WO2022136685A1

WO2022136685A1 - Antibody compositions for treatment of corona virus infection

Info

Publication number: WO2022136685A1
Application number: PCT/EP2021/087602
Authority: WO
Inventors: Xavier Saelens; Nico Callewaert; Bert Schepens; Kenny ROOSE; Loes VAN SCHIE; Catelijne Stortelers
Original assignee: Vib Vzw; Universiteit Gent
Priority date: 2020-12-23
Filing date: 2021-12-23
Publication date: 2022-06-30
Also published as: GB202020502D0; CA3198333A1

Abstract

The present invention relates to antibody compositions targeting multiple epitopes on the Receptor Binding Domain (RBD) of the Corona virus Spike protein. More specifically, the invention relates to compositions comprising antibody fusion proteins which comprise a conventional antibody targeting an anti-SARS-CoV-2 Spike protein epitope and an immunoglobulin single variable domain (ISVD) fused to said antibody, which targets a different epitope on the RBD domain. Even more specifically, the invention relate to an antibody composition comprising a fusion between the S309 anti-SARS-CoV-2 RBD-specific antibody (parent of VIR-7831/ sotrovimab) and the VHH72-based ISVD, and its use in treatment of corona virus infections, especially Covid19.

Description

ANTIBODY COMPOSITIONS FOR TREATMENT OF CORONA VIRUS INFECTION

FIELD OF THE INVENTION

The present invention relates to antibody compositions targeting multiple epitopes on the Receptor Binding Domain (RBD) of the Corona virus Spike protein. More specifically, the invention relates to compositions comprising antibody fusion proteins which comprise a conventional antibody targeting an anti-SARS-CoV-2 Spike protein epitope and an immunoglobulin single variable domain (ISVD) fused to said antibody, which targets a different epitope on the RBD domain. Even more specifically, the invention relate to an antibody composition comprising a fusion between the S309 anti-SARS-CoV-2 RBD-specific antibody (parent of VIR-7831/ sotrovimab) and the VHH72-based ISVD, and its use in treatment of corona virus infections, especially Covidl9.

BACKGROUND

Coronaviruses such as the p-coronaviruses have been the cause of previous major human outbreaks, in 2002 by the SARS-CoV originating in Guangdong in China, and in 2012, by the novel MERS-CoV, though fortunately, the outbreaks did not spread to a global extent. In December 2019, SARS-CoV-2 causing COVID-19 was first detected in the city of Wuhan in China and spread quickly throughout the world by person-to-person transmission leading to the worst pandemic since the Spanish flu in 1918. As there were neither antiviral drugs nor vaccines available, the pandemic has forced countries to take extreme measures. Vaccines were developed and launched in record times, and the World health organisation took strategic approaches to install the goal on the global community to vaccinate 70 % of the world's population in less than a year time, with the aim to substantially increase population immunity globally to prevent and protect from disease, reduce pressure on the health system, reactivate economies, and lower the risk of new variants. Since the first outbreak in 2019, several variants of concern of SARS-CoV- 2, including B.1.1.761 (Alpha), B.1.351 (Beta), B.1.1.28 (P.l, Gamma), and B.1.617.2 (Delta), among others, each having varying numbers of substitutions in the N-terminal domain and the RBD of the SARS- CoV-2 spike. In addition, the recent emergence of the highly-transmissible B.1.1.529 Omicron variant with a high number of mutations, deletions, and insertions in the spike protein, even increased the urgency to accelerated development of novel treatments and prevention of COVID-19. In view of limiting the risk that novel corona virus mutants are resistant to currently developed treatments, COVID19 treatments would benefit from a composition that combines more than one SARS-CoV-2 neutralizing agent, as to broaden the neutralizing potential and improve efficacy of pharmaceutical compositions in treatment of corona virus infections. Moreover, the availability of a number of Corona Spike protein binders that recognize a conserved binding site present in SARS-CoV (also called SARS-CoV-1) and SARS- CoV-2, and in addition with the capacity to compete for binding of the RBD with the human ACE2 receptor, allows to anticipate for treatments broadly targeting corona viruses, including some that may appear in the future.

The reported VHH72² as well as a number of conventional antibodies such as the S309 monoclonal antibody¹, provide for a set of candidates fulfilling the criteria as part of a combinatorial therapy, and with VHH72 being a Nanobody, the specific combination of antibodies and Nbs may as well provide for certain benefits in a combination treatment. VHH72 is known to target a conserved region of the Receptor-binding domain (RBD) of the spike protein of SARS-CoV-1 and -2 betacorona viruses, and upon binding to the RBD, this Nb competes for human ACE2 receptor binding. Moreover, this VHH in an efused bivalent format showed equally strong binding to WT SARS-CoV-2 as well as to SARS-CoV-2 RBD N501Y, K417N, E484K, and K417N + E484K + N501Y SARS-CoV-2 RBD mutants expressed on the surface of mammalian cells in the context of the complete spike of SARS-CoV-1, which was confirmed by an equal neutralization potency against authentic SARS-CoV-2 BetaCov/Belgium/GHB03021/2020, a B.1.1.7. (Alpha) variant with N501Y and a B.1.351 (Beta) variant carrying K417N, E484K, and N501Y mutations (Schepens et al. 2021, Science Translational Medicine, PMID: 34609205). XVR011, the clinical candidate of Exevir Bio based on VHH72, demonstrated strong in vitro neutralization potency against the variants of concern delta (B.1.617.2) and gamma (P.l).

The S309 mAb¹ also cross-reacts with SARS-Cov-1 and -2 RBD to outcompete human ACE2 receptor binding to the Spike protein. From the published data, the binding of S309 and VHH72 to the RBD seem not the be competing with each other, which positions these Corona Spike protein targeting biologicals as good candidates to further investigate a combination therapy. Moreover, VIR-7831 and VIR-7832, both derived from the parent antibody (S309) isolated from memory B cells of a 2003 severe acute respiratory syndrome coronavirus (SARS-CoV-1) survivor, and containing an "LS" mutation in the Fc region to prolong serum half-life; in addition, VIR-7832 encoding an Fc GAALIE mutation that has been shown previously to evoke CD8+ T-cells in the context of an in vivo viral respiratory infection; their epitope does not overlap with mutational sites in variants of concern and continues to be highly conserved among circulating sequences consistent with the high barrier to resistance observed in vitro (Cathart et al., 1 Dec 2021, bioRxiv preprint, doi:10.1101/2021.03.09.434607). Finally, this monoclonal antibody also seems minimally affected in its neutralization capacity of the prevailing, infectious B.1.1.529 Omicron isolate (VanBlargan et al., 17 Dec 2021, bioRxiv preprint doi: 10.1101/2021.12.15.472828).

Whether and how the combination of Spike protein targeting moieties to create multispecificity would prevent infection and under which treatment conditions this would allow to establish a superior drug product is still unknown, but would certainly be of benefit in controlling current and future outbreaks of corona virus infections.

SUMMARY OF THE INVENTION

The present invention discloses antibodies and antibody-based polypeptide fusions or substances which target the Corona virus Spike protein via at least two binding sites on its Receptor Binding domain (RBD), wherein the binding to one epitope was shown to enhance the binding affinity to the second epitope binder. These antibody compositions thus provide for improved treatment options for Covid-19, by targeting multiple epitopes in a single composition to overcome viral escapes as well as to provide for an improved neutralization potential for treatment of corona virus infections.

In a first aspect, the invention relates to an antibody composition specifically binding the Corona virus Spike protein comprising a conventional antibody specifically binding an epitope on the RBD domain via its paratope composed of the Vi and VH chains, through 6 CDRs, and wherein the light and/or heavy chain of said conventional antibody further comprises an N- or C-terminal fusion to a protein binding agent specifically binding the Corona virus Spike protein at another epitope which is thus different from the antibody its antigen-binding site defined by VH and VL CDRS, and wherein said epitope of said fused protein binding agent is defined by binding to the amino acid residues Leu355, Tyr356, Ser358, Ser362, Thr363, Phe364, Lys365, Cys366 and Tyr494 of SEQ ID NO:18, which forms the epitope on the SARS-CoV- 1 RBD of VHH72. A more specific embodiment further includes binding the residue R426 of the RBD of SARS-CoV-1 spike protein as depicted in SEQ. ID NO:18.

In an alternative embodiment, said antibody composition comprises a multispecific antibody, which comprises a conventional antibody specifically binding an epitope on the Spike RBD domain via its antigen-binding site containing VL and VH chains, and which has a protein binding agent fused to its light and/or heavy chain that specifically binds the Spike protein at another epitope, which is defined by binding to the amino acid residues L368, Y369, S371, S375, T376, F377, K378, C379 and Y508 as set forth in SEQ ID NO: 19, which forms the epitope on the SARS-CoV-2 RBD of VHH72.

More specifically, since the exemplified protein binding agent used herein is provided by VHH72, which is specifically binds said epitope on SARS-CoV-1 and SARS-CoV-2 since it concerns a conserved epitope among sarbecoviruses. So in a specific embodiment, said protein binding agent of the antibody composition described herein specifically binds at least both epitopes: Leu355, Tyr356, Ser358, Ser362, Thr363, Phe364, Lys365, Cys366, and Tyr494 of SEQ ID NO:18 , and the corresponding binding site comprising amino acids L368, Y369, S371, S375, T376, F377, K378, C379 and Y508 as set forth in SEQ ID NO: 19, so providing for a protein binding agent that is at least cross-reactive to the RBD of SARS-CoV-1 and SARS-CoV-2.

In a further embodiment, said antibody composition comprising a conventional antibody specifically binding the SARS-CoV-2 spike protein RBD domain of which at least one chain is fused to a protein binding agent which specifically binds said epitope of the RBD as described herein, wherein said protein binding agent comprises an immunoglobulin single variable domain (ISVD). More specifically, said antibody fusion comprising an ISVD, comprises said ISVD comprising at least the binding region of VHH72, VHH72-S56A, or VHH72h5, which contains at least the CDR1, CDR2, and CDR3 of VHH72, or of VHH72-S56A, or of VHH72h5. Specifically, said CDR1, 2, and 3 sequences may be annotated according to Kabat, MacCallum, IMGT, AbM, aHo, Chothia, Gelfand, or Honegger. More specifically, said antibody fusion comprising an ISVD, relates to an ISVD comprising VHH72 as depicted in SEQ ID NO: 9 or a humanized variant thereof, such as SEQ. ID NO: 10 or 11, or the VHH72-S56A mutant variant as depicted in SEQ ID NO: 1, or another variant thereof, such as SEQ ID NO: 12 or 13, or the affinity-matured variant of VHH72 called VHH72h5, or VHH72hl_ElD_R27L_E31D_Y32l_S56G_L97A which contains 5 additional substitutions over the VHH72hl-ElD sequence of SEQ ID NO:11, specifically R27L, E31D, Y32I, S56G, and L97A (according to Kabat numbering) as shown in SEQ ID NQ:20, or a further humanized variant of any one thereof. In a further specific embodiment, the composition comprises a monoclonal antibody, specifically an antibody that is also cross-reactive to the RBD of the Spike protein of coronavirus SARS- CoV-1 (SEQ ID NO:18) and SARS-CoV-2 (SEQ ID NO:19), more specifically said antibody may comprise the S309 monoclonal antibody as described in Pinto et ai.¹, or a mutant variant thereof, or a further improved variant thereof, such as the VIR-7831 and VIR-7832 antibodies. Said variants may alternatively contain a further mutation to reduce or abolish binding to the Fey receptors as to avoid immune effector function, a mutation that increases binding to the Fc neonatal receptor to increase the half-life in circulation, or a humanized variant of any one thereof.

In a further embodiment the conventional antibody is connected to the protein binding agent, specifically the ISVD, at the N- or C-terminus of the light chain of the antibody, and/or at the N- or C- terminus of the heavy chain of the antibody, preferably at the N-terminus of the light chain and/or the C-terminus of the heavy chain.

In another embodiment, the composition comprises a light chain fusion with a VHH72 variant, as exemplified in SEQ ID NO: 5, SEQ ID NO: 22, or a variant thereof, and/or comprises a heavy chain a fusion with a VHH72 variant as exemplified in SEQ ID NO: 4, SEQ ID NO: 21, or a variant thereof, or wherein said heavy chain fusion comprises a heavy chain which is an Fc receptor mutant variant, as exemplified in SEQ ID NO: 7, SEQ ID NO: 23, or a further humanized variant thereof. A further specific embodiment provides for a composition as described herein, produced in and/or purified from a host cell, wherein the antibody comprises the S309-Hc-72 fusion, composed of SEQ ID NO:2 as light chain, and SEQ. ID NO:4, as heavy chain; or the S309-LC-72 fusion, composed of SEQ ID NO:5 as light chain, and SEQ ID NO:3 as heavy chain; or the double fusion S309-Lc-72 Hc-72, composed of SEQ ID NO:5 as light chain, and SEQ ID NO:4 as heavy chain; or any further (humanized) variant of any one thereof; alternatively, the antibody composition is composed of conventional or monoclonal antibody with a reduced or abolished Fc receptor binding Fc region, such as the well-known LALA or LALAPG Fc regions⁶, for instance, the antibody compositions comprising the S309-LALA-Hc-72 fusion, composed of SEQ ID NO:2 as light chain, and SEQ ID NO:7 as heavy chain; or the S309-LALA-LC-72 fusion composed of SEQ ID NO:5 as light chain, and SEQ ID NO:6 as heavy chain; or the double S309-LALA-LC-72 Hc-72 fusion composed of SEQ ID NO:5 as light chain, and SEQ ID NO:7 as heavy chain, or any further (humanized) variant of any one thereof.

