Antibodies directed to ace2 and their use as medicine

Abstract

The present invention relates to anti-ACE2 peptidase domain single domain antibodies. Further, the present invention relates to anti-ACE2 single domain antibodies comprising the CDR regions of the present single domain antibodies, polypeptides comprising thereof such as multimers thereof, and fusion proteins comprising thereof, as well as pharmaceutical compositions comprising the single domain antibodies, the polypeptides comprising thereof such as the multimeric single domain antibodies thereof, or the fusion proteins comprising thereof, and their use for treating diseases caused by a virus having ACE2 as receptor.

Description

[0001] ANTIBODIES DIRECTED TO ACE2 AND THEIR USE AS MEDICINE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to anti-ACE2 peptidase domain single domain antibodies. Further, the present invention relates to multimers thereof, fusion proteins comprising thereof, as well as a pharmaceutical composition comprising the present single domain antibodies, the multimers or the fusion proteins, and their use for treating a disease caused by a virus having ACE2 as receptor, preferably by a coronavirus.

[0004] BACKGROUND

[0005] Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. So far, it has infected more than 750 million people with more than 6.9 million deaths worldwide. The majority of infected individuals experience mild or no symptoms, however severe forms of COVID-19 are associated with respiratory failure, shock and pneumonia [1]. To date, multiple COVID-19 vaccines, effective against severe forms, have been authorized in Europe. Depending on the SARS-CoV-2 variants, the vaccines differ in their protection against transmission and loose effectiveness over time. Hence, multiple vaccination boost protocols have been in use [2]. Vaccine protection may also be insufficient in individuals with altered immune status. For these reasons, in addition to vaccines, researchers and manufacturers have been conducting large-scale clinical trials to evaluate various therapies. Unfortunately, to date there are no effective antiviral therapies for COVID-19, although some treatments have shown some benefits in certain patients [3]. In this context, passive immunization with monoclonal antibodies neutralizing SARS-CoV-2 protein Spike has been suggested as prophylactic intervention against infection. However, their effectiveness differs according to the variants and most of those currently in used prove to be ineffective against the latest Omicron variants due to accumulation of mutations in the Spike protein [4-6]. Therefore, other strategies are needed to complete the arsenal against COVID-19.

[0006] SUMMARY OF THE INVENTION

[0007] As shown in the Examples, the inventors have found camelid single domain antibodies (VHH) that target the angiotensin-converting enzyme 2 (ACE2), the receptor with which SARS-CoV-2 interacts to infect cells and then drives fusion of the viral membrane and the cell membrane. Both processes, receptor binding and membrane fusion, are mediated by the viral envelope protein Spike present at the surface of the virus [7]. These single domain antibodies (sdAbs) bind to ACE2 without interfering with its enzymatic activity. By targeting ACE2, and not the spike protein, they are less susceptible to viral escape and thus have the potential to inhibit SARS-CoV-2 infection by all SARS-CoV-2 variants. These inhibitors are therefore particularly useful in the actual context, where neutralizing antibodies in used showed poor effective effect on current variants. Moreover, these single domain antibodies represent new tools, offering the possibility to specifically detect and follow ACE2, especially in SARS-CoV-2 infected cells.

[0008] In a first aspect, an isolated single domain antibody that binds to the peptidase domain of the human angiotensin-converting enzyme 2 (ACE2) having the amino acid sequence SEQ ID NO: 2is provided.

[0009] The isolated anti-ACE2 single domain antibody according to this first aspect comprises the Complementary Determining Regions (CDRs):

[0010] - CDR1 having the amino acid sequence selected from SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 and variants thereof having up to 6, 5, 4, 3, 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11;

[0011] - CDR2 having the amino acid sequence selected from SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17 and variants thereof having up to 5, 4, 3, 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17; and

[0012] - CDR3 having the amino acid sequence selected from SEQ ID NO: 18 and SEQ ID NO: 19 and variants thereof having up to 2, or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 18 or SEQ ID NO: 19.

[0013] In some embodiments, the isolated anti-ACE2 single domain antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0014] The present invention also relates to a polypeptide comprising an anti-ACE2 single domain antibody according to this first aspect.

[0015] In a second aspect of the invention, it is provided multimeric single domain antibodies comprising at least one anti-ACE2 single domain antibody according to the first aspect of the invention linked to at least one other single domain antibody. In some embodiments, the multimeric single domain antibody comprises at least 2, 3 or 4 anti-ACE2 single domain antibodies according to the invention. In some embodiments, the multimeric single domain antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 45, 46 and 47or that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 45, 46 and 47. In a third aspect of the invention, it is provided a fusion protein comprising an anti-ACE2 single domain antibody according to the first aspect of the invention covalently linked to a second polypeptide which is not a single domain antibody.

[0016] In some embodiments the fusion protein is the fusion protein comprising an anti-ACE2 single domain antibody according to the first aspect, fused at its C-terminus to a human Fc fragment. In some embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 32, 33 and 34 or that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 32, 33 and 34.

[0017] In a fourth aspect of the invention, it is provided a pharmaceutical composition comprising an isolated anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention and / or the fusion protein of the third aspect of the invention.

[0018] The pharmaceutical compositions may be for use to treat or ameliorate a disease caused by a virus having ACE2 as receptor, preferably coronavirus caused disease, more preferably a disease selected from the group consisting of severe acute respiratory syndrome (SARS) and COVID-19 (coronavirus disease 2019), in a patient in need thereof. The pharmaceutical compositions may also be used prophylactically to reduce the likelihood of development of such disease in a patient in need thereof, such as an immunocompromised patient.

[0019] A further aspect of the invention provides methods for inhibiting binding of virus having ACE2 as receptor, preferably of coronavirus, to host cells with an isolated anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention and / or a fusion protein of the third aspect of the invention.

[0020] Also provided is an isolated anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention, a fusion protein of the third aspect of the invention or a pharmaceutical composition comprising thereof for use as a medicine.

[0021] Also provided is an isolated anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention, a fusion protein of the third aspect of the invention or a pharmaceutical composition comprising thereof for use in treating a disease caused by a virus having ACE2 as receptor, preferably a coronavirus, more preferably a disease selected from the group consisting of severe acute respiratory syndrome (SARS), and COVID-19 (coronavirus disease 2019).

[0022] Also provided is a method of reducing a viral load of a virus having ACE2 as receptor, preferably a coronavirus, in a subject in need thereof, comprising administering to the subject an isolated anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention, a fusion protein of the third aspect of the invention or a pharmaceutical composition comprising thereof.

[0023] Also provided is a method of reducing the likelihood of development of a disease caused by a virus having ACE2 as receptor, preferably a coronavirus, more preferably a disease selected from the group consisting of severe acute respiratory syndrome (SARS), and COVID-19 (coronavirus disease 2019), in a subject in need thereof, comprising administering to the subject an isolated anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention, a fusion protein of the third aspect of the invention or a pharmaceutical composition comprising thereof.

[0024] Also provided are detection agents comprising the anti-ACE2 single domain antibodies according to the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention or a fusion protein of the third aspect of the invention and a label.

[0025] Also provided are isolated nucleic acid sequences that encode any of the anti-ACE2 single domain antibodies according to the first aspect, or that encodes any of the multimeric single domain antibodies of the second aspect or that encodes any of the fusion proteins of the third aspect. Recombinant cells comprising these isolated nucleic acid sequences are also provided.

[0026] Also provided are methods of producing the single domain antibodies, the multimeric single domain antibodies or the fusion proteins according to the invention. The methods comprise culturing the recombinant cells under conditions sufficient for production of the single domain antibody, of multimeric single domain antibody or of the fusion protein.

[0027] Methods for detection of ACE2, in particular ACE2 peptidase domain, in a sample are also provided. In certain embodiments the methods comprise: providing a detection agent comprising the anti-ACE2 single domain antibody according to the first aspect of the invention, the multimeric single domain antibody according to the second aspect of this invention, or the fusion protein according to the third aspect of this invention; providing a sample; contacting the detection agent with the sample; and visualizing the antigen-detection agent complexes formed.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1: Sequence of anti-sACE2 VHHs obtained by phage display methods. The three variable regions (CDR1, CDR2, CDR3) are overlined. Sequence changes in variables regions relative to B07 are in bold. Figure 2: Kinetic analysis of sACE2 on His-VHH-B07 by BioLayer Interferometry (BLI) using various concentrations of sACE2 (0.3 ; 0.625 ; 1.25 ; 2.5 ; 5 ; 10 ; 20 pg / ml). Ni-NTA biosensors were used to immobilize 5 pg / ml of His-VHH. One representative experiment out of two. Results are the following: B07

[0030] - KD=350 nM, Kon=37.07 pM^.s1, Koff=0.13 s -1.

[0031] Figure 3: Kinetic analysis of sACE2 on His-VHH-B09 by BioLayer Interferometry (BLI) using various concentrations of sACE2 (0.3 ; 0.625 ; 1.25 ; 2.5 ; 5 ; 10 ; 20 pg / ml). Ni-NTA biosensors were used to immobilize 5 pg / ml of His-VHH. One representative experiment out of two. Results are the following: B09

[0032] - KD=353 nM, Kon=37.7 pM1.^1, Koff=0.133 s -1.

[0033] Figure 4: Kinetic analysis of sACE2 on His-VHH-BlO by BioLayer Interferometry (BLI) using various concentrations of sACE2 (0.3 ; 0.625 ; 1.25 ; 2.5 ; 5 ; 10 ; 20 pg / ml). Ni-NTA biosensors were used to immobilize 5 pg / ml of His-VHH. One representative experiment out of two. Results are the following: B10

[0034] - KD=10 nM, Kon=50.97 pM1.^1, Koff=0.005186 s -1.

[0035] Figure 5: Schema of the competition assay. Following a baseline step, the sACE2-Fc (10 pg / ml) was immobilized onto the biosensors. After a second baseline step, a first VHH was applied (VHH1; 5 pg / ml) and the sensor was dipped in a mixture of VHH1 and the competitor VHH2 at the same concentration (5 pg / ml). An anti-IgM specific single domain antibody was used as a control (VHH-ctl).

[0036] Figure 6: Competition assay with immobilized sACE2 pre-bound to B07 (VHH1) and VHH2 being B10, B09 or lgM (VHH-ctl).

[0037] Figure 7: Competition assay with immobilized sACE2 pre-bound to B09 (VHH1) and VHH2 being B07, B10 or lgM (VHH-ctl).

[0038] Figure 8: Competition assay with immobilized sACE2 pre-bound to B10 (VHH1) and VHH2 being B07, B09 or lgM (VHH-ctl).

[0039] Figure 9: VHH binding on cells expressing ACE2. Cells were incubated with the different VHHs (10 pg / ml), stained with an anti-myc antibody and a 488-conjugated anti-mouse antibody, before being analyzed by flow cytometry. Bars represent staining efficiency on HEK293 ACE2-expressing cells. Data are means SD of four independent experiments.

[0040] Figure 10: B07 binding efficacy on different cell lines expressing exogenous (HEK293-ACE2, A549-ACE2, U20S-ACE2) or endogenous ACE2 (IGROV-1, Vero E6). Data are means SD of two experiments.

