SINGLE DOMAIN ANTIBODIES AGAINST HACE2 AND THEIR USE TO PREVENT SARS-COV-2 INFECTION

FIELD OF THE INVENTION

The present invention relates to molecules with therapeutic activity against a coronavirus infection.

BACKGROUND TO THE INVENTION

In December 2019, a novel coronavirus (SARS-CoV-2, COVID-19 or 2019-nCoV) crossed species barriers to infect humans and was effectively transmitted from person to person, leading to a pneumonia outbreak first reported in Wuhan, China. This virus causes coronavirus disease-19 (COVID-19) with influenza like symptoms ranging from mild disease to severe lung injury and multi-organ failure, eventually leading to death, especially in older patients with other co-morbidities. The WHO has declared that COVID-19 is a public health emergency of pandemic proportions. The SARS-CoV-2 pandemic is not only an enormous burden to public health but has already markedly affected civil societies and the global economy.

SARS-CoV-2 is currently considered a pandemic infection involving 190 countries, with more than 150 million confirmed cases and more than 3.2 million confirmed deaths worldwide, as of 4 ^th May 2020. Trials are currently ongoing for the antiviral reagents Remdesivir (Gilead), Chloroquine and hydroxychloroquine, and Ritonavir/Lopinavir (Kaletra, Abb Vie). Other companies such as EliLilly/ AbCeller, Takeda, and Regeneron have also announced intentions to test cocktails of neutralising antibodies, while more than 35 vaccine strategies are currently being investigated. Presently, no specific anti-viral treatment has been approved.

Recent structural data has elucidated the mechanism by which SARS-CoV-2 viral particles enter cells. SARS-CoV-2 has been shown to bind to angiotensin-converting enzyme 2 (ACE2) via the spike protein (S protein) on its surface (Figure 1 A). In a recent publication, Lei at al. described a recombinant ACE2-Fc fusion protein able to neutralize SARS-CoV-2 (Lei et al., 2020, bioRxiv 2020.02.01.929976; https://doi.org/10.1101/2020.02.01.929976). A similar construct was also effective against SARS-CoV in 2003 (Moore et al., 2004, J Virol 78:10628-35). A pilot clinical trial from GSK (NCT01597635) using a recombinant version of ACE2 (GSK2586881) proved to be well tolerated in patients with acute respiratory distress syndrome (Khan et al., 2017, Crit Care 21:234). However, it is not clear whether the catalytic activity of ACE2 will have an adverse effect on the renin-angiotensin system and provoke detrimental and possibly long-term effects on patients.

Although SARS-CoV and SARS-CoV-2 S proteins share a high amino acid homology (76.5%), neutralising antibodies against SARS-CoV have been shown to decrease SARS- CoV-2 infectivity but did not block it entirely (Hoffmann et al., 2020, Cell, in press; Walls et al., 2020, Cell, in press). Moreover, due to the nature of SARS-CoV-2, a high mutation rate is expected as a result of selective pressure. Several strain variants have already been described in the literature, which in some cases presented mutations within the receptor binding domain (RDB) of the S protein (Ou et al., 2020, bioRxiv 2020.03.15.991844; doi: 10.1101/2020.03.15.991844; Wang et al., 2020, J Med Virol, early view; doi: 10.1002/jmv.25762). Some of the RDB mutations resulted in a higher binding affinity for ACE2 (Ou et al., 2020, bioRxiv 2020.03.15.991844; doi: 10.1101/2020.03.15.991844). This occurrence may prevent successful application of neutralising antibodies in clinical therapy.

Paradoxically, non-neutralizing antibodies to variable S domains may enable an alternative infection pathway via Fc receptor-mediated uptake. These antibodies can act to enhance viral infection by aiding viral entry into cells which don’t express the target receptor. This mechanism of improved virus uptake, termed antibody-dependent enhancement (ADE) of infection. Prior studies involving anti-spike protein antibodies (Yip et al., 2014, Virol J 11:82) and vaccine candidates for SARS-CoV (Wang et al., 2014, Biochem Biophys Res Commun 451:208-14) and Middle East Respiratory Syndrome coronavirus (MERS-CoV) (Agrawal et al., 2016, Hum Vaccine Immunother 12:2351-6) demonstrate vaccination- induced ADE of disease, including infection of phagocytic antigen presenting cells (APC) - so-called extrinsic ADE. Once internalised, these immunocomplexes may modulate innate antiviral cells responses to increase virus production substantially in each cell, a process termed intrinsic ADE. Together, extrinsic and intrinsic ADE are thought to prompt the massive release of inflammatory and vasoactive mediators that ultimately contribute to disease severity. Sequence and structural conservation of S motifs suggests that SARS and MERS vaccine ADE risks may foreshadow the risks of SARS-CoV-2 S-based vaccine or antibody mediated approaches.

To date, a limited number of therapeutics and vaccines have been approved against any human-infecting coronaviruses, including SARS-CoV-2. Therefore, there is a need in the art to provide effective therapeutics for the treatment of human-infecting coronaviruses and, especially, SARS-CoV-2.

SUMMARY OF ASPECTS OF THE INVENTION

The present inventors have generated a series of antiviral agents with ability to block infection by coronaviruses, and SARS-CoV and SARS-CoV-2 in particular. These molecules are based on antibodies that are specific for ACE2 that have been fused to serum half-life extending moieties. The ability of these molecules to block coronavirus virions entry into host cells is increased by combining antibodies to different epitopes on ACE2. Through stronger interactions with the ACE2 receptor, the inefficient neutralisation capacity of previous targeting approaches is improved. Furthermore, these fusion proteins do not interfere with the catalytic activity of ACE2 and thus do not interfere with its key role in maintaining blood pressure via the Renin- Angiotensin System (RAS).

Targeting ACE2 is also advantageous because the host receptor does not change and thus viral escape from binding the therapeutic agent is prevented. Moreover, if the binding site for the virus is occluded the virus will not have the ability to mutate and bind an entirely new host receptor in the time frame of the current SARS-CoV-2 outbreak.

Thus, in a first aspect, the present invention provides a polypeptide comprising: a) an antigen-binding domain that binds specifically to the human angiotensin converting enzyme type 2 (hACE2) ectodomain; and b) a half-life extending domain.

The antigen-binding domain and the half-life extending domain may be joined by a linker. The polypeptide may further comprise at least one additional antigen-binding domain that binds specifically to the hACE2 ectodomain, wherein the antigen-binding domain and the at least one additional antigen-binding domain bind to different epitopes of the hACE2 ectodomain.

The antigen-binding domain and/or the at least one additional antigen-binding domain, where present, may compete with a coronavirus spike protein (S protein) for binding to hACE2.

The binding of the antigen-binding domain and the at least one additional antigen-binding domain, where present, to hACE2 may not inhibit its enzymatic activity.

The antigen-binding domain and the at least one additional antigen-binding domain, where present, may bind to an epitope in hACE2 which does not comprise one or more of amino acids R169, R273, H345, P346, T371, H374, Q375, H378, E402, W477, K481, H505, or Y515 of the sequence shown as SEQ ID NO: 1.

The antigen-binding domain and/or at least one additional antigen-binding domain, where present, may bind to the cell-bound hACE2 ectodomain.

The antigen-binding domain and the at least one additional antigen-binding domain, where present, may comprise the CDR1, CDR2, and CDR3 from one of the sequences shown as SEQ ID NO: 5-11 or 50-97.

The antigen-binding domain and the at least one additional antigen-binding domain, where present, are selected from a scFv or a domain antibody selected from a VH or a dAb, or a single-domain antibody (sdAb), or a VHH or a nanobody.

The half-life extending domain is selected from albumin, or an albumin domain, or an antigen-binding domain that binds specifically to albumin, or an albumin-binding-peptide, or an albumin-binding domain of a Streptococcus protein, or transferrin, or a hetero oligomerization domain, or a polyethylene glycol moiety. In a second aspect, the present invention provides a nucleic acid encoding the polypeptide according to the first aspect of the invention.

In a third aspect, the present invention provides an expression cassette comprising the nucleic acid according to the second aspect of the invention.

In a fourth aspect, the present invention provides a vector comprising the nucleic acid according to the second aspect of the invention or the expression cassette according to the third aspect of the invention.

In a fifth aspect, the present invention provides a cell comprising the nucleic acid according to the second aspect of the invention, or the expression cassette according to the third aspect of the invention, or the vector according to the fourth aspect of the invention.

In a sixth aspect, the present invention provides a method for making a polypeptide according to the first aspect of the invention by culturing a cell according to the fifth aspect of the invention and purifying the polypeptide from the supernatant.

In a seventh aspect, the present invention provides a pharmaceutical composition comprising the polypeptide according to the first aspect of the invention and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

In an eighth aspect, the present invention provides a polypeptide according to the first aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention for use in medicine.

In a ninth aspect, the present invention provides a polypeptide according the first aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention for use in the treatment of a coronavirus infection or a condition or disorder resulting from this infection.

In a tenth aspect, the present invention provides a use of a polypeptide according to the first aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention in the manufacturing of a medicament for the treatment of a coronavirus infection or a condition or disorder resulting from this infection.

In an eleventh aspect, the present invention provides a method for treating a coronavirus infection or a condition or disorder resulting from this infection in a subject in need thereof comprising a step of administering a polypeptide according to the first aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention to the subject.

In a twelfth aspect, the present invention provides a method of neutralising a coronavirus infection, comprising a step of contacting a polypeptide according to the first aspect of the invention or a pharmaceutical composition according to the seventh aspect of the invention with a cell expressing hACE2.

The coronavirus in the ninth, tenth, eleventh, or twelfth aspects of the invention may be SARS-CoV-2.

DESCRIPTION OF THE FIGURES

Figure 1. A. X-ray crystal structure of the ACE2-S1 complex. A) ACE2 (bottom, light grey), SI (top, dark grey) complex X-ray crystal structure (6M0J). Catalytic, substrate binding and ion binding residues of ACE 2 highlighted in black. B) X-ray crystal structure of ACE2. Catalytic, substrate binding and ion binding residues of ACE 2 highlighted in black. Dashed line indicating area for blocking antibody intervention.

Figure 2. Half-life extended anti ACE2 fusion constructs. A) dAb-albumin based fusion proteins. B) dAb-Fc hetero-oligomer based fusion proteins.

Figure 3. Schematic of naive llama library display. Potential hits identified are shown in right hand box.

Figure 4. Characterisation of the binding specificity of dAbs specific for the human ACE2 protein. Black bars show binding to human ACE2; white bars show non-specific binding control to unrelated B7H3 peptides; grey bars show non-specific binding to milk.

Figure 5. Binding of anti-ACE2 dAbs (VHH) to human ACE2.

Binding to hACE2 was detected by ELISA on ACE2-Fc-coated wells. Individual anti-ACE2 dAb (VHH) clones in VHH-Fc format (VHH-MuIgG2a Fc) were evaluated. Bars show binding to human ACE2.

Figure 6. Blocking of SI binding to human ACE2 by anti-ACE2 dAbs (VHH).

A. Blocking of the binding of SARS-CoV2 SI protein to hACE2 by anti-ACE2 dAb (VHH) clones in VHH-Fc format (VHH-MuIgG2a Fc) was assessed by ELISA on ACE2-Fc-coated wells. Bars show binding of SARS-CoV2 SI protein to human ACE2. B. Blocking activity of three dAb (VHH) clones shown as the percentage of binding of SARS-CoV2 SI protein to hACE2, relative to no antibody condition (Baseline - no SI - subtracted).

Figure 7. ACE2 enzymatic activity in the presence of anti-ACE2 dAbs (VHH).

Enzymatic activity of active ACE2-Fc for substrate Mca-APK(Dnp) in the presence of individual anti-ACE2 dAb (VHH) clones in VHH-Fc format (VHH-MuIgG2a Fc). Bars show half-maximal activity or reaction half-life or tl/2 (s) of human ACE2. For clones C76 and C82, enzymatic activity was strongly inhibited and a tl/2 value could not be determined.

Figure 8. SARS-CoV-2 pseudovirus neutralization

Neutralization capacity for anti-ACE2 dAb (VHH) clones C55, C66, C72 and C84 on pseudotyped viral vectors expressing Wuhan (wild type) and B.l.1.7 spike protein. Neutralisation % expressed relative to buffer condition. ACE2-Fc was used as positive control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides recombinant fusion proteins which have the ability to block viral entry of coronavirus, and SARS-CoV-2 virus in particular, and viral re-entry. These fusion proteins are based on antibodies binding to ACE2. 1. Coronavirus

Three coronaviruses have crossed the species barrier to cause deadly pneumonia in humans since the beginning of the 21 ^st century: severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS- CoV-2. In 2002-2003, SARS-CoV, a lineage B beta-CoV, emerged from bat and palm civet, and infected over 8,000 people and caused about 800 deaths. In 2012, MERS-CoV, a lineage C beta-CoV, was discovered as the causative agent of a severe respiratory syndrome in Saudi Arabia, currently with 2,494 confirmed cases and 858 deaths, it remains endemic in Middle East, and dromedary camel is considered as the zoonotic reservoir host of MERS-CoV. At the end of 2019, a novel coronavirus, named SARS-CoV-2, was found in patients with severe pneumonia in Wuhan, China. Viruses were isolated from patients and sequenced. Phylogenetical analysis revealed that it is a lineage B beta-CoV and closely related to a SARS-like (SL) CoV, RaTG13, discovered in a cave of Yunnan, China, in 2013. They share about 96% nucleotide sequence identities, suggesting that SARS-CoV-2 might have emerged from a Bat SL-CoV. However, the intermediate host or whether there is an intermediate host remains to be determined.

In addition to the highly pathogenic zoonotic pathogens SARS-CoV, MERS-CoV, and SARS-CoV-2, all belonging to the b-coronavirus genus, four low-pathogenicity coronaviruses are endemic in humans: HCoV-OC43, HCoV-HKUl, HCoV-NL63, and HCoV-229E.

The coronaviruses (order Nidovirales , family Coronaviridae, genus Coronavirus) are a diverse group of large RNA viruses that cause varieties of diseases in humans and other animals, including respiratory, enteric, renal, and neurological diseases. Coronaviruses are enveloped viruses that contain a large single-stranded RNA genome of positive polarity. At ~30,000 nucleotides (nt), their genome is the largest found in any of the RNA viruses. Their envelope accommodates three or four membrane proteins of which the membrane (M), envelope (E), and spike (S) proteins are common to all. The S protein is a relatively large, about 180kDa type I glycoprotein, turners of which form the petal-shaped projections on the surface of the virion that give rise to the characteristic corona solis-like appearance. It has been suggested that the SI subunit constitutes the globular head, while the S2 subunit forms the stalk-like region of the spike.

The two functions of the coronavirus S protein appear to be spatially separated. The SI subunit (or the equivalent part in viruses with uncleaved S protein) is responsible for receptor binding, and the S2 subunit is responsible for membrane fusion. In the structure, N- and C- terminal portions of SI fold as two independent domains, N-terminal domain (NTD) and C- terminal domain (C-domain). Depending on the virus, either NTD or C-domain can serve as the receptor-binding domain (RBD). While RBD of mouse hepatitis virus (MHV) is located at the NTD, most of other CoVs, including SARS-CoV and MERS-CoV use C-domain to bind their receptors. MHV uses mouse carcinoembryonic antigen related cell adhesion molecule la (mCEACAMla) as the receptor, and the receptors for SARS-CoV and MERS- CoV are human angiotensin-converting enzyme 2 (hACE2) and dipeptidyl peptidase 4 (DPP4), respectively. In terms of sequence identity, S proteins of SARS-CoV-2 share about 76% and 97% of amino acid identity with SARS-CoV and RaTG13, respectively, and the amino acid sequence of potential RBD of SARS-CoV-2 is about 74% and 90.1% homologous to that of SARS-CoV and RaTG13, respectively.

The ectodomain of the S2 subunit contains two heptad repeat (HR) regions, HRl and HR2, characteristic of coiled coils, while the fusion peptide (FP) is predicted to be located amino terminally of the first HR region (HRl). Binding of the SI subunit to the (soluble) receptor has been shown to trigger conformational changes that supposedly facilitate virus entry by activation of the fusion function of the S2 subunit. The conformational changes are thought to expose the fusion peptide and to lead to the formation of a heterotrimeric six-helix bundle by the two HR regions, a characteristic of class I viral fusion proteins, resulting in the close locations of the fusion peptide and the transmembrane domain in the process of membrane fusion.

Coronavirus S proteins are typical class I viral fusion proteins, and protease cleavage is required for activation of the fusion potential of S protein. A two-step sequential protease cleavage model has been proposed for activation of S proteins of SARS-CoV and MERS- CoV, priming cleavage between SI and S2 and activating cleavage on S2’ site. Depending on virus strains and cell types, CoV S proteins may be cleaved by one or several host proteases, including furin, trypsin, cathepsins, transmembrane protease serine protease-2 (TMPRSS-2), TMPRSS-4, or human airway trypsin-like protease (HAT). Availability of these proteases on target cells largely determines whether coronaviruses enter cells through plasma membrane or endocytosis. It has been reported that SARS-CoV-2 S protein is capable of triggering protease-independent and receptor-dependent syncytium formation. Such a mechanism might enhance virus spreading through cell-cell fusion and this might partially explain rapid progress of the disease.

The coronavirus may be one of the following coronaviruses: SARS-CoV-2, SARS-CoV, SARS-like CoV RaTG13, MERS-CoV, HCoV-OC43, HCoV-HKUl, HCoV-NL63, and HCoV-229E.

The coronavirus may be selected from SARS-CoV-2, SARS-CoV or SARS-like CoV RaTG13.

The coronavirus may be SARS-CoV-2.

The coronavirus S protein may be the S protein of one of the following coronavirus: SARS- CoV-2, SARS-CoV, SARS-like CoV RaTG13, MERS-CoV, HCoV-OC43, HCoV-HKUl, HCoV-NL63, and HCoV-229E.

The coronavirus S protein may be the S protein of SARS-CoV-2, SARS-CoV, or SARS-like CoV RaTG13.

The coronavirus S protein may be the S protein of SARS-CoV-2

The coronavirus S protein may be the S protein of SARS-CoV-2 depicted under Uniprot accession number P0DTC2 (sequence version 1, as of 22 ^nd April 2020).