A further specific embodiment provides for a composition as described herein, produced in and/or purified from a host cell, wherein the antibody comprises the S309-Hc-72h5 fusion, composed of SEQ ID NO:2 as light chain, and SEQ ID NO:21, as heavy chain; or the S309-Lc-72h5 fusion, composed of SEQ ID NO:22 as light chain, and SEQ ID NO:3 as heavy chain; or the double fusion S309-Lc-72h5 Hc-72h5, composed of SEQ ID NO:22 as light chain, and SEQ ID NO:21 as heavy chain; or any further (humanized) variant of any one thereof; alternatively, the antibody composition is composed of conventional or monoclonal antibody with a reduced or abolished Fc receptor binding Fc region, such as the well-known LALA or LALAPG Fc regions⁶, for instance, the antibody compositions comprising the S309-LALA-Hc-72h5 fusion, composed of SEQ ID NO:2 as light chain, and SEQ ID NO:23 as heavy chain; or the S309-LALA-LC- 72h5 fusion composed of SEQ ID NO:22 as light chain, and SEQ ID NO:6 as heavy chain; or the double S309-LALA-Lc-72h5 Hc-72h5 fusion composed of SEQ ID NO:22 as light chain, and SEQ ID NO:23 as heavy chain, or any further (humanized) variant of any one thereof.

Another aspect of the invention relates to a composition comprising a protein binding agent comprising VHH72 or a variant thereof, such as VHH72-S56A, or VHH72h5, or an alternative humanization variant of any one thereof, for which multiple options are disclosed herein, or an Fc fusion of any one thereof, and the S309 antibody or a variant thereof. Said combination of at least two spike protein binding agents with different specificity may further be represented as a combination of S309 or a variant thereof, and the VHH72-S56A-Fc variant as depicted in SEQ ID NO:8. In a further embodiment, said composition comprises the S309 monoclonal antibody comprising the light and heavy chain as set forth in SEQ ID NO:2 and 3, respectively, or a variant with at least 90 % identity over the full length thereof, retaining the CDR sequences. Said compositions of the present invention may also be envisaged as being a pharmaceutical composition comprising any of the antibody compositions as described herein.

In a final aspect, said antibody composition or pharmaceutical composition as described herein may be envisaged for use as a medicament, more specifically, for use in treatment of a corona virus infection.

A specific embodiment relates to the use of the antibody composition, or combination composition or pharmaceutical compositions as described herein for treating a subject in need thereof in a curative or prophylactic manner. More specifically, the invention relates to the use of the antibody composition, or combination of antibodies in the composition or pharmaceutical compositions as described herein for treating a subject to cure or prevent from Covidl9 infection.

DESCRIPTION OF THE FIGURES

The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes.

Figure 1. S309-VHH72 fusions neutralize SARS-CoV-2 S VSV pseudotypes better than S309 or VHH72- Fc alone.

VSV SARS-CoV-2 spike pseudotype GFP reporter virus was incubated with different concentrations of the indicated antibody constructs and used to inoculate confluent monolayers of VeroE6 cells in a 96-well plate. For each dilution series, the GFP signals were normalized to the lowest and highest values of that dilution series and plotted as percentage. The left graph shows the normalized GFP signals ± SD (n=8) obtained for the VHH72-Fc WT IgG constructs. The right graph shows the normalized GFP signals ± SD (n=4) obtained for the S309-VHH72 LALA IgG constructs and PB9683 (n =8). The IC50 values in ug/ml are indicated below each graph. Left: S309 constructs with wt heavy chain. Right: S309 constructs with LALA mutations⁴ in the Fc domain.

Figure 2. S309-VHH72 fusion neutralization of SARS-CoV-2 S VSV pseudotypes versus the combination of the single antibodies.

VSV SARS-CoV-2 spike pseudotype GFP reporter virus was incubated with different concentrations of the indicated antibody constructs, including the 1:1 mixture of S309 with VHH72-Fc (as PB9683; at half the concentration of S309, resulting in nearly equimolar amounts as S309), and used to inoculate confluent monolayers of VeroE6 cells in a 96-well plate. For each dilution series the GFP signals were normalized to the lowest and highest values of that dilution series and plotted as percentage. The left graph shows the normalized GFP signals ± SD (n=4) obtained for the WT IgG constructs and PB9683 (n =4). The right graph shows the normalized GFP signals ± SD (n=8) obtained for the LALA IgG constructs and PB9683 (n =4). The IC₅o values in ug/ml are indicated below each graph. Left: S309 constructs with wt heavy chain. Right: S309 constructs with LALA mutations⁴ in the Fc domain. Figure 3. Light and heavy chain combined fusions neutralize SARS-CoV-2 S VSV pseudotypes better than the single S309 mAb.

VSV SARS-CoV-2 spike pseudotype GFP reporter virus was incubated with different concentrations of the indicated antibody constructs, and used to inoculate confluent monolayers of VeroE6 cells in a 96-well plate. For each dilution series the GFP signals were normalized to the lowest and highest values of that dilution series and plotted as percentage. The left graph shows the normalized GFP signals ± SD (n=2) obtained for the WT IgG constructs. The right graph shows the normalized GFP signals ± SD (n=2) obtained for the LALA IgG constructs. The IC50 values expressed in ug/ml are indicated below each graph. Left: S309 constructs with wt heavy chain. Right: S309 constructs with LALA mutations⁴ in the Fc domain.

Figure 4. The capacity of RBD-binding antibody formats to compete with monovalent VHH72_hl (S56A) binding to SARS-CoV-2 RBD protein in competition AlphaLISA assay.

A, The S309 antibody enhanced the interaction of VHH72 with the RBD domain in a dose-dependent manner, with an EC50 value for S309 of 287 pM; B, VHH72-FC formats (PB9683 batch, called XVR011 herein, comprising VHH72_hl_ElD_S56A-Fc as depicted in SEQ. ID NO:8 , and a WT VHH72-FC batch), as well CB6 anti-SARS-CoV-2 RBD antibody, inhibit the interaction of the VHH72_hl (S56A)-flag3-His6 with the RBD; while the Synagis negative control shows no inhibition, and the S309 antibody increases the VHH72 binding to RBD; C, VHH72-FC format (D72-23 batch comprising the VHH72_hl_S56A-Fc), as well as the conventional CR3022 antibody inhibit the interaction between monovalent VHH72_hl (S56A)- flag3-His6 and RBD.

Figure 5. Purification of S309_D construct.

(A) a HiTrap MAbSelect SuRe 5mL column (affinity chromatography step) followed by (B) a 50mL G-25 desalting column to switch the buffer to PBS (desalting chromatography step).

Figure 6. Coomassie-stained reducing SDS-PAGE of purified S309_A, S309_B, S309_C, and S309_D.

Ten microgram of purified protein was loaded per lane.

Figure 7. S309-VHH72 fusion constructs S309 A to H side by side comparison in a pseudotype neutralization assay.

VSV SARS-CoV-2 spike pseudotype GFP reporter virus was incubated with different concentrations of the indicated antibody constructs (see Table 1) and used to inoculate confluent monolayers of VeroE6 cells in a 96-well plate. For each dilution series, the GFP signals were normalized to the lowest and highest values of that dilution series and plotted as percentage. The graph shows the relative infection percentage ± SEM (N=3). syn, synagis negative control. Figure 8. Comparison of the EC₅o values of S309 armed with either no (S309_A and S309_B), two (S309_C, S309_D, S309_E, S309_F) or four (S309_G and S309_H; see Table 1) VHH72hl_ElD-S56A building blocks compared with D72-53 in the SARS-CoV-2 S VSV pseudotype neutralization assay.

D72-53 corresponds to VHH72_hl_ElD_S56A-(G4S)2-hlgGlhinge_EPKSCdel-hlgGlFc_LALA_K477del, as described in Schepens et al. (2021, Science Translational Medicine, PMID: 34609205; corresponding to SEQ. ID NO:8).

Figure 9. S309-VHH72 fusion constructs neutralize VSV pseudotyped with the spike of Wuhan SARS- CoV-2 more potently than a cocktail of D72-53 and S309.

The graph shows the average (±SD, N=3) relative infection of VeroE6 by SARS-CoV-2 S VSV pseudotypes in the presence of the indicated antibodies. The IC₅o values in nM are indicated below the graph.

Figure 10. S309-VHH72 fusion constructs neutralize VSV pseudotyped with the spike of SARS-CoV-2 beta (B.1.351) more potently than a cocktail of D72-53 and S309.

The graph shows the average (±SD, N=2) relative infection of VeroE6 by SARS-CoV-2 S VSV pseudotypes in the presence of the indicated antibodies. The IC₅o values in nM are indicated below the graph.

Figure 11. Escape map of the SARS-CoV-2 receptor binding-domain (RBD) of S309 antibody, of S309_C (VHH72 fused at the C-terminus of the S309 heavy chain), and of the cocktail of S309 with VHH72-Fc based on deep mutational scanning.

(A) The profile of the RBD amino acid positions involved in the binding of S309 applied at a concentration of 10 pg/ml to the RBD yeast-displayed library are shown in black lines in the graphs. (B) The profile of the RBD amino acid positions involved in the binding of S309_C (S309 with VHH72hl-ElD-S56A fused to the C-terminus of the heavy chain) applied at a concentration of 10 pg/ml to the RBD yeast-displayed library are shown in black lines in the graphs (C) The profile of the RBD amino acid positions involved in the binding of S309, applied at a concentration of 10 pg/ml, mixed with D72-53 (antibody made from SEQ. ID NO: 8) applied at a concentration of 18.5 pg/ml to the RBD yeast-displayed library are shown in black lines in the graphs. Note the fivefold lower scale of the Y-axis in B compared to A and C.

Figure 12. VHH72h5-Fc in a side by side comparison to D72-53 in a pseudotype neutralization assay.

VSV SARS-CoV-2 spike pseudotype GFP reporter virus was incubated with different concentrations of the each antibody constructs and used to inoculate confluent monolayers of VeroE6 cells in a 96-well plate. For each dilution series, the GFP signals were normalized to the lowest and highest values of that dilution series and plotted as percentage. The graph shows the relative infection percentage ± SEM (N=2). DETAILED DESCRIPTION

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. Of course, it is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein. The invention, both as to organization and method of operation, together with features and advantages thereof, may best be understood by reference to the following detailed description when read in conjunction with the accompanying drawings. The aspects and advantages of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases 'in one embodiment' or 'in an embodiment' in various places throughout this specification are not necessarily all referring to the same embodiment but may.

Definitions

Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments, of the invention described herein are capable of operation in other sequences than described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4^th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in molecular biology, biochemistry, structural biology, and/or computational biology). The terms "protein", "polypeptide", and "peptide" are interchangeably used further herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. A "peptide" may also be referred to as a partial amino acid sequence derived from its original protein, for instance after tryptic digestion. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. This term also includes posttranslational modifications of the polypeptide, such as glycosylation, phosphorylation and acetylation. Based on the amino acid sequence and the modifications, the atomic or molecular mass or weight of a polypeptide is expressed in (kilo)dalton (kDa). By "isolated" or "purified" is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated polypeptide" or "purified polypeptide" refers to a polypeptide which has been purified from the molecules which flank it in a naturally-occurring state, e.g., an antibody or nanobody as identified and disclosed herein which has been removed from the molecules present in the sample or mixture, such as a production host, that are adjacent to said polypeptide. An isolated protein or peptide can be generated by amino acid chemical synthesis or can be generated by recombinant production or by purification from a complex sample.

"Homologue", "Homologues" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. The term "amino acid identity" as used herein refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met, also indicated in one-letter code herein) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. A "substitution", or "mutation", or "variant" as used herein, results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a parental protein or a fragment thereof.

"Binding" means any interaction, be it direct or indirect. A direct interaction implies a contact between the binding partners. An indirect interaction means any interaction whereby the interaction partners interact in a complex of more than two molecules. The interaction can be completely indirect, with the help of one or more bridging molecules, or partly indirect, where there is still a direct contact between the partners, which is stabilized by the additional interaction of one or more molecules. By the term "specifically binds," as used herein is meant a binding domain which recognizes a specific target, but does not substantially recognize or bind other molecules in a sample. Specific binding does not mean exclusive binding. However, specific binding does mean that proteins have a certain increased affinity or preference for one or a few of their binders. The term "affinity", as used herein, generally refers to the degree to which a ligand, chemical, antibody, protein or peptide binds to another (target) protein or peptide so as to shift the equilibrium of single protein monomers toward the presence of a complex formed by their binding. As used herein, the term "protein complex" or "complex" or "assembled protein(s)" refers to a group of two or more associated macromolecules, whereby at least one of the macromolecules is a protein. A protein complex, as used herein, typically refers to associations of macromolecules that can be formed under physiological conditions. Individual members of a protein complex are linked by non-covalent interactions.