[0041] Figure 11: Enzymatic activity of soluble ACE2 (sACE2) in the presence of VHHs B07, B09, B10 or absence (-) as assayed using the SensoLyte 390 ACE2 Activity Assay Kit, which measures fluorogenic peptide cleavage. An ACE2 inhibitor, provided with the kit, was used as control (DX600, lpM). VHHs were used at 10 pM (3 independent experiments). Figure 12: Enzymatic activity of soluble ACE2 (sACE2) in the presence the ACE2 inhibitor DX600 at different doses (2 experiments).

[0042] Figure 13: Enzymatic activity of soluble ACE2 (sACE2) in the presence of VHHs B07, B09, BIO used at different doses (one representative experiment out of two).

[0043] Figure 14: Inhibition of fusion (S-Fuse assay) by B07, B09, BIO VHHs after infection of S-Fuse cells with different SARS-CoV-2 variants: (A) Delta B.l.617.2, (B) Omicron BA.l, (C) Omicron subvariant BQ.1.1 and (D) XBB.1.5. Data are mean ± SD of 2 independent experiments. The dashed line indicates the limit of detection. An anti-IgM (or IgE) specific single domain antibody was used as a control (VHH-ctl).

[0044] Figure 15: VHH B07-Fc binding on cells expressing exogenous (HEK293-ACE2, A549-ACE2, U2OS-ACE2) or endogenous ACE2 (IGROV-1, Vero E6). Experiments were performed as in Figure 10 but using 0.1 pg / ml of VHH B07-Fc.

[0045] Figure 16: Kinetic analysis of VHH-B07-FC on ACE2-His by BioLayer Interferometry (BLI) using various concentrations of VHH B07-Fc :0.08 ; 0.254 ; 0.76 ; 2.3 ; 6.9 ; 21 ; 62 nM (The curves corresponding to 21 and 62 nM of VHH are not shown. They are saturating and biphasic. HIS2 biosensors were used to immobilize 1 pg / ml of ACE2-His. Results are the following: B07-Fc - KD=0.11 nM, KOn=1.950 pM ^.s1, KOff=2.14xl0-4s’1.

[0046] Figure 17: Enzymatic activity of soluble ACE2 (sACE2) in the presence of VHH B07-Fc or absence (-) using the SensoLyte 390 ACE2 Activity Assay Kit, as performed in Figure 13. DX600 was an inhibitor control of the kit (one representative experiment).

[0047] Figure 18: Inhibition of fusion (S-Fuse assay) by B07-Fc after infection of S-Fuse cells with different SARS- CoV-2 variants Delta B.l.617.2, Omicron BA.l, Omicron subvariant BQ.1.1, Omicron subvariant XBB.1.5. The dashed line indicates the limit of detection.

[0048] Figure 19: Enzymatic activity of cell surface ACE2 in the presence of saturated concentration of VHHs (> 20 pM) or 1 pM DX600. Data are means ± SD of three independent experiments.

[0049] Figure 20: Inhibition of fusion (S-Fuse assay) by B07, B09, B10 VHHs after infection of S-Fuse cells with different SARS-CoV-2 variants: (A) XBB.1.16.1, (B) EG.5.1.3; and (C) BA.2.86.1. Data are mean ± SD of 2 independent experiments. The dashed line indicates the limit of detection. An anti-IgM (or IgE) specific single domain antibody was used as a control (VHH-ctl).

[0050] Figure 21: Inhibition of 5 pg / ml spike binding to ACE2-expressing HEK293 cells by increasing concentrations of VHHs B07, B09 or B10. HEK293-ACE2 cells pretreated or not with the VHHs and incubated with soluble spike (S) protein (Wuhan) were stained with an anti-S antibody. Results were normalized for nonspecific (0%) and specific binding in the absence of inhibitor (100%). Experiments were fitted to a one-site competitive binding model. Data are mean ± SD of at least two independent experiments.

[0051] Figure 22: Displacement of S binding by B07 for various concentrations of S (5 pg / ml, 8 pg / ml, 10 pg / ml).

[0052] Experiments were carried out as in Figure 21 (one representative experiment out of at least two).

[0053] Figure 23: Displacement of S binding by B09 for various concentrations of S (5 pg / ml, 8 pg / ml, 10 pg / ml).

[0054] Experiments were carried out as in Figure 21 (one representative experiment out of at least two).

[0055] Figure 24: Relationship between the observed IC50 values for B07 or B09 displacement of S binding and initial concentration of S. Results represent means ± SD of at least two independent experiments. Linear regression analysis: R2 B07=0.9005; R2 B09=0.7896.

[0056] Figure 25: Inhibition of fusion (S-Fuse assay) by B07-Fc after infection of S-Fuse cells with different SARS- CoV-2 Omicron subvariants XBB.l.16.1., EG.5.1.3, BA.2.86.1. performed as in Fig. 18.

[0057] Figure 26: Inhibition of SARS-CoV-2 replication by B07-Fc measured by RT-qPCR. Vero E6 cells preincubated with B07-Fc were infected with (A) Delta B.l.617.2, (B) BA.l, (C) BQ.1.1, (D) XBB.l.5 and (E) XBB.l.16.1 (MOI between 0.07 and 0.33) during 48 or 66 hours and viral replication was measured by quantitative RT-qPCR (one experiment in triplicates).

[0058] Figure 27: Impact of ACE2 variation on VHH B07-Fc binding. Cells were transfected with hACE2 polymorphism constructs and incubated with the B07-Fc at 0.1 pg / ml, stained with an AF647-conjugated anti-human antibody before being analyzed by flow cytometry. Data are mean ± SD of at least two independent experiments.

[0059] Figure 28: Impact of ACE2 variation on VHH B07-Fc binding. Cells were transfected with bat, human, hamster, or mouseACE2 and incubated with the B07-Fc at different doses, stained with an AF647- conjugated anti-human antibody before being analyzed by flow cytometry. Data are mean ± SD of at least three independent experiments.

[0060] Figure 29: Prophylaxis of VHH B07-Fc for SARS-CoV-2 infection in mice. K18 hACE2 micereceived intranasally (i.n.) 7 mg / kg VHH-B07-Fc (B07-Fc) or 7 mg / ml VHH-Fc ctl (ctl-Fc). Twenty four hours later, they were infected with 105 PFU XBB.l.5. intranasally (i.n.). Three days postinfection, lungs were collected for analysis. RNA load measured by RT-qPCR of SARS-CoV-2 in lung.

[0061] Figure 30: Prophylaxis of VHH B07-Fc for SARS-CoV-2 infection in K18 hACE2 mouse. Relationship between RNA load measured in the lungs by RT-qPCR and B07-Fc dose quantified in the lungs by ELISA. ELISA plates coated with sACE2 and incubated with homogeneate of lower lung lobes of B07-Fc treated animals or a range of purified B07-Fc as a control were stained with peroxidase anti-human IgG. Figure 31: Effect of VHH B07-Fc on hamster ACE2. RNA Ioad measured by RT-qPCR of SARS-CoV-2 in lung. Data are mean ± SD of 4 animals. Mann-Witney test: P value 0.0286*.

[0062] Figure 32: B07-Fc staining and toxicity. Vero E6 cells viability in the presence of B07-Fc measured by quantitating ATP using the CellTiter-Glo assay (triplicates).

[0063] Figure 33: Binding efficacy compared to a commercial anti-ACE2 antibody. ACE2-expressing cells were incubated with a commercial mAb (R&D system, AF933), B07, BIO, B07-Fc, or BlO-Fc at different doses and stained with an anti-myc antibody and a AF488-conjugated anti-mouse antibody for the monomeric VHHs B07 and BIO; with an AF488-conjugated anti-human antibody for the dimeric VHHs B07-Fc and B10- Fc, and with a AF488-conjugated anti-goat antibody for the commercial mAb, before being analyzed by flow cytometry. Data are mean ± SD of at least two independent experiments.

[0064] Figure 34: BlO-Fc binding efficacy on HEK293-ACE2 cells. Experiments were performed using 0.1 pg / ml of VHH BlO-Fc. Data are mean ± SD of at least two independent experiments.

[0065] Figure 35: BlO-Fc binding efficacy on IGROV-1 cells. Experiments were performed using 0.1 pg / ml of VHH BlO-Fc. Data are mean ± SD of at least two independent experiments.

[0066] Figure 36: BlO-Fc binding efficacy on Vero E6 cells. Experiments were performed using 0.1 pg / ml of VHH BlO-Fc. Data are mean ± SD of at least two independent experiments.

[0067] Figure 37: Enzymatic activity of soluble ACE2 (sACE2) in the presence of VHH BlO-Fc or absence using the SensoLyte 390 ACE2 Activity Assay Kit. DX600 was an inhibitor control of the kit (one representative experiment out of two).

[0068] Figure 38: Inhibition of fusion (S-Fuse assay) by B07-Fc. S-Fuse assay in the presence B07-Fc, BlO-Fc, or ctl-Fc after infection with different SARS-CoV-2 variants (A) BA.l, (B) BQ.1.1, (C) XBB.1.16.1, (D) EG.5.1.3, (E) BA.2.86.1. The dashed line indicates the limit of detection. One or two experiments (mean ± SD).

[0069] Figure 39: Impact of ACE2 variation on B07-Fc binding. Cells were transfected with hACE2 constructs (myc-ACE2-E35A or myc-ACE2-H34Q), incubated with an anti-myc antibody or the B07-Fc at 0.1 pg / ml, and stained with an AF488-conjugated anti-mouse antibody or an AF647-conjugated anti-human antibody before being analyzed by flow cytometry. Data are mean ± SD of two experiments.

[0070] Figure 40: Enzymatic activity of cell surface ACE2 in the presence of saturated concentration of B07-Fc (> 20 pM) or 1 pM DX600. Data are means ± SD of three independent experiments.

[0071] DETAILED DESCRIPTION

[0072] A. Anti-ACE2 single Domain Antibodies

[0073] As reported in the examples, isolated single domain antibodies that bind to the angiotensin-converting enzyme 2 (also called herein ACE2) are provided. ACE2 is a type I integral membrane glycoprotein widely expressed in almost all tissues [8], which regulates the Renin-Angiotensin-System [9].

[0074] Human ACE2 (hACE2) is depicted under Accession No. Q9BYF1 in the Uniprot database on 30th March 2020. hACE2 is an 805 amino acids transmembrane protein with a processed ectodomain that spans amino acids 19-740 comprising a peptidase domain at residues 19-615.

[0075] The amino acid sequence of hACE2 (full length) is as set forth in SEQ ID NO: 1.

[0076] Human ACE2 (full-length)

[0077] MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWS AFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQE CLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYWLKNEMARANHYEDYGDYWRGDYEVN GVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYS LTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWD LGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKS IGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGWEP VPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRL GKSEPWTLALENWGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGD KAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEV EKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSIWLIVFGWMGVIWGIVILIFTGIRDRKK KNKARSGENPYASIDISKGENNPGFQNTDDVQTSF (SEQ ID NO: 1)

[0078] In some embodiments, the anti-ACE2 single domain antibodies of the invention bind to the ACE2 having the amino acid sequence SEQ ID NO: 1 or to a variant thereof having an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereof.