The sequences of S protein (SEQ ID NO: 2; signal sequence underlined), subunit SI (SEQ ID NO: 3) and subunit S2 (SEQ ID NO: 4; HR1 region is underlined and HR2 region is in bold) of coronavirus SARS-CoV-2 are shown below. SARS-CoV-2 S protein (SEQ ID NO: 2):

MF VFLVLLPLV S SOC VNLTTRTOLPPAYTN SFTRGVYYPDKVFRS S VLHSTODLFL PFF SNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYF ASTEKSNIIRGWIF GTTLDSK TQSLLIVNNATNVVIKVCEF QF CNDPFLGVYYHKNNKSWMESEFRVY S S ANNCTF EYVSQPFLMDLEGKQGNFKNLREF VFKNIDGYFKIY SKHTPINLVRDLPQGF SALE PLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYN ENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPF GEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFT NVY AD SF VIRGDEVRQIAPGQTGKI AD YN YKLPDDFTGC VI AWN SNNLD SK V GGN YNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVG Y QP YRV VVL SFELLH AP AT VCGPKK S TNL VKNKC VNFNFN GLT GT GVLTESNKKF LPF QQF GRDI ADTTD AVRDPQTLEILDITPC SF GGV S VITPGTNTSNQ VAVL Y QD VN CTEVP VAIHADQLTPTWRVYSTGSNVF QTRAGCLIGAEHVNN S YECDIPIGAGIC A S Y QTQTN SPRRARS VASQ SIIAYTMSLGAEN S VAY SNN SIAIPTNFTIS VTTEILP V S MTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQV KQFYKTPPIKDF GGFNF SQILPDP SKP SKRSFIEDLLFNK VTL AD AGFIKQ Y GDCLGD I A ARDLIC AQKFN GLT VLPPLLTDEMI AQ YT S ALL AGTIT S GWTF GAGA ALQIPF A MQM A YRFN GIGVTQNVL YEN QKLI AN QFN S AIGKIQD SL S S T AS ALGKLQD VVN Q NAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQ LIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVT YVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFV SGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNI QKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLC CMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

SARS-CoV-2 Spike protein, SI subunit (SEQ ID NO: 3):

QCVNLTTRTQLPP AYTNSFTRGVYYPDKVFRS SVLHSTQDLFLPFF SNVTWFHAIH VSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNV VIKVCEF QF CNDPFLGVYYHKNNKSWMESEFRVY S S ANNCTFEYVSQPFLMDLEG KQGNFKNLREF VFKNIDGYFKIY SKHTPINL VRDLPQGF S ALEPL VDLPIGINITRF Q TLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALD PLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYA WNRKRISNC VAD Y S VLYN S ASF STFKC Y GV SPTKLNDLCFTNVY AD SF VIRGDE V RQI APGQTGKI AD YNYKLPDDF T GC VI A WN SNNLD SK V GGNYN YL YRLFRK SNLK PFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELL HAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTT DAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQL TPTWRVYSTGSNVF QTRAGCLIGAEHVNN S YECDIPIGAGIC AS Y QTQTN SP

SARS-CoV-2 Spike protein, S2 subunit (SEQ ID NO: 4):

RRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDC T MYICGD STEC SNLLLQ Y GSF CTQLNRALT GI AVEQDKNTQEVF AQ VKQIYKTPPIK DFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICA QKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFN GIGVT ONVL YEN OKLI AN OFN S AIGKIOD SLS STAS ALGKLOD VVNONAOALNTL

VKOLSSNFGAISSVLNDILSRLDKVEAEVOIDRLITGRLOSLOTYVTOOLIRAAEIR A

SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKN

FTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVI

GIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRL

NEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSC

C S CLKGCC S CGS C CKFDEDD SEP VLKGVKLH YT

Several variants carrying mutations in S-protein, including in its receptor-binding domain (RBD), have emerged, likely due to the rapid dissemination of the virus coupled with pressure from the patients’ immune response. Of note is the identification of the D614G (Nextstrain clade 20A) in early March 2020 that has rapidly become the dominant strain globally. Additional variants have also gained partial dominance in different regions of the globe. The variants A222V (Nextstrain clade 20A.EU1) and S477N (Nextstrain clade 20A.EU2) have emerged in the summer of 2020 in Spain and have rapidly shown diffusion within Europe. Recently, a new variant (clade 20B/501Y.V1, B.l.1.7) characterised by multiple mutations in S-protein (deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H) has been associated with a rapid surge in COVID-19 cases in the UK between December 2020 and January 2021. In the same period, a new variant in South Africa (clade 20C/501Y.V2, B.1.351), also carrying the N501Y mutation in the RBD (L18F, D80A, D215G, R246I, K417N, E484K, N501Y, D614G and A701V), has been associated with increased transmissibility and reduction of serum neutralisation capacity. Two variants that emerged in Brazil (B.1.1.28 and P.1) contain mutational hallmarks of both the UK and South Africa variants (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I), suggesting convergent evolution in SARS-CoV-2 due to similar selective pressures. Finally, new lineages (e.g. B.1.617, B.1.1617.1, B.1.617.2 and B.1.617.3) are emerging in India strongly suggesting that new variants are expected to arise. The mutation numbering refers to the SARS-CoV-2 S protein as shown in SEQ ID NO: 2.

The coronavirus may be SARS-CoV-2. The SARS-CoV-2 coronavirus may have the S protein of wild type SARS-CoV-2 (SEQ ID NO: 1) or a variant thereof having one or more mutations from the sequence shown as SEQ ID NO: 2. Variants of one the S protein of wild type SARS-CoV-2 include, without limitation, variants D614G; A222V; S477N; clade 20B/501Y.V1 or UK variant B.1.1.7; clade 20C/501Y.V2, B.1.351 or South African variant; Brazilian variants B.1.1.28, P.l and P.2; Indian variants B.1.617, B.1.617.1, B.1.617.2 and B.1.617.3; variants B.1.526 and B.1.526.1.

Variants of wild type SARS-CoV-2 include, without limitation, variants D614G; A222V; S477N; clade 20B/501Y.V1 or UK variant B.l.1.7; and clade 20C/501Y.V2, B.1.351 or South African variant; Brazilian variant B.1.1.28, P.l and P.2; Indian variants B.1.617, B.1.617.1, B.1.617.2 and B.1.617.3; variants B.1.526 and B.1.526.1.

It will be immediately understood that the present invention is useful with any other SARS- CoV-2 variants existent at the time of filing or with any future variants that may emerge, as well as any other coronaviruses that use ACE2 as a receptor to infect cells. Thus, the coronavirus S protein may be the S protein of any of these variants.

2. Polypeptide

In a first aspect, the present invention provides a polypeptide, hereinafter “the polypeptide of the invention” comprising: a) an antigen-binding domain that binds specifically to the human angiotensin converting enzyme type 2 (hACE2) ectodomain, and b) a half-life extending domain. The term polypeptide, as used herein, refers to natural, synthetic, and recombinant proteins or peptides generally having more than 10 amino acids.

2.1. Antigen-binding domain

The polypeptide of the invention comprises an antigen-binding domain that binds specifically to the human angiotensin converting enzyme type 2 (hACE2) ectodomain.

The terms “angiotensin converting enzyme type 2”, “ACE2”, “hACE2”, “ACE-related carboxypeptidase”, “angiotensin-converting enzyme homolog”, “ACEH”, “metalloprotease MPROT15”, and “processed angiotensin-converting enzyme 2” are used indistinctly in the present invention. Human ACE2 is depicted under Accession No. Q9BYF1 in the Uniprot database on 30 ^th March 2020. hACE2 is an 805 aa transmembrane protein with a processed ectodomain that spans aa 18-740 of the sequence shown under Uniprot Accession No. Q9BYF1.

Full ACE2 ectodomain (SEQ ID NO: 1; signal peptide is in bold and underlined):

MSS S S WLLL SL VA VT A AO S TIEEO AKTFLDKFNHEAEDLF Y O S SL AS WNYNTNIT EENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLS EDK SKRLNTILNTM S TI Y S T GK V CNPDNPQECLLLEPGLNEIM AN SLD YNERL W A WESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYD Y SRGQLIED VEHTFEEIKPL YEHLH A YVRAKLMN A YP S YI SPIGCLP AHLLGDMW G RFWTNLY SLTVPF GQKPNID VTDAMVDQ AWD AQRIFKEAEKFF V S VGLPNMTQG FWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHI Q YDM A Y A AQPFLLRN GANEGFHE A V GEIMSL S A ATPKHLK SIGLL SPDF QEDNET EINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGV VEP VPHDET Y CDP ASLFH V SND Y SFIRYYTRTL Y QF QF QE ALCQ AAKHEGPLHKC DISNS TE AGQKLFNMLRLGK SEP WTL ALEN V V GAKNMN VRPLLNYFEPLF T WLK DQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYA MRQ YFLK VKN QMILF GEED VR V ANLKPRI SFNFF VT APKN V SDIIPRTE VEK AIRM SRSRIND AFRLNDN SLEFLGIQPTLGPPNQPP V S For reference, the sequence of hACE2 depicted under Accession No. Q9B YF 1 in the Uniprot database on 30 ^th March 2020 is shown as SEQ ID NO: 42. In this sequence the leader peptide spans aa 1-17, the processed ectodomain spans aa 18-740, the transmembrane domain spans aa 741-761, and the cytoplasmic domain spans 762-805.

Human ACE2 (full-length) (SEQ ID NO: 42)

MS SS SWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLF YQS SLASWNYNTNITE

ENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSE

DKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAW

ESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDY

SRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGR

FWTNL Y SLTVPF GQKPNID VTDAMVDQ AWD AQRIFKEAEKFF V S VGLPNMTQGF

WEN SMLTDPGNV QK AV CHPT AWDLGKGDFRILMCTK VTMDDFLTAHHEMGHIQ

YDM A Y A AQPFLLRN GANEGFHE A V GEIMSL S A ATPKHLK SIGLL SPDF QEDNETEI

NFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVE

PVPHDET Y CDP ASLFHV SND Y SFIRYYTRTL Y QF QF QE ALCQ AAKHEGPLHKCDIS

NSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQN

KNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQ

YFLK VKN QMILF GEED VRV ANLKPRI SFNFF VT APKN V SDIIPRTE VEK AIRM SRSRI

ND AFRLNDN SLEFLGIQPTLGPPNQPP V SIWLIVF GVVMGVIVVGIVILIFTGIRDRK

KKNKARSGENPYASIDISKGENNPGFQNTDDVQTSF

Human ACE2 has been identified as a functional receptor for the S protein of human coronavirus NL63 (HCoV-NL63) and of SARS coronavirus (SARS-CoV). More recently, it has also been shown to be a receptor for SARS-CoV-2. The epitopes on hACE2 that are recognised by the S protein have been reported to comprise one or more of the regions corresponding to positions 19-41, 82-84 and 353-357 of the full ACE2 ectodomain (SEQ ID NO: 1).

ACE2 is a metalloprotease involved in the Renin- Angiotensin System (RAS), which controls blood pressure, electrolytes and intravascular fluid volume. A key function of hACE2 is believed to be the cleavage of Angiotensin II (Ang II) to Ang (1-7), which have opposing physiological roles. Elevated levels of Ang II are associated with vasoconstriction, inflammation, fibrosis, vascular leak, and sodium absorption. Ang (1-7) appears to be a counterregulatory protein in the RAS; associated with vasodilation, anti-proliferation, antiinflammation, and reduced vascular leak. hACE2 has also been reported to have a protective role in acute lung injury, providing a molecular explanation for the severe lung failure and death due to SARS-CoV infections.

In a normal adult human lung, hACE2 is expressed primarily in alveolar epithelial type II cells, which can serve as a viral reservoir. These cells produce surfactant which reduces surface tension, thus preventing alveoli from collapsing, and hence are critical to the gas exchange function of the lung. Injury to these cells could explain the severe lung injury observed in COVID-19 patients. hACE2 is also expressed in multiple extrapulmonary tissues including heart, kidneys, blood vessels, and intestine. The ACE2 tissue distribution in these organs may explain the multiorgan dysfunction observed in patients. There have been reports of patients presenting pulmonary embolism and kidney damage, probably caused by blood clots damaging the pulmonary and renal capillaries, as well as myocarditis, increased blood pressure, abdominal pain, diarrhoea and nausea.

The term “antigen-binding domain”, as used herein, refers to a polypeptide having an antigen binding site which comprises at least one complementarity determining region or CDR. The antigen-binding domain may comprise 3 CDRs and have an antigen binding site which is equivalent to that of a single domain antibody (dAb), heavy chain antibody (VHH) or a nanobody. Alternatively, the antigen-binding domain may comprise 6 CDRs and have an antigen binding site which is equivalent to that of a classical antibody molecule. The remainder of the polypeptide may be any sequence which provides a suitable scaffold for the antigen binding site and displays it in an appropriate manner for it to bind the antigen.

A full-length antibody or immunoglobulin typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N terminal variable (VH) region and three C-terminal constant (CHi, CEE and CEE) regions, and each light chain contains one N- terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. They are characterised by the same general structure constituted by relatively preserved regions called frameworks (FR) joined by three hyper-variable regions called complementarity determining regions (CDR) (Rabat et al., 1991, Sequences of Proteins of Immunological Interest, 5 ^th Ed., NIH Publication No. 91-3242, Bethesda, MD.; Chothia & Lesk, 1987, J Mol Biol 196:901-17). The term “complementarity determining region” or “CDR”, as used herein, refers to the region within an antibody that complements an antigen’s shape. Thus, CDRs determine the protein’s affinity and specificity for specific antigens. The CDRs of the two chains of each pair are aligned by the framework regions, acquiring the function of binding a specific epitope. Consequently, in the case of VH and VL domains both the heavy chain and the light chain are characterised by three CDRs, respectively CDRH1, CDRH2, CDRH3 and CDRLl, CDRL2, CDRL3.

A number of definitions of the CDRs are commonly in use. The Rabat definition is based on sequence variability and is the most commonly used (see http://www.bioinf.org.uk/abs/). The ImMunoGeneTics information system (IMGT) (see http://www.imgt.org) can also be used. According to this system, a complementarity determining region (CDR-IMGT) is a loop region of a variable domain, delimited according to the IMGT unique numbering for V domain. There are three CDR-IMGT in a variable domain: CDR1-IMGT (loop BC), CDR2- IMGT (loop C'C"), and CDR3-IMGT (loop FG). Other definitions of the CDRs have also been developed, such as the Chothia, the AbM and the contact definitions (see http://www.imgt.org). Unless stated otherwise, the CDRs described herein are derived using the IMGT system.

The terms “antigen-binding fragment” and “antigen-binding portion” are used interchangeably herein and refer to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen. The antibody fragment may comprise, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations thereof. Examples of antibody fragments include, but are not limited to, a Fab fragment, a F(ab’)2 fragment, an Fv fragment, a single chain Fv (scFv), or a domain antibody [VH or dAb, single domain antibody (sdAb), VHH, and nanobody].

The antigen-binding domain may be selected from a scFv or a domain antibody selected from a VH or a dAb, single domain antibody (sdAb), VHH, or nanobody. The term “VH” refers to the variable heavy domain of an IgG. The term “VHH” or “dAb” or “sdAb” or “nanobody” refers to the variable domain (single domain) of a camelid antibody.

The antigen-binding domain of the polypeptide of the invention may comprise an antigenbinding domain which is based on a non-immunoglobulin scaffold, also known as antibody mimetic. These antibody-binding domains are also called antibody mimetics. Non-limiting examples of non-immunoglobulin antigen-binding domains include an affibody, a fibronectin artificial antibody scaffold, an anticalin, an affilin, a DARPin, a VNAR, an iBody, an affimer, a fynomeran, abdurin/nanoantibody, a centyrin, an alphabody, a nanofitin, and a D domain.

The antigen-binding domain may be non-human, such as murine, rat or camelid, chimeric, humanised or fully human. The antigen-binding domain may be synthetic.

Preparation of antibodies

Preparation of antibodies may be performed using standard laboratory techniques. Antibodies may be obtained from animal serum, or, in the case of monoclonal antibodies or fragments thereof, produced in cell culture. Recombinant DNA technology may be used to produce the antibodies according to established procedure, in bacterial or mammalian cell culture.

Methods for the production of monoclonal antibodies are well known in the art. Briefly, monoclonal antibodies are typically made by fusing myeloma cells with the spleen cells from a mouse or rabbit that has been immunised with the desired antigen. Herein, the desired antigen is the ectodomain of hACE2 or a peptide thereof. The antigen-binding domain will be readily obtained from monoclonal antibodies by means of molecular biology techniques that are conventional in the art.

Alternatively, antibodies and related molecules, particularly scFvs, may be made outside the immune system by combining libraries of VH and VL chains in a recombinant manner. Libraries of VH or VHH, or dAb, or sdAb or nanobodies may also be generated. Such libraries may be constructed and screened using phage-display technology as described in Example 1.

The antibody libraries may be immune or non-immune.

Approaches to generate antibody mimetics having a particular specificity are similar and well-known in the art. Methods to generate and screen antibody mimetics against a particular target are described, for example, in Binz et al., 2005 (Binz et al., 2005, Nat Biotech 23 : 1257- 68).

Identification of coronavirus S protein-specific antibodies

Antibodies which are selective for hACE2 or a peptide thereof may be identified using methods which are standard in the art. Methods for determining the binding specificity of an antibody include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), and competitive ELISA, western blot, immunofluorescent techniques such as immunohistochemistry (IHC), fluorescence microscopy, and flow cytometry; surface plasmon resonance (SPR), radioimmunoassay (RIA), Forster resonance energy transfer (FRET), phage display libraries, yeast two-hybrid screens, co-immunoprecipitation, bimolecular fluorescence complementation and tandem affinity purification. Additionally, the infectivity neutralisation ability of antibodies may be determined by incubating the antibodies with relevant virus, e.g. lentiviral vectors pseudotyped with coronavirus S protein, and cultured onto ACE2-expressing cells. These methods are further described in Example 3.

It has been reported that coronaviruses have high mutation rates, some of them in the S protein which may translate in stronger interactions with hACE2. Binding to different epitopes on hACE2 is advantageous for blocking the interaction of the S protein more efficiently. This may in turn also prevent viral entry.

The polypeptide of the invention may further comprise at least one additional antigen binding domain that binds specifically to the hACE2 ectodomain, wherein the antigen binding domain and the at least one additional antigen-binding domains bind to different epitopes of the hACE2 ectodomain. Thus, the polypeptide of the invention may have two, three, or more antigen-binding domains, each binding to a different epitope of the hACE2 ectodomain.