A "binding agent" relates to a molecule that is capable of binding to another molecule, wherein said binding is preferably a specific binding, recognizing a defined binding site, pocket or epitope. The binding agent may be of any nature or type and is not dependent on its origin. The binding agent may be chemically synthesized, naturally occurring, recombinantly produced (and purified), as well as designed and synthetically produced. Said binding agent may hence be a small molecule, a chemical, a peptide, a polypeptide, an antibody, or any derivatives thereof, such as a peptidomimetic, an antibody mimetic, an active fragment, a chemical derivative, among others. The term "binding pocket" or "binding site" refers to a region of a molecule or molecular complex, that, as a result of its shape and charge, favourably associates with another chemical entity, compound, proteins, peptide, antibody or Nb. The term "pocket" includes, but is not limited to cleft, channel or site. The term "part of a binding pocket/site" refers to less than all of the amino acid residues that define the binding pocket, or binding site. For example, the portion of residues may be key residues that play a role in ligand binding, or may be residues that are spatially related and define a three-dimensional compartment of the binding pocket. The residues may be contiguous or non-contiguous in primary sequence. For antibody-related molecules, the term "epitope" is also used to describe the binding site, as used interchangeably herein. An "epitope", refers to an antigenic determinant of a polypeptide, constituting a binding site or binding pocket on a target molecule, such as the Corona virus Spike protein, more specifically a binding pocket on the RBD domain accessible for the antibodies or binding agents as presented herein. An epitope could comprise 3 amino acids in a spatial conformation, which is unique to the epitope. Generally, an epitope consists of at least 4, 5, 6, 7 such amino acids, and more usually, consists of at least 8, 9, 10 such amino acids. Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, X-ray crystallography and multi-dimensional nuclear magnetic resonance. A "conformational epitope", as used herein, refers to an epitope comprising amino acids in a spatial conformation that is unique to a folded 3-dimensional conformation of a polypeptide. Generally, a conformational epitope consists of amino acids that are discontinuous in the linear sequence but that come together in the folded structure of the protein. However, a conformational epitope may also consist of a linear sequence of amino acids that adopts a conformation that is unique to a folded 3- dimensional conformation of the polypeptide (and not present in a denatured state). In protein complexes, conformational epitopes consist of amino acids that are discontinuous in the linear sequences of one or more polypeptides that come together upon folding of the different folded polypeptides and their association in a unique quaternary structure. The term "conformation" or "conformational state" of a protein refers generally to the range of structures that a protein may adopt at any instant in time. A conformational epitope may thus comprise amino acid interactions from different protein domains of the Spike protein. One of skill in the art will recognize that determinants of conformation or conformational state include a protein's primary structure as reflected in a protein's amino acid sequence (including modified amino acids) and the environment surrounding the protein. The conformation or conformational state of a protein also relates to structural features such as protein secondary structures (e.g., a-helix, p-sheet, among others), tertiary structure (e.g., the three dimensional folding of a polypeptide chain), and quaternary structure (e.g., interactions of a polypeptide chain with other protein subunits). Posttranslational and other modifications to a polypeptide chain such as ligand binding, phosphorylation, sulfation, glycosylation, or attachments of hydrophobic groups, among others, can influence the conformation of a protein. Furthermore, environmental factors, such as pH, salt concentration, ionic strength, and osmolality of the surrounding solution, and interaction with other proteins and co-factors, among others, can affect protein conformation. The conformational state of a protein may be determined by either functional assay for activity or binding to another molecule or by means of physical methods such as X-ray crystallography, NMR, or spin labeling, among other methods. For a general discussion of protein conformation and conformational states, one is referred to Cantor and Schimmel, Biophysical Chemistry, Part I: The Conformation of Biological. Macromolecules, W.H. Freeman and Company, 1980, and Creighton, Proteins: Structures and Molecular Properties, W.H. Freeman and Company, 1993.

The term "antibody", "antibody fragment" and "active antibody fragment" as used herein refer to a protein comprising an immunoglobulin (Ig) domain or an antigen binding domain capable of specifically binding the antigen, in this case the Spike protein. 'Antibodies' can further be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The term "active antibody fragment" refers to a portion of any antibody or antibody-like structure that by itself has high affinity for an antigenic determinant, or epitope, and contains one or more complementarity- determining-regions (CDRs) accounting for such specificity. Non-limiting examples include immunoglobulin domains, Fab, F(ab)'2, scFv, heavy-light chain dimers, immunoglobulin single variable domains, Nanobodies, domain antibodies, and single chain structures, such as a complete light chain or complete heavy chain. An additional requirement for "activity" of said fragments in the light of the present invention is that said fragments are capable of binding the RBD spike protein, and preferably are competing for the human ACE2 binding to the RBD in a subject. The term "immunoglobulin (Ig) domain", or more specifically "immunoglobulin variable domain" (abbreviated as "IVD") means an immunoglobulin domain essentially consisting of four "framework regions" which are referred to in the art and herein below as "framework region 1" or "FR1"; as "framework region 2" or "FR2"; as "framework region 3" or "FR3"; and as "framework region 4" or "FR4", respectively; which framework regions are interrupted by three "complementarity determining regions" or "CDRs", which are referred to in the art and herein below as "complementarity determining region 1" or "CDR1"; as "complementarity determining region 2" or "CDR2"; and as "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. It is the immunoglobulin variable domain(s) (IVDs) that confer specificity to an antibody for the antigen by carrying the antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation. In view of the above definition, the antigenbinding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, with binding to the respective epitope of an antigen by a pair of (associated) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen. An immunoglobulin single variable domain (ISVD) as used herein, refers to a protein with an amino acid sequence comprising 4 Framework regions (FR) and 3 complementary determining regions (CDR) according to the format of FR1-CDR1-FR2-CDR2-FR3-CDR3- FR4. An "immunoglobulin single variable domains" (abbreviated as "ISVD"), as used herein, is equivalent to the term "single variable domains", and defines molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from "conventional" immunoglobulins or their fragments, wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. The binding site of an immunoglobulin single variable domain is formed by a single VH/VHH or VL domain. Hence, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDR's. As such, the single variable domain may be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit). In particular, the immunoglobulin single variable domain may be a Nanobody® (as defined herein) or a suitable fragment thereof. Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx N.V. (a Sanofi Company). For a general description of Nanobodies, reference is made to the further description below, as well as to the prior art cited herein, such as e.g. described in W02008/020079. "VHH domains", also known as VHHs, VHH domains, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen binding immunoglobulin (Ig) (variable) domain of "heavy chain antibodies" (i.e., of "antibodies devoid of light chains"; Hamers-Casterman et al (1993) Nature 363: 446-448). The term "VHH domain" has been chosen to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VH domains") and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VL domains"). For a further description of VHHs and Nanobody , reference is made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001), as well as to the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and Ablynx N.V.; WO 01/90190 by the National Research Council of Canada; WO 03/025020 (= EP 1433793) by the Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the further published patent applications by Ablynx N.V. As described in these references, Nanobody (in particular VHH sequences and partially humanized Nanobody) can in particular be characterized by the presence of one or more "Hallmark residues" in one or more of the framework sequences. A further description of the Nanobody, including humanization and/or camelization of Nanobody, as well as other modifications, parts or fragments, derivatives or "Nanobody fusions", multivalent or multispecific constructs (including some non-limiting examples of linker sequences) and different modifications to increase the half-life of the Nanobody and their preparations can be found e.g. in WO 08/101985 and WO 08/142164. Nanobodies form the smallest antigen binding fragment that completely retains the binding affinity and specificity of a full-length antibody. Nbs possess exceptionally long complementarity-determining region 3 (CDR3) loops and a convex paratope, which allow them to penetrate into hidden cavities of target antigens.

As used herein, the terms "determining," "measuring," "assessing,", "identifying", "screening", and "assaying" are used interchangeably and include both quantitative and qualitative determinations.

The term "subject", "individual" or "patient", used interchangeably herein, relates to any organism such as a vertebrate, particularly any mammal, including both a human and another mammal, for whom diagnosis, therapy or prophylaxis is desired, e.g., an animal such as a rodent, a rabbit, a cow, a sheep, a horse, a dog, a cat, a lama, a pig, or a non-human primate (e.g., a monkey). The rodent may be a mouse, rat, hamster, guinea pig, or chinchilla. In one embodiment, the subject is a human, a rat or a non-human primate. Preferably, the subject is a human. In one embodiment, a subject is a subject with or suspected of having a disease or disorder, in particular a disease or disorder as disclosed herein, also designated "patient" herein. However, it will be understood that the aforementioned terms do not imply that symptoms are present. The term "treatment" or "treating" or "treat" can be used interchangeably and are defined by a therapeutic intervention that slows, interrupts, arrests, controls, stops, reduces, or reverts the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related signs, symptoms, conditions, or disorders.

The term "medicament", as used herein, refers to a substance/composition used in therapy, i.e., in the prevention or treatment of a disease or disorder. According to the invention, the terms "disease" or "disorder" refer to any pathological state, in particular to the diseases or disorders as defined herein.

Detailed description

The present invention relates to an antibody composition or polypeptide composed of an antibody fused to another antigen-binding protein via its N- or C-terminal end of its light and/or heavy chain. The composition provides for specific binding to the RBD domain of the Corona virus Spike protein at sites that are conserved in at least SARS-Cov and SARS-Cov-2 virus strains. Moreover, the specific binding is conferred by binding at least two different epitopes on the RBD domain, which may enhance for the effect of binding to the RBD domain and/or thereby improve blocking the ACE2 binding to the RBD domain. This effect translates further in an enhanced viral neutralization, as compared to targeting just a single binding site on the RBD domain, and moreover, provides for at least similar or improved neutralization as compared to the combination of the single binders. A further advantage of the use of said antibody composition lies in the simultaneous targeting of multiple epitopes, anticipating a viral escape for the treatment. Indeed, combining (monoclonal) antibodies that are binding to the same target in a non-competing manner is commonly used in a treatment strategy, via applying a mixture or cocktail of said biologicals as to prevent viral infection by escape mutants. The antibody composition of the present invention comprising the fusion proteins however provides for a single drug substance that is capable of targeting more than one target site or epitope, allowing to anticipate on viral escape mutants of the Spike protein, and/or to increase the efficacy of antiviral coronavirus treatments without a need for combination therapy. Moreover, an escape mutant profiling analysis revealed that the presence of the multispecific antibodies targeting SARS-CoV-2 spike protein as a fusion results in a significantly lower risk to escape mutations as compared to a combination of several antibodies in a composition.

A composition as described herein relates to a substance containing at least the 'antibody', 'antibody fusion', or 'antibody composition' of the invention, as interchangeably used herein. The composition provides for at least one antibody that is composed of two light chains and two heavy chains, as typically known for conventional antibodies, wherein at least one of said light of heavy chain is present as a fusion protein of the chain, linked via a linker or directly, at its N- and/or C-terminus, to another antigen-binding protein that specifically binds the Corona virus Spike protein RBD domain.

The 'composition' as described herein may further comprise other antibodies or binding agents, or may comprise other components, for instance important for providing a physiologically stable conditions for the antibody. The composition may thus also be provided in the form of a kit comprising a first container comprising lyophilised antibody or antibody fusion protein and a second container comprising a solution for resuspension of the lyophilised proteins. The protein powder may comprise one or more lyoprotectant such as sucrose, dextran, sorbitol and amino acids to stabilise the protein during lyophilisation. Alternatively, the composition is provided in a single container comprising the antibody or antibody fusion protein(s) in suspension or solution. Either solution or the composition may contain one or more excipient(s). The solutions are typically water-based. Therefore, purified water may form the main excipient. For example, dilution of the protein to give the desired final concentration will usually be performed with water for injection (WFI). The solution typically contains a buffer. Therefore, further excipients include buffering agents and pH regulators such as sodium citrate, sodium dihydrogen phosphate monohydrate, and sodium hydroxide. In some instances, a thickening agent such as xanthan may be present as a further excipient. A surfactant, in particular a non-ionic surfactant such as polysorbate 80, may also be present. Other excipients include sucrose, sorbitol, inorganic salts, amino acids and vitamins.

In one embodiment, said antibody composition contains at least two paratopes for specifically binding to different binding sites of the RBD of the corona spike protein, wherein one paratope is formed by the light and heavy chains of the antibody, similar to the binding domain of a conventional antibody, comprising residues of at least the CDRs of the heavy and the CDRs of the light chain, and appearing in a bivalent format in the antibody composition, and wherein the second paratope is formed by the antigenbinding protein linked to the conventional antibody its light and/or heavy chain. Since conventional antibodies are formed by dimerization at the Fes of the two heavy chains, each in contact with a light chain, the linked antigen-binding protein will thus also be present in the fused antibody in bivalent format, the latter specifically binding the epitope or binding site on the RBD comprising residues Leu355, Tyr356, Ser358, Ser362, Thr363, Phe364, Lys365, Cys366 and Tyr494 of SEQ ID NO:18, which forms the epitope on the SARS-Cov RBD of VHH72 as described in Wrap et al.², and corresponds to that epitope comprising residues L368, Y369, S371, S375, T376, F377, K378, C379 and Y508 of the SARS-Cov2 spike protein as depicted in SEQ. ID NO:19.