[0079] ACE2 via its peptidase domain catalyzes the cleavage of small circulating peptides, thus playing a central role in the control of the renin-angiotensin system. ACE2 processes angiotensin I into angiotensin 1-9, and angiotensin II into angiotensin 1-7, thus converting vasoconstrictor hormone in vasodilator hormone. It contributes to the regulation of the blood pressure and has a critical role in vascular, renal, and myocardial physiology.

[0080] ACE2 peptidase domain (residues 19-615 of ACE2 Uniport :Q9BYF1) :

[0081] STIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEI QNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDY NERLWAWESWRSEVGKQLRPLYEEYWLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEH TFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMV DQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDD FLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINF LLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGWEPVPHDETYCDPASLFHVSN DYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENWGAKN MNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD (SEQ ID N0:2)

[0082] In preferred embodiments, the single domain antibodies of the invention binds to the ACE2 peptidase domain having the amino acid sequence SEQ ID NO: 2 or to a variant thereof having an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 2.

[0083] The single domain antibodies (sdAb), also known as nanobodies®, are ten times smaller than conventional antibodies and they present numerous advantages: they are well produced, they are easily engineered, they can diffuse in tissues, they can bind to "non-conventional epitopes" or poorly accessible epitopes for conventional antibodies, they can penetrate into cells and bind intracellular antigens. To sum up, they combine the advantages of binding of antibodies with the advantages of small molecules.

[0084] The sdAb small size allows them to bind to epitopes that could be inaccessible to an IgG Fab arm.

[0085] The sdAb may be a variable region of camelid heavy-chain-only antibody (also called camelid single domain antibody, single domain VHH antibody or VHH) or a variable region of cartilaginous fish heavy- chain-only antibody (also called VNAR).

[0086] In a preferred embodiment, the anti-ACE2 single domain antibody is a camelid single domain antibody.

[0087] Single domain antibodies comprise four framework regions (FR1 to FR4, respectively) and three complementary determining regions (CDR1 to CDR3, respectively).

[0088] Specifically, the Examples describe isolation and characterization of the following anti-ACE2 single domain antibodies: B07 (also named VHH-B07), B09 (also named VHH-B09) and BIO (also named VHH-B10).

[0089] The amino acid sequences of the VHHs are provided below and the CDR1 to CDR3 and FR1 to FR4 domains are indicated. The following table indicates the amino acid sequence identification numbers of each disclosed sequence.

[0090] The number and location of CDR region amino acid residues herein comply with the known CDR numbering criteria such as Kabat (Kabat, EA, etc. 1991 Sequences of Proteins of Immunological Interest, 5th Ed), IMGT (IMGT®:the international ImMunoGeneTics information system® http: / / www.imgt.org) or Chothia (Chothia C., Lesk A.M. Canonical structures for the hypervariable regions of immunoglobulins.

[0091] Mol. Biol. 1987;196:901-917. doi: 10.1016 / 0022-2836(87)90412-8.), preferably IMGT.

[0092] The three single domain antibodies B07, B09 and B10 all bind to the peptidase domain of ACE2 without interfering with its peptidase activity. Thus, these single domain antibodies share structural and functional properties. The following table indicates the amino acid sequences of CDR1 to CDR3 of B07, B09 and B10 and variants thereof. Variant 1 comprises mutations or deletions in CDR1 and CDR2 amino acid sequences between B07, B09 and B10. Variant 2 comprises mutations in CDR1 and CDR2 amino acid sequences between B07 and B09. CDR3 of B07 and B09 is identical and differ totally from CDR3 of B10.

[0093] Table 2: amino acid sequences of CDRs Therefore, in some embodiments, the isolated anti-ACE2 single domain antibody of the invention comprises the following complementary determining regions (CDR): - CDR1 having the amino acid sequence selected from SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 and variants thereof having up to 6, 5, 4, 3, 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11;

[0094] - CDR2 having the amino acid sequence selected from SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17 and variants thereof having up to 5, 4, 3, 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17; and

[0095] - CDR3 having the amino acid sequence selected from SEQ ID NO: 18 and SEQ ID NO: 19 and variants thereof having up to 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 18 or SEQ ID NO: 19.

[0096] In some embodiments, the isolated anti-ACE2 single domain antibody of the invention comprises:

[0097] - CDR1 having the amino acid sequence selected from SEQ ID NO: 7 and variants thereof having up to 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 7;

[0098] - CDR2 having the amino acid sequence selected from SEQ ID NO: 12 and variants thereof having up to 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 12; and

[0099] - CDR3 having the amino acid sequence selected from SEQ ID NO: 18 and SEQ ID NO: 19 and variants thereof having up to 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 18 or SEQ ID NO: 19.

[0100] In a preferred embodiment, the isolated anti-ACE2 single domain antibody of the invention comprises:

[0101] - CDR1 having the amino acid sequence selected from SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11;

[0102] - CDR2 having the amino acid sequence selected from SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17; and

[0103] - CDR3 having the amino acid sequence selected from SEQ ID NO: 18 and SEQ ID NO: 19.

[0104] The following table indicates the amino acid sequences of FR1 to FR4 of B07, B09 and BIO and variants thereof. Variant 1 comprises mutations or deletions in FR1 to FR4 amino acid sequences between B07, B09 and BIO. Variant 2 comprises mutations in FR1 amino acid sequences between B07 and B09. FR2 to FR4 of B07 and B09 are identical.

[0105] Table 3: amino acid sequences of FRs

[0106] In some embodiments, the isolated anti-ACE2 single domain antibody of the invention comprises the following framework regions (FR):

[0107] - FR1 having the amino acid sequence selected from SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 and variants thereof having up to 5, 4, 3, 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24;

[0108] - FR2 having the amino acid sequence selected from SEQ ID NO: 26 and SEQ ID NO: 27 and variants thereof having up to 4, 3, 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 26 and SEQ ID NO: 27;

[0109] - FR3 having the amino acid sequence selected from SEQ ID NO: 29 and SEQ ID NO: 30 and variants thereof having up to 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 29 and SEQ ID NO: 30,

[0110] - FR4 having the amino acid sequence selected from SEQ ID NO: 31 and variants thereof having up to 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 31.

[0111] In some embodiments, the isolated anti-ACE2 single domain antibody of the invention comprises:

[0112] - FR1 having the amino acid sequence selected from SEQ ID NO: 20 and variants thereof having up to 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 20;

[0113] - FR2 having the amino acid sequence selected from SEQ ID NO: 25 and variants thereof having up to 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 25; and

[0114] - FR3 having the amino acid sequence selected from SEQ ID NO: 28 and variants thereof having up to 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 28,

[0115] - FR4 having the amino acid sequence selected from SEQ ID NO: 31 and variants thereof having up to 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 31.

[0116] In a preferred embodiment, the isolated anti-ACE2 single domain antibody of the invention comprises:

[0117] - FR1 having the amino acid sequence selected from SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24;

[0118] - FR2 having the amino acid sequence selected from SEQ ID NO: 26 and SEQ ID NO: 27; and

[0119] - FR3 having the amino acid sequence selected from SEQ ID NO: 29 and SEQ ID NO: 30, - FR4 having the amino acid sequence selected from SEQ ID NO: 31.

[0120] In a more general way, the person skilled art will understand the modifications in the framework regions that do not affect the functionality of the single domain antibodies. Examples of such modification include modification at their N-terminal end with a sequence (EV, DV or QV) which corresponds to the first amino acids of the VHH domain of mature Camelidae antibodies after removal of the peptide signal sequence.

[0121] The affinity for the soluble peptidase domain of ACE2 (sACE2) of the single domain antibodies of the invention may be specified. As used herein, the affinity for sACE2 of the single domain antibody is measured using the biolayer interferometry (BLI) assay as described in Example 2. Unless otherwise specified, the affinity for sACE2 is the affinity for human sACE2.

[0122] It was shown in Example 2 that VHH B07, VHH B09 and VHH BIO bind sACE2 with a KDcomprised between 10 nM and 350 nM.

[0123] Therefore, in an embodiment, the anti-ACE2 single domain antibody has a biolayer interferometry (BLI)- measured affinity for sACE2 of less than 500 nM, less than 400 nM, less than 375 nM or less than 355 nM. In another embodiment, the anti-ACE2 single domain antibody has a biolayer interferometry (BLI)- measured affinity for sACE2 of less than 15 nM or less than lOnM.

[0124] In some embodiments, the anti-ACE2 single domain antibody has a biolayer interferometry (BLI)- measured affinity for sACE2 between 1 and 355 nM, preferably between 5 and 355 nM.

[0125] The effect of the single domain antibodies on the ACE2 enzymatic activity may be specified. As used herein, the inhibitory activity of the single domain antibody is determined using the ACE2 protease activity as described in Example 4.

[0126] It was shown in Example 4 that none of VHH B07, VHH B09 and VHH B10 affects the ACE2 protease activity even at 40 pM.

[0127] Therefore, in some embodiments, the single domain antibody has no effect on the ACE2 protease activity. Among the single domain antibodies B07, B09 and B10, B07 and B09 which share a close sequence have been both found to neutralize different SARS-CoV-2 variants. The competition assay of Example 2 shows that B07 and B09 bind the same epitope.

[0128] Therefore, in some embodiments, the isolated anti-ACE2 single domain antibody of the invention comprises the following complementary determining regions (CDR):

[0129] - CDR1 having the amino acid sequence selected from SEQ ID NO: 9 and SEQ ID NO: 10 and variants thereof having 1 amino acid addition, deletion, and / or substitution compared to SEQ ID NO: 9 or SEQ ID NO: 10; - CDR2 having the amino acid sequence selected from SEQ ID NO: 15 and SEQ ID NO: 16 and variants thereof having 1 amino acid addition, deletion, and / or substitution compared to SEQ ID NO: 15 or SEQ ID NO: 16; and

[0130] - CDR3 having the amino acid sequence selected from SEQ ID NO: 18 and variants thereof having 1 amino acid addition, deletion, and / or substitution compared to SEQ ID NO: 18.

[0131] In some embodiments, the isolated anti-ACE2 single domain antibody of the invention comprises:

[0132] - CDR1 having the amino acid sequence selected from SEQ ID NO: 8 and variants thereof having up to 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 8;

[0133] - CDR2 having the amino acid sequence selected from SEQ ID NO: 13 and SEQ ID NO: 14 and variants thereof having up to 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 13 and SEQ ID NO: 14; and

[0134] - CDR3 having the amino acid sequence selected from SEQ ID NO: 18 and variants thereof having up to 2 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 18.

[0135] In a preferred embodiment, the isolated anti-ACE2 single domain antibody of the invention comprises:

[0136] - CDR1 having the amino acid sequence selected from SEQ ID NO: 9 and SEQ ID NO: 10;

[0137] - CDR2 having the amino acid sequence selected from SEQ ID NO: 15 and SEQ ID NO: 16; and

[0138] - CDR3 having the amino acid sequence selected from SEQ ID NO: 18.

[0139] In some embodiments, the isolated anti-ACE2 single domain antibody of the invention comprises the following framework regions (FR):

[0140] - FR1 having the amino acid sequence selected from SEQ ID NO: 22 and SEQ ID NO: 23 and variants thereof having up to 4, 3, 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 22 and SEQ ID NO: 23;

[0141] - FR2 having the amino acid sequence selected from SEQ ID NO: 26 and variants thereof having up to 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 26;

[0142] - FR3 having the amino acid sequence selected from SEQ ID NO: 29 and variants thereof having up to 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 29,

[0143] - FR4 having the amino acid sequence selected from SEQ ID NO: 31 and variants thereof having up to 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 31.