The two or more antigen-binding domains may be fused to the half-life extending domain in tandem. Where the half-life extending domain is a hetero-oligomer, each of the two or more antigen-binding domains may be fused to a different monomer.

The antigen binding domain and/or the at least one additional antigen-binding domain, where present, may compete with a coronavirus S protein for binding to hACE2. Competition for the binding site on hACE2 may be direct by binding to the same or to an overlapping epitope. Competition may also be achieved by steric hindrance when the epitope recognised by the binding antigen(s) is not the same as the one recognised by the S protein. Both types of competition may be achieved when the polypeptide of the invention comprises two or more antigen-binding domains, each binding to a different epitope of the hACE2 ectodomain.

Since hACE2 has a key role in the Renin-Angiotensin System (RAS), as previously explained, the binding of the antigen-binding domain and, where present, the at least one additional antigen-binding domain, to hACE2 may not inhibit its enzymatic activity. In other words, the ability of hACE2 to cleave Ang II to Ang (1-7) may not be prevented or inhibited by binding of the one, two or more antigen-binding domains to hACE2. Other functions of hACE2 that may be later discovered may also not be prevented or inhibited.

The antigen binding domain and/or, where present, the at least one additional antigen binding domain may not inhibit the catalytic activity of hACE2 compared to that of wild- type hACE2. The catalytic or enzymatic activity of hACE2 may be considered to be inhibited when it is less than 80%, or 70%, or 60%, or 50%, or lower of the catalytic activity of hACE2 in the presence of a substrate and in the absence of any inhibitor. Alternatively, the antigen binding domain and/or, where present, the at least one additional antigen-binding domain may maintain, substantially maintain or increase the catalytic activity of hACE2 compared to that of wild-type hACE2. The catalytic or enzymatic activity of hACE2 may be considered to be maintained or substantially maintained when it is between about 80% and about 120%, or between about 90% and about 110%, or between about 95% and 105%, or about 100% of the catalytic activity of hACE2 in the presence of a substrate and in the absence of any inhibitor. The catalytic or enzymatic activity of hACE2 may be considered to be increased when it is more than 120%, or 130%, or 140%, or 150%, or higher of the catalytic activity of hACE2 in the presence of a substrate and in the absence of any inhibitor.

The enzymatic or catalytic activity of hACE2 may be determined by any method available in the art. Non-limiting examples include methods that use chromogenic and fluorogenic substrates for ACE2, i.e. by incubating the protein with a surrogate fluorogenic substrate for ACE2, such as Mca-APK(Dnp) (Examples 3 or 6). Briefly, ACE2 activity is measured using Mca-Ala-Pro-Lys(Dnp)-OH substrate [Mca-APK(Dnp)], and a Mca control peptide is used as a negative control. Mca fluorescence is quenched by the Dnp group until cleavage by ACE2 (at Pro-Lys) separates them enabling inherent fluorescence from Mca. The fluorescence is monitored over time.

The antigen-binding domain and the at least one additional antigen-binding domain, where present, may bind to an epitope in hACE2 which does not comprise one or more of amino acids R169, R273, H345, P346, T371, H374, Q375, H378 , E402, W477, K481, H505, or Y515 of the sequence shown as SEQ ID NO: 1. The present inventors have hypothesised that these residues form part of the active site and/or allosteric sites of hACE2 (Figure IB).

The antigen-binding domain and the at least one additional antigen-binding domain, where present, may bind to an epitope in hACE2 which comprises or consists of one or more of the following amino acid regions on SEQ ID NO: 1: amino acids 19-89, amino acids 319-340, amino acids 352-357, amino acids 386-389, and/or amino acid 393.

Protease mediated shedding of the ectodomain of hACE2 has been reported and a soluble form of ACE2, lacking its cytosolic and transmembrane domains, has been shown to block binding of the SARS-CoV spike protein to its receptor. This may be exploited by the present antiviral approach by using binders that recognise cell-bound hACE2. The antigen-binding domain and/or at least one additional antigen-binding domain, where present, may bind to the cell-bound hACE2 ectodomain.

The antigen-binding domain and/or at least one additional antigen-binding domain, where present, may comprise the CDR1, CDR2 and CDR3 of one of the sequences shown as SEQ ID NO: 5-11 and 50-97.

The antigen-binding domain and/or at least one additional antigen-binding domain, where present, may comprise the CDR1, CDR2 and CDR3 of one of the sequences shown as SEQ ID NO: 81, 91 and 96.

The antigen-binding domain and/or at least one additional antigen-binding domain, where present, may comprise or consist of one of the sequences shown as SEQ ID NO: 5-11 and 50-97.

The antigen-binding domain and/or at least one additional antigen-binding domain, where present, may comprise or consist of one of the sequences shown as SEQ ID NO: 81, 91 and 96.

The CDRs in the following sequences are identified in bold and underlined:

Clone 3 dAb (SEQ ID NO: 5)

O V OLOE S GGGL V O AGGSLRL S C A AS GSIF SIN AMGW YRO APGKOREL V A AIT SN

GRTEYADSVKGRFTISRDYGKNTVYLOMNNLKPEDTAVYYCKRYSTWGPGTOV

TVSS

Clone 5 dAb (SEQ ID NO: 6)

OVOLOOSGGRSVOAGGSLRLSCVASGRIFNNHAMAWFROAPGKEREFVAGISRS

SSNTYYTGSVKGRFTISRDNAENTLYLOMNSLKLEDTAVYYCAAOSRWYGGAY

YSRPGEYAYWGOGTOVTVAS

Clone 6 dAb (SEQ ID NO: 7) OVOLOESGGGLAOPGGSLTLSCATSGFTFRSAAMSWYHOAPGKEYELVAAISPG

GRGTQVTVSS

Clone 11 dAb (SEQ ID NO: 8)

OVOLOOSGGGLVOPGGSLRLSCAASGRTFSRHVMGWFROVSGKEREFVATISWS

Clone 12 dAb (SEQ ID NO: 9)

O V OLOE S GGGL V O AGGSLRL AC A AS GRTLS S Y AMGWFRRAPGKDREF V ATISW DGRSTSYADAVKGRFTISRDNAKNLLYLEMNNVKPEDTAVYYCAARRGFVSISS

Clone 16 dAb (SEQ ID NO: 10)

OVOLOESGGGLVOAGNSMTLACTGSGRTIRGYAMGWFROAPGKEREFVAAISVS

Clone 20 dAb (SEQ ID NO: 11)

OVOLOESGGGLVOPGGSLRLSCAASGFTFSSYTMNWYROVPGKERELVARISND

Clone 21 (SEQ ID NO: 50)

OVOLOESGGGLVOAGESLRLSCAVYGGTFNRYNMGWFROAPGKEREFVAGISKS

C22 (SEQ ID NO: 51)

OVOLOESGGGLVOAGESLRLSCAVSGGSFNRYNMGWFROAPGKEREFVAGISKS GTTIDYLDSVKGRFTISRDNAKNTMYLOINSLKPEDTAVYYCAADYMPWTISRA TSRYNYWGQGTQVTVS C23 (SEQ ID NO: 52)

O V OLOE S GGGL V O AGGSLRL S C V V S GNRLSIGAMGW YRO APGKOREL V ASITRG GSTNYADSVKGRFTISRDNALDTVSLOMNSLKPEDTAVYYCNAHYLISDYWGOG TQVTVS

C24 (SEQ ID NO: 53)

O V OLOE S GGGL V O AGGSLRL S C A AS GRTF VT Y V AGWFRO ALGKEREF V A AISW S GGSTYYADSVKGRFTISRDNAKDTVYLOMNTLTPEDTAVYYCAVKPRPWLRSYS DYERTEEYDYWGOGTOVTV S

C25 (SEQ ID NO: 54)

Q V QLQE S GGGL V Q AGGSLRL S CAY S GRTFSTYAMGWFRQTPGKEREF V ATISGS GSITNYADSVKGRFTISRDNAKNTVFLOMNSLKPEDTAVYYCAARWLRGVTGNP DEYRYWGOGTOVTVS

C26 (SEQ ID NO: 55)

O V OLOE S GGGL V O AGGSLRL S C A AS GRTFSGY AIGWFRO APGKERDL V AAV S W DGGRIYYKESVKGRFTISKDNAKNTVYLOLDSLKPEDTAVYFCAVADTYTPLVA SGSYD YRGQGTQ VT V S

C27 (SEQ ID NO: 56)

O V OLOE S GGGL V O AGGSLTL S C A AS GNRLSIGVMGW YRO APGKOREL V ATITR GGSTNY AD S VKGRF AISRDNAKDT V SLOMN SLKPEDT AVYY C SAHYLISDLW GO GTQVTVS

C29 (SEQ ID NO: 57)

O V OLOE S GGGL V O AGGSLTL S C A AS GNRL SIGTMGW YRO APGKOREL V ATITRG GSTNYADSVKGRFTISRDNAEDTVSLKMNSLKPEDTAVYYCTAHYLISDLWGOG TQVTVS

C30 (SEQ ID NO: 58) Q VQLQQ S GGGL V Q AGGSLTL SCATS GNRLSVGAMGW YRQ APGKQREL V ATITR

GGSTNYADSVKGRFTISRDNAEDTVSLOMTSLKPDDTAVYYCYAHYLISDRWGO

GTQVTVS

C31 (SEQ ID NO: 59)

O V OLOE S GGGL V O S GGSLRL S C A AS GRPF SAY AMGWFRO APGKEREF V S TIS WD GVTAGYADSVKGRFTISRDNAKNTVYLOMNSLOPEDTAIYFCAAKTGFSSNLRS OYNYW GOGT O VT V S

C32 (SEQ ID NO: 60)

O V OLOE S GGGL V O AGGSLKL AC S AS GRPF SAY AMGWFRO APGKEREFL S TIS WN GATTLYADSVKGRFTISRDNAENTVYLOMNSLKPEDTAIYYCAAKMGFTSNLRS OYVYWGOGTOVTVS

C33 (SEQ ID NO: 61)

O V OLOE S GGGL V O AGP SLRL S C A AS GRTIS S Y AMGWFRO APGKEREF V AT STRS GGRAYYADSVKGRFTISRDNAKNTVNLOMNSLKPEDTAVYYCAATYSDSDYVIR S VHGTD Y W GKGTL VT V S

C34 (SEQ ID NO: 62)

OVOLOESGGGLVOAGRSLRLSCAHSGRPFSGYAMGWFROAPGKEREFVSTISWD GATT YY AD S VKGRFTISRDNAENT VYLQMN SLKPEDT AI YY C A AKTGFTSNLRS OYNYWGOGTOVSVS

C35 (SEQ ID NO: 63)

OVOLOESGGGLVOSGGSLRLSCAASGRPFSAY AMGWFRO APGKEREFVSTISWD GVT AGY AD SVKGRFTISRDNAKNT VYLQMN SLQPEDT AIYF C AAKTGFSSNLRS OYKYWGOGTOVTVS

C36 (SEQ ID NO: 64)

O V OLOE S GGGL V O AGGSLRL S C A AS GRTFS A Y AMGWFRO APGKEREF V S TIS WD GVATGYADSVKGRFTISRDNAKNTMYLOMNSLOPEDTAIYFCAAKMGFTSNLRS OYNYW GOGT O VT V S C37 (SEQ ID NO: 65)

OVOLOESGGGLVOSGGSLRLSCAASGRPFSAYAMGWFROAPGKEREFVSTISWD GVTAGY AD S VKGRFTISRDNAKNT VYLQMN SLQPEDT AIYF C AAKMGFSSNI^RS OYNYWGOGTOVTVS

C38 (SEQ ID NO: 66)

Q V QL QE S GGGL V Q AGGSLRL S C A AS GRPFS A Y AMGWFR Q APGKEREF V S TIS WD GVTTDYADSVKGRFTISRDNAKNTVYLOMNTLOPEDTAIYFCAAKMGFSSNVRS OYNYWGOGTOVTVS

C39 (SEQ ID NO: 67) Q V OLOE S GGGL V O AGGSLRL S C A AS GRPFS AY AMGWFRO APGKEREF V S TIS WD GATTDSADSVKGRFTIFRDNAKNTVYLOMNSLKAEDTAIYFCAAKTGFTSNLRS OYRYW G QGT O VT V S

C40 (SEQ ID NO: 68)

Q V OLOE S GGGL V O AGGSLRL S C A AS GRTIS S Y AMGWFRO APGKEREF V AT STRS GDRA YYADS VKGRFTISRDNAKNTVNLOMN SLKPEDTAVYY C AATY SDSDYVIK S VHGTD Y W GKGTL VT V S

C42 (SEQ ID NO: 69)

QV QLOESGGGLVOSGGSLRLSCAASGRPFSAY AMGWFRO APGKEREFVSTISWD GVTAGY AD S VKGRFTISRDNAKNT VYLQMN SLQPEDT AIYF C AAKTG FSSNLRG OYNYWGOGTOVTVS

C43 (SEQ ID NO: 70)

OVOLOESGGGLVOAGGSLRLSCAASGSIFSINAMGWYROAPGKEREPVADITNG GSTNYKDS VKGRFTISRDNAKNTV SLOMN SLKPEET AVYY GYARVTTYY GSREY VYWGQGTQVTVS

C44 (SEQ ID NO: 71) O V OLOE S GGGL V O AGGSLRL S C A AS GGTFS A YTMGWFRO APGKEREF V A A V SW

C45 (SEQ ID NO: 72)

O V OLOE S GGGL V O AGGSLRL S C A AS GRP S SN Y AMGWFRO APGKEREF V S AITW S

C46 (SEQ ID NO: 73)

O V OLOE S GGGL V O AGGSLRL S C AHF GRPFN GY AMGWFRO APGKEREF V S TIS W

C48 (SEQ ID NO: 74)

O V OLOE S GGGL V O AGGSLKL AC S AS GRPFS A Y AMGWFRO APGKEREFL S TIS WD

C49 (SEQ ID NO: 75)

O V OLOE S GGGL V O AGGSLRL S C A AS GRPFS AY AMGWFRO APGKEREF V S TIS WD

C50 (SEQ ID NO: 76)

OVOLOESGGGLVOAGESLRLSCAVSGGTFNRYNMGWFROAPGKEREFVAGISKS

C51 (SEQ ID NO: 77)

O V OLOE S GGGL V O AGP SLRL S C AGS GGTFST Y VTGWFRO VPGKEREF V A AITW S GGSTYYADSVKGRFTISRDNAKTTVYLOMNSLKPEDTAVYYCALKPRPWLRTSS DYERTEEYDYWGQGTQVTV S C52 (SEQ ID NO: 78)

OVOLOESGGGLVOVGESLRLSCAVSGGSFNRYNMGWFROASGKEREFVAGISKS

DSTIDYLDSVKGRFTISRDNAKNTMYLOMNSLKPEDTAVYYCAADYMPWSISRM

TSRYHYWG QGT QVTVS

C53 (SEQ ID NO: 79)

OVOLOESGGGLVOVGESLRLSCAVSGGSFNRYNMGWFROAPGKEREFVAGISKS

GTTIDYLDSVKGRFTISRDNAKNTMYLOINSLKPEDTAVYYCAADYMPWTISRA

TSRYNYWGOGTOVTVS

C54 (SEQ ID NO: 80)

O V OLOE S GGGL V O AGGSLRL S C A ASKRTF ST Y VMGWFRO APGKEREF V A AITW S GKST YY AD S VKGRFTISRDNAKNT VYLQMN SLKPEDT AVYY C AYKPGPWLRTS RD YERTEE YD YW GOGT O VT V S

C55 (SEQ ID NO: 81)

OVOLOESGGGLVOAGGSLRHSCAASGLTFGIYOMGWYROAPGKERELVAAATS RGDT YY AD SVKGRFTISRDGAKNT VYLQMN SLRPEDT AVYY C VADPTSVRVGN PD YW GO GT Q VT V S

C56 (SEQ ID NO: 82)

O V OLOE S GGGL V O AGGSLRL S C A AS GRTFTTY VMGWFRO APGKEREF V A AISW S GKST YY AD S VKGRFTISRDNAKNT VYLQMN SLKPEDT AVYY C AVKPGPWLRSY SDYERTEEYDYWG QGT QVTVS

C57 (SEQ ID NO: 83)

O V OLOE S GGGL V O AGGSLRL S C AGS GGTFVTY VMGWFRO APGKEREF V A AITW SGGSTYYADSVKGRFTISRDNAKSTVYLOMNSLKPEDTAVYYCALKPRPWLSSS SDYERTEEYDYWGOGTOVTVS

C58 (SEQ ID NO: 84) O V OLOE S GGGL V O AGGSLTL S C A AS GNRL SIGTMGW YRO APGKOREL V ATITRA

TQVTVS

C60 (SEQ ID NO: 85)

OVOLOESGGGLVOVGESLRLSCAVSGGSFNRYNMGWFROASGKEREFVAGISKS

C61 (SEQ ID NO: 86)

O V OLOE S GGGL V O AGGSLTL S C A AS GNRL SIGTMGW YRO APGKOREL V ATITRG

TPVTVS

C62 (SEQ ID NO: 87)

OVOLOESGGGLVOAGESLRLSCEVSGYAFNRYHMGWFROAPGKEREFVAGISKS

C63 (SEQ ID NO: 88)

OVOLOESGGGLVOAWGSLRLSCTASGRTIRNYVMGWFROAPGKEREFVARISWS

C64 (SEQ ID NO: 89)

O V OLOE S GGGL V O AGGSLTL SCAT S GNRL S V GAMGW YRO APGKOREL V ATITR

GTQVTVS

C65 (SEQ ID NO: 90)

O V OLOE S GGGL V O AGGSLTL S C A V S GNRLSIGAMGW YRO APGKOREM V ATITR GGSTNYADSVKGRFTISRDNAKDTVSLOMNSLKPEDTAVYYCNAHYLISDRWGO

GTQVTVS C66 (SEQ ID NO: 91)

OVOLOESGGGLVOAGGSLRHSCAASGLTFGIYOMGWYROAPGKEREFVAAATS RGDT YY GPS VKGRFTISRDGAKNTVYL QMN SLRPEDT AVYY C VADRTS VRVGN PDYWG QGT QVTVS

C67 (SEQ ID NO: 92)

O V OLOE S GGGL V O AGGSLRL S C A AS GGTFIT Y VMGWFRO APGKEREF V A AITW S GSTTYYADSVKGRFTISRDNAKSTVYLOMNSLKPEDTAVYYCALKPRPWLRTSS D YERTDE YD YW GO GT O VT V S

C68 (SEQ ID NO: 93)

O V OLOE S GGGL V O AGGSLRL S C A AS GRP SRTYLMGWFRO APGKEREF V A AIGW STDTK Y GD S AKGRFTI ARDN SKNM VYLOMN SLKPEDT AVYY C AGRGGLTMKY DAGDYDYWGOGTOVTVS

C70 (SEQ ID NO: 94)

O V OLOE S GGGL V O AGGSLRL S C A V S GSIF S VNDMGW YRO APGREREW V ATITSG GRTNYAESVKGRFTISRDNVNKT V SLOMN SLEAEDT AVYY CDAFLRPSRGSREY VYWGQGIQVTVS

C71 (SEQ ID NO: 95)

O V OLOE S GGGL V O AGGSLRL S CT AS GRAF SAY VMGWFRO APGKEREF V A AIRGS GLITKYADSVKGRFTISRDNAKNTVYLOMNSLKPEDTAVYYCAADRNVGFSSST YDGNY GYW GOGT O VT V S

C72 (SEQ ID NO: 96)

OVOLOESGGGLVOTGGSLRLSCAASGRTFSSLAMGWFROAPGKERVIVAATGW

GGVGTYYADSVKGRFTISRDNAKNTLYLOMGSLKPEDTAVYYCAADRTSVRVG

APDYWGQGTQVTVS

C73 (SEQ ID NO: 97) OVOLOESGGGLVOAGGSLRLSCOASGRTFSTYVMGWFROAPGKDREFVAAISW TGRNINY GD S VKGRFTISRDNAKNT VYLQMN SLKPEDT AVYY C SAKTYESGPEV AYWG QGT QVTVS

The term antigen-binding domain of the polypeptide of the invention is also intended to embrace functionally equivalent variants of the antigen-binding domain, variants which have been modified in the amino acid sequence without adversely affecting, to any substantial degree, (i) its capacity to block the binding of the S protein of coronavirus to hACE2 relative to that of the parent antigen-binding domain, and/or (ii) the enzymatic activity of hACE2. Said modifications include the conservative (or non-conservative) substitution of one or more amino acids for other amino acids, the insertion and/or the deletion of one or more amino acids into the antigen-binding domain, provided that the capacity to interact with hACE2 of the variant is substantially maintained and/or the enzymatic activity of hACE2 is not substantially affected at physiological conditions.

