutralizing variant of ACE2-728H-Fc (mutACE2-728H-Fc) does not neutralize SARS-CoV-2 even at the highest concentration tested (10 pg / ml) . In contrast, less than 0.15 pg / ml wild-type ACE2-728H-Fc are required for 50% inhibition in plaque-reduction neutralization tests.

[0180] Treatment of virus-infected Syrian hamsters with ACE2-728H-Fc

[0181] Golden Syrian hamsters are currently the best non-transgenic small animal model for studies of SARS-CoV-2 infection and countermeasure development (Imai et al., Proc. Natl. Acad. Sci. USA 2020; 117,16587-16595. doi: 10.1073 / pnas .2009799117 ) . Hamsters (6-8 weeks of age; three female and three male animals per group) were intranasally inoculated with 104TCID50 (50% tissue culture infectious doses) of SARS-CoV-2 (Wuhan strain) per animal (100 pl total, 50 pl / nostril) . Animals from the untreated control group (negative control) received the same volume of phosphate-buff ered saline solution (PBS) . At day 1, 2, 3, and 4 post-challenge animals were treated intranasally as described above with 100 pl 2.5 mg / ml ACE2-728H-Fc, mutACE2-728H-Fc or PBS. The body weight was recorded every day until the end of the experiment. At 5 days postchallenge, all animals were euthanised and lung extracts tested for the presence of infectious SARS-CoV-2.

[0182] Figure 22 shows that intranasal application of 2.5 mg / ml ACE2- 728H-Fc significantly reduced weight loss whereas weight loss could not be prevented by treatment with 2.5 mg / ml of the nonneutralizing variant mutACE2-728H-Fc . Furthermore, the median infectious virus levels were significantly lower in the lungs of hamsters treated with ACE2-728H-Fc than in those receiving PBS or mutACE2-728H-Fc instead.

[0183] Generation of an ACE2-728H-Fc (IgA) variant Passive delivery of antibodies to mucosal sites may be a valuable adjunct to conventional antiviral vaccination approaches. Using therefore the Fc part of IgA, the antibody class predominantly found at these sites, may provide unique structural and functional properties that may enhance its stability and persistence in the mucosal environment.

[0184] Since the biophysical properties of ACE2-728H-Fc make it the preferred variant from the manufacturing point of view, the Fc part of human IgAl (SEQ ID NO: 4) was linked to ACE2-728H. Affinity- purified unfractionated ACE2-728H-Fc ( IgA) produced in N. benthamiana exhibited comparable binding properties to the receptor binding domain (RBD) as the IgG-fusion variant (Figure 23) . The virus neutralization activity was tested on VeroE6 cells with different concentrations (starting with 20pg / ml) of ACE2- 728H-Fc(IgG) and ACE2-728H-Fc ( IgA) (Figure 24) . It revealed that less than 0.25 pg / ml of ACE2-728H-FC ( IgG) and 0.57 pg / ml ACE2- 728H-Fc(IgA) are required for 50% inhibition in plaque-reduction neutralization tests.

Claims

Claims1 . An isolated polypeptide comprising an ACE2 moiety and a stability-increasing polypeptide moiety, wherein the ACE2 moiety consists of amino acid residues 18 to 728 of the ACE2 protein ( SEQ ID NO : 2 ) or a biologically active ACE2 mutant or ACE2 variant polypeptide thereof ; and wherein the stability-increasing polypeptide moiety increases the stability of the ACE2 moiety in the course of administration to a patient .2 . A polypeptide according to claim 1 , wherein the ACE2 moiety and the stability-increasing polypeptide moiety are linked by a linker peptide and / or a hinge region of a human immunoglobulin, preferably a hinge region of human IgGl , human IgA or human IgM .3 . A polypeptide according to claim 1 or 2 , wherein the stability- increasing polypeptide is an Fc moiety, preferably an Fc moiety selected from a human IgGl or a human IgA or a human IgM .4 . A polypeptide according to any one of claims 1 to 3 , wherein the stability-increasing polypeptide is a Fc moiety selected from SEQ ID NO : 3 , SEQ ID NO : 4 , SEQ ID NO : 5 or SEQ ID NO : 6 or a fragment or variant thereof capable of extending hal f-li fe of ACE2 , preferably a fragment consisting of amino acid residues 106 to 330 , with the exchange of P01857- 1 ( 329 and 330 being GK instead of EL ) ; or a suitable IgAl fragment or a suitable IgA2 fragment or a suitable IgM fragment .5 . A polypeptide according to any one of claims 1 to 4 , wherein the ACE2 variant or ACE2 mutant has a KDof 50 nM or less as measured by surface plasmon resonance ( SPR) , preferably a KDof 20 nM or less as measured by SPR, more preferred a KDof 10 nM or less as measured by SPR, especially a KDof 5 nM or less as measured by SPR .6 . A polypeptide according to any one of claims 1 to 5 , where- in the ACE2 variant or ACE2 mutant has a KD of 1 nM or less as measured by SPR, preferably a KD of 800 pM or less as measured by SPR .7 . A polypeptide according to any one of claims 1 to 6 , wherein the ACE2 moiety comprises an exchange at an amino acid corresponding to amino acid position 19 ( S ) and / or at amino acid position 92 ( T ) of SEQ ID NO : 1 , preferably wherein the ACE2 moiety comprises an S 19P and / or a T92 I exchange .8 . A polypeptide according to any one of claims 1 to 7 , wherein the ACE2 moiety comprises a substitution selected from N90Q, T92Q, N322Q, H374N and H378N .9 . A nucleic acid molecule encoding a polypeptide according to any one of claims 1 to 8 .10 . A nucleic acid molecule according to claim 9 further comprising a signal peptide encoding region, wherein the signal coding region is not an ACE2 signal peptide , or an IgGl , IgAl , IgA2 or IgM signal peptide , wherein the signal peptide is preferably a plant signal peptide, more preferred a signal peptide of barley, especially barley a-amylase signal peptide of barley .11 . A method for producing a polypeptide according to any one of claims 1 to 8 , wherein a nucleic acid according to any one of claims 9 or 10 is expressed in a host cell , preferably in a plant host , especially in Ni cotiana benthamiana .12 . A polypeptide according to any one of claims 1 to 8 for use in neutralising SARS-CoV- 1 and SARS-CoV-2 .13 . A pharmaceutical preparation comprising a polypeptide according to anyone of claims 1 to 8 or a nucleic acid according to any one of claims 9 or 10 and a pharmaceutically acceptable carrier, preferably provided and finished for inhalation or nasal delivery, especially in an aerosol formulation .14 . A pharmaceutical preparation according to claim 13 further comprising an antiviral agent , preferably an antiviral agent selected from the group convalescent plasma, fusion inhibitors , monoclonal or polyclonal antibodies , nucleoside analogs , polymerase inhibitors , receptor decoys , protease inhibitors , translationinhibitors, endocytosis inhibitors, interferons, kinase inhibitors, and lipidomic drugs, especially 3, 3 ' , 4, 4 ' , 5, 5 ' -hexahydroxy- trans-stilbene, nirmatrelvir , ritonavir, remdesivir, dexamethasone or combinations thereof, either as a physical combination or as a set of at least two separate pharmaceutical preparations.

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