[0144] In some embodiments, the isolated anti-ACE2 single domain antibody of the invention comprises:

[0145] - FR1 having the amino acid sequence selected from SEQ ID NO: 21 and variants thereof having up to 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 21; - FR2 having the amino acid sequence selected from SEQ ID NO: 26 and variants thereof having up to 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 26; and

[0146] - FR3 having the amino acid sequence selected from SEQ ID NO: 29 and variants thereof having up to 2 or 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 29,

[0147] - FR4 having the amino acid sequence selected from SEQ ID NO: 31 and variants thereof having up to 2 or

[0148] 1 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 31.

[0149] In a preferred embodiment, the isolated anti-ACE2 single domain antibody of the invention comprises:

[0150] - FR1 having the amino acid sequence selected from SEQ ID NO: 22 and SEQ ID NO: 23;

[0151] - FR2 having the amino acid sequence SEQ ID NO: 26; and

[0152] - FR3 having the amino acid sequence SEQ ID NO: 29,

[0153] - FR4 having the amino acid SEQ ID NO: 31.

[0154] It was shown in Example 2 that VHH B07 and VHH B09 bind sACE2 with a KDcomprised between 350 nM and 355 nM.

[0155] Therefore, in some embodiments, the anti-ACE2 single domain antibody has a biolayer interferometry (BLI)-measured affinity for sACE2 of less than 500 nM, preferably less than 400, more preferably less than 375 nM, most preferably less than 355 nM.

[0156] In some embodiments, the anti-ACE2 single domain antibody has a biolayer interferometry (BLI)- measured affinity for sACE2 between 1 and 355 nM.

[0157] It was shown in Example 5 that VHH B07 and B09 have a SARS-CoV-2 neutralization titer of less than 30 pg / ml.

[0158] In some embodiments the anti-ACE2 single domain antibody has a SARS-CoV-2 neutralization titer of less than 50 pg / ml, preferably less than 30 pg / ml most preferably less than 10 pg / ml.

[0159] The neutralization may be determined as disclosed in Planas, D., et al., Considerable escape of SARS-CoV-

[0160] 2 Omicron to antibody neutralization. Nature, 2022. 602(7898): p. 671-675. This method is disclosed in details in the Examples (Measurement of the effect of VHH on SARS-CoV-2 induced cell-cell fusion). Briefly, SARS-CoV-2 neutralization titer of each VHH was expressed as the IC50. The percentage of neutralization was calculated using the number of syncytia as value with the following formula: 100 x (1 - (value with VHH -value in 'non-infected') / (value in 'control VHH' - value in 'non-infected')). The number of syncytia is quantified by measuring the number of object GFP+ (S-Fuse cells, become GFP+ upon fusion after being productively infected by SARS-CoV-2, and expressing S at their surface).

[0161] In some embodiments the anti-ACE2 single domain antibody has a SARS-CoV-2 neutralization titer between 1 pg / ml and 30 pg / ml. The single domain antibodies of the invention by targeting ACE2, and not the spike protein, are less susceptible to viral escape and thus have the potential to inhibit SARS-CoV-2 infection by all SARS-CoV-2 variants. It was also shown in Example 5 that VHH B07 is able to neutralize various SARS-CoV-2 variants. In some embodiments the anti-ACE2 single domain antibody has a neutralization titer against at least 1, at least 2, at least 3, at least 4, at least 5 or any SARS-CoV-2 variants of concern (VOCs) of less than 50 pg / ml, preferably less than 30 pg / ml, most preferably less than 10 pg / ml.

[0162] VOCs may be for example selected from the group consisting of Alpha or Alpha-like, Beta or Beta-like, Gamma or Gamma-like, Delta or Delta-like and Omicron or Omicron-like, more preferably selected from the group consisting of Alpha, Beta, Gamma, Delta and Omicron.

[0163] Unless it was specified otherwise SARS-CoV-2 VOCs are as defined by World Health Organization (https: / / www.who.int / en / activities / tracking-SARS-CoV-2-variants / as well as https: / / www.pango.network / and https: / / cov-lineages.org / for further details on their lineages).

[0164] Single domain antibody B07

[0165] The single domain antibody B07 (also named herein VHH B07, B07 or VHH ACE2-B07) has the following amino acid sequence: EVQLQASGGGLVQAGGSLRLSCAASRSTFSTNTIGWYRQAPGKQRELVASISTSGNTYYPDSVKGRFAIS RDNAENTVYLQMNSLKPEDTAVYYCNTPSRRISNFWGQGTQVTVSS (SEQ ID NO: 4).

[0166] VHH antibody B07 comprises the following CDR domains (indicated in bold in the preceding paragraphs):

[0167] CDR1: SRSTFSTNTIG (SEQ ID NO: 9),

[0168] CDR2: ASISTSGNTYYPDSVKG (SEQ ID NO: 15), and

[0169] CDR3: NTPSRRISNF (SEQ ID NO: 18).

[0170] E. coli comprising a vector coding for the VHH antibody B07 was deposited, according to the Budapest Treaty, at CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur - 25 rue du Docteur Roux, 75724 Paris Cedex 15, France) on 7 September 2023 with the number CNCM 1-5982.

[0171] Therefore, in some embodiments this invention provides an anti-ACE2 single domain antibody that comprises an amino acid sequence that is SEQ ID NO: 4 or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4.

[0172] In some embodiments this invention provides an anti-ACE2 single domain antibody that consists of an amino acid sequence that is SEQ ID NO: 4 or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4.

[0173] In some embodiments this invention provides an anti-ACE2 single domain antibody that comprises:

[0174] - a CDR1 domain having the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having up to two amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 9,

[0175] - a CDR2 domain having the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having up to two amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 15, and

[0176] -a CDR3 domain having the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having up to two amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 18.

[0177] In some embodiments this invention provides an anti-ACE2 single domain antibody that comprises:

[0178] - a CDR1 domain having the amino acid sequence of SEQ ID NO: 9,

[0179] - a CDR2 domain having the amino acid sequence of SEQ ID NO: 15, and

[0180] -a CDR3 domain having the amino acid sequence of SEQ ID NO: 18.

[0181] It was shown in Example 2 that VHH B07 binds sACE2 with a KDof 350 nM.

[0182] Therefore, in some embodiments, the anti-ACE2 single domain antibody has a biolayer interferometry (BLI)-measured affinity for sACE2 of less than 500 nM, preferably less than 400 nM, more preferably less 375 nM, most preferably less than 350 nM.

[0183] In some embodiments, the anti-ACE2 single domain antibody has a biolayer interferometry (BLI)- measured affinity for sACE2 between 1 and 350 nM.

[0184] It was shown in Example 5 that VHH B07 has a SARS-CoV-2 neutralization titer between 30 pg / ml and 9 pg / ml depending on the variant.

[0185] In some embodiments the anti-ACE2 single domain antibody has a SARS-CoV-2 neutralization titer of less than 50 pg / ml, preferably less than 30 pg / ml, most preferably less than 10 pg / ml.

[0186] In some embodiments the anti-ACE2 single domain antibody has a SARS-CoV-2 neutralization titer between 1 pg / ml and 30 pg / ml.

[0187] In some embodiments the anti-ACE2 single domain antibody has a neutralization titer against at least 1, at least 2, at least 3, at least 4, at least 5 or any SARS-CoV-2 variants of concern (VOCs) of less than 50 pg / ml, preferably less than 30 pg / ml most preferably less than 10 pg / ml.

[0188] It was shown in Example 8 that VHH B07 neutralizes SARS-CoV-2 variants with a competitive mechanism. Therefore, in some embodiments, the anti-ACE2 single domain antibody, as well as multimer or fusion protein comprising thereof, is a competitive inhibitor of SARS-CoV-2 protein Spike. It was shown in Example 10 that VHH B07-Fc, and therefore B07, binds to variants of hACE2 having the amino acid sequence SEQ ID NO: 1 selected from the group consisting of S19P, I21T, K26R, T27A, E37K, K68E, M82I and P84T variants.

[0189] Therefore, in some embodiments, the anti-ACE2 single domain antibody, as well as multimer or fusion protein comprising thereof, binds to variants of hACE2 having the amino acid sequence SEQ ID NO: 1 selected from the group consisting of S19P, I21T, K26R, T27A, E37K, K68E, M82I and P84T variants, preferably with at least 75%; more preferably 100% compared to the binding to hACE2 having amino acid sequence SEQ ID NO: 1.

[0190] The binding to hACE2 having the amino acid sequence SEQ ID NO: 1 or variant thereof may be measured by flow cytometry, for example as disclosed in the example (Measurement of VHH binding on ACE2 expressing cells).

[0191] It was shown in Example 10 that the epitope of VHH B07-Fc comprises H34 and E35 of hACE2 having the amino acid sequence SEQ ID NO: 1.

[0192] Therefore, in some embodiments, the anti-ACE2 single domain antibody, as well as multimer or fusion protein comprising thereof binds to an epitope that comprises H34 and / or E35 of ACE2 when numbered in accordance with SEQ ID NO: 1.

[0193] Single domain antibody B09

[0194] The single domain antibody B09 (also named herein VHH B09, B09 or VHH ACE2-B09) has the following amino acid sequence: QVQLVE SGGGLVQAGGSLRLSCATSRSTFSSNTIGWYRQAPGKQRELVASISTSGNTYYADSVKGRFAI SRDNAENT VYLQMNSLKPEDTAVYYCNTPSRRISNFWGQGTQVTVSS (SEQ ID NO: 5).

[0195] VHH antibody B09 comprises the following CDR domains (indicated in bold in the preceding paragraphs): CDR1: SRSTFS SNTIG (SEQ ID NO: 10),

[0196] CDR2: ASI STSGNTYYADSVKG (SEQ ID NO: 16), and

[0197] CDR3: NTP SRRI SNF (SEQ ID NO: 18).

[0198] E. coli comprising a vector coding for the VHH antibody B09 was deposited, according to the Budapest Treaty, at CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur - 25 rue du Docteur Roux, 75724 Paris Cedex 15, France) on 7 September 2023 with the number CNCM 1-5984.

[0199] Therefore, in some embodiments this invention provides an anti-ACE2 single domain antibody that comprises an amino acid sequence that is SEQ ID NO: 5 or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5.

[0200] In some embodiments this invention provides an anti-ACE2 single domain antibody that consists of an amino acid sequence that is SEQ ID NO: 5 or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5.

[0201] In some embodiments this invention provides an anti-ACE2 single domain antibody that comprises:

[0202] - a CDR1 domain having the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having up to two amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 10,

[0203] - a CDR2 domain having the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having up to two amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 16, and

[0204] -a CDR3 domain having the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having up to two amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 18.

[0205] In some embodiments this invention provides an anti-ACE2 single domain antibody that comprises:

[0206] - a CDR1 domain having the amino acid sequence of SEQ ID NO: 10,

[0207] - a CDR2 domain having the amino acid sequence of SEQ ID NO: 16, and

[0208] -a CDR3 domain having the amino acid sequence of SEQ ID NO: 18.