The term “variant” or “mutant”, as used herein, refers to a polypeptide differing from a specifically recited polypeptide, i.e. reference or parent polypeptide by amino acid insertions, deletions, and/or substitutions, created using, for example, recombinant DNA techniques or by de novo synthesis. Variant and mutant are used indistinctly in the context of the present invention. The variants or mutants of the antigen binding domain comprising one of the amino acid sequences shown as SEQ ID NO: 5-11 and 50-97 may have at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the corresponding sequences shown as SEQ ID NO: 5-11 and 50-97, provided that the capacity to interact with hACE2 of the variant is substantially maintained and/or the enzymatic activity of hACE2 is not substantially affected upon binding relative to those of the parent antigen-binding domain.

Variants of the antigen-binding domain comprising one of the amino acid sequences shown as SEQ ID NO: 5-11 and 50-97 typically have at least about 75%, for example at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding sequences given as SEQ ID NO: 5-11 and 50-97. Typically, variants may contain one or more conservative amino acid substitutions compared to the original amino acid or nucleic acid sequence. Conservative substitutions are those substitutions that do not substantially affect or decrease the capacity to interact with hACE2 of the variant and/or the enzymatic activity of hACE2 upon binding. For example, an antigen-binding domain variant that specifically binds hACE2 may include up to 1, up to 2, up to 5, up to 10, or up to 15 conservative substitutions compared to any of the sequences given as SEQ ID NO: 5-11 and 50-97 and retain capacity to interact with hACE2 of the parent antigen-binding domain and/or the enzymatic activity of hACE2 upon binding.

Variants of the antigen-binding domain have at least about 75%, for example at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequences given as SEQ ID NO: 5-11 and 50-97.

Functionally similar amino acids which may be exchanged by way of conservative substitution are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The percentage of sequence identity may be determined by comparing two optimally aligned sequences over a comparison window. The aligned sequences may be polynucleotide sequences or polypeptide sequences. For optimal alignment of the two sequences, the portion of the polynucleotide or amino acid sequence in the comparison window may comprise insertions or deletions (i.e., gaps) as compared to the reference sequence (that does not comprise insertions or deletions). The percentage of sequence identity is calculated by determining the number of positions at which the identical nucleotide residues, or the identical amino acid residues, occurs in both compared sequences to yield the number of matched positions, then dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Sequence identity between two polypeptide sequences or two polynucleotide sequences can be determined, for example, by using the Gap program in the WISCONSIN PACKAGE version 10.0-UNIX from Genetics Computer Group, Inc. based on the method of Needleman and Wunsch (1970, J Mol Biol 48:443-53) using the set of default parameters for pairwise comparison (for amino acid sequence comparison: Gap Creation Penalty=8, Gap Extension Penalty=2; for nucleotide sequence comparison: Gap Creation Penalty=50; Gap Extension Penalty=3), or using the TBLASTN program in the BLAST 2.2.1 software suite (Altschul et al., 1997, Nucleic Acids Res 25:3389-402), using BLOSUM62 matrix (Henikoff & Henikoff, 1992, ProcNatl Acad Sci USA 89:10915-9) and the set of default parameters for pair-wise comparison (gap creation cost=l 1, gap extension cost=l).

The percentage of sequence identity between polypeptides and their corresponding functions may be determined, for example, using a variety of homology -based search algorithms that are available to compare a query sequence, to a protein database, including for example, BLAST, FASTA, and Smith-Waterman. BLASTX and BLASTP algorithms may be used to provide protein function information. A number of values are examined in order to assess the confidence of the function assignment. Useful measurements include “E-value” (also shown as “hit JD”), “percent identity”, “percent query coverage”, and “percent hit coverage”. In BLAST, the E-value, or the expectation value, represents the number of different alignments with scores equivalent to or better than the raw alignment score, S, that are expected to occur in a database search by chance. Hence, the lower the E value, the more significant the match. Since database size is an element in E-value calculations, the E-values obtained by doing a BLAST search against public databases, such as GenBank, have generally increased over time for any given query/entry match. Thus, in setting criteria for confidence of polypeptide function prediction, a “high” BLASTX match is considered as having an E- value for the top BLASTX hit of less than IE-30; a medium BLASTX is considered as having an E-value of IE-30 to IE-8; and a low BLASTX is considered as having an E- value of greater than IE-8. Percent identity refers to the percentage of identically matched amino acid residues that exist along the length of that portion of the sequences which is aligned by the BLAST algorithm. In setting criteria for confidence of polypeptide function prediction, a “high” BLAST match is considered as having percent identity for the top BLAST hit of at least 70%; a medium percent identity value is considered from 35% to 70%; and a low percent identity is considered of less than 35%. Of particular interest in protein function assignment is the use of combinations of E- values, percent identity, query coverage and hit coverage. Query coverage refers to the percent of the query sequence that is represented in the BLAST alignment, whereas hit coverage refers to the percent of the database entry that is represented in the BLAST alignment. For the purpose of defining the polypeptides functionally covered by the present invention, the function of a polypeptide is deduced from the function of a protein homolog, such as one of the sequences shown as SEQ ID NO: 5-11, wherein a polypeptide of the invention is one that either (1) results in hit_p<le-30 or % identity >35% AND query _coverage>50% AND hit_coverage>50%, or (2) results in hit p<le-8 AND query _coverage>70% AND hit_coverage>70%.

Variants of the antigen-binding domain comprising one of the amino acid sequences shown as SEQ ID NO: 5-11 and 50-97 may maintain at least about 75%, for example at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the capacity to interact with hACE2 of the parent antigen-binding domain. Variants of the antigen-binding domain comprising one of the amino acid sequences shown as SEQ ID NO: 5-11 and 50-97 may have an increased capacity to interact with hACE2 of about 105%, for example at least about 110%, 115%, 120%, 125%, 130%, 140%, 150% or more compared with that of the parent molecule.

The interaction between the antigen-binding domain or the variants thereof and hACE2 and coronavirus S protein can be determined by conventional methods. For example, the interaction may be determined by ELISA, surface plasmon resonance (SPR), or by flow cytometry using ACE2-expressing cells. Additionally, the ability of the antigen-binding domain or the variants thereof to block viral entry on hACE2-expressing cells may be determined by incubating the antigen-binding domain or the variants thereof or the polypeptide of the invention with relevant viruses, e.g. lentiviral vectors pseudotyped with coronavirus S protein, and cultured onto ACE2-expressing cells. These methods are further described in Example 3.

Variants of the antigen-binding domain comprising one of the amino acid sequences shown as SEQ ID NO: 5-11 and 50-97 may maintain at least about 75%, for example at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the enzymatic activity of hACE2 upon binding to hACE2. Methods to determine the enzymatic activity of hACE2 have been described previously. 2.2. Half-life extending domain

The antigen-binding domain of the polypeptide of the invention may be short-lived in the bloodstream because of its low molecular weight may be below the permeability threshold of the kidney glomerular membrane. Additionally or alternatively, its pharmacokinetic properties may need to be enhanced. Therefore, the antigen-binding domain of the polypeptide of the invention is coupled either genetically or chemically to a half-life extending domain.

The term “half-life”, as used herein, refers to the time where 50% of an administered polypeptide of the invention are eliminated through biological processes, e.g. metabolism, excretion, etc. Methods for pharmacokinetic analysis and determination of molecule half- life will be familiar to those skilled in the art.

The term “half-life-extending domain” or “half-life extension domain”, as used herein, refers to any moiety which extends the half-life of an antigen-binding domain. Such domains are contemplated to include, but are not limited, to plasma proteins, plasma protein binding proteins or peptides, binders to the neonatal Fc receptor (FcRn), and other half-life extension domains known in the art. Plasma proteins and plasma protein binding proteins/peptides can be an effective means of improving the pharmacokinetic properties of any molecule. One of these plasma proteins is albumin, which has been extensively investigated for extending the half-life of therapeutic molecules in blood. Other plasma proteins are transferrin and immunoglobulin.

The half-life extending domain may be selected from albumin, or an albumin domain, or an antigen-binding domain that binds specifically to albumin, or an albumin-binding-peptide, or an albumin-binding domain of a Streptococcus protein, or transferrin, or a homo oligomerization domain, or a hetero-oligomerization domain, or a polyethylene glycol moiety.

Albumin and related moieties Albumin or human serum albumin (HS A) can be exploited in one of two ways. One approach is to directly couple the therapeutic protein to has or to one of its domains. A second approach is to use an albumin-binding domain.

Albumin is the most abundant protein in plasma, present at 50 mg/ml (600 mM), and has a half-life of 19 days in humans. With a molecular mass of about 67 kDa, albumin serves to maintain plasma pH, contributes to colloidal blood pressure, functions as carrier of many metabolites and fatty acids, and serves as a major drug transport protein in plasma. Human albumin is depicted under Accession No. P02768 in the Uniprot database on 30 ^th March 2020. Albumin is formed by three domains, i.e. albumin domain I spanning aa 19-210, albumin domain II spanning aa 211-403, and albumin domain III spanning aa 404-601.

The half-life extending domain may comprise the full sequence of HSA shown as SEQ ID NO: 12. Alternatively, it may comprise a mutated HSA, such as the ones described in W02010059315.

Wild-type human serum albumin; processed protein (SEQ ID NO: 12) RGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEF AKTC VADES AEN CDK SLHTLF GDKLC TV ATLRET Y GEMADC C AKQEPERNECFL QHKDDNPNLPRL VRPEVD VMCT AFHDNEETFLKKYLYEI ARRHP YF YAPELLFF A KRYKAAFTECCQ AADKAACLLPKLDELRDEGKAS S AKQRLKC ASLQKF GERAFK AWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICE NQD SIS SKLKECCEKPLLEKSHCIAEVENDEMP ADLP SL AADF VESKD V CKNY AE A KDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDE FKPLVEEPQNLIKQNCELFEQLGEYKF QNALLVRYTKKVPQ VSTPTLVEV SRNLGK VGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRP CFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE QLK A VMDDF A AF VEKC CK ADDKET CF AEEGKKL V A AS Q AALGL

The half-life extending domain may comprise one domain of HSA, i.e. domain I of HSA (residues 1-192 of SEQ ID NO: 12), domain II of HSA (residues 193-385 of SEQ ID NO: 12), or domain III of HSA (residues 386-591 of SEQ ID NO: 12). The half-life extending domain may comprise a combination of HSA domains, such as domains I and II, I and III, or II and III.

Noncovalent association with albumin extends the elimination half-time of short-lived proteins.

The half-life extending domain may comprise the sequence of an antigen-binding domain that binds specifically to albumin.

The term “antigen-binding domain” has been described previously, and its definition and particular features apply equally herein. Where necessary, the skilled person will readily be able to adapt the previous description to albumin.

Numerous anti-HSA antigen-binding domains have been described in the art, such as scFvs, single domain antibodies (Nanobody™, AlbudAb™) and Fabs, as well as albumin-binding domains based on antibody mimetics, such as anti-albumin DARPins. Anti-serum albumin binding single variable domains have been described, for example, in Holt et al., Protein Eng Des Sel 21:283-8, W004003019, W02008096158, WO05118642, W020060591056, WO201 1/006915, which are incorporated herein by reference.

The half-life extending domain may comprise the sequence of an albumin-binding-peptide. A non-limiting example of an albumin-binding-peptide includes peptides having the core sequence DICLPRWGCLW (SEQ ID NO: 13), which was generated using peptide phage display to specifically bind to albumin.

The half-life extending domain may comprise the sequence of an albumin-binding domain of a Streptococcus protein. The Streptococcal protein may be Protein G. The albuminbinding domain of Streptococcal protein G may be the albumin-binding domain B2A3 (BA) and/or B1A2B2 A3 (BABA).

Exemplary structures of polypeptides of the invention comprising a half-life extending domain selected from albumin, or an albumin domain, or an antigen-binding domain that binds specifically to albumin, or an albumin-binding-peptide, or an albumin-binding domain of a Streptococcus protein are depicted in Figure 2A.

Transferrin

The half-life extending domain may be transferrin.

Human transferrin is a 698 amino acid protein, of approximately 75kDa (not accounting for glycosylation), with two main domains, N (about 330 amino acids) and C (about 340 amino acids), which appear to originate from a gene duplication. Transferrins are responsible for the transport of iron from sites of absorption and heme degradation to those of storage and utilization. Serum transferrin may also have a further role in stimulating cell proliferation. Human transferrin is depicted under Accession No. P02787 in the Uniprot database on 15 ^th May 2020.

The half-life extending domain of the polypeptide of the invention may comprise the amino acid sequence of human transferrin as shown in SEQ ID NO: 14.

Processed human transferrin (SEQ ID NO: 14)

VPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRAIAAN EADAVTLDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVKKDSGFQ MNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANFFSGSCAPCAD GTDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAFVKHSTIFENLANKA DRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARSMGGKEDLIWELLNQAQE HF GKDK SKEF QLF S SPHGKDLLFKD S AHGFLK VPPRMD ARM YLGYE Y VT AIRNLR EGT CPEAPTDECKP VKWC ALSHHERLKCDEW S VN S VGKIEC V S AETTEDCIAKIM N GE AD AM SLDGGF V YI AGKC GL VP VL AENYNK SDN CEDTPE AGYF AI A VVKK S A SDLTWDNLKGKKSCHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKK D S SLCKLCMGSGLNLCEPNNKEGYY GYT GAFRCL VEKGD VAF VKHQT VPQNT GG KNPDPWAKNLNEKDYELLCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEACV HKILRQQQHLFGSNVTDCSGNFCLFRSETKDLLFRDDTVCLAKLHDRNTYEKYLG EE YVK A V GNLRKC STS SLLE ACTFRRP Homo-olieomerisation domain

The term “oligomerisation domain”, as used herein, refers to a protein sequence, polypeptide or oligopeptide that self-assembles to form an oligomer. The oligomer may be a dimer, trimer, tetramer, pentamer, hexamer and so on depending on the number of monomers that assemble together, i.e. two, three, four, five, six and so on, respectively. Oligomers can be homo-oligomers, when all the monomers are the same, or heterooligomers, when the monomers are different.

The oligomer may be a homo-oligomer. The oligomerisation domain of the homo-oligomer may be any homo-oligomerisation domain that is suitable for making fusion proteins.

The oligomerisation domain of the homo-oligomer may be selected from an IgG Fc region or a variant thereof which does not interact with FcyRI, FcyRIIa and FcyRIII, an IgM Fc region, an IgA Fc region, a collagen XVIII trimerizing structural element, a collagen XV trimerizing structural element, a foldon domain, a TenC domain and a homo-oligomerising coiled-coil domain.

The Fc region is the tail region of an antibody that is formed by the CH2 and the CH3 domains of an antibody. There are several different Fc regions, according to the antibody isotype and subclass, and these are the Fc regions of an IgGl, an IgG2, an IgG3, an IgG4, an IgM, an IgA, an IgE, and an IgD. The Fc regions dimerise, but in the case of IgM and IgA these dimers additionally form pentamers or further dimers, respectively. The additional oligomerisation may be particularly advantageous for increasing the valency of the first polypeptide of the invention, or when an avidity effect is to be obtained. The Fc region may be the Fc region of an IgGl, an IgG2, an IgG3, an IgG4, an IgM, or an IgA.

The Fc region may be the Fc region of an IgGl depicted under Uniprot Accession No. P01857 as of 8 ^th April 2020 or a sequence shown as SEQ ID NO: 61. The Fc region may be the Fc region of an IgG2 (SEQ ID NO: 62).