In a specific embodiment, the antigen-binding protein that is fused to the light or heavy chain comprises an ISVD, or may also be a single domain antibody, a VHH, or a Nanobody. Said ISVD is fused via its C- terminus to the N-terminus of the light or heavy chain, or alternatively is fused via its N-terminus to the C -terminus of the light or heavy chain of the conventional antibody. The resulting antibody product after co-expression of the fusion product and the complementary light or heavy chain of the same wild type antibody, also with or without fusion, in a host, leads to the formation of antibody compositions with at least two different binding sites on the RBD target, each of which binding site can be bound via at least 2 paratopes present on the resulting antibody of the antibody composition described herein. Moreover, the bivalent nature of the antibody (in a conventional manner thus with 2 (identical) paratopes formed by the light and heavy chain in each arm of the antibody), will lead to at least 4 binding sites when the light or heavy chain is present as a fusion in the antibody composition, wherein each paratope formed by a light+heavy chain binds the RBD domain, as 2 identical paratopes formed by the conventional dimeric antibody structure, and wherein in addition 2 identical binding sites are formed by the antigenbinding ISVD paratopes fused to light or heavy chain, as present on the antibody composition. When more than one fusion is applied, for instance one antigen-binding protein on the light chain and one on the heavy chain, at least 6 binding sites, defined by at least 2 different paratopes are provided for the SARS-CoV-2 spike protein binding by said antibody composition, more specifically with two identical paratopes from the conventional antibody binding site provided by VL+VH, including 6 CDRs each, two identical paratopes from the ISVD fused at the light chain, provided by 3 CDRs each, and two identical paratopes formed by the ISVD fused at the heavy chain, provided by 3 CDRs each. Even 8 or 10 paratopes may be present on a single antibody composition, when light and heavy chain are fused at N- and C- terminal ends. Said antigen-binding proteins fused to the light and/or heavy chain may be identical or may be variants or different binding agents. So any combination of a light and heavy chain in dimeric format may be possible for combining the paratopes as described herein.

With an 'antibody comprising an N-terminal fusion to a protein binding agent' as used herein is meant that the antibody is fused at the N-terminus of its light or heavy chain to the protein binding agent, which is then fused to the antibody chain at is C-terminus. Alternatively, an 'antibody comprising a C-terminal fusion to the protein binding agent' relates to the antibody's light or heavy chain its C-terminus which is connected to the N-terminus of the protein binding agent, in particular the ISVD. Said fusions or connections can be direct fusions, made via peptide bonds between amino acid residues of the chain and ISVD itself, or indirect fusions made by a linker. Said fusion sites preferably being designed to result in flexible fusion protein, wherein the different paratopes do not interfere with each other. Preferred "linker molecules", "linkers", or "short polypeptide linkers" are peptides with a length of about ten amino acids. Non-limiting examples of suitable linker sequences are described in the Example section, and are known by the skilled person. Linkers may be selected to keep a fixed distance between the structural domains, as well as to maintain the fusion partners their independent functions (e.g. antigenbinding). Fusions of conventional antibodies are known in the art, though not frequently applied, and depending on the aimed target, the function of the fusion, and on the structural relation between the antibody and the fusion, the outcome of applying a fusion as to expect an additive or synergistic effect over its single part is not predictable.

In a further specific embodiment, the composition as described herein relates to said conventional or monoclonal antibody recognizing the corona virus spike protein, or specifically RBD domain, further containing a protein binding agent connected to its light and/or heavy chain comprising an ISVD comprising the CDR1, 2 and 3 sequence of VHH72, VHH72-S56A, or VHH72h5. Determination of CDR regions may be done according to different methods, such as the designation based on contact analysis and binding site topography as described in MacCallum et al. (J. Mol. Biol. (1996) 262, 732-745). Or alternatively the annotation of CDRs may be done according to AbM (AbM is Oxford Molecular Ltd.'s antibody modelling package as described on http://www.bioinf.org.uk/abs/index.html), Chothia (Chothia and Lesk, 1987; Mol Biol. 196:901-17), Kabat (Kabat et al., 1991; 5^th edition, NIH publication 91- 3242), IMGT (LeFranc, 2014; Frontiers in Immunology. 5 (22): 1-22), and/or alternative annotations including aHo, Gelfand, and Honegger; see, e.g., Dondelinger et al. 2018, Front Immunol 9:2278 for a review). Said annotations further include delineation of CDRs and framework regions (FRs) in immunoglobulin-domain-containing proteins, and are known methods and systems to a skilled artisan who thus can apply these annotations onto any immunoglobulin protein sequences without undue burden. These annotations differ slightly, but each intend to comprise the regions of the loops involved in binding the target. The main difference between the CDRs of VHH72 and VHH72-S56A and VHH72h5 is found in CDR2 which contains a Serine, Alanine of Glycine in position 56 (Kabat numbering).

In a further specific embodiment, the composition as described herein relates to said conventional or monoclonal antibody recognizing the corona virus spike protein, or specifically RBD domain, further containing a protein binding agent connected to its light and/or heavy chain comprising an ISVD consisting of VHH72 (as depicted in SEQ ID NO:9, also called VHH72 WT) or an ISVD variant thereof. With 'variant' or 'variant of an ISVD', specifically 'VHH72 variant' or 'VHH72-S56A variant', or 'VHH72h5 variant' as used herein is meant that the VHH72, the VHH72-S56A, or the VHH72h5 ISVD its binding epitope, defined as the residues L368, Y369, S371, S375, T376, F377, K378, C379 and Y508 of SEQ. ID NO:19, is retained in the function of the variant, though that the amino acid sequence of the VHH72, VHH72-S56A, or VHH72h5 variant may be altered in several other ways. For instance, said sequence may be altered in order to increase the affinity to the RBD epitope, or may be altered to humanize the building block, or both, as known from the art, and as described herein. For instance, the initially identified VHH72 selected for specifically binding the RBD from screening a llama immunization library contained the sequence as provided in SEQ ID NO:9 or VHH72 WT (Wrapp et al., 2020). Though after testing a number of VHH72 mutant variants for their binding affinity and/or neutralization potential when fused to an Fc (as shown for VHH72 in Wrapp et al.), a particularly interesting mutant variant of VHH72, the VHH72- S56A mutant (as depicted in SEQ ID NO: 12), with a CDR2 mutation in residue 56 (according to Kabat numbering), was identified. Moreover, in view of the identification and characterization of further VHH72 family members, such as VHH3.115 (WQ2021/156490A2), further affinity-improved variants were designed and shown to provide for enhanced neutralization capacity as compared to the wild type form (as demonstrated in Figure 12). Indeed, the VHH72h5 variant (SEQ ID NQ:20) as used herein has 5 additional substitutions over the VHH71hl-ElD humanized VHH72 variant, which were based on the sequence properties of VHH3.115, resulting in a further variant of VHH72.

It should be noted that - as is well known in the art for VH domains and for VHH domains - the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in a VH domain and a VHH domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein. Humanization of VHHs, as described herein further resulted in a number of exemplified VHH72 variants (e.g. SEQ. ID NO: 1, 10, 11, 13, 20). Those all comprise VHH72, VHH72-S56A, VHH72h5 humanized variants. The term 'humanized variant' of an immunoglobulin single variable domain such as a domain antibody and Nanobody® (including VHH domain) refers to an amino acid sequence of said ISVD representing the outcome of being subjected to humanization, i.e. to increase the degree of sequence identity with the closest human germline sequence. In particular, humanized immunoglobulin single variable domains, such as Nanobody® (including VHH domains) may be immunoglobulin single variable domains in which at least one amino acid residue is present (and in particular, at least one framework residue) that is and/or that corresponds to a humanizing substitution (as defined further herein). Potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring VHH sequence with the corresponding framework sequence of one or more closely related human VH sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said VHH sequence (in any manner known per se, as further described herein) and the resulting humanized VHH sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other or further suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person. In general, humanized variants as described herein are not substituted in their CDR sequences beyond the CDR mutation described herein. Also, based on what is described before, (the framework regions of) an immunoglobulin single variable domain, such as a Nanobody® (including VHH domains) may be partially humanized or fully humanized. Humanized immunoglobulin single variable domains, in particular Nanobody, may have several advantages, such as a reduced immunogenicity, compared to the corresponding naturally occurring VHH domains. In summary, the humanizing substitutions should be chosen such that the resulting humanized amino acid sequence of the ISVD and/or VHH still retains the favourable properties, such as the antigen-binding capacity, and allosteric modulation capacity. The skilled person will be able to select humanizing substitutions or suitable combinations of humanizing substitutions which optimize or achieve a desired or suitable balance between the favourable properties provided by the humanizing substitutions on the one hand and the favourable properties of naturally occurring VHH domains on the other hand. Such methods are known by the skilled addressee. A human consensus sequence can be used as target sequence for humanization, but also other means are known in the art. One alternative includes a method wherein the skilled person aligns a number of human germline alleles, such as for instance but not limited to the alignment of IGHV3 alleles, to use said alignment for identification of residues suitable for humanization in the target sequence. Also, a subset of human germline alleles most homologous to the target sequence may be aligned as starting point to identify suitable humanisation residues. Alternatively, the VHH is analyzed to identify its closest homologue in the human alleles, and used for humanisation construct design. A humanisation technique applied to Camelidae VHHs may also be performed by a method comprising the replacement of specific amino acids, either alone or in combination. Said replacements may be selected based on what is known from literature, are from known humanization efforts, as well as from human consensus sequences compared to the natural VHH sequences, or the human alleles most similar to the VHH sequence of interest. As can be seen from the data on the VHH entropy and VHH variability given in Tables A-5-A-8 of WO 08/020079, some amino acid residues (i.e. hallmark residues, Figure 10) in the framework regions are more conserved between human and Camelidae than others. Generally, although the invention in its broadest sense is not limited thereto, any substitutions, deletions or insertions are preferably made at positions that are less conserved. Also, generally, amino acid substitutions are preferred over amino acid deletions or insertions. For instance, a human-like class of Camelidae single domain antibodies contain the hydrophobic FR2 residues typically found in conventional antibodies of human origin or from other species, but compensating this loss in hydrophilicity by other substitutions at position 103 that substitutes the conserved tryptophan residue present in VH from double-chain antibodies. As such, peptides belonging to these two classes show a high amino acid sequence homology to human VH framework regions and said peptides might be administered to a human directly without expectation of an unwanted immune response therefrom, and without the burden of further humanisation. Indeed, some Camelidae VHH sequences display a high sequence homology to human VH framework regions and therefore said VHH might be administered to patients directly without expectation of an immune response therefrom, and without the additional burden of humanization. Suitable mutations, in particular substitutions, can be introduced during humanization to generate a polypeptide with reduced binding to pre-existing antibodies (reference is made for example to WO 2012/175741 and WO2015/173325), for example in at least one of the positions: 11, 13, 14, 15, 40, 41, 42, 82, 82a, 82b, 83, 84, 85, 87, 88, 89, 103, or 108. The amino acid sequences and/or VHH of the invention may be suitably humanized at any framework residue(s), such as at one or more Hallmark residues (as defined herein) or preferably at one or more other framework residues (i.e. non-Hallmark residues) or any suitable combination thereof. Another example of humanization includes substitution of residues in FR 1, such as position 1, 5, 11, 14, 16, and/or 23, and/or 28; in FR2 such as positions 40 and/or 43; in FR3, such as positions 60-64, 73, 74, 75, 76, 78, 79, 81, 82b, 83, 84, 85, 93 and/or 94; and in FR4, such as position 103, 104, 105, 108 and/or 111 (see W02008/020079 Tables A-05 -A08; all numbering according to the Kabat).

In one embodiment said humanized variant includes at least one further substitution in any one of the

ISVDs comprising SEQ. ID NO:1, and 9 to 13 selected from the group of substitutions at the following positions (according to Kabat N°): residue 1 substitution to E or D; residue 14 to P; residue 23 to A; 40 to A; 43 to K; 60 to A; 61 to D; 62 to S; 63 to V; 64 to K; 73 to A; 76 to N; 81 to Q; 83 to R; 85 to E; 103 to W; 105 to Q and/or 108 to L. More preferably, said humanized variant includes at least one substitution in any one of the VHH72-S56A or VHH72h5 ISVDs, or a combination of humanization substitutions as described herein above, at the proposed positions. Specifically, the substitutions provided herein as the VHH72humanized 1, VHH72hl (SEQ ID NO:10), contains humanization substitutions over the VHH72 wild type sequence (SEQ ID NO:9) and thus relates to one specific humanization variant. Additionally, as specifically disclosed herein, humanization is further provided by replacing E at position 1 with D, as present in SEQ. ID NO:1, 11, 20. Finally, as specifically disclosed herein in SEQ ID NO:1 and SEQ ID NQ:20, combined humanization substitutions may also be present in the humanized or further humanized variant.

Depending on the host organism used to express the amino acid sequence, ISVD, VHH or polypeptide of the invention, such deletions and/or substitutions may also be designed in such a way that one or more sites for posttranslational modification (such as one or more glycosylation sites at asparagine to be replaced with G, A, or S; and/or Methionine oxidation sites) are removed, as will be within the ability of the person skilled in the art.