[0209] It was shown in Example 2 that VHH B09 binds sACE2 with a KD of 353 nM.

[0210] Therefore, in some embodiments, the anti-ACE2 single domain antibody has a biolayer interferometry (BLI)-measured affinity for sACE2 of less than 500 nM, preferably less than 400 nM, more preferably less 375 nM, most preferably less than 355 nM.

[0211] In some embodiments, the anti-ACE2 single domain antibody has a biolayer interferometry (BLI)- measured affinity for sACE2 between 1 and 355 nM.

[0212] It was shown in Example 5 that VHH B07 has a SARS-CoV-2 neutralization titer between 23 pg / ml and 8.3 pg / ml depending on the variant.

[0213] In some embodiments the anti-ACE2 single domain antibody has a SARS-CoV-2 neutralization titer of less than 25 pg / ml, preferably less than 15 pg / ml most preferably less than 10 pg / ml.

[0214] In some embodiments the anti-ACE2 single domain antibody has a SARS-CoV-2 neutralization titer between 1 pg / ml or 23 pg / ml.

[0215] In some embodiments the anti-ACE2 single domain antibody has a neutralization titer against at least 1, at least 2, at least 3, at least 4, at least 5 or any SARS-CoV-2 variants of concern (VOCs) of less than 25 pg / ml, preferably less than 15 pg / ml most preferably less than 10 pg / ml. It was shown in Example 8 that VHH B09 neutralizes SARS-CoV-2 variants with a competitive mechanism. Therefore, in some embodiments, the anti-ACE2 single domain antibody, as well as multimer or fusion protein comprising thereof, is a competitive inhibitor of SARS-CoV-2 protein Spike.

[0216] Single domain antibody BIO

[0217] The single domain antibody BIO (also named herein VHH BIO, BIO or VHH ACE2-B10) has the following amino acid sequence: QVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQALGKEREFVAVISGSGTSTYYTDSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCARAKGFSTIYNQDEGRYDYWGQGTQVTVSS (SEQ ID NO: 6).

[0218] VHH antibody BIO comprises the following CDR domains (indicated in bold in the preceding paragraphs): CDR1: SGRTFSSYAMG (SEQ ID NO: 11),

[0219] CDR2: AVISGSGTSTYYTDSVKG (SEQ ID NO: 17), and

[0220] CDR3: ARAKGFSTIYNQDEGRYDY (SEQ ID NO: 19).

[0221] E. coli comprising a vector coding for the VHH antibody BIO was deposited, according to the Budapest Treaty, at CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur - 25 rue du Docteur Roux, 75724 Paris Cedex 15, France) on 7 September, 2023 with the number CNCM 1-5985.

[0222] Therefore, in some embodiments this invention provides an anti-ACE2 single domain antibody that comprises an amino acid sequence that is SEQ ID NO: 6 or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6.

[0223] In some embodiments this invention provides an anti-ACE2 single domain antibody that consists of an amino acid sequence that is SEQ ID NO: 6 or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6.

[0224] In some embodiments this invention provides an anti-ACE2 single domain antibody that comprises:

[0225] - a CDR1 domain having the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having up to two amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 11,

[0226] - a CDR2 domain having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having up to two amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 17, and

[0227] -a CDR3 domain having the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having up to two amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 19. In some embodiments this invention provides an anti-ACE2 single domain antibody that comprises:

[0228] - a CDR1 domain having the amino acid sequence of SEQ ID NO: 11,

[0229] - a CDR2 domain having the amino acid sequence of SEQ ID NO: 17, and

[0230] -a CDR3 domain having the amino acid sequence of SEQ ID NO: 19.

[0231] It was shown in Example 2 that VHH BIO binds sACE2 with a KDof 10 nM.

[0232] Therefore, in some embodiments, the single domain antibody has a biolayer interferometry (BLI)- measured affinity for sACE2 of less than 25 nM, preferably less than 15nM, preferably less than lOnM.

[0233] In some embodiments, the single domain antibody has a biolayer interferometry (BLI)-measured affinity for sACE2 between 1 and 10 nM.

[0234] The present invention also relates to the polypeptides comprising any anti-ACE2 single domain antibody according to the invention. The polypeptides may be multimeric single domain antibodies or fusion proteins.

[0235] B. Multimeric single domain antibodies

[0236] Multimeric single domain antibodies are also provided. The multimeric single domain antibodies (multi- sdAb) comprise at least one single domain antibody of the invention linked to at least one other single domain antibody, the at least one other single domain antibody being preferably a single domain antibody of the invention.

[0237] Each single domain antibody present in the multimeric single domain antibody may be the same. Alternatively, each single domain antibody present in the multimeric single domain antibody may be different than one, several or all the other single domain antibodies present in the multimeric single domain antibody.

[0238] In some embodiments, the multimeric single domain antibody comprises:

[0239] - at least one, two, three or four single domain antibodies as disclosed in section A, preferably a single domain antibody comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, more preferably the amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0240] In certain embodiments the single domain antibodies may be linked together via one or more type of linker. The linker may be a peptide, peptide nucleic acid, or polyamide linkage. Suitable peptide linkers may include a plurality of amino acid residues, for example, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 amino acids, such as (Gly)4(SEQ ID NO: 35), (Gly)s (SEQ ID NO: 36), (Gly)4Ser (SEQ ID NO: 37), (Gly)4(Ser)(Gly)4(SEQ ID NO: 38), or combinations thereof or a multimer thereof (for example a dimer, a trimer, or a tetramer, or greater of [(G ly)4(Ser)] such as [(G ly)4(Se r)]5(SEQ ID NO: 39) or [(Gly)4(Ser)]3(SEQ ID NO: 40)).

[0241] Also included is a peptide linker sequence, which has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% identity thereto, using the default parameters of the BLAST computer program provided by HGMP.

[0242] In some embodiments the multimeric single domain antibody is a dimer. In some embodiments the dimer comprises two copies of the same single domain antibody (homodimer).

[0243] Advantageously, homodimers of the single domain antibodies B07, B09 and B10 are provided.

[0244] The homodimer may comprise two copies of the amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.

[0245] The homodimer of VHH-B07 (also named B07-linker-B07) has the following amino acid sequence (with a

[0246] "MA" residues at its N terminus (the "MA" residues are part of a cloning site which is cleaved during protein export) and a polyA at its C-terminus, the linker is in bold):

[0247] MAEVQLQASGGGLVQAGGSLRLSCAASRSTFSTNTIGWYRQAPGKQRELVASISTSGNTYYPDSVKGRFAISRDNAE NTVYLQMNSLKPEDTAVYYCNTPSRRI SNFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLQASGGGLVQAGG SLRLSCAASRSTFSTNTIGWYRQAPGKQRELVASISTSGNTYYPDSVKGRFAISRDNAENTVYLQMNSLKPEDTAVY YCNTPSRRI SNFWGQGTQVTVSSAAA ( SEQ ID NO : 45 )

[0248] Its nucleic acid sequence is the following: ccATGGCCGAGGTGCAGCTGCAGGCGTCTGGAGGAGGCTTGGTGCAGGCTGGGGGGTCTCTCAGACTCTCCTGTGCA

[0249] GCCTCTAGAAGCACCTTCAGTACCAATACCATCGGCTGGTACCGTCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGC

[0250] ATCTATTAGTACTAGTGGTAACACTTACTATCCAGACTCCGTGAAGGGCCGGTTCGCCATCTCCAGAGACAACGCCG

[0251] AGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATACTCCATCAAGA

[0252] CGGATTTCTAACTTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAggtggtggtggcagtggtggcggcggttc aggtggcggtggctccggtggtggcggtagtGAGGTGCAGCTGCAGGCtTCcGGcGGcGGaTTGGTGCAGGCaGGGG

[0253] GGagcCTCcggCTCTCCTGTGCcGCaagcAGAtccACCTTCAGTACtAATACtATCGGtTGGTACCGgCAGGCaCCA

[0254] GGtAAGCAGCGgGAGcTGGTCGCcagtATTtcTACTtcTGGgAACACcTAtTAcCCAGACagtGTGAAaGGtagaTT

[0255] CGCtATCagtAGAGACAACGCtGAGAACACtGTGTAcCTGCAAATGAACtctCTGAAgCCTGAGGACACcGCaGTCT

[0256] AcTAtTGTAATACcCCAagtcggagaATTagTAACTTCTGGGGaCAGGGaACCCAGGTCACCGTCTCaTCcgcggcc gca ( SEQ ID NO : 48 )

[0257] The homodimer of VHH-B09 (also named B09-linker-B09) has the following amino acid sequence (with a

[0258] "MA" at its N terminus and a polyA at its C-terminus, the linker is in bold):

[0259] MAQVQLVESGGGLVQAGGSLRLSCATSRSTFSSNTIGWYRQAPGKQRELVASISTSGNTYYADSVKGRFAISRDNAE NTVYLQMNSLKPEDTAVYYCNTPSRRI SNFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVQAGG SLRLSCATSRSTFSSNTIGWYRQAPGKQRELVASISTSGNTYYADSVKGRFAISRDNAENTVYLQMNSLKPEDTAVY YCNTPSRRI SNFWGQGTQVTVSSAAA ( SEQ ID NO : 46 ) Its nucleic acid sequence is the following:

[0260] ATGGCTCAGGTGCAGCTCGTGGAGTCAGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTCAGACTCTCCTGTGCAAC CTCTAGAAGCACCTTCAGTAGCAATACCATCGGCTGGTACCGTCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCAT CTATTAGTACTAGTGGTAACACTTACTATGCAGACTCCGTGAAGGGCCGGTTCGCCATCTCCAGAGACAACGCCGAG AACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATACTCCATCAAGACG GATTTCTAACTTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAggtggtggtggcagtggtggcggcggttcag gtggcggtggctccggtggtggcggtagtCAGGTGCAGCTCGTGGAGTCAGGGGGAGGCTTGGTGCAGGCTGGGGGG TCTCTCAGACTCTCCTGTGCAACCTCTAGAAGCACCTTCAGTAGCAATACCATCGGCTGGTACCGTCAGGCTCCAGG GAAGCAGCGCGAGTTGGTCGCATCTATTAGTACTAGTGGTAACACTTACTATGCAGACTCCGTGAAGGGCCGGTTCG CCATCTCCAGAGACAACGCCGAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTAT TACTGTAATACTCCATCAAGACGGATTTCTAACTTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA ( SEQ ID NO : 52 )

[0261] The homodimer of VHH-B10 (also named BlO-linker-BlO) has the following amino acid sequence (with a "MA" at its N terminus and a polyA at its C-terminus, the linker is in bold):

[0262] MAQVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQALGKEREFVAVISGSGTSTYYTDSVKGRFTI SRDNA KNTVYLQMNSLKPEDTAVYYCARAKGFSTIYNQDEGRYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSQVQLVE SGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQALGKEREFVAVI SGSGTSTYYTDSVKGRFTISRDNAKNTVYLQM NSLKPEDTAVYYCARAKGFSTIYNQDEGRYDYWGQGTQVTVSSAAA ( SEQ ID NO : 47 )

[0263] Its nucleic acid sequence is the following: ccATGGCTCAGGTGCAGCTCGTGGAGTCAGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCA GCCTCTGGACGCACCTTCAGTAGCTACGCt ATGGGCTGGTTCCGCCAGGCTCTAGGGAAGGAGCGTGAGTTTGTAGC AGTTATTAGCGGGAGTGGTACTAGCACATACTATACAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACG CCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCCCGAGCTAAG GGATTTAGTACTATATACAATCAAGACGAAGGTCGGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTC AggtggtggtggcagtggtggcggcggttcaggtggcggtggctccggtggtggcggtagtCAGGTcCAGCTaGTcG AGagtGGaGGcGGgTTGGTcCAGGCaGGcGACagcCTGcgcCTCTCtTGTGCcGCgagcGGgagaACCTTCtcaAGt TACGCt ATGGGgTGGTTCagaCAGGCaCTcGGcAAaGAGagaGAGTTTGTgGCtGTTATTAGtGGcAGcGGgACTtc aACATAtTAcACAGACagcGTGAAaGGgCGcTTtACCATCagCcgcGACAACGCtAAaAACACtGTGTAcCTcCAAA TGAACtctCTGAAgCCTGAGGACACcGCtGTTTAcTAtTGTGCtaGAGCaAAaGGgTTcAGcACcAttTACAATCAA GACGAgGGTagaTAcGACTACTGGGGaCAGGGaACtCAGGTaACgGTtTCaTCcgcggccgca ( SEQ ID NO : 49 )

[0264] In some embodiment, the multimeric single domain antibody comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47, more preferably comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47, most preferably consists of the amino acid sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47. In some embodiments the dimer comprises one copy of each of two different single domain antibodies (heterodimer), preferably one copy of two different single domain antibodies of the invention.