The Fc region may comprise the hinge region. The Fc region may not comprise the hinge region. The homo-oligomerisation domain may comprise the sequence shown as SEQ ID NO: 45 or SEQ ID NO: 46, shown below (hinge region is underlined):

Hinge-IgGl Fc region (SEQ ID NO: 45)

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVV S VLTVLHQDWLNGKEYKCKV SN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFL Y SKLTVDK SRWQQGNVF SC S VMHE ALHNH YTQKSLSLSPGK

IgG2 Fc (SEQ ID NO: 46)

ERKCCVECPPCP APP V AGPS VFLFPPKPKDTLMISRTPEVT C VVVD V SHEDPEV QF NWYVDGVEVHNAKTKPREEQFNSTFRVV S VLT VVHQDWLNGKEYKCKV SNKGL PAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNG QPENNYKTTPPMLD SDGSFFL Y SKLTVDK SRWQQ GNVF S C S VMHE ALHNH YT QK SLSLSPGK

Reports from SARS-CoV have highlighted that the natural immune response and seroconversion in affected patients correlated with decreasing viral loads from day 10. However, it was also associated to severe clinical worsening, probably due to an overexuberant host response. Similarly, there is evidence of a detrimental role of anti-S protein antibodies in SARS-CoV infection, causing lung injuries, abrogating TGFp production and promoting inflammatory macrophage accumulation. This activity could be counteracted by FcyR blockade.

Thus, the present invention also contemplates using a variant of an Fc region of an IgG which does not interact with FcyRI, FcyRIIa and FcyRIII. Mutations that abrogate the effector function of the Fc region have been extensively investigated and are well-known in the art. For example, the Fc region of an IgG may contain one or more of the following mutations or mutation combinations:

- Leu235Glu;

Leu234Ala and Leu235Ala (LALA); - Leu234Ala, Leu235Ala (LALA) and Pro329Gly (PG);

- Pro331 Ser, Leu234Glu and Leu235Phe;

Asp265Ala;

- Gly237Ala;

- Glu318Ala;

- Glu233Pro;

Gly236Arg and Leu328Arg;

Ala330Leu;

Asp270Ala;

- Lys322Ala;

Pro329Ala;

Pro331Ala;

- Val264Ala;

- Phe241Ala;

Asn297Ala;

Asn297Gly; and

- Asn297Gln.

The homo-oligomerisation domain may comprise the Fc region of an IgG which contains the mutations Leu234Ala and Leu235Ala (LALA).

The homo-oligomerisation domain may comprise the Fc region of an IgG which contains the mutations Leu234Ala, Leu235Ala (LALA) and Pro329Gly (PG).

HuIgGFc with LALA mutations (SEQ ID NO: 47)

EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFL Y SKLTVDK SRWQQGNVF SC S VMHE ALHNH YTQKSLSLSPGK

HuIgGFc with LALA and PG mutations (SEQ ID NO: 48) EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVV S VLTVLHQDWLNGKEYKCKV SN KALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFL Y SKLTVDK SRWQQGNVF SC S VMHE ALHNH YTQKSLSLSPGK

The Fc region may comprise the hinge region. The Fc region may lack the hinge region.

IgGl hinge (SEQ ID NO: 98) EPK S CDKTHT CP

The Fc region may be truncated. The truncated Fc region may comprise the CH3 domain. The truncated Fc region may comprise the hinge region and, optionally, a flexible linker. The truncated Fc region may comprise the sequence shown as SEQ ID NO: 99 or SEQ ID NO: 100.

Truncated Fc region (Hinge-CH3) (SEQ ID NO: 99)

EPK S CDKTHT CPPC GQPREPQ V YTLPP SREEMTKN Q VSLT CL VKGF YP SDI A VEWE

SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY

TQKSLSLSPGK

Truncated Fc region (Hinge-CH3) (SEQ ID NO: 100)

EPK S CDKTHT CPPCGGGS S GGGS GGQPREPQV YTLPP SREEMTKN Q V SLTCL VKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK

The present invention also contemplates using a variant of the Fc region of an IgG which does not interact with FcyRI, FcyRIIa and FcyRIII and displays improved circulation or serum half-life. One or more of the following mutations or mutation combinations may be combined to the previously described silencing mutations (i.e. mutations that abrogate the effector function of the Fc region) in the IgG Fc region:

Arg435His;

Asn434Ala; - Met252Tyr, Ser 254Thr and Thr256Glu;

Met428Leu and Asn434Ser;

- Thr252Leu, Thr253 Ser and Thr254Phe;

Glu294delta, Thr307Pro and Asn434Tyr;

Thr256Asn, Ala378Val, Ser383Asn, and Asn434Tyr; and Glu294delta.

The variant of the Fc region of an IgG which does not interact with FcyRI, FcyRI la and FcyRI 11 and displays improved circulation or serum half-life may comprise mutations Met252Tyr, Ser 254Thr and Thr256Glu (YTE).

The variant of the Fc region of an IgG which does not interact with FcyRI, FcyRI la and FcyRI 11 and displays improved circulation or serum half-life may comprise the sequence shown as SEQ ID NO: 49.

HuIgGIFc with LALA, PG and YTE mutations (SEQ ID NO: 49) EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVV S VLTVLHQDWLNGKEYKCKV SN KALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFL Y SKLTVDK SRWQQGNVF SC S VMHEALHNH YTQKSLSLSPGK

The homo-oligomerisation domain may be a collagen XVIII trimerizing structural element. The term “collagen XVIII trimerizing structural element” or “XVIIITSE”, as used herein, refers to the portion of collagen XVIII which is responsible for trimerization between monomers of collagen XVIII. The term is also intended to embrace functionally equivalent variants of a XVIIITSE of a naturally occurring collagen XVIII, variants which have been modified in the amino acid sequence without adversely affecting, to any substantial degree, the trimerization properties relative to those of the native collagen XVIII molecule. Said modifications include the conservative (or non-conservative) substitution of one or more amino acids for other amino acids, the insertion and/or the deletion of one or more amino acids, provided that the trimerization properties of the native collagen XVIII protein is substantially maintained, i.e., the variant maintains the ability (capacity) of forming trimers with other peptides having the same sequence at physiological conditions.

The XVIIITSE may be a polypeptide having the amino acid sequence shown in SEQ ID NO: 101

XVIIITSE (SEQ ID NO: 101):

SGVRLWATRQAMLGQVHEVPEGWLIFVAEQEELYVRVQNGFRKVQLEARTPLPR

GTDNE

The oligomerisation domain may be a collagen XV trimerizing structural element. The term “collagen XV trimerizing structural element” or “XVTSE”, as used herein, refers to the portion of collagen XV which is responsible for trimerization between monomers of collagen XV. The term is also intended to embrace functionally equivalent variants of a XVTSE of a naturally occurring collagen XV, variants which have been modified in the amino acid sequence without adversely affecting, to any substantial degree, the trimerization properties relative to those of the native collagen XV molecule. Said modifications include, the conservative (or non-conservative) substitution of one or more amino acids for other amino acids, the insertion and/or the deletion of one or more amino acids, provided that the trimerization properties of the native collagen XV protein is substantially maintained, i.e., the variant maintains the ability (capacity) of forming trimers with other peptides having the same sequence at physiological conditions.

The XVTSE may be a polypeptide having the amino acid sequence shown in SEQ ID NO: 102

XVTSE (SEQ ID NO: 102):

VTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKLQLGELIPIPADSPPP

PALSSNP

The oligomerisation domain may be a foldon domain. The term “foldon domain”, “foldon T4” or “foldon T4 domain”, as used herein, refers to the C-terminal amino acid residues of the trimeric protein fibritin from bacteriophage T4 (SEQ ID NO: 103). The foldon domain promotes folding and trimerisation of fibritin. This feature has been exploited to trimerise other molecules.

Foldon T4 (SEQ ID NO: 103):

GYIPEAPRDGQAYVRKDGEWVLLSTFL

The oligomerisation domain may be a TenC domain. The term “TenC domain”, as used herein, refers to the oligomerisation domain located at the N-terminus of Tenascin C (TN- C). The TenC domain may be human (SEQ ID NO: 104) or from chicken (SEQ ID NO: 105). The TenC domain forms trimers.

Human TenC domain (SEQ ID NO: 104):

ACGC AAAPDVKELLSRLEELENL VS SLREQ

Chicken TenC domain (SEQ ID NO: 105):

ACGC AAAPD VKDLL SRLEELEGL V S SLREQ

The oligomerisation domain may be a coiled coil domain. A “coiled coil” is a structural motif in which two to seven alpha helices are wrapped together like the strands of a rope. Many endogenous proteins incorporate coiled coil domains. The coiled coil domain may be involved in protein folding (e.g. it interacts with several alpha helical motifs within the same protein chain) or responsible for protein-protein interaction. In the latter case, the coiled coil can initiate homo or hetero oligomer structures.

The structure of coiled coil domains is well known in the art. For example, as described by Lupas & Gruber (2007, Advances in Protein Chemistry 70:37-8). Coiled coils usually contain a repeated pattern, hxxhcxc, of hydrophobic (h) and charged (c) amino-acid residues, referred to as a heptad repeat. The positions in the heptad repeat are usually labelled abcdefg, where a and d are the hydrophobic positions, often being occupied by isoleucine, leucine, or valine.

Examples of proteins which contain a homo-oligomerising coiled coil domain include, but are not limited to, cartilage-oligomeric matrix protein (COMP), kinesin motor protein, hepatitis D delta antigen, archaeal box C/D sRNP core protein, mannose-binding protein A, coiled-coil serine-rich protein 1, polypeptide release factor 2, SNAP-25, SNARE, Lac repressor or apolipoprotein E.

The sequence of various coiled coil domains is shown below:

Cartilage oligomeric matrix protein (COMP) homopentamer (SEQ ID NO: 106)

AGSDLGPQMLRELQETNAALQDVRELLRQQVREITFLKNTVMECDACGSGKKDP

K

Kinesin motor protein: parallel homodimer (SEQ ID NO: 107)

MH A AL S TE VVHLRQRTEELLRCNEQQ A AELET CKEQLF Q SNMERKELHNT VMDL RGN

Hepatitis D delta antigen: parallel homodimer (SEQ ID NO: 108) GREDILEQWVSGRKKLEELERDLRKLKKKIKKLEEDNPWLGNIKGIIGKY

Archaeal box C/D sRNP core protein: anti-parallel heterodimer (SEQ ID NO: 109)

RY V V AL VK ALEEIDE SINMLNEKLEDIR A VKE SEITEKFEKKIRELRELRRD VEREIE EVM

Mannose-binding protein A: parallel homotrimer (SEQ ID NO: 110)

AIE VKL ANME AEINTLK SKLELTNKEH AF SM

Coiled-coil serine-rich protein 1 : parallel homotrimer (SEQ ID NO: 111)

EWE ALEKKL A ALE SKLQ ALEKKLE ALEHG

The oligomerisation domain may be a p53 oligomerisation domain. The term “p53 oligomerisation domain” or “p53 domain”, as used herein, refers to the oligomerisation domain located at the C-terminus of p53. The p53 domain may comprise the sequence shown as SEQ ID NO: 112. The p53 domain forms tetramers. p53 oligomerisation domain (SEQ ID NO: 112) KKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPG

Hetero-oligomerisation domain

The half-life extending domain may be a hetero-oligomerisation domain. The use of heterooligomerisation domains may be advantageous when it is intended that the polypeptide of the invention contains two or more different antigen-binding domains. For example, the use of a hetero-oligomerisation domain, such as a heterodimerization domain, enables the fusion of an antibody binding domain having different specificity to each of the hetero-monomers. This results in bispecific molecules, such as the general bispecific scFv-based fusion proteins depicted in Figure 2B.

Different hetero-oligomerisation domains may be used in the context of the present invention. Non-limiting examples of hetero-oligomerisation domains are described in Brinkmann & Kontermann (2017, MAbs 9:182-212), and include the dock-and-lock (DNL) modules, knobs-into-holes modified CH3 domains, SEEDbodies, bispecific tetravalent Fc or IgG fusions, dual variable domain Ig (DVD), the barnase-barstar domains, and hetero- oligomerising coiled coil domains.

The basis of the DNL method is the exploitation of the specific protein-protein interactions occurring in nature between the regulatory (R) subunits of protein kinase (PKA) and the anchoring domain (AD) of A-kinase anchoring proteins (AKAPs). The dimerization domain and AKAP binding domain of human Rlla are both located within the same amino-terminal 44-amino acid sequence, which is termed the dimerization and docking domain (DDD) This platform technology exploits the DDD of human Rlla and the AD of a certain amino acid sequence as a pair of linker modules for docking any two entities into a noncovalent complex, which could be further locked into a stably tethered structure through the introduction of cysteine residues into both the DDD and the AD at strategic positions to facilitate the formation of disulfide bonds.

The general methodology of the DNL approach is as follows. A recombinant protein is constructed by linking a DDD sequence to the compound of interest, for example the ectodomain of hACE2. Because the DDD sequence effects the spontaneous formation of a dimer, the resulting recombinant protein is a divalent compound, for example a divalent ectodomain of hACE2. To make the end product bispecific, a second recombinant protein is prepared by fusing an AD sequence. This second recombinant protein may comprise domain C, domain ABD, or domain ALB, which will be described in more detail in following sections of present invention. The dimeric motif of DDD in the first recombinant protein creates a docking site for binding to the AD sequence, thus facilitating a ready association of the dimeric ectodomain of hACE2 construct with the monomeric domain C, domain ABD, or domain ALB to form a binary, trimeric complex. This binding event is made irreversible with a subsequent reaction to secure the 2 entities covalently via disulfide bridges between the inserted cysteine residues. This reaction occurs very efficiently, because the initial binding interactions bring the reactive thiol groups on both the DDD and AD into proximity to ligate site-specifically. By attaching the DDD and AD away from the functional groups of the 2 precursors, such site-specific ligations preserve the original activities of the 2 precursors. The DNL method was disclosed in US provisional application 60/751196, which is incorporated herein by reference in its entirety.

“Knobs-into-holes” is a design strategy for engineering antibody heavy chain homodimers for heterodimerization. In this approach, a 'knob' variant was first obtained by replacement of a small amino acid with a larger one in the CH3 domain of an IgG: T366Y. The knob was designed to insert into a 'hole' in the CH3 domain of a different IgG created by judicious replacement of a large residue with a smaller one: Y407T. Another example of knobs-into- holes structure (CW-CSAV) comprises mutations S354C, T366W in the CH3 domain of one IgG chain, and Y349C, T366S, L368A, and Y407V in the CH3 domain of other IgG chain. Other paired variant combinations have been developed. Knobs-into-holes fusion proteins consist of [IgGl hinge]-CH2-[Knobs-into-holes CH3], that may be genetically linked to one or more fusion partners. This results in bispecific molecules, such as the general bispecific scFv-based fusion proteins depicted in Figure 2B.

By designing strand-exchange engineered domain (SEED) heterodimers, another way of achieving complementarity in the CH3 interface allowing for a heterodimeric assembly of Fc chains was developed. These SEED CH3 domains or SEEDbodies are composed of alternating segments derived from human IgA and IgG CH3 sequences (AG SEED CH3 and GA SEED CH3) and were used to generate so-called SEEDbodies. Because molecular models suggested that interaction with FcRn is impaired in the AG SEED CH3, residues at the CH2-CH3 junction were returned to IgG sequences. Pharmacokinetic studies confirmed that the half-life of SEEDbodies was comparable to other Fc fusion proteins and IgGl. SEEDbody fusion proteins consist of [IgGl hinge]-CH2-[SEED CH3], that may be genetically linked to one or more fusion partners.

Another immunoglobulin-based architecture that may be used in the context of the present invention consists in fusing antigen binding domains (e.g. scFv or dAb) of different specificity to the constant domain of human k chain (CL) and the first constant domain of human heavy chain (CHI) to form two polypeptides, (ABDl)-CL and (ABD2)-CH1-CH2- CH3, respectively. These molecules are termed bispecific tetravalent Fc fusions. The two polypeptides are co-expressed in cells. Association between the heavy and the light chains forms a covalently linked hetero-tetramer with dual specificity. This approach yields a homogeneous bispecific IgG-like antibody product with each molecule containing four antigen binding sites, two for each of its target antigen. The BsAb retains not only antigen binding efficiency but also the biological activity of its component antibodies.

Sequence examples include human IgGl CHI (SEQ ID NO: 43, human IgGl constant kappa (SEQ ID NO: 44), hinge-IgGl Fc region (SEQ ID NO: 45), and hinge-IgG2 Fc region (SEQ ID NO: 46).

Human IgGl CHI (SEQ ID NO: 43)

ASTKGP S VFPL AP S SK S T S GGT A ALGCL VKD YFPEP VT V S WN S GALT S GVHTFP A V LQ S S GL Y SL S SWT VP S S SLGT Q T YICNVNHKP SNTK VDKK V

Human IgGl constant kappa (SEQ ID NO: 44)

VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQD SKD S T Y SL S S TLTL SK AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC

The skilled person will appreciate that antigen-binding domain(s) according to the invention, CL or CHl-hinge-CH2-CH3 chains may be combined in different arrangements. For example, antigen-binding domains of different specificity may each be fused to one of the CL or CHl-hinge-CH2-CH3 chains, or to only one of the CL or CHl-hinge-CH2-CH3 chains while the other may be left “empty” (i.e. not fused to an antigen binding domain).

Further immunoglobulin-based architectures that form hetero-oligomers include the dual variable domain (DVD or DVD-Ig) (Wu et al., 2007, Nat Biotechnol 25:1290-7). Like a conventional IgG, the DVD molecule is composed of two heavy chains and two light chains. Unlike IgG, however, both heavy and light chains of a DVD molecule contain an additional variable domain (VD) connected via a linker sequence at the N-termini of the VH and VL of an existing monoclonal antibody (mAh). Thus, when the heavy and the light chains combine, the resulting DVD molecule contains four antigen recognition sites (Figure 25B). The outermost or N-terminal variable domain is termed VDl and the innermost variable domain is termed VD2; the VD2 is proximal to the C-terminal CHI or CL.

Variants of the Fc region of an IgG which do not interact with FcyRI, FcyRIIa and FcyRIII and/or which display improved circulation or serum half-life, which have been described in detail in the context of the homo-oligomerisation domain, are equally applicable to the immunoglobulin-based architectures of the hetero-oligomerisation domain.

The barnase-barstar system is a multimerisation module based on the tight interaction between barnase and barstar. Bamase is a 110 aa secreted ribonuclease from Bacillus amyloliquefaciens . Barstar is an 89 aa cytoplasmic barnase inhibitor with which the host protects itself. They rapidly form a complex with a KD of ~10 ^'14 M. Both the N- and C- termini of both proteins are accessible and available for fusions (Deyev et al., 2003, Nat Biotech 21:1486-92).