Moreover, the protein binding agent for fusion to the conventional antibody light or heavy chain may also comprise an ISVD, in particular a VHH72, VHH72-S56A, VHH72h5, or a humanized VHH72, VHH72- S56A, or VHH72h5, that is further a variant in its structural features by containing a post-translational modification, or a label or a tag, or a further functional moiety, which may also be a half-life extension. Substitutions or insertions may be designed so as to introduce one or more sites for attachment of functional groups, for example to allow site-specific pegylation. In some cases, at least one of the typical Camelidae hallmark residues with hydrophilic characteristics at position 37, 44, 45 and/or 47 is replaced (Kabat N°; see WQ2008/020079 Table A-03).

Another embodiment relates to said antibody composition wherein the light and/or heavy chain are composed from the monoclonal S309 SARS-Cov2-specific antibody ⁽¹⁾. Said S309 monoclonal antibody indeed binds the RBD domain, and as well as the VHH72 said S309 antibody is known to bind an epitope on the RBD which is conserved in SARS-Cov and SARS-Cov2, and competes by its binding to the binding of the ACE2 human receptor to the RBD. The S309 antibody binds to an epitope that is not competing with the epitope of VHH72 on RBD, though, remarkably, a competition assay of VHH72-S56A showed that in the presence of S309, the binding of VHH72 is enhanced, suggesting that the RBD protein conformation formed by binding both binders may be extremely valuable in therapeutic targeting of SARS-Cov2. Thus, the antibody composition comprising a paratope of S309, formed by it light and heavy chain, in combination with an ISVD fused to its light and/or heavy chain, which binds the RBD epitope of VHH72 provides for a single drug substance that specifically binds the RBD domains via at least 4 paratopes (2 time two identical paratopes).

In a specific embodiment said antibody composition may comprise the light and heavy chain of S309, or of a variant thereof. With an 'S309 variant' as used herein is meant that the binding specificity is retained, but that one or more amino acid differences may be present with a different anticipated effect, such as for further humanization of the monoclonal antibody chain, or for increasing the affinity to the target, or for altering its binding capacity to the Fc receptor in a subject (e.g. the non-limiting examples of LALA or LALAPG variants as exemplified herein). Specific examples of such an 'S309 variant' as defined herein comprise the VIR-7831 and VIR-7832 antibodies.

Another embodiment relates to the antibody composition as described herein wherein said antibody is composed of an N- or C-terminal fusion of the S309 light chain or variant thereof, more specifically of an N-terminal fusion of the S309 light chain or a variant thereof with a VHH72 variant, more specifically with the VHH72 variant as depicted in SEQ ID NO:1, or a further humanized variant thereof; and the heavy chain of S309, or a variant thereof, such as for instance the variant with abolished Fc receptor binding, as shown by the LALA or LALA PG Fc mutant. A more specific embodiment thereof provides for the composition containing the antibody composed of SEQ. ID NO:5 as light chain and SEQ ID NO:3 as heavy chain, thereby forming the 'S309-Lc-72' fusion as used herein; or alternatively SEQ ID NO:5 as light chain, and SEQ ID NO:6 as heavy chain, thereby forming the S309-LALA-Lc-72 antibody composition as used herein. A further embodiment relates to the composition wherein the antibody is composed of the light chain of S309, specifically as depicted in SEQ ID NO:2; or a variant thereof, in combination with an S309 heavy chain or a variant thereof, fused to the VHH72 or VHH72 variant. More specifically said antibody composition may comprise SEQ ID NO:2 as light chain, and SEQ ID NO:4 as heavy chain, thereby constituting the 'S309-Hc-72' antibody composition as used herein. Alternatively, said composition may also comprise SEQ ID NO:7 as heavy chain, thereby forming the 'S309-LALA-Hc-72' antibody as described herein. The antibody composition as provided herein may also be composed of two fusion proteins, based on a fusion to the light chain, and a fusion to the heavy chain. In a specific embodiment, said double fusions constituting an antibody composition may be provided by co-expressing the sequences encoding the amino acid sequence of SEQ ID NO:5 as a light chain, or a variant thereof, and the amino acid sequence of SEQ ID NO: 4 or 7, or a variant thereof, as a heavy chain.

Said variants as described herein preferably have at least 70 % amino acid identity to the light, heavy or ISVD polypeptide chain, or at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 98% to the light, heavy or ISVD polypeptide chain referred to therein, wherein the CDRs and paratope residues of their SARS-CoV-2 binding sites is retained. Another composition as an aspect of the invention relates to a combination rather than a fusion of antibody and protein binding agents targeting the corona virus spike protein. More specifically, said composition provides for a combination comprising at least two binding agents, one provided by the S309 antibody, or a variant thereof, the second binding agent provided by the VHH72, or a variant thereof, such as the VHH72-S56A variant, or the VHH72h5 variant, or an active Fc fusion thereof, or any further humanized variant of any one thereof. Said composition may also comprise a combination of the antibody fusion composition as described herein, supplemented with the S309 or a variant thereof, and/or the VHH72, the VHH72-S56A, or the VHH72h5 ISVD or multivalent or multi-specific form or variant thereof, and/or the VHH72-Fc or further variant thereof. An Fc variant or Fc fusion as described herein also relates to Fc fusions wherein an Immunoglobulin Fc is mutated, for instance as to avoid Fc receptor binding, as specifically exemplified for Fc-LALA or Fc-LALAPG mutants as known to the skilled person. Said composition comprising S309 and VHH72-Fc antibodies may more specifically provide for a composition comprising or consisting of S309 constituted by the light chain of SEQ ID NO:2 or a variant thereof, and the heavy chain of SEQ. ID NO:3 or a variant thereof, and the VHH72-Fc as depicted in SEQ ID NO:8 or a variant thereof, wherein variants have at least 90 % identity to any one thereof.

This invention also relates to "pharmaceutical compositions" comprising one or more antibodies of the invention, in particular, the antibody composition as described herein and, optionally, a pharmaceutically acceptable carrier or diluent or excipient. These pharmaceutical compositions can be utilized to achieve the desired pharmacological effect by administration to a patient in need thereof. The present invention includes pharmaceutical compositions that are comprised of a pharmaceutically acceptable carrier and a pharmaceutically effective amount of an antibody composition or combination, or salt thereof, of the present invention. A pharmaceutically effective amount of compound is preferably that amount which produces a result or exerts an influence on the particular condition being treated.

A "pharmaceutically or therapeutically effective amount" of compound or binding agent or composition is preferably that amount which produces a result or exerts an influence on the particular condition being treated. The antibodies or the pharmaceutical composition as described herein may also function as a "therapeutically active agent" which is used to refer to any molecule that has or may have a therapeutic effect (i.e. curative or stabilizing effect) in the context of treatment of a disease (as described further herein). Preferably, a therapeutically active agent is a disease-modifying agent, and/or an agent with a curative effect on the disease. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. A pharmaceutically acceptable carrier is preferably a carrier that is relatively non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredient. Suitable carriers or adjuvantia typically comprise one or more of the compounds included in the following non- exhaustive list: large slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. Such ingredients and procedures include those described in the following references, each of which is incorporated herein by reference: Powell, M. F. et al. ("Compendium of Excipients for Parenteral Formulations" PDA Journal of Pharmaceutical Science & Technology 1998, 52(5), 238-311), Strickley, R.G ("Parenteral Formulations of Small Molecule Therapeutics Marketed in the United States (1999)-Part-1" PDA Journal of Pharmaceutical Science & Technology 1999, 53(6), 324-349), and Nema, S. et al. ("Excipients and Their Use in Injectable Products" PDA Journal of Pharmaceutical Science & Technology 1997, 51 (4), 166-171). The term "excipient", as used herein, is intended to include all substances which may be present in a pharmaceutical composition and which are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffer substances, stabilizing agents, flavouring agents or colorants. A "diluent", in particular a "pharmaceutically acceptable vehicle", includes vehicles such as water, saline, physiological salt solutions, glycerol, ethanol, etc. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, preservatives may be included in such vehicles.

A final aspect of the invention relates to the antibody composition or pharmaceutical composition as described herein for use as a medicine. More specifically to treat a subject, in a curative or therapeutic manner, or alternatively in a preventive or prophylactic manner. Said compositions may thus be used for treatment or inhibition or blocking a viral infection. More specifically, said compositions or pharmaceutical compositions or antibodies may be used in treatment of a betacoronavirus infection, or more specifically in treatment of a subject with Covidl9.

It is to be understood that although particular embodiments, specific configurations as well as materials and/or molecules, have been discussed herein for compositions, methods, samples and drug substance or products according to the disclosure, various changes or modifications in form and detail may be made without departing from the scope of this invention. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims. EXAMPLES

Example 1. Design, expression and purification of S309-based VHH72-S56A antibody-fusion constructs.

Through the fusion of a SARS-CoV-1 and SARS-CoV-2 neutralizing monoclonal antibody S309 with broadly neutralizing nanobody VHH72hl-ElD-S56A, either with the light chain, the heavy chain, or both of S309 we investigated whether such multiparatopic targeting of the spike protein would increase the in vitro antiviral potency, increase the broadness of the antiviral activity, and decrease the chance of mutational escape of SARS-CoV-2 viruses, as compared to S309 monoclonal antibody alone, VHH72-Fc fusions, or as compared to a mixture of the combination of both. Specifically, we generated S309 [with wild type heavy chain Fc (as present in SEQ ID NO:3) or the L234A-L235A (LALA) mutations in the heavy chain Fc (as present in SEQ. ID NO: 6)], combinations of S309 and VHH72hl-ElD-S56A (as present in SEQ ID NO:1); with the latter fused N-terminally to the light chain, C-terminally to the heavy chain, or both). The fusions of the VHH building block and S309 light or heavy chains were linked together with a (G4S)2 linker.

Human monoclonal antibody S309 is derived from an individual who was infected with SARS-CoV-1 in 2003 and the antibody was shown to neutralize SARS-CoV-1 and -2¹. Single domain antibody VHH72, also named SARS VHH-72, was reported before². This single domain antibody can bind to a broad range of Sarbecoviruses, neutralize SARS-CoV-1 and-2 pseudotyped viruses in vitro and, when administered prophylactically, strongly reduce SARS-CoV-2 replication in a hamster challenge model³. Here we used a humanized version of VHH72_S56A, named VHH72_hl_ElD_S56A (SEQ ID NO:1), with the serine to alanine mutation at position 56 (S56A) in CDR2, and humanized as the humanization hl variant by substitution of Q5V, A14P, K83R, D85E and Q108L, and in addition an aspartic acid to glutamic acid mutation at position 1 (EID), wherein the numbering is according to Kabat annotation. The S56A mutation was shown to result in a higher affinity for SARS-CoV-1 and -2 spike and receptor-binding domain and an approximately 5-7 fold higher authentic SARS-CoV-2 neutralizing activity when fused to a human IgGl Fc (Schepens et al., 2021, Science Translational Medicine, PMID: 34609205).

VHH72_hl_ElD_S56A (SEQ ID NO:1) was fused either N-terminally to the light chain (Lc) of S309 (SEQ ID NO:2), or C-terminally to the heavy chain (He) of S309 (SEQ ID NO:3), in both cases separated by a (648)2 (also called 10GS) linker. An antibody version with a single fusion of VHH72_hl_ElD_S56A to either the light chain, named 'S309-Lc-72' (SEQ ID NO:5), or the heavy chain of S309, named 'S309-Hc- 72' (SEQ ID NO:4), were obtained, or an antibody version with VHH72_hl_ElD_S56A fusion to both the light chain and the heavy chain of S309, named 'S309-Lc-72 Hc-72'. S309 constructs carried either wild type Fc of Fc with the LALA mutations⁴, this results in a total of 6 different fusion versions (Table 1). In addition, next to the wild type S309, and the S309 wherein the Fc has the LALA mutations, also batch PB9683 (construct D72-53), constituting the human IgGl Fc fusion containing humanized VHH72_hl- E1D-S56A in bivalent format (= VHH72_hl_ElD_S56A-10xGS-hlgGl_EPKSCdel_LALA_K447del; SEQ ID NO:8; also named VHH72-lgGl-Fc or XVR011 herein), was used as a benchmark.

The antibody productions were performed by transiently transfecting ExpiCHO-S cells for expression of the constructs, following purification, and the resulting antibodies formed were numbered as follows:

• S309 A, representing S309 with a wild type Fc domain (Lc: SEQ ID NO:2 and He: SEQ ID NO: 3).

• S309_B (also called S309-LALA ) representing S309 with a L234A-L235A (LALA) mutant Fc domain (Lc: SEQ ID NO:2 and He: SEQ ID NO:6)

• S309_C (also called S309-Hc-72) has a wild type S309 light chain and a S309 heavy chain C- terminally fused with VHH72hl-ElD-S56A spaced by a 10GS linker (GGGGSGGGGS) (Lc: SEQ ID NO:2 and He: SEQ ID NO:4).

• S309_D (also called S309-Lc-72) has a S309 light chain that carries an N-terminal fusion with VHH72hl-ElD-S56A spaced by a 10GS linker (GGGGSGGGGS) and a wild type S309 heavy chain (Lc: SEQ ID NO:5 and He: SEQ ID NO:3).