[0265] In an embodiment, the dimer is a heterodimer comprising a single domain antibody having the amino acid sequence selected from the group consisting of SEQ ID NO: 4 or an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof and a single domain antibody having the amino acid sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 6 or an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.

[0266] In an embodiment, the dimer is a heterodimer comprising a single domain antibody having the amino acid sequence selected from the group consisting of SEQ ID NO: 5 or an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof and a single domain antibody having the amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6 or an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.

[0267] In an embodiment, the dimer is a heterodimer comprising a single domain antibody having the amino acid sequence selected from the group consisting of SEQ ID NO: 6 or an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof and a single domain antibody having the amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 5 or an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.

[0268] The multimeric single domain antibodies may exhibit an enhanced affinity for the soluble peptidase domain of ACE2 (sACE2) and / or neutralization ability.

[0269] Thus, advantageously, in some embodiments, the multimeric single domain antibody has a biolayer interferometry (BLI)-measured affinity for sACE2 of less than 10 nM, preferably less than 5 nM, more preferably less than 3.5 nM, most preferably less than 0.1 nM.

[0270] In some embodiments, the multimeric single domain antibody has a biolayer interferometry (BLI)- measured affinity for sACE2 between 0.1 nM and 3.5 nM.

[0271] In some embodiments, the multimeric single domain antibody has a SARS-CoV-2 neutralization titer of less than 0.5 pg / ml, preferably less than 0.25 pg / ml, more preferably less than 0.15 pg / ml, most preferably less than 0.12 p / ml.

[0272] The SARS-CoV-2 neutralization titer may be against at least 1, at least 2, at least 3, at least 4, at least 5 or any SARS-CoV-2 variants of concern (VOCs), VOCs being preferably selected from the group consisting of Alpha or Alpha-like, Beta or Beta-like, Gamma or Gamma-like, Delta or Delta-like and Omicron orOmicron- like, more preferably selected from the group consisting of Alpha, Beta, Gamma, Delta and Omicron.

[0273] C. Fusion Proteins

[0274] In a third aspect, a fusion protein comprising an anti-ACE2 single domain antibody of the first aspect of the invention or a multimeric single domain antibody according to the second aspect of the invention covalently linked to at least a second polypeptide that is not a single domain antibody is provided. The single domain antibody may be any antibody described in Section A above. The multimeric single domain antibody may be any antibody described in Section B above.

[0275] As used herein a "fusion protein" refers to a polypeptide having two or more portions covalently linked together, where each of the portions is a polypeptide having at least one different property. The property may be a biological property, such as activity in vitro or in vivo. The property may also be a simple chemical or physical property, such as binding to a target antigen, catalysis of a reaction, etc. The two portions may be linked directly by a single peptide bond or through a peptide linker containing one or more amino acid residues. Generally, the at least two portions and the linker will be in reading frame with each other. Preferably, the at least two portions of the polypeptide are obtained from heterologous or different polypeptides. In the context of this invention, one of the portions is a single domain antibody or a multimeric single domain antibody of the invention.

[0276] In the fusion protein of the present invention, the anti-ACE2 single domain antibody or the multimeric single domain antibody may be directly fused or linked via a linker moiety to the at least another element of the fusion protein.

[0277] The linker may be selected from the group consisting of AAA, ( Ala)3(H is)6(SEQ ID NO: 41) or AAARS (SEQ ID NO: 42), AAARSDKTHTCPPCPAPELLGG (SEQ ID NO: 43), multimers thereof or any other linker disclosed in section B regarding multimeric single domain antibodies.

[0278] Also included is a sequence, which has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% identity thereto, using the default parameters of the BLAST computer program provided by HGMP.

[0279] Other examples of linkers are given in Xiaoying Chen et al. Fusion protein linkers: property, design and functionality, Adv Drug Deliv Rev. 2013 Oct;65(10):1357-69.

[0280] In some embodiments of the fusion protein, the second polypeptide is selected from a Fab, Fc, F(ab')2(including chemically linked F(ab')2chains), Fab', scFv (including multimer forms thereof, i.e. di-scFv, or tri-scFv), or BiTE (bi-specific T-cell engager). In some embodiments the second polypeptide is a Fc fragment of a mammalian immunoglobulin. In some embodiments the mammal is a human. In some embodiments, the Fc fragment is a Fc fragment from IgGl and has the amino acid sequence of SEQ ID NO: 44.

[0281] PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ( SEQ ID NO : 44 ) .

[0282] Preferred linkers, in particular to fuse a sdAb to a Fc fragment, are linkers derived from the linker AAARS (SEQ ID NO: 42), more preferably the linker AAARSDKTHTCPPCPAPELLGG (SEQ ID NO: 43).

[0283] Advantageously, fusion proteins of the single domain antibodies B07, B09 and BIO with a human Fc sequence are also provided.

[0284] Single domain antibody B07 was expressed fused at its C-terminus to the human Fc fragment (huFc). The VHH-B07- Fchu fusion molecule (also named VHH-B07-Fc, B07-Fc or VHH ACE2-B07- Fc hu) has the following amino acid sequence (the Fc portion is underlined):

[0285] EVQLQASGGGLVQAGGSLRLSCAASRSTFSTNTIGWYRQAPGKQRELVASISTSGNTYYPDSVKGRFAIS RDNAENTVYLQMNSLKPEDTAVYYCNTPSRRISNFWGQGTQVTVSSAAARSDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 32).

[0286] E. coli comprising a vector coding for the VHH-B07- Fchu was deposited, according to the Budapest Treaty, at CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur - 25 rue du Docteur Roux, 75724 Paris Cedex 15, France) on 7 September, 2023 with the number CNCM 1-5983.

[0287] Single domain antibody B09 was expressed fused at its C-terminus to the human Fc fragment. The VHH- B09-Fchu fusion molecule (also named VHH-B09-Fc, B09-Fc, VHH ACE2-B09-Fc hu) has the following amino acid sequence (the Fc portion is underlined):

[0288] QVQLVESGGGLVQAGGSLRLSCATSRSTFSSNTIGWYRQAPGKQRELVASISTSGNTYYADSVKGRFAIS RDNAENTVYLQMNSLKPEDTAVYYCNTPSRRISNFWGQGTQVTVSSAAARSDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 33). Single domain antibody BIO was expressed fused at its C-terminus to the human Fc fragment. The VHH- BlO-huFc fusion molecule (also named VHH-BlO-Fc, BlO-Fc, VHH ACE2-B10-Fc hu) has the following amino acid sequence (the Fc portion is underlined):

[0289] QVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQALGKEREFVAVISGSGTSTYYTDSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCARAKGFSTIYNQDEGRYDYWGQGTQVTVSSAAARSDKTHTCPPC P AP E L L G GPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 34).

[0290] Thus, in an embodiment of the invention it is provided a fusion protein comprising an anti-ACE2 single domain antibody according to the invention fused at its C-terminus to a human Fc fragment. In some embodiments, the fusion protein comprises and / or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 34.

[0291] In some embodiments the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 34.

[0292] In some embodiments the fusion protein consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 34.

[0293] Fusion proteins of multimeric single domain antibodies with a Fc sequence are also provided. In particular it is provided fusion proteins comprising a dimer (homodimer or heterodimer, preferably homodimer) comprising at least one of B07, B09 or BIO fused with a Fc sequence, preferably a human Fc sequence, more preferably a human Fc sequence from IgGl.

[0294] In some embodiments, fusion protein of dimers comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47 fused with a Fc sequence, preferably a human Fc fragment, more preferably a human Fc fragment from IgGl are provided. As for the fusion protein comprising sdAb and Fc, the fusion protein comprising the multimeric sdAb and the Fc may be directly fused or linked via a linker moiety to the Fc fragment.

[0295] Advantageously, the Fc portion of the fusion proteins comprising Fc fragment is responsible for linking these fusion proteins together into a homodimeric form. This homodimeric form comprises two fusion proteins (sdAb or multimeric sdAb fused to a Fc fragment) linked together through their Fc portion.

[0296] Thus, when the sdAb-Fc fusion protein or the multi-sdAb-Fc fusion protein is expressed in a suitable recombinant cell type, homodimer of the fusion proteins are formed. Therefore, the present invention encompasses both the fusion proteins comprising a Fc fragment (sdAb- Fc or multi-sdAb-Fc) in their monomeric and in their homodimeric forms.

[0297] Attaching an Fc fragment has the advantage of allowing immune effector functions that increase viral clearance (such as antibody-dependent cell cytotoxicity, ADCC or complement activation).

[0298] Moreover, the fusion of the single domain antibody or the multimeric single domain antibody with a Fc fragment enhances its affinity for ACE2 and / or its neutralization potency.

[0299] As shown in Examples 6 and 12, the affinity of B07-Fc and BlO-Fc is respectively 0.11 nM and 0.5 nM.

[0300] Thus, the fusion proteins exhibit an enhanced affinity as well as, in some embodiments enhanced neutralization and / or replication inhibition abilities . Affinity is at least 20 times, preferably at least 100 times, more preferably between 100 to 3500 times, most preferably between 500 and 3500 times increased.

[0301] Thus, it is also provided herein a method for increasing the neutralization potency of a single domain antibody of the invention, preferably from 10 to 1000, comprising fusing the C-terminus of single domain antibody to a Fc fragment, preferably a human Fc fragment.

[0302] Thus, advantageously, in some embodiments, the fusion protein has a biolayer interferometry (BLI)- measured affinity for sACE2 of less than 10 nM, preferably less than 5 nM, more preferably less than 3.5 nM, even more preferably less than 0.5nM, most preferably less than 0.12 nM, even most preferably less than 0.1 nM.