The hetero-oligomerisation domain may be a coiled coil domain. A “coiled coil” is a structural motif in which two to seven alpha helices are wrapped together like the strands of a rope. Many endogenous proteins incorporate coiled coil domains. The coiled coil domain may be involved in protein folding (e.g. it interacts with several alpha helical motifs within the same protein chain) or responsible for protein-protein interaction. In the latter case, the coiled coil can initiate homo or hetero oligomer structures. The structure of coiled coil domains is well known in the art. For example, as described by Lupas & Gruber (2007, Advances in Protein Chemistry 70:37-8). Coiled coils usually contain a repeated pattern, hxxhcxc, of hydrophobic (h) and charged (c) amino-acid residues, referred to as a heptad repeat. The positions in the heptad repeat are usually labelled abcdefg, where a and d are the hydrophobic positions, often being occupied by isoleucine, leucine, or valine.

Examples of proteins which contain a hetero-oligomerising coiled coil domain include, but are not limited to, polypeptide release factor 2, SNAP-25, SNARE, Lac repressor and apolipoprotein E. Non-limiting examples of heterooligomerising coiled coil domains include

Polypeptide release factor 2: anti-parallel heterotrimer

Chain A: INPVNNRIQDLTERSDVLRGYLDY (SEQ ID No. 15)

Chain B :

VVDTLDQMKQ GLED V S GLLEL A VE ADDEETFNE A V AELD ALEEKL AQLEFR (SEQ ID NO: 16)

SNAP-25 and SNARE: parallel heterotetramer Chain A:

IETRHSEIIKLENSIRELHDMFMDMAMLVESQGEMIDRIEYNVEHAVDYVE (SEQ ID NO: 17)

Chain B :

ALSEIETRHSEIIKLENSIRELHDMFMDMAMLVESQGEMIDRIEYNVEHAVDYVER AVSDTKKAVKY (SEQ ID NO: 18)

Chain C:

ELEEMQRRADQLADESLESTRRMLQLVEESKDAGIRTLVMLDEQGEQLERIEE GMDQINKDMKEAEKNL (SEQ ID NO: 19)

Chain D:

IETRHSEIIKLENSIRELHDMFMDMAMLVESQGEMIDRIEYNVEHAVDYVE (SEQ ID NO: 20)

Apolipoprotein E: anti-parallel heterotetramer SGQRWEL ALGRF WD YLRW VQTL SEQ V QEELL S S Q VT QELRALMDETMKELK A Y KSELEEQLTARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRV RLASHLRKLRKRLLRD ADDLQKRLAVY Q A (SEQ ID No. 21)

The person skilled in the art will immediately appreciate that a hetero-oligomerisation domain provides a possibility for multiple structures of the polypeptide of the invention, since the one or more antigen-binding domains may be fused to only one or all of the different hetero-oligomerisation domains. For example, where the polypeptide of the invention comprises two antigen binding domains and a heterodimerisation domain (e.g. knob-into-holes domains), the two antigen binding domains may be fused in tandem to only one of the heterodimerisation domains, or only one antigen-binding domain fused to each of the heterodimerisation domains (Figure 2B). This is advantageous in embodiments where exact conformations are required for binding to various epitopes on a single hACE2 molecule while preventing cross-linking with other hACE2 molecules.

The ability of the polypeptide of the invention to form dimers, trimers or higher heterooligomers can be determined by conventional methods known by the skilled person in the art. For example, by way of a simple illustration, the ability of a polypeptide of the invention comprising a trimerising domain, such as the DNL system, to form a trimer can be determined by using standard chromatographic techniques. Thus, the variant to be assessed is put under suitable trimerisation conditions and the complex is subjected to a standard chromatographic assay under non denaturing conditions so that the eventually formed complex (trimer) is not altered. If the variant trimerises properly, the molecular size of the complex would be three times heavier than the molecular size of a single molecule of the variant. The molecular size of the complex can be revealed by using standard methods such as analytical centrifugation, mass spectrometry, size-exclusion chromatography, sedimentation velocity, etc.

Other modifications used to extend half-life that are currently known in the art, or that will be developed in the future, also form part of the present invention. For example, the first polypeptide of the invention may be conjugated to polyethylene glycol (PEG), or pegylated. 2.3. Linker

The antigen-binding domain and the half-life extending domain may be joined by a linker.

The linker provides spatial separation between antigen-binding domain and the half-life extending domain. The linker may be a flexible linker. This type of linkers allows for torsion of the antigen-binding domain respective of the half-life extending domain, which may be beneficial when the antigen-binding domain interacts with hACE2. Non-limiting examples of flexible linkers that may be used in the polypeptide of the invention include:

- (Gly ₄ Ser)2 (SEQ ID NO: 22: GGGGSGGGGS);

- (Gly ₄ Ser)3 (SEQ ID NO: 23 : GGGGS GGGGS GGGGS) ;

- (Gly ₄ Ser) ₄ (SEQ ID NO: 24: GGGGSGGGGS GGGGS GGGGS) ;

- (Gly ₄ Ser) ₅ (SEQ ID NO: 25 : GGGGSGGGGSGGGGSGGGGSGGGGS);

- S GGGGS GGGGS GGGGS (SEQ ID NO : 26);

- GGGGS GGGGS GGGGS (SEQ ID NO: 27); and

- GGGGSGGGGSGGGAS (SEQ ID NO: 28).

The linker may be the (Gly ₄ Ser)3 linker (SEQ ID NO: 23).

Alternatively, it may be preferable that the movement of the antigen-binding domain respective of the half-life extending domain is constrained. This may be achieved using a short linker or an inflexible linker. Non-limiting examples of short linkers that may be used in the first polypeptide of the invention include:

- Gly ₄ Ser (SEQ ID NO: 29: GGGGS);

- GlyiSer (GGS);

- GlySer (GS); and

- GlyAla (GA).

The linker may be inflexible or rigid. Inflexible linkers that may be used in the first polypeptide of the invention include those forming part of the families of linkers (EAAAK)n (SEQ ID NO: 114), (EP) _n , (KP) _n , and (TRP) _n . Non-limiting examples of inflexible or rigid linkers include: - LEAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAA KALE (SEQ ID NO: 37),

- GSGSEPEPEPEPGSGS (SEQ ID NO: 38),

- GSGSEPEPEPEPEPEPGSGS (SEQ ID NO: 39),

- GSGSKPKPKPKPGSGS (SEQ ID NO : 40), and

- GSGSKPKPKPKPKPKPGSGS (SEQ ID NO: 41).

Restricted movement and torsion may be particularly useful where the polypeptide of the invention comprises two or more antigen-binding domains to ensure that binding occurs on the same hACE2 molecule, and thus prevent cross-linking of hACE2.

Where the polypeptide of the invention comprises two or more antigen-binding domains in tandem, these may be joined by a linker as described herein.

2.4. Orientation

The different components of the polypeptide of the invention, i.e. the antigen-binding domain and the half-life extending domain and, optionally, the at least one additional antigen binding domain, may take any orientation.

For example, where the half-life extending domain comprises a domain which forms monomers, i.e. albumin, an albumin domain, an antigen-binding domain that binds specifically to albumin, an albumin-binding-peptide, an albumin-binding domain of a Streptococcus protein, or transferrin, the polypeptide of the invention may have one of the following domain orientations:

- ABD-HLED

- HLED-ABD

- ABD-ABDx-HLED

- ABD-HLED-ABDx

- ABD-ABDx-ABDy-HLED

- ABD-ABDx-HLED-ABDy

- ABD-HLED-ABDx-ABDy wherein ABD is the antibody-binding domain that binds specifically to the hACE2 ectodomain;

HLED is the half-life extending domain;

ABDx is an additional antibody-binding domain that binds specifically to the hACE2 ectodomain,

ABDy is an additional antibody-binding domain that binds specifically to the hACE2 ectodomain, and

ABD, ABDx and, where present, ABDy recognise different epitopes on the hACE2 ectodomain.

The skilled person will immediately recognise all the possible different polypeptide structures available for any given domain combinations where the half-life extending domain comprises a domain which forms hetero-oligomers, i.e. hetero-oligomerisation domains.

3. Signal peptide

The polypeptide of the present invention may comprise a signal peptide at its N-terminus so that when the polypeptide is expressed inside a cell, the nascent protein is directed to the endoplasmic reticulum and subsequently secreted.

The core of the signal peptide or leader sequence may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix. The signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation. At the end of the signal peptide there is typically a stretch of amino acids that is recognised and cleaved by signal peptidase. Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. The free signal peptides are then digested by specific proteases.

The signal peptide may be at the amino terminus of the molecule. The signal peptide may comprise the SEQ ID NO: 30-34 or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions) provided that the signal peptide still functions to cause cell surface expression of the protein.

SEQ ID NO: 30: MGTSLLCWMALCLLGADHADG

The signal peptide of SEQ ID NO: 30 is compact and highly efficient. It is predicted to give about 95% cleavage after the terminal glycine, giving efficient removal by signal peptidase.

SEQ ID NO: 31 : MSLP VT ALLLPLALLLHAARP

The signal peptide of SEQ ID NO: 31 is derived from IgGl.

SEQ ID NO: 32: MA VPT Q VLGLLLLWLTD ARC

The signal peptide of SEQ ID NO: 32 is derived from CD8.

SEQ ID NO: 33: METDTLLLWVLLLWVPGSTGD

The signal peptide of SEQ ID NO: 33 is derived from mouse Ig Kappa.

SEQ ID NO: 34: MGW S CIILFL V AT AT GVHS

The signal peptide of SEQ ID NO: 34 is derived from human IgG2 heavy chain.

4. Nucleic acid

In another aspect, the present invention also provides a nucleic acid encoding the polypeptide of the invention, hereinafter “the nucleic acid of the invention”.

In particular embodiments where the half-life domain comprises a hetero-oligomeric domain, the nucleic acid construct encoding such a polynucleotide may have one of the following structure: ABD-linker 1 -HDEL 1 -coexpr-ABDx-linker2-HDEL2 or

HDEL 1 -linker 1 -ABD-coexpr-HDEL2-linker2-ABDx in which:

ABD is a nucleic acid encoding the antibody-binding domain that binds specifically to the hACE2 ectodomain;

- HLED1 is a nucleic acid encoding one hetero-oligomerisation domain;

- HLED2 is a nucleic acid encoding the other hetero-oligomerisation domain; ABDx is a nucleic acid encoding an additional antibody-binding domain that binds specifically to the hACE2 ectodomain, linker is a nucleic acid encoding a linker; coexpr is a nucleic acid encoding a sequence enabling co-expression of the first and second polypeptides; and

ABD and ABDx recognise different epitopes on the hACE2 ectodomain.

For the structures mentioned above, nucleic acid sequences encoding the two or more polypeptides may be in either order in the construct. Additionally, for structures mentioned above, nucleic acid sequences encoding each of the different domains may be in any orientation.

The above nucleic acid structures are not intended to be exhaustive but merely an indication for the skilled person. Different construct structures may be needed to encode different polypeptide structures as described previously in the context of the first aspect of the invention.

Alternatively, where the half-life extending domain comprises a heterooligomeric domain the different monomers may be encoded by separate nucleic acids.

The term “polypeptide of the invention” has been described in detail in the context of the first aspect of the invention and its definitions, features and embodiments apply equally to this aspect of the invention. As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other.

It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described here to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed.

The nucleic acid sequences and constructs of the invention may contain alternative codons in regions of sequence encoding the same or similar amino acid sequences, in order to avoid homologous recombination.

Nucleic acids according to the invention may comprise DNA or RNA. They may be single- stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.

The terms “variant”, “homologue” or “derivative” in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence.

In the structure above, “coexpr” is a nucleic acid sequence enabling co-expression of two polypeptides as separate entities. It may be a sequence encoding a cleavage site, such that the nucleic acid construct produces both polypeptides, joined by a cleavage site(s). The cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into individual peptides without the need for any external cleavage activity.

The cleavage site may be any sequence which enables the two polypeptides to become separated.

The term “cleavage” is used herein for convenience, but the cleavage site may cause the peptides to separate into individual entities by a mechanism other than classical cleavage. For example, for the Foot-and-Mouth disease virus (FMDV) 2A self-cleaving peptide (see below), various models have been proposed for to account for the “cleavage” activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect (Donnelly et al., 2001, J Gen Virol 82:1027-41). The exact mechanism of such “cleavage” is not important for the purposes of the present invention, as long as the cleavage site, when positioned between nucleic acid sequences which encode proteins, causes the proteins to be expressed as separate entities.

The cleavage site may, for example be a furin cleavage site, a Tobacco Etch Virus (TEV) cleavage site or encode a self-cleaving peptide.

A ‘self-cleaving peptide’ refers to a peptide which functions such that when the polypeptide comprising the proteins and the self-cleaving peptide is produced, it is immediately “cleaved” or separated into distinct and discrete first and second polypeptides without the need for any external cleavage activity.

The self-cleaving peptide may be a 2A self-cleaving peptide from an aphtho- or a cardiovirus. The primary 2A/2B cleavage of the aptho- and cardioviruses is mediated by 2A “cleaving” at its own C-terminus. In apthoviruses, such as foot-and-mouth disease viruses (FMDV) and equine rhinitis A virus, the 2A region is a short section of about 18 amino acids, which, together with the N-terminal residue of protein 2B (a conserved proline residue) represents an autonomous element capable of mediating “cleavage” at its own C- terminus (Donelly et al (2001) as above). “2A-like” sequences have been found in picomaviruses other than aptho- or cardioviruses, ‘picornavirus-like’ insect viruses, type C rotaviruses and repeated sequences within Trypanosoma spp and a bacterial sequence (Donnelly et al (2001) as above).

The cleavage site may comprise the 2A-like sequence shown as SEQ ID NO: 113 (RAEGRGSLLTCGDVEENPGP).

The nucleic acid of the invention can contain a regulatory sequence operatively linked for the expression of the nucleotide sequence encoding the first polypeptide of the invention, thereby forming a gene construct, hereinafter the “gene construct of the invention”. As used herein, the term “operatively linked” means that the antibody encoded by the nucleic acid sequence of the invention is expressed in the correct reading frame under control of the expression control or regulating sequences. Therefore, in another aspect, the invention provides an expression cassette, hereinafter “the expression cassette of the invention”, comprising the gene construct of the invention operatively linked to an expression control sequence. The gene construct of the invention can be obtained through the use of techniques that are widely known in the art.

The expression cassette may comprise one or more control sequences. Control sequences are sequences that control and regulate transcription and, where appropriate, the translation of said antibody, and include promoter sequences, transcriptional regulators encoding sequences, ribosome binding sequences (RBS) and/or transcription terminating sequences. The expression cassette of the present invention may additionally include an enhancer, which may be adjacent to or distant from the promoter sequence and can function to increase transcription from the same. The expression control sequence may functional in prokaryotic cells or in eukaryotic cells and organisms, such as mammalian cells. The expression cassette may comprise a promoter. Any promoter may be used in this methodology.

It will be appreciated that different nucleic acids are required to encode the polypeptide of the invention where the half-life extending domain comprises a hetero-oligomerisation domain. The skilled person will readily know how to make the necessary modifications to obtain the different nucleic acids encoding for these heterooligomeric first, second or third polypeptides of the invention. 5. Vector

In another aspect, the present invention also provides a vector, or kit of vectors, which comprises a nucleic acid of the invention, or an expression cassette of the invention. Such a vector may be used to introduce the nucleic acid or expression cassette into a host cell so that it expresses the polypeptide of the invention.

The term “polypeptide of the invention”, “nucleic acid of the invention” and “expression cassette or the invention” have been described in detail in the context of previous aspects of the invention and their features and embodiments apply equally to this aspect of the invention.

The vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon-based vector or synthetic mRNA.

6. Cell

Another aspect of the present invention relates to a cell, hereinafter “the cell of the invention”, comprising the nucleic acid of the invention, the nucleic acid of the invention, the expression cassette of the invention, the vector of the invention, or the vector of the invention.

The cell may comprise a nucleic acid, or an expression cassette, or a vector according to the present invention, and may express the polypeptide of the invention.

The term “polypeptide of the invention”, “nucleic acid of the invention”, “expression cassette or the invention”, and “vector of the invention” have been described in detail in the context of previous aspects of the invention and their features and embodiments apply equally to this aspect of the invention.

The cell may be prokaryotic or eukaryotic. Cells suitable for performing the invention include, without limitation, mammalian, plant, insect, fungal and bacterial cells. Mammalian cells suitable for the present invention include epithelial cell lines, osteosarcoma cell lines, neuroblastoma cell lines, epithelial carcinomas, glial cells, hepatic cell lines, CHO (Chinese Hamster Ovary) cells, COS, BHK cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, 293 and 293T cells, PER.C6 cells, NTERA-2 human ECCs cells, D3 cells of the mESCs line, human embryonic stem cells such as HS293, hMSCs and BGVOl, SHEF1, SHEF2 and HS181, NIH3T3 cells, REH and MCF-7 cells. Bacterial cells include, without limitation, cells from Gram positive bacteria such as species of the genus Bacillus , Streptomyces and Staphylococcus and Gram-negative bacterial cells such as cells of the genus Escherichia and Pseudomonas. Fungal cells preferably include yeast cells such as Saccharomyces, Pichia pastoris and Hansenula polymorpha. Insect cells include, without limitation, Drosophila cells and Sf9 cells. Plant cells include, among others, cells of crop plants such as cereals, medicinal, ornamental or bulbs.

The polypeptide of the invention may be produced by culturing the host cells for a period of time sufficient to allow for expression of the polypeptide in the host cells or, more preferably, secretion of the polypeptide into the culture medium in which the host cells are grown. The polypeptide of the invention can be recovered from the culture medium using standard protein purification methods. For example, where the half-life extending domain is albumin or an albumin domain the purification can be advantageously carried out by affinity chromatography using albumin-binding resins and columns.

7. Pharmaceutical composition

In another aspect, the present invention also relates to a pharmaceutical composition containing the polypeptide of the invention, hereinafter “the pharmaceutical composition of the invention”.

The pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds. Such a formulation may, for example, be in a form suitable for intravenous infusion. The term “polypeptide of the invention” has been described in detail in the context of previous aspects of the invention and its definitions and particular features apply equally to this aspect of the invention.

As used herein, “pharmaceutically acceptable carrier” means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the polypeptide of the invention.

The pharmaceutical compositions may be in a variety of forms, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g. injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, and liposomes. The preferred form depends on the intended mode of administration and therapeutic application.