• S309_E (also called S309-LALA-Lc-72 ) has a S309 light chain that carries an N-terminal fusion with VHH72hl-ElD-S56A spaced by a 10GS linker (GGGGSGGGGS) and a S309 heavy chain with LALA mutations (Lc: SEQ ID NO:5 and He: SEQ ID NO:6).

• S309_F (also called S309-LALA-HC-72 ) has a wild type S309 light chain and a LALA mutant S309 heavy chain C-terminally fused with VHH72hl_S56A spaced by a 10GS linker (GGGGSGGGGS) (Lc: SEQ ID NO:2 and He: SEQ ID NO:7).

• S309_G (also called S309-Lc-72 Hc-72) has a S309 light chain that carries an N-terminal fusion with VHH72hl-ElD-S56A spaced by a 10GS linker (GGGGSGGGGS) and a S309 heavy chain C- terminally fused with VHH72h3_S56A spaced by a 10GS linker (GGGGSGGGGS) (Lc: SEQ ID NO:5 and He: SEQ ID NO:4).

• S309_H (also called S309-LALA-Lc-72 Hc-72 ) has a S309 light chain that carries an N-terminal fusion with VHH72hl-ElD-S56A spaced by a 10GS linker (GGGGSGGGGS) and a LALA mutant S309 heavy chain C-terminally fused with VHH72hl-ElD-S56A spaced by a 10GS linker (GGGGSGGGGS) (Lc: SEQ ID NO:5 and He: SEQ ID NO:7). Table 1. Overview of S309 monoclonal antibody constructs without or with genetically fused SARS- Coronavirus-1 and -2 neutralizing VHHs produced in and purified from transiently transfected ExpiCHO- S cells.

The column on the right lists the yields obtained after production in ExpiCHO-S cells and purification of the respective proteins, calculating the yield back to the original transfection volume.

The proteins were produced by transient transfection of ExpiCHO cells followed by protein A affinity purification and desalting, as described previously². In brief, synthetic DNA sequences coding for the different constructs were ordered at IDT. Upon arrival, synthetic DNA was dissolved in ultraclean water at a concentration of 20 ng/pL. DNA fragments were then A-tailed using the NEBNext-dA-tailing module (NEB), purified using CleanPCR magnetic beads (CleanNA) and inserted in pcDNA3.3-TOPO vector (ThermoFisher). The ORF of positive clones was fully sequenced, and pDNA of selected clones was prepared using the NucleoBond Xtra Midi kit (Machery-Nagel). The different antibody constructs were expressed in ExpiCHO-S™ cells (ThermoFisher Scientific), according to the manufacturer's protocol. Briefly, a 25 mL culture of 6 x 10^s cells per mL, grown at 37°C and 8 % CO2, was transfected with a total of 20 pg of pcDNA3.3 plasmid DNA using ExpiFectamine™ CHO reagent. Heavy and light chain coding plasmids were mixed in a ratio of 1:2. One day after transfection, 150 pL ExpiCHO™ enhancer and 4 mL ExpiCHO™ feed was added to the cells, and cultures were further incubated at 32°C and 5 % CO2. Cells were fed a second time on day 5 after transfection. Productions were collected as soon as cell viability dropped below 75%.

For purification of the different antibody constructs, supernatants were loaded on a 5 mL MAbSelect SuRe column (GE Healthcare). Unbound proteins were washed away with Mcllvaine buffer pH 7.2, and bound proteins were eluted using Mcllvaine buffer pH 3 (Figure 5A). Immediately after elution, proteincontaining fractions were neutralized using a saturated NasPC buffer. Next, these fractions were pooled, and loaded on a HiPrep Desalting column for buffer exchange to storage buffer (PBS, pH 7.4) (Figure 5B).

Figure 6 shows a reducing SDS-PAGE for purified S309 antibody A & B, and the purified antibody fusions S309 C and D, indicating that the expected antibody heavy and light chain fusions were formed and obtained after purification.

Example 2. Pseudotype virus neutralization activity of antibodies containing S309-based VHH72 fusions.

Replication-deficient VSV pseudotyped with SARS-CoV-2 S and coding for GFP or firefly luciferase were generated as described previously⁵. For the VSV pseudotype neutralization experiments, the pseudoviruses were incubated for 30 min at 37 degrees C with different dilutions of purified antibody constructs. The incubated pseudoviruses were subsequently added to confluent monolayers of Vero E6 cells in a 96-well plate. Sixteen hours later, the transduction efficiency was quantified by measuring the GFP fluorescence (MFI) of each well using an Infinite 200 Pro Tecan fluorimeter. For each dilution series the GFP signals were normalized to the lowest and highest values of that dilution series and plotted as percentage. The IC₅o values were determined by non-linear regression (log(inhibitor) vs. response -- Variable slope (four parameters), expressed in pg/ml.

In a first experiment, the S309-Hc-72 and S309-Lc-72 antibody fusions were assayed as compared to the S309 mAb or VHH72-lgGl-Fc (PB9683; as provided in SEQ. ID NO:8) alone. The fusions were shown to neutralize SARS-CoV-2 S VSV pseudotypes 3-4 fold better than S309 monoclonal antibody and about 5 fold better than VHH72-lgGl-Fc (Figure 1, left). When LALA Fc variants were used in the Fes of the heavy chains, the IC₅o values were for the fusions were 2-3-fold better in neutralization as compared to S309- LALA, or 3-4-fold better than VHH72-lgGl-Fc (Figure 1, right).

A second experiment for testing the S309-Hc-72 and S309-Lc-72 antibody fusions also included the mixture of monoclonal S309 antibody with VHH72-lgGl-Fc (PB9683; at half the concentration of S309). The choice was made to compare the performance of the fusions with a cocktail of S309 and VHH72- IgGl-Fc instead of bivalent VHH72, because the Fc variant has a better neutralization activity, and is therefore the more relevant comparison to make. Furthermore, the concentration of S309(LALA) mAb (starting at 5 pg/ml ) and the half concentration of VHH72-lgGl-Fc(LALA) (starting at 2.5 pg/ml) provides for nearly equimolar amounts of binders present in the cocktails, namely 68 nM versus 63 nM.

The IC₅o values resulted in a better or equal performance of the mixture as compared to the LALA mutant or wild-type S309 alone (Figure 2). Importantly, the fusions S309-Hc-72 and S309-Lc-72 were not only outperforming the single antibody treatments, but also the mixture of the wild type S309 and VHH72- IgGl-Fc (PB9683). The fusion construct wherein the heavy chain of S309 is fused with VHH72_hl_ElD_S56A showed the best performance in the neutralization assay over the other constructs. Finally, also as compared to the mixture of the LALA-S309 and VHH72-lgGl-Fc, the fusions with LALA mutations in the Fc domain performed at least as good in neutralization activity as the fusions which did not have the LALA mutations.

Thirdly, the double fusion, i.e. S309-LC-72 Hc-72 and S309-LALA-LC-72 Hc-72 constructs were tested in comparison to the S309 alone, or the S309 LALA, respectively (Figure 3). Although no side-by-side comparison with the single fusions or the mix of the single fusions is shown in this experiment, the IC₅o values provide an indication that the double fusion construct has at least an additive effect on neutralization potential of SARS-Cov-2 pseudoviruses.

Furthermore, from a comparative neutralization assay using VSV pseudotyped with the Wuhan SARS- CoV-2 spike protein using S309 constructs A to H, we conclude that S309 constructs with VHH72 fused to both light chains and both heavy chains (S309 G and H) have higher SARS-CoV-2 neutralizing activity as compared to S309 constructs with VHH72 fused to either the light chains or to the heavy chains (S309 C-F), WT-Fc (S309A) and LALA-Fc (S309B) antibodies. Figure 7 and 8 illustrate that S309 constructs fused with four copies of VHH72 (S309_H) have considerably higher neutralizing activity as compared to S309 constructs armed with either no or two copies of VHH72.

To investigate how the neutralizing activity of S309H compares to that of a cocktail of S309_B and VHH72-FC (D72-53, SEQ ID NO: 8) we tested the potency of S309_H and of a cocktail of S309_B and D72_53 at 1:1 and 1:2 molar ratio to neutralize VSV-delG pseudotyped with the spike of Wuhan SARS- CoV-2 (Figure 9) or with the spike of SARS-CoV-2 beta (B.1.351) (Figure 10). Figure 9 illustrates that the 1:1 and the 1:2 molar cocktail of S309_B and D72-53 are not more potent in neutralizing VSV-delG pseudotyped with the spike of Wuhan SARS-CoV-2 than S309_B whereas S309_H has a tenfold higher neutralizing potency. Figure 10 illustrates that the 1:1 and the 1:2 molar cocktail of S309_B and D72_53 are not more potent in neutralizing VSV-delG pseudotyped with the spike of SARS-CoV-2 beta (B.1.351) than S309_B whereas S309_H has a fourfold higher neutralizing potency.

Example 3. The VHH72 interaction with the Sars-Cov-2 RBD increases upon addition of S309 antibody. Dose-dependent inhibition of the interaction of SARS-CoV-2 RBD protein with monovalent nanobody VHH72_hl (S56A)-flag3-His6 was assessed in a competition AlphaLISA (amplified luminescent proximity homogeneous assay). In here, 2019-nCoV S protein RBD (corresponding with the amino acid residues 320-502 derived from GenBank ID: NP_828851.1, as set forth in SEQ. ID NO: 18) that was biotinylated through an Avi-tag (AcroBiosystems, Cat nr. SPD-C82E9) was loaded on streptavidin coated Alpha Donor beads (Perkin Elmer, Cat nr. 6760002). Monovalent nanobody VHH72_hl(S56A) (SEQ ID NO: 13) was captured on anti-Flag AlphaLISA acceptor beads (Perkin Elmer, Cat nr. AL112C). Binding of VHH72 and RBD captured on the beads leads to an energy transfer from one bead to the other, ultimately producing a luminescent/fluorescent signal.

Serial dilutions of anti-SARS-CoV-2 antibodies (CR3022, CB6, S309) and VHH-Fc (XVR011, WT-VHH72-FC, and D72-23 batches, all providing for the same antibody corresponding to SEQ. ID NO:8) (final concentration ranging between lOOnM - 0,001 nM) were made in assay buffer (PBS containing 0.5% BSA and 0.05% Tween-20), and mixed with VHH72-hl (S56A)-flag3-His6 (final concentration 0.6 nM) and biotinylated RBD protein (final concentration 0.6 nM) in white low binding 384well microtitre plates (F- bottom, Greiner Cat nr 781904). After an incubation for 1 hour at room temperature, donor and acceptor beads were added to a final concentration of 20 pg/mL for each in a final volume of 0,025 ml for an additional incubation of 1 hour at room temperature in the dark. Interaction between beads was assessed after illumination at 680 nm and reading at 615 nm of on an Ensight instrument.

As isotype control antibody Synagis hlgGl (Palivizumab, Medlmmune) was included. Anti-SARS-CoV-1 RBD antibody CR3022 was commercially purchased (Absolute Antibody, Cat nr Ab01680-10.0). S309 antibody was generated (hlgGl) based on SEQ. ID NO: 14 and 15, from Pinto et al. 2020, using standard mammalian productions. CB6 antibody was generated according to the public sequence (Genbank MT470196 and MT470197), corresponding to the sequence in SEQ ID NO:16 and 17, for the light and heavy chain resp. and including an additional signal peptide.

As shown in Figure 4, the VHH72-FC formats (XVR011 batch comprising VHH72_hl_ElD_S56A-Fc (SEQ ID NO:8), and the WT VHH72-FC batch in Fig. 4B, and the D72-23 batch comprising the VHH72_hl_S56A- Fc in Figure 4C) inhibit the interaction between monovalent VHH72_hl (S56A)-flag3-His6 and RBD, as well as the CR3022 antibody, which recognizes a partially overlapping epitope (Figure 4C). The CB6 anti- SARS-CoV-2 RBD antibody, which has a binding site that is competing with the VHH72 binding site, has a mild inhibitory impact. However, the anti-SARS-CoV-2 RBD S309 antibody, with a binding site identified as not to compete with the VHH72 binding site, enhanced the interaction of VHH72 with the RBD domain in a dose-dependent manner, with an EC₅o value of 287 pM (Figure 4A-C).

So, the alphaLISA immunoassay allows to conclude that the S309 conventional antibody upon binding to the RBD allows for an improved binding of the VHH72_hl (S56A) to the RBD. Based on this observation, a combination treatment of VHH72_hl (S56A)-Fc and S309 antibodies may provide for a synergistically higher viral neutralization activity as compared to the treatment with single compounds. Example 4. Escape mutant analysis of the SARS-CoV-2 receptor binding-domain (RBD) for binding with the S309 antibody, the S309_C (VHH72 fused at the C-terminus of the S309 heavy chain), and the cocktail of S309 with VHH72-Fc, based on deep mutational scanning.

To get insights into the mutational constraints of the binding sites in the RBD of the spike that are targeted by the fusion antibodies, binding was analyzed to a library of RBD mutants for one representative fusion antibody. The binding of S309 C, wherein the VHH72hl-ElD-S56A building block is fused to the heavy chain of the S309 antibody, was compared to the binding profiles of S309 monoclonal antibody, or as compared to a mixture of said monoclonal S309 and the D72-53 (VHH72hl-ElD-S56A-Fc, as presented in SEQ ID NO:8).