[0303] In some embodiments, the fusion protein has a biolayer interferometry (BLI)-measured affinity for sACE2 between 0.1 nM and 3.5 nM, preferably between 0.1 nM and 0.5 nM, more preferably between 0.1 nM and 0.25 nM, most preferably between 0.1 nM and 0.125 nM.

[0304] In some embodiments, the fusion protein has a SARS-CoV-2 neutralization titer of less than 0.5 pg / ml, preferably less than 0.25 pg / ml, more preferably less than 0.15 pg / ml, most preferably less than 0.12 p / ml.

[0305] The neutralization titer may be determined as disclosed above.

[0306] The SARS-CoV-2 neutralization titer may be against at least 1, at least 2, at least 3, at least 4, at least 5 or any SARS-CoV-2 variants of concern (VOCs), VOCs being preferably selected from the group consisting of Alpha or Alpha-like, Beta or Beta-like, Gamma or Gamma-like, Delta or Delta-like and Omicron orOmicron- like, more preferably selected from the group consisting of Alpha, Beta, Gamma, Delta and Omicron.

[0307] In some embodiments, the fusion protein has a SARS-CoV-2 replication inhibition titer of less than 5 pg / ml, preferably less than 2.5 pg / ml, more preferably less than 1 pg / ml, most preferably less than 0.75 p / ml. The replication inhibition titer may be determined as disclosed in details in the Examples (SARS-CoV-2 Viral replication).

[0308] Briefly, replication inhibition titer of each VHH was expressed as the IC50. Raw data (Cq) are normalized against appropriate negative (0%) and positive controls (100%) and are expressed in % of viral replication inhibition. The curve fit is performed using the variable Hill slope model or the four parameters logistic curve using GraphPad Prism: Response = Baseline response + (Maximum response - baseline response) / 1+ 10 (L°8EC50-iogconcentration)+Hiiisiopew(-,ereResponse is the measured response on Y axis; Baseline response is the minimum response at the bottom of the plateau; Maximum response is the maximum response at the top of the plateau; IC50 is the concentration at 50 % response; concentration is the measured drug concentration on the X axis. The number of SARS-CoV-2 copies was measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR).

[0309] In some embodiments, the fusion protein has a SARS-CoV-2 replication inhibition titer of between 0.5 and 5 pg / ml, preferably of between 0.5 and 4 pg / ml.

[0310] The SARS-CoV-2 replication inhibition titer may be against at least 1, at least 2, at least 3, at least 4, at least 5 or any SARS-CoV-2 variants of concern (VOCs), VOCs being preferably selected from the group consisting of Alpha or Alpha-like, Beta or Beta-like, Gamma or Gamma-like, Delta or Delta-like and Omicron or Omicron-like, more preferably selected from the group consisting of Alpha, Beta, Gamma, Delta and Omicron.

[0311] In some embodiments, the fusion protein, preferably the fusion protein comprising and / or consisting of an amino acid sequence SEQ ID NO: 32 or SEQ ID NO: 33, more preferably SEQ ID NO: 32, or that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereof, is a competitive inhibitor of SARS-CoV-2 protein Spike.

[0312] In some embodiments, the fusion protein, preferably the fusion protein comprising and / or consisting of an amino acid sequence SEQ ID NO: 32 or SEQ ID NO: 33, more preferably SEQ ID NO: 32, or that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereof, binds to variants of hACE2 having the amino acid sequence SEQ ID NO: 1 selected from the group consisting S19P, I21T, K26R, T27A, E37K, K68E, M82I and P84T variants, preferably with at least 75%, more preferably at least 100% compared to the binding to hACE2 having the amino acid sequence SEQ ID NO: 1.

[0313] In some embodiments, the fusion protein, preferably the fusion protein comprising and / or consisting of an amino acid sequence SEQ ID NO: 32 or SEQ ID NO: 33, more preferably SEQ ID NO: 32, or that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereof, binds to an epitope that comprises H34 and / or E35 of ACE2 when numbered in accordance with SEQ ID NO: 1.

[0314] In another aspect of the invention, the fusion protein comprising anti-ACE2 single domain antibody or the multimeric single domain antibody according to the invention is fused to a 2nd polypeptide, which is a label.

[0315] Polypeptide labels may be for example fluorescent, luminescent, enzymatic, magnetic or radioactive polypeptide labels. More details about labels are given in section H below.

[0316] In some embodiments, "CSA" residues may be directly fused or linked via a linker moiety to the sdAb or the multi-sdAb of the invention. Thus, a fluorescent label, such as an Alexa fluorescent protein, may be conjugated to the sdAb or the multi-sdAb of the invention via the cysteine of the "CSA" residues.

[0317] The fusion protein comprising BIO and the CSA residues (also named B10-CSA) has the following amino acid sequence (with at its N terminus "AM" residues which are part of a cloning site which is cleaved during protein export, a linker GSS and a histidine tag, and "**" at its C-terminus represent stop codon, the linkers are in bold):

[0318] AMGSSHHHHHHSGQVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQALGKEREFVAVISGSGTSTYYTDSV KGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCARAKGFSTIYNQDEGRYDYWGQGTQVTVSSGGGGSCSA** ( SEQ ID NO : 50 )

[0319] Its nucleic acid sequence is the following:

[0320] GccatgggcagcagccatcatcatcatcatcacagcggcCAGGTGCAGCTCGTGGAGTCAGGGGGAGGATTGGTGCA GGCTGGGGACTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGCTACGCtATGGGCTGGTTCCGCC AGGCTCTAGGGAAGGAGCGTGAGTTTGTAGCAGTTATTAGCGGGAGTGGTACTAGCACATACTATACAGACTCCGTG AAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGA CACGGCCGTTTATTACTGTGCCCGAGCTAAGGGATTTAGTACTATATACAATCAAGACGAAGGTCGGTATGACTACT GGGGCCAGGGGACCCAGGTCACCGTCTCCTCAggtggtggaggcagctgtagcgcttagtaa ( SEQ ID NO : 51 )

[0321] D. Nucleic Acids and Cells

[0322] In another aspect an isolated nucleic acid sequence that encodes the anti-ACE2 single domain antibody of the first aspect of the invention, or the multimeric single domain antibody of the second aspect of the invention or the fusion protein of the third aspect is provided. The anti-ACE2 single domain antibody may be any anti-ACE2 single domain antibody described in Section A above. The multimeric single domain antibody may be any multimeric single domain antibody described in Section B above. The fusion protein may be any fusion protein described in Section C above.

[0323] Also provided are recombinant vectors comprising the isolated nucleic acid sequence. The recombinant vector can be a vector for eukaryotic or prokaryotic expression, such as a plasmid, a phage for bacterium introduction, a YAC able to transform yeast, a viral vector and especially a retroviral vector, or any expression vector. An expression vector as defined herein is chosen to enable the production of single domain antibody, multimeric single domain antibody or fusion protein comprising single domain antibody either in vitro or in vivo.

[0324] In one embodiment, the expression vector is pHEN6, pET23, pET22, pASK-IBA2 or pCL In one embodiment, the expression vector encodes a protease cleavage site, such as TEV cleave site, inserted between the single domain antibody protein coding sequence and a protein purification Tag, such as polyHis tag or strep tag. In a preferred embodiment, the expression vector encodes a His tag or a strep tag. In one embodiment, a TEV cleavage site is positioned to remove the His tag, for example, after purification.

[0325] The expression vector can comprise transcription regulation regions (including promoter, enhancer, ribosome binding site (RBS), polyA signal), a termination signal, a prokaryotic or eukaryotic origin of replication and / or a selection gene. The features of the promoter can be easily determined by the man skilled in the art in view of the expression needed, i.e., constitutive, transitory or inducible (e.g. IPTG), strong or weak. The vector can also comprise sequences enabling conditional expression, such as sequences of the Cre / Lox system or analogue systems.

[0326] The nucleic acid molecules according to the invention can be obtained by conventional methods, known per se, following standard protocols such as those described in Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc., Library of Congress, USA). For example, they may be obtained by amplification of a nucleic sequence by PCR or RT-PCR or alternatively by total or partial chemical synthesis.

[0327] The vectors are constructed and introduced into host cells by conventional recombinant DNA and genetic engineering methods, which are known per se. Numerous vectors into which a nucleic acid molecule of interest may be inserted in order to introduce it and to maintain it in a host cell are known per se; the choice of an appropriate vector depends on the use envisaged for this vector (for example replication of the sequence of interest, expression of this sequence, maintenance of the sequence in extrachromosomal form or alternatively integration into the chromosomal material of the host), and on the nature of the host cell.

[0328] In another aspect a recombinant cell comprising the isolated nucleic acid sequence of the invention is provided. In some embodiments the cell is a eukaryotic cell.

[0329] In another aspect methods of producing the single domain antibody, the multimeric single domain antibody or the fusion protein are provided. The methods comprise culturing the recombinant cell comprising the isolated nucleic acid sequence that encodes the single domain antibody, the multimeric single domain antibody or the fusion protein under conditions sufficient for production of the single domain antibody, the multimeric single domain antibody or the fusion protein.

[0330] E. Pharmaceutical Compositions, Uses, and Methods

[0331] Also provided are pharmaceutical compositions of an isolated anti-ACE2 single domain antibody according to the first aspect of the invention, a multimeric single domain antibody according to the second aspect of the invention or a fusion protein according to the third aspect of the invention. The pharmaceutical composition may comprise one or a plurality of anti-ACE2 single domain antibodies according to the invention; and / or one or a plurality of multimeric single domain antibodies according to the invention; and / or one or a plurality of fusion proteins according to the invention.

[0332] Such compositions may comprise any suitable and pharmaceutically acceptable carrier, diluent, adjuvant or buffer solution. The composition may comprise a further pharmaceutically active agent. Such carriers may include, but are not limited to, saline, buffered saline, dextrose, liposomes, water, glycerol, ethanol and combinations thereof.

[0333] Such compositions may comprise a further pharmaceutically active agent as indicated. The additional agents may be therapeutic compounds, e.g. anti-inflammatory drugs, cytotoxic agents, cytostatic agents or antibiotics. Such additional agents may be present in a form suitable for administration to patient in need thereof and such administration may be simultaneous, separate or sequential. The components may be prepared in the form of a kit, which may comprise instructions as appropriate.

[0334] The pharmaceutical compositions may be administered in any convenient manner effective for treating a patient's disease including, for instance, administration by intravenous, intramuscular, or intranasal routes among others. In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.

[0335] The terms "subject" and "patient", are used interchangeably herein and refer to any animals for whom treatment is desired, particularly humans. Animals includes humans, domestic and farm animals, and zoo, sports, or pet animals. Preferably, the patients are mammals, most preferably humans.

[0336] For administration to mammals, and particularly humans, it is expected that the daily dosage of the active agent will be from 0.01 mg / kg body weight, typically around 1 mg / kg, 2 mg / kg or up to 4 mg / kg or up to 10 mg / kg or up to 50 mg / kg. The physician in any event will determine the actual dosage, which will be most suitable for an individual which will be dependent on factors including the age, weight, sex and response of the individual. The above dosages are exemplary of the average case. There can, of course, be instances where higher or lower dosages are merited, and such are within the scope of this invention. The pharmaceutical compositions may be for use to treat or ameliorate the condition of a patient in need thereof. The pharmaceutical compositions may also be used prophylactically to reduce the likelihood of development of a disease caused by a virus having ACE2 as receptor, preferably by a coronavirus, in a patient in need thereof, such as an immunocompromised patient.