ADMINISTRATION

The administration of the polypeptide of the invention can be accomplished using any of a variety of routes that make the active ingredient bioavailable. For example, the agent can be administered via an oral, mucosal, buccal, intranasal, inhalable, intravenous, subcutaneous, transcutaneous, intramuscular, intraperitoneal, parenteral or topical route. Oral administration may be by inhalation, by nebulisation or nasally. The polypeptide of the invention may be administered locally, for example by catheter or stent, or systemically.

The pharmaceutical compositions comprising the polypeptide of the invention may be administered to the subject in a variety of pharmaceutically acceptable dosing forms, which will be familiar to those skilled in the art. For example, the polypeptide of the invention may be administered via the nasal route using a nasal insufflator device. Examples of these are already employed for commercial powder systems intended for nasal application (e.g. Fisons Lomudal System). Details of other devices are well-known in the art.

Other delivery routes for the polypeptide of the invention include via the pulmonary route using a powder inhaler or metered dose inhaler, via the buccal route formulated into a tablet or a buccal patch, and via the oral route in the form of a tablet, a capsule or a pellet (which compositions may administer agent via the stomach, the small intestine or the colon), all of which may be formulated in accordance with techniques which are well known to those skilled in the art.

Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient.

8. Method of treatment

The specificity for the coronavirus S protein of the polypeptides of the invention makes these molecules capable of neutralising coronavirus virions and prevent cell infection. This can be exploited for therapeutic purposes.

Thus, in another aspect, the present invention provides a polypeptide of the invention for use in medicine.

The invention provides a method for neutralising a coronavirus by administering a polypeptide of the invention to a patient in need thereof. The coronavirus may be a coronavirus that uses ACE2 as a receptor to infect cells. The coronavirus may be SARS- CoV-2 or SARS-CoV. The SARS-CoV-2 strain may be selected from wild-type, variant D614G, variant A222V, variant S477N, variant B.l.1.7, variant B.1.351, variant B.1.1.28, variant P.1, variant P.2, variant B.1.526, variants B.1.526.1, variant B.1.617, variant B.1.617.1, variant B.1.617.2, variant B.1.617.2, and variant B.1.617.3. In another aspect, the present invention provides a method for treating a coronavirus infection or a condition or disorder resulting from this infection in a subject, hereinafter “the method of treatment of the invention”, which comprises the step of administering a polypeptide of the invention to a subject in need thereof. The administration step may be in the form of a pharmaceutical composition as described above.

This aspect of the invention may be alternatively formulated as a polypeptide of the invention for use in the treatment a coronavirus infection or a condition or disorder resulting from this infection.

This aspect of the invention may be alternatively formulated as the use of a polypeptide of the invention in the manufacture of a medicament for treating a coronavirus infection or a condition or disorder resulting from this infection.

The terms “polypeptide of the invention” and “pharmaceutical composition of the invention” have been described in detail in the context of previous aspects of the invention and its definitions and particular features apply equally to this aspect of the invention.

A method for treating a coronavirus infection or a condition or disorder resulting from this infection relates to the therapeutic use of the polypeptide of the invention, which may be administered to a subject who has been infected with a coronavirus, or is suspected to have been infected with a coronavirus, or has tested positive for a coronavirus in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.

The method for preventing a coronavirus infection or a condition or disorder resulting from this infection relates to the prophylactic use of the polypeptide of the invention. Herein such polypeptide may be administered to a subject who has not yet contracted the coronavirus infection or condition or disorder resulting from this infection and/or who is not showing any symptoms of the coronavirus infection or condition or disorder resulting from this infection to prevent or impair the coronavirus from infecting the cells of the subject or to reduce or prevent development of at least one symptom associated with the coronavirus infection or condition or disorder resulting from this infection. The subject may have a predisposition for or be thought to be at risk of contracting a coronavirus infection or a condition or disorder resulting from this infection.

In another aspect, the present invention provides a method for treating a subject having COVID-19 of unknown SARS-CoV-2 strain, comprising a step of administering the polypeptide of the invention or a pharmaceutical composition of the invention to the subject. This aspect may be alternatively formulated as a polypeptide of the invention or a pharmaceutical composition of the invention for use in the treatment of COVID-19 of unknown SARS-CoV-2 strain. This aspect may be alternatively formulated as the use of a polypeptide of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for treating COVID-19 of unknown SARS-CoV-2 strain.

In another aspect, the present invention provides a method for treating a subject previously immunised with a vaccine based on S protein depicted under Uniprot accession number P0DTC2, comprising a step of administering a polypeptide of the invention or a pharmaceutical composition of the invention to the subject. This aspect may be alternatively formulated as a polypeptide of the invention or a pharmaceutical composition of the invention for use in the treatment of a subject previously immunised with a vaccine based on S protein depicted under Uniprot accession number P0DTC2. This aspect may be alternatively formulated as the use of a polypeptide of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for treating a subject previously immunised with a vaccine based on S protein depicted under Uniprot accession number P0DTC2.

In another aspect, the present invention provides a method for treating a subject previously treated with antibodies specific to S protein depicted under Uniprot accession number P0DTC2, comprising a step of administering a polypeptide of the invention or a pharmaceutical composition of the invention to the subject. This aspect may be alternatively formulated as a polypeptide of the invention or a pharmaceutical composition of the invention for use in the treatment of a subject previously treated with antibodies specific to S protein depicted under Uniprot accession number P0DTC2. This aspect may be alternatively formulated as the use of a polypeptide of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for treating a subject previously treated with antibodies specific to S protein depicted under Uniprot accession number P0DTC2.

In another aspect, the present invention provides a method for treating a subject previously infected with a first SARS-CoV-2 strain who is currently infected with a second SARS- CoV-2 strain, wherein the first and second SARS-CoV-2 strains are different, comprising a step of administering a polypeptide of the invention or a pharmaceutical composition of the invention to the subject. This aspect may be alternatively formulated as a polypeptide of the invention or a pharmaceutical composition of the invention for use in the treatment of a subject previously infected with a first SARS-CoV-2 strain who is currently infected with a second SARS-CoV-2 strain, wherein the first and second SARS-CoV-2 strains are different. This aspect may be alternatively formulated as the use of a polypeptide of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for treating a subject previously infected with a first SARS-CoV-2 strain who is currently infected with a second SARS-CoV-2 strain, wherein the first and second SARS-CoV-2 strains are different.

The first and second SARS-CoV-2 strains may be selected from wild-type, variant D614G, variant A222V, variant S477N, variant B.l.1.7, variant B.1.351, variant B.1.1.28, variant P.1, variant P.2, variant B.1.526, variants B.1.526.1, variant B.1.617, variant B.1.617.1, variant B.1.617.2, variant B.1.617.2, and variant B.1.617.3. It will be appreciated that these aspects are not limited to the SARS-CoV-2 strains described above since the present invention is useful in the treatment of any other SARS-CoV-2 variants existent at the time of filing or of any future variants that may emerge. It will also be immediately understood that these aspects are not limited to SARS-CoV-2 and that the polypeptide of the invention or the pharmaceutical composition of the invention may be used therapeutically in the treatment and/or prevention of a coronavirus infection or a condition or disorder resulting from this infection caused by a different coronavirus that uses ACE2 as a receptor to infect cells.

In another aspect, the present invention provides a method for treating a coronavirus infection of one SARS-CoV-2 strain selected from wild-type, variant D614G, variant A222V, variant S477N, variant B.l.1.7, variant B.1.351, variant B.1.1.28, variant P.1, variant P.2, variant B.1.526, variants B.1.526.1, variant B.1.617, variant B.1.617.1, variant B.1.617.2, variant B.1.617.2, and variant B.1.617.3, comprising a step of administering a polypeptide of the invention or a pharmaceutical composition of the invention to the subject. This aspect may be alternatively formulated as a polypeptide of the invention or a pharmaceutical composition of the invention for use in the treatment of a coronavirus infection of one SARS-CoV-2 strain selected from wild-type, variant D614G, variant A222V, variant S477N, variant B.l.1.7, variant B.1.351, and variant B.1.1.28. This aspect may be alternatively formulated as the use of a polypeptide of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for treating a coronavirus infection of one SARS-CoV-2 strain selected from wild-type, variant D614G, variant A222V, variant S477N, variant B.l.1.7, variant B.1.351, variant B.1.1.28, variant P.1, variant P.2, variant B.1.526, variants B.1.526.1, variant B.1.617, variant B.1.617.1, variant B.1.617.2, variant B.1.617.2, and variant B.1.617.3.

It will be appreciated that this aspect is not limited to the SARS-CoV-2 strains described above since the present invention is useful in the treatment of any other SARS-CoV-2 variants existent at the time of filing or of any future variants that may emerge. It will also be immediately understood that these aspects are not limited to SARS-CoV-2 and that the polypeptide of the invention or the pharmaceutical composition of the invention may be used therapeutically in the treatment and/or prevention of a coronavirus infection or a condition or disorder resulting from this infection caused by a different coronavirus that uses ACE2 as a receptor to infect cells.

These therapeutic applications will comprise the administration of a therapeutically effective amount of the polypeptide of the invention.

The terms “polypeptide of the invention” and “pharmaceutical composition of the invention” have been described in detail in the context of previous aspects of the invention and their definitions and particular features apply equally to these aspects of the invention.

The treatment of a coronavirus disease in a subject may comprise the step of administrating the polypeptide of the invention to the subject, to cause complete or partial neutralisation of the coronaviruses. In another aspect, the present invention provides a method of neutralising a coronavirus infection, comprising a step of contacting a polypeptide of the invention with a cell infected with said coronavirus. The polypeptide of the invention may be in the form of a pharmaceutical composition as described above.

The terms “polypeptide of the invention” and “pharmaceutical composition of the invention” have been described in detail in the context of previous aspects of the invention and their definitions and particular features apply equally to this aspect of the invention.

The term “subject” or “individual”, as used in the context of the present invention, refers to members of mammalian species. The subject may be a human patient of any gender, age or race.

Alternatively, the subject may be a non-human mammal infected with coronavirus. The polypeptide of the invention or the pharmaceutical composition of the invention may be administered to a non-human mammal infected with coronavirus for veterinary purposes or as an animal model of human disease. Such animal models may be useful for evaluating the therapeutic efficacy of the polypeptides of this invention. Non-limiting examples of nonhuman mammal that may be subject to treatment according to the invention include a cat or any other feline, a dog or any other canid, a mouse, a rat or any other rodent, a pig, a primate, a camelid, and a bat.

The term “therapeutically effective amount”, as used herein, refers to the amount of the polypeptide of the invention which is required to achieve an appreciable prevention, neutralisation, cure, delay, reduction of the severity of, or amelioration of one or more symptoms of a coronavirus disease.

The term “a coronavirus infection or a condition or disorder resulting from this infection”, as used herein, refers to an infection, condition or disorder caused by a coronavirus. The coronaviruses can cause varieties of diseases in humans and other animals, including respiratory, enteric, renal, and neurological diseases. Particularly important are the diseases caused by SARS-CoV and SARS-CoV-2 coronaviruses because of the severe acute respiratory syndrome that they cause.

The coronavirus condition or disorder may be coronavirus disease 2019 (COVID-19). The disease was first identified in December 2019 in Wuhan, the capital of China's Hubei province, and has since spread globally, resulting in the ongoing 2019-20 coronavirus pandemic. Common symptoms include fever, cough and shortness of breath. Other symptoms may include fatigue, muscle pain, diarrhoea, nausea, sore throat, loss of smell and abdominal pain. While the majority of cases result in mild symptoms, some progress to viral pneumonia and multi-organ failure.

Reports have shown that COVID-19 manifests as a clotting disorder, which may cause pulmonary embolism and hypoxia. Pulmonary vasculature affected by pulmonary embolism is not fully restored and can cause permanent fibrosis of the lining of blood vessels. Pulmonary fibrosis may also be the result of prolonged mechanical ventilation; even prolonged use of high concentration oxygen can lead to lung injury and result in fibrosis. Permanent fibrosis may lead to chronic thromboembolic pulmonary hypertension (CTEPH). Additionally, the clotting disorder causes end organ damage, primarily kidney. Kidney injury does not fully recover and may lead to chronic kidney disease (CKD) in post- COVID19 patients.

Direct infection of SARS-CoV-2 of ACE2-expressing cells has a number of consequences. Infection of the heart muscle cells leads to myocarditis. Patients who have no or minimal pulmonary symptoms but presented with fatigue may experience myocarditis as the primary disease. Myocardial injury may also explain the increase incidence of cardiac arrest in COVID-19 patients. Because ACE2 receptors play a key role in the renin-angiotensin system, which is a primary regulatory mechanism for blood pressure, viral infection of ACE2-expressing cells may lead to malfunction of the system and increased blood pressure.

Severe COVID-19 presents with a cytokine storm or cytokine release syndrome (CRS), which is an immediate and intense response of the immune system to viral infection. However, there are indications that the immune response may not just be temporary. One example is the case reports of Kawasaki disease symptoms in children infected with SARS- CoV-2. Kawasaki disease is an autoimmune disease in which blood vessels throughout the body become inflamed. It is considered a “post-viral” autoimmune disease. Several reports have described COVID-19 patients suffering from Guillain-Barre syndrome Guillain-Barre syndrome is a neurological disorder where the immune system responds to an infection and ends up mistakenly attacking nerve cells, resulting in muscle weakness and eventually paralysis. Thus, severe COVID-19 may also cause an incidence of other more prevalent autoimmune diseases in recovered patients.

The loss of the sense of smell is a direct result of the virus infecting the olfactory neurons. It has been suggested that this may enable the virus to spread from the respiratory tract to the brain. Cells in the human brain express the ACE2 protein on their surface. ACE2 is also found on endothelial cells that line blood vessels. Infection of endothelial cells may allow the virus to pass from the respiratory tract to the blood and then across the blood-brain barrier into the brain. Once in the brain, replication of the virus may cause neurological disorders. Larger studies from China and France have also investigated the prevalence of neurological disorders in COVID-19 patients. These studies have shown that 36% of patients have neurological symptoms. Many of these symptoms were mild and include headache or dizziness that could be caused by a robust immune response. Other more specific and severe symptoms were also seen and include loss of smell or taste, muscle weakness, stroke, seizure and hallucinations. Case studies have described severe COVID-19 encephalitis and stroke in healthy young people with otherwise mild COVID-19 symptoms. These symptoms are seen more often in severe cases, with estimates ranging from 46% to 84% of severe cases showing neurological symptoms. Changes in consciousness, such as disorientation, inattention and movement disorders, were also seen in severe cases and found to persist after recovery. Therefore, brain inflammation in severe COVID-19 might also indirectly cause neurological damage, such as through brain swelling, which is associated with neurodegenerative diseases.

The virus is mainly spread during close contact and by small droplets produced when those infected cough, sneeze or talk. These small droplets may also be produced during breathing, but rapidly fall to the ground or surfaces and are not generally spread through the air over large distances. People may also become infected by touching a contaminated surface and then their face. The virus can survive on surfaces for up to 72 hours. It is most contagious during the first three days after onset of symptoms, although spread may be possible before symptoms appear and in later stages of the disease. The time from exposure to onset of symptoms is typically around five days, but may range from two to 14 days. The standard method of diagnosis is by real-time reverse transcription polymerase chain reaction (RT- PCR) from a nasopharyngeal swab. The infection can also be diagnosed from a combination of symptoms, risk factors and a chest CT scan showing features of pneumonia.

Currently, there is no vaccine or specific antiviral treatment for COVID-19. Management involves treatment of symptoms, supportive care, isolation and experimental measures.

If any of the above-mentioned effects of COVID-19 is related to the immune response, and not only to the viral infection itself, then there is a risk that it may also be triggered by a vaccine, since a vaccine could produce the same immune responses. Therefore, the method of treatment of the present invention poses a significant advantage over vaccine and other antibody -mediated therapeutic approaches.

The method of treatment may comprise a step of administering the polypeptide of the invention to the subject. The skilled person will be able to determine by conventional methods the amount of the polypeptide of the invention that are able to exert a therapeutic effect on the patient.

The polypeptide of the invention or the pharmaceutical composition of the invention may be administered once, but it may be administered multiple times. The polypeptide of the invention may be administered from three times daily to once every six months or longer. The administering may be on a schedule such as three times daily, twice daily, once daily, once every two days, once every three days, once weekly, once every two weeks, once every month, once every two months, once every three months and once every six months. The polypeptide of the invention or the pharmaceutical composition of the invention may also be administered continuously via a minipump. The polypeptide of the invention or the pharmaceutical composition of the invention may be administered via an oral, mucosal, buccal, intranasal, inhalable, intravenous, subcutaneous, intramuscular, parenteral, or topical route. The polypeptide of the invention may be administered locally or systemically. The polypeptide of the invention or the pharmaceutical composition of the invention may be administered once, at least twice or for at least the period of time until the condition is treated, palliated or cured. The polypeptide of the invention will generally be administered as part of a composition as described supra. The dosage of polypeptide of the invention will generally be in the range of 0.1-100 mg/kg, more preferably 0.5-50 mg/kg, more preferably 1-20 mg/kg, and even more preferably 1-10 mg/kg. The serum concentration of the polypeptide of the invention may be measured by any method known in the art.

The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

EXAMPLES

Example 1: Generation of binders specific for the ectodomain of hACE2 by phage display

Single domain antibodies (dAb or VHH) with specificity for human ACE2 were isolated from a combined naive llama phage display library using a combination of immobilised and solution phase antigen selection. Screening assays were initially carried out on recombinant protein.

Library preparation

Peripheral blood lymphocytes (PBLs) from eight non-immunised llamas were isolated from 150 ml blood samples. RNA was isolated from PBLs using the RNeasy maxi kit (Qiagen). cDNA was synthesised from each llama using oligo-dT priming and Superscript III reverse transcriptase. dAb-encoding cDNA was amplified by PCR using specific primers, analysed to ensure presence of 400bp fragment and purified (Zymoclean). The resulting clean PCR product was cut by restriction enzyme digest (PST1 and NOT1) and ligated into M13 phage vector viral coat protein (pRL144). Multiple electroporations were performed into TGI E. coli and selections carried out on ampicillin agar plates before pooling and freezing samples. Phage with antibodies expressed as protein coat were amplified from TGI E. coli. Briefly, TGI cells were inoculated into 2TY media supplemented with glucose (2%)/ ampicillin (1 pg/ml) and cultured at 37 °C with agitation to an ODeoonm of 0.5 before addition of M13K07 helper phage. Cells were resuspended 40 min later into 2YT media supplemented with kanamycin and ampicillin (100 pg/70 pg/ml) and incubated at 30 °C overnight with agitation. Phage were purified using PEG based precipitation and titrated into TGI E. coli to determine PFUs/ml.