The results indicate that the fusion antibody S309 C has better binding profile breadth to a large variety of RBD mutants as compared to the combination of the single compounds.

Deep mutational scanning analysis was performed as described in the method below, and the fraction of escape mutants for each amino acid position in the RBD of the spike protein as depicted in SEQ. ID NO:19 is shown is shown in Figure 11, wherein the peaks indicate the fraction of possible escape mutant substitutions per amino acid.

The peak height or fraction of possible escape mutants is much lower for the binding profile shown in Figure 11 B as compared to Figure 11 A & Figure 11 C (fivefold lower scale of the Y-axis in B compared to A and C), indicating that the escape potential for binding to a mutant RBD domain of the SARS-CoV-2 spike protein is much lower for the S309 C (with VHH72hl-ElD-S56A fused to the heavy chain of S309; Figure 11B) as compared to the escape potential for S309 monoclonal antibody (Figure 11A) or as even as compared to the cocktail of S309 and D72-53 (VHH72-Fc) (Figure 11C).

Method used for Deep mutational scanning analysis

Transformation of deep mutational SARS-CoV2 RBD libraries to E. coli

Plasmid preps of two independently generated deep mutational SARS-CoV2 RBD libraries in the pETcon vector were generously provided by Dr. Jesse Bloom (Starr et al. 2020, Cell 182, 1295-1310.e20). Ten ng of these preps were transformed to E. coli TOP10 strain via electroporation, and allowed to recover for one hour in SOC medium at 37°C. The transformation mixture was divided and plated on ten 24.5 cm x 24.5 cm large bio-assay dishes containing low salt LB medium supplemented with carbenicillin, at an expected density of 100.000 clones per plate. After growing overnight, all colonies were scraped from the plates and resuspended into 300 ml low salt LB supplemented with carbenicillin. The cultures were grown for 2 hours and a half before pelleting. The cell pellet was washed once with sterile MQ, and plasmid was extracted via the QI Afilter plasmid Giga prep kit (Qiagen) according to the manufacturer's instructions.

Transformation of deep mutational SARS-CoV2 RBD libraries to S. cerevisiae

Ten pg of the resulting plasmid preps were transformed to Saccharomyces cerevisiae strain EBY100, according to the large-scale protocol by (Gietz et al. Nature Protocols 2007, 2, 31-345) Gietz and Schiestl. Transformants were selected in 100 ml liquid yeast drop-out medium (SD -trp -ura) for 16 hours. Then the cultures were back-diluted into 100m L fresh SD -trp -ura at 1 ODsoofor an additional 9 hours passage. Afterwards, the cultures were flash frozen in le8 cells aliquots in 15% glycerol and stored at -80°C.

Cloning and transformation of WT RBD of SARS-CoV2

The CDS of the RBD of SARS-CoV2 was ordered as a yeast codon-optimized gBIock and cloned into the pETcon vector by Gibson assembly. The cloning mixture was similarly electroporated into E. coli TQP10 cells, and plasmid was extracted via a Miniprep kit (Promega) according to the manufacturer's instructions. The plasmid was Sanger sequenced with primers covering the entire RBD CDS. Finally, the plasmid was transformed to Saccharomyces cerevisiae strain EBY100, according to the small-scale protocol by Gietz et al. (Nature Protocols 2007, 2, 31-34) . Transformants were selected via a yeast colony PCR.

Presorting of deep mutational SARS-CoV2 RBD libraries on ACE2

One aliquot of each library was thawed and grown overnight in 10 ml liquid repressive medium (SRaf - ura -trp) at 28°C. Additionally, the control EBY100 strain containing the pETcon plasmid expressing WT RBD from SARS-CoV2 was inoculated in 10 ml liquid repressive medium and grown overnight at 28°C. These precultures were then back-diluted to 50 ml liquid inducing medium (SRaf/Gal -ura -trp) at an QD600 of 0.67/ml and grown for 16 hours before harvest.

The cells pellets were washed thrice with washing buffer (IX PBS + 1 mM EDTA, pH 7.2 + 1 Complete Inhibitor EDTA-free tablet (Roche) per 50ml buffer), and stained at an ODsoo of 8/ml with 9.09 nM hACE2- muFc (Sino Biological) in staining buffer (washing buffer + 0.5 mg/ml of Bovine Serum Albumin) for one hour at 4°C on a rotating wheel. Cells were washed thrice with staining buffer and stained with 1:100 anti-cmyc-FITC (Immunology Consultants Lab), 1:1000 anti-mouse-lgG-AF568 (Molecular Probes) and 1:2000 L/D eFluor506 (Thermo Fischer Scientific) for one hour at 4°C on a rotating wheel. Cells were washed thrice with staining buffer, and filtered over 35 pm cell strainers before sorting on a FACS Melody (BD Biosciences). A selection gate was drawn that captures the ACE2+ cells, such that, after compensation, max. 0.1% of cells of unstained and single stained controls appeared above the background. Approximately 2.5 million ACE2+ cells were collected per library, each in 5 ml polypropylene tubes coated with 2X YPAD + 1% BSA. Sorted cells were recovered by growth in liquid SD -trp -ura medium with 100 U/ml penicillin and 100 pg/ml streptomycin (Thermo Fisher Scientific) for 48 hours at 28°C, and flash frozen at -80°C in 9 ODsoo unit aliquots in 15% glycerol.

Nanobody escape mutant sorting on ACE2-sorted deep mutational SARS-CoV2 RBD libraries

One ACE2-sorted aliquot of each library was thawed and grown in 15 ml liquid repressive medium (SRaf -ura -trp) at 28°C for 36 hours. Additionally, the control EBY100 strain containing the pETcon plasmid expressing WT RBD from SARS-CoV-2 (SEQ ID NO:19) was inoculated in 15 ml liquid repressive medium and grown at 28°C for 36 hours. These precultures were then back-diluted to 50 ml liquid inducing medium (SRaf/Gal --ura -trp) at an OD600 of 0.67/ml and grown for 16 hours before harvest.

The cells pellets were washed thrice with washing buffer (IX PBS + 1 mM EDTA, pH 7.2 + 1 Complete Inhibitor EDTA-free tablet (Roche) per 50ml buffer, freshly made and filter sterile) and stained at an ODsoo of 8/ml with a specific concentration per stained antibody in staining buffer (washing buffer + 0.5 mg/ml of Bovine Serum Albumin) for one hour at 4°C on a rotating wheel. Specifically, we added 10 pg/ml S309, 10 pg/ml S309 C, and for the mixture 10 pg/ml S309 and 18.5 pg/ml D72-53 (VHH72-FC antibody made from SEQ ID NO: 8) to the RBD yeast-displayed library.

Cells were washed thrice with staining buffer and stained with 1:100 anti-c-myc-FITC (Immunology Consultants Lab), 1:1000 anti-human IgG AF594 (Molecular Probes) and 1:2000 L/D eFluor506 (Thermo Fischer Scientific) for one hour at 4°C on a rotating wheel. Cells were washed thrice with staining buffer, and filtered over 35 pm cell strainers before sorting on a FACS Melody (BD Biosciences). Gating was chosen as such that, after compensation, max. 0.1% of cells of the fully stained WT RBD control appeared in the selection gate. Between 50.000 and 70.000 escaped cells were collected per library, each in 5 ml polypropylene tubes coated with 2X YPAD + 1% BSA.

Sorted cells were recovered by growth in liquid SD -trp -ura medium supplemented with 100 U/ml penicillin and 100 pg/ml streptomycin (Thermo Fisher Scientific) for 16 hours at 28°C.

DNA extraction and Illumina sequencing of nanobody escape sorted deep mutational SARS-CoV2 RBD libraries

Plasmids were extracted from sorted cells using the Zymoprep yeast plasmid miniprep II kit (Zymo Research) according to the manufacturer's instructions, but with the exception of a longer (2 hour) incubation with the Zymolyase enzyme, and with the addition of a freeze-thaw cycle in liquid nitrogen after Zymolyase incubation.

A PCR was performed on the extracted plasmids using KAPA HiFi HotStart ReadyMix to add sample indices and remaining Illumina adaptor sequences using NEBNext UDI primers (20 cycles). PCR samples were purified once using CleanNGS magnetic beads (CleanNA), and once using AMPure magnetic beads (Beckman Coulter). Fragments were eluted in 15 pl O.lx TE buffer. Size distributions were assessed using the High Sensitivity NGS kit (DNF-474, Advanced Analytical) on a 12-capillary Fragment Analyzer (Advanced Analytical). Hundred bp single-end sequencing was performed on a NovaSeq 6000 by the VIB Nucleomics core (Leuven, Belgium).

Analysis of sequencing data and epitope calculation using mutation escape profiles

Deep sequencing reads were processed as described by Greaney et al. (2021, Cell Host Microbe) using the code available at https://github.com/jbloomlab/SARS-CoV-2-RBD MAP Crowe antibodies, with adjustments. Briefly, nucleotide barcodes and their corresponding mutations were counted using the dms_variants package (0.8.6). Escape fraction for each barcode was defined as the fraction of reads after enrichment divided by the fraction of reads before enrichment of escape variants. The resulting variants were filtered to remove unreliably low counts and keep variants with sufficient RBD expression and ACE2 binding (based on published data Starr et al. (2020, Cell 182, 1295-1310.e20)). For variants with several mutations, the effects of individual mutations were estimated with global epistasis models, excluding mutations not observed in at least one single mutant variant and two variants overall. The resulting escape measurements correlated well between the duplicate experiments and the average across libraries was thus used for further analysis. To determine the most prominent escape sites for each nanobody, RBD positions were identified where the total site escape was > lOx the median across all sites, and was also at least 10% of the maximum total site escape across all positions for a given nanobody.

Sequence listing

> SEQ ID NO:1: VHH72_hl_ElD_S56A

DVQLVESGGGLVQPGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGATYYTDSVKGRFTISRDNAK NTVYLQM N S LR P E DTAVYYCAAAG LGTVVS E WDYDYDYWGQGTLVTVSS

> SEQ ID NO:2: Light chain (Lc) of S309 antibody

EIVLTQSPGTLSLSPGERATLSCRASQTVSSTSLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPE DFAVYYCQQHDTSLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

> SEQ ID NO:3: Heavy chain (He) of S309 antibody

DVQLVQSGAEVKKPGASVKVSCKASGYPFTSYGISWVRQAPGQGLEWMGWISTYNGNTNYAQKFQGRVTMTTDT STTTGYMELRRLRSDDTAVYYCARDYTRGAWFGESLIGGFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

> SEQ ID NO:4: VHH72_hl_ElD_S56A C-terminally fused to Heavy chain of S309 antibody (Hc-72) DVQLVQSGAEVKKPGASVKVSCKASGYPFTSYGISWVRQAPGQGLEWMGWISTYNGNTNYAQKFQGRVTMTTDT STTTGYMELRRLRSDDTAVYYCARDYTRGAWFGESLIGGFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD

KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST

YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV

EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGG

GGSDVQLVESGGGLVQPGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGATYYTDSVKGRFTISRDN

AKNTVYLQMNSLRPEDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTLVTVSS

> SEQ ID N0:5: VHH72_hl_ElD_S56A N-terminally fused to Light chain of S309 antibody (Lc-72)

DVQLVESGGGLVQPGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGATYYTDSVKGRFTISRDNAK

NTVYLQMNSLRPEDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTLVTVSSGGGGSGGGGSEIVLTQSPGTLSLSPGE

RATLSCRASQTVSSTSLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHDTSLT

FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

> SEQ ID NO:6: Heavy chain (He) of S309 antibody containing the LALA mutations (in bold)

DVQLVQSGAEVKKPGASVKVSCKASGYPFTSYGISWVRQAPGQGLEWMGWISTYNGNTNYAQKFQGRVTMTTDT

STTTGYMELRRLRSDDTAVYYCARDYTRGAWFGESLIGGFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD

KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS

TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG

> SEQ ID NO:7: VHH72_hl_ElD_S56A C-terminally fused to Heavy chain of S309 antibody containing the LALA mutations (in bold) (Hc-72-LALA)

DVQLVQSGAEVKKPGASVKVSCKASGYPFTSYGISWVRQAPGQGLEWMGWISTYNGNTNYAQKFQGRVTMTTDT

STTTGYMELRRLRSDDTAVYYCARDYTRGAWFGESLIGGFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD

KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS

VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKGGGGSG

GGGSDVQLVESGGGLVQPGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGATYYTDSVKGRFTISRD NAKNTVYLQMNSLRPEDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTLVTVSS

> SEQ ID NO:8: VHH72_hl_ElD_S56A-10xGS-hlgGl_EPKSCdel_LALA_K447del (PB9683 batch; construct D72-53) DVQLVESGGGLVQPGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGATYYTDSVKGRFTISRDNAK

NTVYLQM NSLRPEDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTLVTVSSGGGGSGGGGSDKTHTCPPCPAPEAA

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD

WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

> SEQ ID NO: 9: amino acid sequence of VHH72 (CDRs underlined)

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGSTYYTDSVKGRFTISRDNAK

NTVYLQM NSLKPDDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSS

> SEQ ID NO: 10: amino acid sequence of VHH72-hl (humanized variant 1 of SEQ. ID NO: 9)

EVQLVESGGGLVQPGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGSTYYTDSVKGRFTISRDNAKN

TVYLQMNSLRPEDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTLVTVSS

> SEQ ID NO: 11: amino acid sequence of VHH72-hl (EID)

DVQLVESGGGLVQPGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGSTYYTDSVKGRFTISRDNAK

NTVYLQM N S LR P E DTAVYYCAAAG LGTVVS E WDYDYDYWGQGTLVTVSS

> SEQ ID NO: 12: amino acid sequence of VHH72-S56A variant (S56A, according to Kabat annotation, substitution in bold)

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGATYYTDSVKGRFTISRDNAK

NTVYLQM NSLKPDDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSS

> SEQ ID NO:13: amino acid sequence of VHH72_hl(S56A)

EVQLVESGGGLVQPGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGATYYTDSVKGRFTISRDNAKN

TVYLQMNSLRPEDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTLVTVSS

>SEQ ID NO:14: S309 light chain sequence including a signal peptide (underlined)

MGWSCIILFLVATATGVHSEIVLTQSPGTLSLSPGERATLSCRASQTVSSTSLAWYQQKPGQAPRLLIYGASSRATGIPD

RFSGSGSGTDFTLTISRLEPEDFAVYYCQQHDTSLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR

EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

>SEQ ID NO:15: S309 heavy chain sequence including a signal peptide (underlined)

MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYPFTSYGISWVRQAPGQGLEWMGWISTYNG

NTNYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYCARDYTRGAWFGESLIGGFDNWGQGTLVTVSSASTK

GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN

VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV

DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE

LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK

>SEQ ID NO:16: CB6 light chain sequence including a signal peptide (underlined) MGWSCIILFLVATATGVHSDIVMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYAASSLQSGVPS

RFSGSGSGTDFTLTISSLQ.PEDFATYYCQ.Q.SYSTPPEYTFGQ.GTKLEIKRTVAAPSVFIFPPSDEQ.LKSGTASVVCLLNNF YPREAKVQ.WKVDNALQ.SGNSQ.ESVTEQ.DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ.GLSSPVTKSFNRGEC >SEQ ID NO:17: CB6 heavy chain sequence including a signal peptide (underlined)

MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKGLEWVSVIYSGGSTF YADSVKGRFTISRDNSM NTLFLQMNSLRAEDTAVYYCARVLPMYGDYLDYWGQ.GTLVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ.SSGLYSLSSVVTVPSSSLGTQ.TYICNVNHKPSNTKVD

KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK

PREEQ.YNSTYRVVSVLTVLHQ.DWLNGKEYKCKVSNKALPAPIEKTISKAKGQ.PREPQ.VYTLPPSRDELTKNQ.VSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K

> SEQ ID N0:18: amino acid sequence of Corona virus SARS Spike protein (corresponds with GenBank accession NP_828851.1).

MFIFLLFLTLTSGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFH

TINHTFGNPVIPFKDGIYFAATEKSNVVRGWVFGSTM NNKSQ.SVIIINNSTNVVIRACNFELCDNPFFAV SKPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQ.PIDVVRDLP SGFNTLKPIFKLPLGINITNFRAILTAFSPAQDIWGTSAAAYFVGYLKPTTFMLKYDENGTITDAVDCSQ.

NPLAELKCSVKSFEIDKGIYQ.TSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVA

DYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQ.IAPGQ.TGVIADYNYKLPDDFI\/IGCV

LAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIG

YQ.PYRVVVLSFELLNAPATVCGPKLSTDLIKNQ.CVNFNFNGLTGTGVLTPSSKRFQ.PFQ.Q.FGRDVSDFTD SVRDPKTSEILDISPCAFGGVSVITPGTNASSEVAVLYQ.DVNCTDVSTAIHADQ.LTPAWRIYSTGNNVFQ.

TQAGCLIGAEHVDTSYECDIPIGAGICASYHTVSLLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTNF

SISnTEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAAEQDRNTREVFAQVKQM

YKTPTLKYFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQ.KFNGL

TVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQ.KQ.IANQ.FN KAISQ.IQ.ESLTTTSTALGKLQ.DVVNQ.NAQ.ALNTLVKQ.LSSNFGAISSVLNDILSRLDKVEAEVQ.IDRLIT GRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYV PSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQHTTDNTFVSGNCDVVIGIINNTVY DPLQ.PELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQ.KEIDRLNEVAKNLNESLIDLQ.ELGKYEQ.

YIKWPWYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKGACSCGSCCKFDEDDSEPVLKGVKLHYT

> SEQ ID NO: 19: amino acid sequence of Spike protein of SARS-Cov2 (Wuhan seafood market pneumonia virus (nCo2019-virus). (Genbank Accession: QHQ82464, version QHQ82464.1). The RBD sequence is between amino acid 330-518.

MFVFLVLLPLVSSQ.CVNLTTRTQ.LPPAYTNSFTRGVYYPDKVFRSSVLHSTQ.DLFLPFFSNVTWFHAIHV SGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPF

LGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPI

NLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYN

ENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASV

YAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD

YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYF

PLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFL

PFQ.Q.FGRDIADTTDAVRDPQ.TLEILDITPCSFGGVSVITPGTNTSNQ.V AVLYQ.DVNCTEVPVAIHADQ.LT

PTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLG

AENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGI

AVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDC

LGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAIVIQIVIAYRFNGIG

VTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDI

LSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLIVl

SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNT

FVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVA

KNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIIVILCCIVITSCCSCLKGCCSCGSCCKFDEDD

SEPVLKGVKLHYT

>SEQ ID NO:20: VHH72_ h5 variant (VHH72hl_ElD_R27L_E31D_Y32l_S56G_L97A)

DVQLVESGGGLVQPGGSLRLSCAASGLTFSDIAMGWFRQAPGKEREFVATISWSGGGTYYTDSVKGRFTISRDNAKN TVYLQMNSLRPEDTAVYYCAAAGAGTVVSEWDYDYDYWGQGTLVTVSS

> SEQ ID N0:21: VHH72_h5 C-terminally fused to Heavy chain of S309 antibody (Hc-72h5)

DVQLVQSGAEVKKPGASVKVSCKASGYPFTSYGISWVRQAPGQGLEWMGWISTYNGNTNYAQKFQGRVTMTTDT STTTGYMELRRLRSDDTAVYYCARDYTRGAWFGESLIGGFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGG GGSDVQLVESGGGLVQPGGSLRLSCAASGLTFSDIAMGWFRQAPGKEREFVATISWSGGGTYYTDSVKGRFTISRDN AKNTVYLQM NSLRPEDTAVYYCAAAGAGTVVSEWDYDYDYWGQGTLVTVSS

> SEQ ID NO:22: VHH72_ h5 N-terminally fused to Light chain of S309 antibody (Lc-72h5)

DVQLVESGGGLVQPGGSLRLSCAASGLTFSDIAMGWFRQAPGKEREFVATISWSGGGTYYTDSVKGRFTISRDNAKN TVYLQMNSLRPEDTAVYYCAAAGAGTVVSEWDYDYDYWGQGTLVTVSSGGGGSGGGGSEIVLTQSPGTLSLSPGER ATLSCRASQTVSSTSLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHDTSLTF GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > SEQ ID NO:23: VHH72_h5 C-terminally fused to Heavy chain of S309 antibody containing the LALA mutations (in bold) (Hc-72h5-LALA) DVQ.LVQ.SGAEVKKPGASVKVSCKASGYPFTSYGISWVRQAPGQ.GLEWI\/IGWISTYNGNTNYAQ.KFQ.GRVTI\/ITTDT STTTGYMELRRLRSDDTAVYYCARDYTRGAWFGESLIGGFDNWGQ.GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQ.DWLNGKEYKCKVSNKALPAPIEKTISKAKGQ.PREPQ.VYTLPPSRDELTKNQ.VSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVIV1 HEALHNHYTQ.KSLSLSPGKGGGGSG GGGSDVQLVESGGGLVQPGGSLRLSCAASGLTFSDIAMGWFRQAPGKEREFVATISWSGGGTYYTDSVKGRFTISRD NAKNTVYLQ.MNSLRPEDTAVYYCAAAGAGTVVSEWDYDYDYWGQ.GTLVTVSS

REFERENCES

1. Pinto, D. et al. Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody. Nature 583, 290-295 (2020).

2. Wrapp, D. et al. Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies. Cell 181, 1004-1015.el5 (2020).

3. Boudewijns, R. et al. STAT2 signaling restricts viral dissemination but drives severe pneumonia in SARS-CoV-2 infected hamsters. Nat. Commun. 11, 5838 (2020).

4. Wines, B. D., Powell, M. S., Parren, P. W., Barnes, N. & Hogarth, P. M. The IgG Fc contains distinct Fc receptor (FcR) binding sites: the leukocyte receptors Fc gamma Rl and Fc gamma Rlla bind to a region in the Fc distinct from that recognized by neonatal FcR and protein A. J. Immunol. Baltim.

Md 1950 164, 5313-5318 (2000).

5. Berger Rentsch, M. & Zimmer, G. A vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type I interferon. PloS One 6, e25858 (2011).

6. Schlothauer T, Herter S, Koller CF, Grau-Richards S, Steinhart V, Spick C, Kubbies M, Klein C, Umana P, Mbssner E. Novel human IgGl and lgG4 Fc-engineered antibodies with completely abolished immune effector functions. Protein Eng Des Sel.;29:457-66 (2016).

Claims

1. A composition comprising an antibody specifically binding the Corona virus Spike protein wherein the light and/or heavy chain of said antibody comprises an N- or C-terminal fusion to a protein binding agent specifically binding the Corona virus Spike protein at an epitope which is different from the antibody binding site, and wherein said protein binding agent's epitope comprises the amino acid residues Leu355, Tyr356, Ser358, Ser362, Thr363, F364, K365, C366 and Y494 of SEQ ID NO:18.

2. The composition of claim 1, wherein the protein binding agent's epitope further comprises amino acid residue R426 of SEQ ID NO:18.

3. The composition of claim 1 or 2, wherein the protein binding agent's epitope comprises residues L368, Y369, S371, S375, T376, F377, K378, C379 and Y508 as set forth in SEQ. ID NO: 19.

4. The composition of claims 1 to 3, wherein the protein binding agent comprises an immunoglobulin single variable domain (ISVD).

5. The composition of claim 4, wherein the ISVD comprises CDR1, CDR2 and CDR3 of VHH72, or the corresponding CDRs of VHH72-S56A, or of VHH72h5, or the ISVD comprises the VHH72 variant as depicted in SEQ ID NO:1 or a variant thereof, such as SEQ ID NO: 9 to 13, or 20, or a further humanized variant of any one thereof.

6. The composition of claims 1 to 5, wherein the antibody is S309 or a variant thereof.

7. The composition of claims 1 to 6, wherein the protein binding agent is fused to the N-terminus of the light chain of the antibody, and/or the protein binding agent is fused to the C-terminus of the heavy chain of the antibody.

8. The composition of claims 4 to 7, comprising a fusion as set forth in SEQ ID NO:4, SEQ ID NO: 21, SEQ ID NO:5, SEQ ID NO:22, SEQ ID NO:7, or SEQ ID NO:23 or a variant thereof.

9. The composition of claims 4 to 8, wherein the antibody comprises:

- SEQ ID NO:2 as light chain, and SEQ ID NO:4 as heavy chain, or

- SEQ ID NO:5 as light chain, and SEQ ID NO:3 as heavy chain, or

- SEQ ID NO:5 as light chain, and SEQ ID NO:4 as heavy chain, or

- SEQ ID NO:2 as light chain, and SEQ ID NO:7 as heavy chain, or

- SEQ ID NO:5 as light chain, and SEQ ID NO:6 as heavy chain, or

- SEQ ID NO:5 as light chain, and SEQ ID NO:7 as heavy chain, or - SEQ ID NO:2 as light chain, and SEQ ID N0:21 as heavy chain, or

- SEQ. ID NO:22 as light chain, and SEQ ID NO:3 as heavy chain, or

- SEQ ID NO:22 as light chain, and SEQ ID NO:21 as heavy chain, or

- SEQ ID NO:2 as light chain, and SEQ ID NO:23 as heavy chain, or

- SEQ ID NO:22 as light chain, and SEQ ID NO:6 as heavy chain, or

- SEQ ID NO:22 as light chain, and SEQ ID NO:23 as heavy chain, or a variant of any one thereof.

10. A composition comprising a protein binding agent comprising VHH72 or a variant thereof, or an Fc fusion of any one thereof, and the S309 antibody or a variant thereof.

11. The composition of claim 10, wherein the VHH72 variant comprises SEQ ID NO:8 or a variant thereof, and/or the S309 antibody comprises the light and heavy chain as set forth in SEQ ID NO:2 and 3, respectively.

12. A pharmaceutical composition comprising the composition of any one of claims 1 to 11.

13. The composition of any of claims 1 to 11, or the pharmaceutical composition of claim 12, for use as a medicament.

14. The composition of any of claims 1 to 11, or the pharmaceutical composition of claim 12, for use in treatment of a corona virus infection.

15. The composition of any of claims 1 to 11, or the pharmaceutical composition of claim 12, for use in treatment of Covidl9.

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