Phage Display Panning

Recombinant human ACE2 conjugated to a human IgG-Fc was immobilised on a Nunc immunotube at 1 pg/ml overnight at 4 °C before blocking with a 2% milk PBS solution. Phage were blocked in 2% milk PBS with 1 pg/ml of a Human IgG-Fc tagged protein included for further blocking (2ml) for 1 h before addition to the Human ACE2 coated immunotube. After 1 h at room temperature, the tube was washed 10 times using PBS 0.05% tween (4 ml/wash). Elution of specific phage was performed by addition of re-warmed trypsin (2 ml) to the tube and incubation at 37°C for 10-15 min. Eluted phage were amplified by reinfection into log phase TGI cells (5 ml) and plating out on Amp/Gluc agar plates. Titrations were performed to establish phage numbers and enrichment.

Further panning selection rounds were performed as above except alterations to panning antigen and elution method were made (see Figure 3). Namely, in Pan 2 separately coated immunotubes were eluted either by addition of trypsin as described above or using a 10-fold excess molar concentration of recombinant SARS-CoV-2 spike protein (SI subunit) in order to elute RBD blocking specific binders only.

Screening

Individual bacterial colonies were picked and cultured at 37 °C in 2TY media supplemented with glucose (2%)/ ampicillin (1 pg/ml) until OD600nm of 0.5 before addition of M13K07 helper phage to induce phage expression, resuspension in kanamycin and ampicillin supplemented media and overnight culture at 30 °C with agitation. The cells were pelleted by centrifugation and supernatant used directly for screening ELISAs. After assessment of initial panning enrichment (data not shown), monoclonal phage expressing TGI colonies were selected from titration plates, cultured and phage expression induced by helper phage addition and kanamycin selection with glucose starvation.

ELISA for detection of ACE 2 binding of selected bacteriophages

All incubations were 1 h with agitation at room temperature whilst washing was 3 times with 0.05% PBS. tween. Human recombinant ACE2-Fc was coated on a 96 well plate at 0.5ug/ml (50 mΐ) and incubated at room temp for 1 h, a further 2 plates were coated with human Fc control protein (B7H3) or blocking reagent (2% milk), washed and blocked with 2% milk for 1 h and washed again before supernatants were added. Detection antibody was anti -Ml 3- HRP conjugate (Sino biologicals 11973-MM05T-H), and the plate was developed with TMB substrate before being read at 450nm.

ELISA analysis showed a mix of positive clones and background binders. These were further analysed in ELISAs against human Fc as well as target ACE2 and background proteins. Results are shown in Figure 4.

Sequencing

All sequences were obtained after PCR amplification of dAb coding region from phage expressing TGI cells using primers M13Rvs: caggaaacagctatgac (SEQ ID NO: 35) and M13 Fwd: gtcgtctttccagacgttagt (SEQ ID NO: 36). Primer annealing temperature was 48 °C. M13Rvs primer was used as sequencing primer at Source Bioscience (Cambridge, UK).

Sequencing yielded seven unique clones with distinct CDR profiles, i.e. clones 3, 5, 6, 11, 12, 16 and 20 (Figure 4). The sequences obtained are as shown as SEQ ID NO: 5-11.

Example 2: Generation of fusion proteins based on anti-hACE2 binders

Fusions with human serum albuminThe hACE2-specific dAbs (VHH) obtained in Example 1 are cloned in a protein expression vector using a murine IgKappa leader sequence. dAbs are fused to a human serum albumin. The plasmid vector is transiently transfected onto suspension Freestyle HEK293 using polyethylenimine (PEI), and onto ExpiCHO using Expifectamine transfection reagent. Transfected cells are cultured for 5 days in a shaker incubator at 37 °C, 8% CO2 to allow for protein secretion. Culture supernatant is filtered using 0,22pm filter units to remove large contaminants (cells and cellular debris).

Fusion proteins are purified using an AKTA™ pure system (GE Healthcare) using a HiTrap Blue HP 1 ml column (GE Healthcare). Briefly, columns are equilibrated with 5 column volumes of PBS pH 7.4. Supernatant is applied to the column at a flow rate of 1 mL/min. Following application of supernatant, the column is washed with 20 column volumes of PBS. Sample is then eluted from the column with 3 ml of PBS, 2M NaCl at 1 mL/min and is directly loaded onto 2 HiTrap 5 ml desalting columns, previously equilibrated in PBS, and is collected on a 96-well plate using a fraction collector unit. Proteins are characterised via SDS-PAGE under reducing and non-reducing conditions to assess molecular weight and purity.

Fusions with IgG Fc

The hACE2-specific dAbs (VHH) obtained in Example 1 were cloned in a protein expression vector using a murine IgKappa leader sequence. dAbs were fused to a Murine IgG 2A-Fc via a hinge spacer within the DNA coding vector. dAb-Fc conjugates were expressed as above in HEK293 or CHO cells and purified via HiTrap Protein A column (GE Healthcare) using an AKTA™ pure system (GE Healthcare) following manufacturer’s instructions is sufficient. Desalting was performed as above using HiTrap 5ml desalting columns before SDS-PAGE visualisation again described above.

Example 3: Characterisation of albumin-based fusion proteins based on anti-hACE2 binders

ELISA assay

The anti hACE2 dAb (VHH)-albumin based fusion proteins generated in Example 2 are characterised by ELISA to assess binding capacity to hACE2. Briefly, Nunc MaxiSorp flat- bottom 96-well plates are coated with 0.5 pg/ml of recombinant hACE2-Fc. As control, 0.5 pg/ml of B7H3-Fc peptides are coated at 1 pg/ml. Plates are blocked with a solution of 2% BSA in PBS for lh at RT. Antibodies (dAb (VHH)-albumin fusions) are incubated at a range of concentrations, diluted in 0.5% BSA, and allowed to bind for lh at room temperature. Non-specific interactions and un-bound fusion proteins are washed away by 4 PBS 0.05% Tween20 buffer washes. Bound fusion proteins are detected via anti-HSA-HRP conjugated secondary antibody. Un-bound fusion proteins are washed away using PBS 0.05% Tween20 buffer washes. Plates are incubated with substrate reagent (1-Step Ultra TMB, Thermo Scientific) and blocked with 1M H2SO4. Signal is acquired using a Varioskan plate reader at 450nm.

SPR assay

Recombinant hACE2-Fc fusion proteins are immobilised on individual flow cells on a Series S CM5 sensor chip (GE Healthcare) previously functionalised with anti-human capture kit or protein A using a Biacore SPR instrument. HBS-P+ buffer is used as running buffer in all experimental conditions. Recombinant purified anti hACE2 dAb (VHH)-albumin-based fusion proteins (Example 2) at known concentrations are used as the ‘analyte’ and injected over the respective flow cells with 150 s contact time and 600 s dissociation at 30 mΐ/min of flow rate with a constant temperature of 25 °C. In each experiment, flow cell 1 is unmodified and used for reference subtraction. A ‘0 concentration’ sensorgram of buffer alone is used as a double reference subtraction to factor for drift. Data are fit to a 1:1 Langmuir binding model using local Rmax.

Fluorescence-based receptor blocking assay

The anti hACE2 dAb (VHH)-albumin-based fusion proteins generated in Example 2 are serially diluted from the mM to the pM range and the decreased concentrations fractions are incubated with hACE2 expressing cells and incubated for 30 min at 37 °C. Biotinylated recombinant soluble SI spike protein domain is then added at a constant concentration for 30 min at 37 °C. Cells are then washed with PBS to remove any unbound proteins and stained with Streptavidin conjugated to a fluorophore for 30 min at room temperature. After another PBS wash, cells are analysed for SI spike protein domain binding to ACE2 in the presence of recombinant therapeutics by flow cytometry.

ACE 2 enzymatic assay

The enzymatic activity of active ACE2 in the presence of anti-hACE2 dAb (VHH)-albumin- based fusion proteins generated in Example 2 is measured by using Mca-APK(Dnp) (Enzo Life Science) as substrate in 96-well black microtiter plates. ACE2-Fc (ACRO biosystems) is initially incubated in the presence of a 10-fold excess of each anti-hACE2 dAb (VHH)- albumin-based fusion protein clone for 20 min at RT. Samples are then diluted in reaction buffer (50 mM 4-morpholineethanesulfonic acid, pH = 6.5, 300 mM NaCl, 10 mM ZnCh and 0.01% Triton X-100) to an ACE2-Fc concentration of 0.1 pg/ml and an VHH-albumin concentration of 10 pg/mL in the presence of 20 pM of Mca- APK(Dnp) or control peptide BML-P127 (Enzo Life Sciences) in a final volume of 100 pl/well. The reaction is performed at room temperature for lh. Activity is measured as fluorescence intensity at 328 nm/393 nm (Ex/Em) wavelength at 1 -minute intervals using a Varioskan LEIX instrument (Thermo Scientific). Half maximal activity (t ½) (s) is calculated using non-linear fit in GraphPad Prism v8 (GraphPad software).

Infectivity neutralisation assay

The anti hACE2 dAb (VHH)-albumin-based fusion proteins generated in Example 2 are serially diluted to cover a broad range of concentrations. Each dilution is then mixed 1:1 with lentiviral vectors pseudotyped with SARS-CoV-2 spike protein to a final volume of 200 pL and incubated at 37 °C for 30 min-1 h. The lentiviral vectors also encode eGFP. Mixtures of fusion proteins and virus are then cultured onto hACE2 expressing cell line for 48-72 h. Viral titers are then quantified by eGFP expression in target cells and infectivity of all dilution is determined as a percentage of viral titers in the absence of the recombinant protein.

Example 4: Generation of binders specific for the ectodomain of hACE2 by immunising Llama

Two llamas were immunised with recombinant human ACE2 protein (400 pg in two separate doses) and 150 ml blood samples obtained from each animal. An enriched lymphocyte population was obtained via Ficoll discontinuous gradient centrifugation. From these cells, total RNA was isolated by acid guanidium thiocyanate extraction. After first strand cDNA synthesis DNA fragments encoding HC-V fragments and part of the long or short hinge region were amplified by PCR. The amplified pool of dAb (VHH) antibody sequences was digested using the restriction enzymes Pstl and Notl, and ligated into the phagemid vector PRL114. Single domain antibodies (dAbs, VHHs) were expressed on phage after infection with M13K07. The phage library was panned for the presence of binders respectively on solid- phase ACE2 in wells of a microtitre plates or in solution with 100 nM biotinylated ACE2 in combination with streptavidin-coated magnetic beads.

ELISA for detection of ACE2 binding of selected bacteriophages and sequencing was carried out as described in Example 1.

Example 5: Generation of fusion proteins based on anti-hACE2 binders and IgG Fc

Anti-ACE2 dAbs (VHH) obtained in Example 4 were formatted into an IgG Fc as described in Example 2.

Briefly, hACE2-specific dAbs (VHH) obtained in Example 4 were cloned in a protein expression vector using a murine IgKappa leader sequence. dAbs were fused to a Murine IgG 2A-Fc via a hinge spacer within the DNA coding vector. The dAb (VHH)-MuIgG2a Fc (dAb-Fc or VHH-Fc) conjugates were expressed as above in HEK293 or CHO cells and purified via HiTrap Protein A column (GE Healthcare) using an AKTA™ pure system (GE Healthcare) following manufacturer’s instructions is sufficient. Desalting was performed as above using HiTrap 5ml desalting columns before SDS-PAGE visualisation again described above.

Example 6: Characterisation of IgG-based fusion proteins based on anti-hACE2 binders (dAbs/VHHs)

ELISA for detection of ACE2 binding

Nunc Maxisorp clear 96-well plates were coated with 1 pg/ml (in PBS) of ACE2-Fc (ACRO Biosystems) overnight at 4 °C. The next day, plates were blocked with PBS 2% BSA. Anti- ACE2 dAb (VHH)-MuIgG2a Fc clones obtained in Example 5 were each added at 10 pg/ml (in PBS with 0.5% BSA), in duplicates, and plates were incubated for lh at RT. Bound antibodies were detected with anti-mouse HRP-conjugated secondary antibodies (Jackson ImmunoResearch) at 1 :5000 dilution in PBS with 0.5% BSA. Incubation was allowed for lh at RT. Specific interaction was revealed with l-step TMB Ultra reagent (Thermo Fisher) and the reaction was blocked with 1M H2SO4. Plates were acquired on a Varioskan Lux instrument at a wavelength of 450 nm. Data analysed with GraphPad Prism 8 (GraphPad software).

Results shown in Figure 5 revealed that 59 out of the 64 tested anti-ACE2 dAb (VHH)- MuIgG2a Fc clones appeared to bind ACE2-Fc.

ELISA for assessing blocking of SI binding to ACE 2

Nunc Maxisorp clear 96-well plates were coated with 1 pg/ml (in PBS) of ACE2-Fc (ACRO Biosystems) overnight at 4°C. The next day, plates were blocked with PBS 2% BSA. Anti- ACE2 dAb (VHH)-MuIgG2a Fc clones obtained in Example 5 were each added at 10 pg/ml (in PBS with 0.5% BSA), in duplicates, and plates were incubated for lh at RT. Following a subsequent round of washes, recombinant dual His6-tagged SARS-CoV2 SI D614G expressed in expi293 cells was added to the plate at 1 pg/ml (in PBS with 0.5% BSA) and allowed to incubate for lh at RT. Bound SI was detected with HRP-conjugated anti-His-tag secondary antibody (Santa Cruz Biotechnology) at 1:200 dilution in PBS with 0.5% BSA. All washes were performed in PBS 0.05% Tween20. Specific interaction was revealed with 1-step TMB Ultra reagent (Thermo Fisher) and blocked with 1M H2SO4. Plates were acquired on a Varioskan Lux instrument at a wavelength of 450 nm. Data analysed with GraphPad Prism 8 (GraphPad software).

Results shown in Figure 6 revealed that 3 out of the 64 tested anti-ACE2 dAb (VHH)- MuIgG2a Fc clones seemed to block ACE2-Fc to SI protein. These three clones showed >90% blocking of SI binding to ACE2-Fc (Figure 6B):

Clone C55 - 99.2% blocking Clone C66 - 98.0% blocking Clone C72 - 93.9% blocking

Clones 74, 75, 76, 77 and 82 seemed to significantly potentiate/stabilise the binding of SI to ACE2-Fc. ACE 2 enzymatic assay

The enzymatic activity of active ACE2 in the presence of each anti-ACE2 dAb (VHH)- MuIgG2a Fc clone obtained in Example 5 was measured by using Mca-APK(Dnp) (Enzo Life Science) as substrate in 96-well black microtiter plates. ACE2-Fc (ACRO biosystems) was initially incubated in the presence of a 10-fold excess of each anti-ACE2 dAb (VHH)- MuIgG2a clone for 20 min at RT. Samples were then diluted in reaction buffer (50 mM 4- morpholineethanesulfonic acid, pH = 6.5, 300 mMNaCl, 10 pMZnCh and 0.01% Triton X- 100) to an ACE2-Fc concentration of 0.1 pg/ml and an VHH-IgG concentration of 10 pg/mL in the presence of 20 pM of Mca-APK(Dnp) or control peptide BML-P127 (Enzo Life Sciences) in a final volume of 100 pl/well. The reaction was performed at room temperature for lh. Activity was measured as fluorescence intensity at 328 nm/393 nm (Ex/Em) wavelength at 1 -minute intervals using a Varioskan LEIX instrument (Thermo Scientific). Half maximal activity (t ½) (s) was calculated using non-linear fit in GraphPad Prism v8 (GraphPad software).

Results are shown in Figure 7 and Table 1. The results revealed that most anti-ACE2 dAb (VHH)-MuIgG2a Fc clones did not significantly impair the catalytic activity of ACE2. It is surprising that the three VHH-IgG clones that blocked the binding of S 1 to ACE2-Fc (Clones C55, C66 and C72) triggered a slight enhancement in the enzymatic activity of ACE2, seen as a decrease in ti/2 (Figure 7B, Table 1). Additionally, the same 5 clones that significantly potentiate/stabilised the binding of SI to ACE2-Fc (Clones 74, 75, 76, 77 and 82) were also the ones that strongly impaired the enzymatic activity of ACE2, which is seen as an increase in ti/2 (Figure 7B).

Table 1. Anti-ACE2 dAb (VHH)-MuIgG2a clones showing no inhibitory effect on the enzymatic activity (ti/2) of ACE2-Fc. Clones causing a decrease in enzymatic activity below 80% of the reference activity (or an increase in ti/2 over 20%) were not included as these were considered to inhibit ACE2.

Neutralisation of SARS-CoV-2 infectivity

Clones 55, 66, 72 and control clone 84, obtained from Example 5 were tested for neutralisation of viral infectivity using lentiviral vectors pseudotyped to express the SARS- CoV-2 spike glycoprotein from the WT (Wuhan) or B.1.1.7 variants.

Selected anti-ACE2 clones, and the control ACE2-Fc protein, were diluted in PBS to a concentration of 100 pg/mL. Each antibody was then mixed 1:1 with lentiviral vectors pseudotyped with SARS-CoV S glycoproteins normalised to 2.5 x 10 ⁵ IU/ml, to a final volume of 200 pL and incubated at 37 °C for 30 minutes. Antibody -virus mixtures were then cultured for 72 h with 2.5 x 10 ⁴ HEK-293T cells previously genetically engineered to express human ACE2 and TMPRSS2. Viral titers were then quantified by eGFP expression in target cells using MacsQuant cell analyser and infectivity of all fractions was determined as a percentage of viral titers in the buffer only control.

Results in Figure 8 show that clones 55, 66 and 72 efficiently neutralise the SARS-CoV-2 pseudoviruses tested, with neutralisation levels over 90% for clones 55 and 66 against both viruses and clone 72 against WT spike, and above 80% for clone 72 against B.l.1.7 spike expressing virus. Neutralisation levels were comparable to the ACE2-Fc receptor decoy molecule. No neutralisation was detected for the clone 84 which failed to show ACE2 binding in Figure 5 and SI competition in Figure 6.

This application claims the benefit of United Kingdom application No. 2007441.5 filed on 19 ^th May 2020. This application is incorporated herein by reference in their entirety.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.