[0337] In some embodiments of the methods, the pharmaceutical composition is administered to a subject that has been tested positive for disease caused by a virus having ACE2 as receptor, in particular by a coronavirus, but has not yet manifested symptoms of the disease. In some embodiments of the methods administering the pharmaceutical composition to the subject reduces a virus having ACE2 as receptor, preferably a coronavirus, viral load of the subject.

[0338] In some embodiments of the methods, the pharmaceutical composition is administered to a patient that has manifested symptoms of a disease caused by a virus having ACE2 as receptor, in particular by a coronavirus. In some embodiments of the methods administering the pharmaceutical composition to the patient reduces the severity of the disease state and / or reduces the duration of the disease state in the subject.

[0339] F. Methods of inhibiting binding

[0340] The invention encompasses methods for inhibiting virus having ACE2 as receptor, preferably coronavirus, binding to host cells with an isolated anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention or a fusion protein of the third aspect of the invention.

[0341] In one embodiment, the method comprises contacting an isolated anti-ACE2 single domain antibody of the invention, a multimeric single domain antibody of the invention or a fusion protein of the invention with host cells, prior to or at the same time that the cells are contacted with a virus having ACE2 as receptor, in particular a coronavirus.

[0342] Preferably, the anti-ACE2 single domain antibody, multimeric single domain antibody and / or fusion protein reduces binding of the virus having ACE2 as receptor, in particular of the coronavirus to the host cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

[0343] In a preferred embodiment, the virus having ACE2 as receptor is a coronavirus. The coronavirus may be selected from the group consisting of the severe acute respiratory syndrome coronavirus 1 and 2 (SARS- CoVl and SARS-CoV 2), the human seasonal coronavirus NL63. Preferably, the coronavirus is SARS-CoV-2. In some embodiments the method utilizes an anti-ACE2 single domain antibody, a multimeric single domain antibody or a fusion protein that binds to ACE2 peptidase domain. In some embodiments, the anti-ACE2 single domain antibody is selected from B07, B09 or BIO, multimer thereof such as B07-B07, B09-B09, B10-B10 or B07-B10 or the fusion protein selected from VHH B07-Fc, VHH B09-Fc or VHH BlO-Fc.

[0344] In some preferred embodiments, the anti-ACE2 single domain antibody is selected from B07 or B09, multimer thereof such as B07-B07 or B09-B09, B07-B10 or the fusion protein selected from VHH B07-Fc or VHH B09-Fc.

[0345] In some more preferred embodiments, the anti-ACE2 single domain antibody is B07, multimer thereof such as B07-B07 or the fusion protein is VHH B07-Fc.

[0346] G. Methods of treatment

[0347] Also provided is an isolated anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention, a fusion protein of the third aspect of the invention or a pharmaceutical composition comprising thereof for use as a medicament.

[0348] The present invention also relates to the use of an isolated anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention, a fusion protein of the third aspect of the invention or a pharmaceutical composition comprising thereof in the manufacture of a medicament.

[0349] The present invention also relates to an anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention, a fusion protein of the third aspect of the invention or a pharmaceutical composition comprising thereof for use in treating a disease caused by a virus having ACE2 as receptor.

[0350] A person skilled in the art will know the tests to carry out in order to identify whether a virus is a virus having ACE2 as receptor, one of these tests being the comparison of viral replication in cell lines expressing or not ACE2.

[0351] In a preferred embodiment, the virus having ACE2 as receptor is a coronavirus. The coronavirus caused disease may be a disease selected from the group consisting of severe acute respiratory syndrome (SARS), and COVID-19 (coronavirus disease 2019. Preferably, the coronavirus caused disease is COVID-19.

[0352] The present invention also relates to a method of treating a disease caused by a virus having ACE2 as receptor, preferably a coronavirus, in a patient in need thereof, comprising administering to the patient an anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention, a fusion protein of the third aspect of the invention or a pharmaceutical composition comprising thereof. The present invention also relates to the use of an anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention, a fusion protein of the third aspect of the invention or a pharmaceutical composition comprising thereof in the manufacture of a medicament for treatment of a disease caused by a virus having ACE2 as receptor, preferably a coronavirus.

[0353] Also provided is a method of reducing a viral load of a virus having ACE2 as receptor in a subject in need thereof, comprising administering to the subject an anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention, a fusion protein of the third aspect of the invention or a pharmaceutical composition comprising thereof.

[0354] In some embodiment, the viral load is reduced of at least 1 log, more preferably of at least 2 log.

[0355] The present invention also relates to an anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention, a fusion protein of the third aspect of the invention or a pharmaceutical composition comprising thereof for use in reducing a coronavirus viral load in a subject.

[0356] The present invention also relates to the use of an anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention, a fusion protein of the third aspect of the invention or a pharmaceutical composition comprising thereof in the manufacture of a medicament for reducing a viral load of a virus having ACE2 as receptor in a subject. Preferably, the virus having ACE2 as receptor is a coronavirus. The coronavirus may be selected from the group consisting of the severe acute respiratory syndrome coronavirus 1 and 2 (SARS-CoVl and SARS-CoV 2), the human seasonal coronavirus NL63 ; more preferably, the coronavirus is SARS-CoV-2.

[0357] Also provided is a method of reducing the likelihood of development of a disease caused by a virus having ACE2 as receptor, preferably a coronavirus, in a subject in need thereof, comprising administering to the subject an anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention, a fusion protein of the third aspect of the invention or a pharmaceutical composition comprising thereof.

[0358] The present invention also relates to an anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention, a fusion protein of the third aspect of the invention or a pharmaceutical composition comprising thereof for use in reducing the likelihood of development of a disease caused by a virus having ACE2 as receptor, preferably a coronavirus. The present invention also relates to the use of an anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention, a fusion protein of the third aspect of the invention or a pharmaceutical composition comprising thereof in the manufacture of a medicament for reducing the likelihood of development of a disease caused by a virus having ACE2 as receptor, preferably a coronavirus.

[0359] In some embodiments, the anti-ACE2 single domain antibody is selected from B07, B09 or BIO, the homodimers selected from B07-B07, B09-B09 or B10-B10 or the heterodimers selected from B07-B10, B09-B10 or B07-B09 or the fusion protein selected from VHH B07-Fc, VHH B09-Fc or VHH BlO-Fc.

[0360] In some preferred embodiments, the single domain antibody is selected from B07 or B09, the multimer is selected from the homodimers such as B07-B07 or B09-B09 or the heterodimers such as B07-B10, B09- B10 or B07-B09 or the fusion protein is selected from VHH B07-Fc or VHH B09-Fc.

[0361] In some more preferred embodiments, the single domain antibody is B07, the homodimer is B07-B07, the fusion protein is VHH B07-Fc.

[0362] H. Methods of detection of ACE2

[0363] In another aspect, methods for detection of ACE2, preferably ACE2 peptidase domain, are provided.

[0364] The methods may comprise providing a detection agent comprising an anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention or a fusion protein of the third aspect of the invention; providing a biological sample; contacting the detection agent with the biological sample; and visualizing the antigen-detection agent complexes formed.

[0365] In an embodiment, the detection agent further comprises a label.

[0366] Preferred labels include a fluorescent label, such as FITC or AlexaFluor647NHS, a chromophore label, an affinity-ligand label, an enzyme label, such as alkaline phosphatase or a luciferase, horseradish peroxidase, or P galactosidase, an enzyme cofactor label, a hapten conjugate label, such as digoxigenin or dinitrophenyl, a Raman signal generating label, a magnetic label, a spin label, an epitope label, such as the FLAG or HA epitope, a luminescent label, a heavy atom label, a nanoparticle label, an electrochemical label, a light scattering label, a spherical shell label, semiconductor nanocrystal label, wherein the label can allow visualization with or without a secondary detection molecule.

[0367] Preferred labels include suitable enzymes such as horseradish peroxidase, alkaline phosphatase, betagalactosidase, luciferase or acetylcholinesterase; members of a binding pair that are capable of forming complexes such as streptavidin / biotin, avidin / biotin or an antigen / antibody complex including, for example, rabbit IgG and anti-rabbit IgG; fluorophores such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, tetramethyl rhodamine, eosin, green fluorescent protein, erythrosin, coumarin, Alexa fluorescent protein, methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow, Cascade Blue, Texas Red, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, fluorescent lanthanide complexes such as those including Europium and Terbium, cyanine dye family members, such as Cy3 and Cy5, molecular beacons and fluorescent derivatives thereof, as well as others known in the art; a luminescent material such as luminol; light scattering or plasmon resonant materials such as gold or silver particles or quantum dots; or radioactive material include4C,123l,124l,125l,32P,33P,35S, or3H.

[0368] More preferably, the label is selected from a chemiluminescent label, an enzyme label, a fluorescence label, and a radioactive (e.g., iodine) label.

[0369] In an embodiment, the step of visualizing the antigen-detection agent complexes formed comprises visualizing the label.

[0370] In one embodiment, the detection agent is attached to an appropriate support, in particular a microplate or a bead.

[0371] The detection agent according to the invention is useful for the direct detection of ACE2, in particular the peptidase domain of ACE2; the detection of ACE2 can be carried out by an appropriate technique, in particular EIA, ELISA, RIA, immunofluorescence, luminescence in a biological sample.

[0372] Therefore, the present invention encompasses the use of the detection agent for the detection of ACE2, in particular the peptidase domain of ACE2.

[0373] A skilled artisan will appreciate that the detection agent can be used in any suitable assay format known in the art that is designed to utilize antibodies.

[0374] In some embodiments, the detection agent comprises the anti-ACE2 single domain antibody selected from B07, B09 or BIO, the homodimers selected from B07-B07, B09-B09 or B10-B10 or the heterodimers selected from B07-B10, B09-B10 or B07-B09 or the fusion protein selected from VHH B07-Fc, VHH B09-Fc, VHH BlO-Fc or B10-CSA. In some embodiments, the detection agent comprises the anti-ACE2 single domain antibody BIO, the homodimer B10-B10 or the fusion protein selected from VHH BlO-Fc or B10- CSA. BIO, which selectively binds ACE2 without interfering with SARS-CoV-2 entry, offers notably the possibility to specifically detect and follow ACE2 in SARS-CoV-2 infected cells.

[0375] The invention encompasses a composition comprising a detection agent comprising an anti-ACE2 single domain antibody of the first aspect of the invention, a multimeric single domain antibody of the second aspect of the invention or a fusion protein of the third aspect of the invention for detection of ACE2, preferably ACE2 peptidase domain, in a biological sample.

[0376] Preferably, the method comprises comparing the results obtained with a positive and / or negative control. In various embodiments, the method comprises quantification of the ACE2 detected.

[0377] EXAMPLES

[0378] Materials and Methods

[0379] Expression and purification of ACE2 peptidase domain (soluble ACE2, sACE2)

[0380] For alpaca immunization the ACE2 peptidase domain (residues 19-615, Uniprot: Q9BYF1; SEQ ID NO: 2) was cloned in pcDNA3.1. The signal peptide from the IgK chain (METDTLLLWVLLLWVPGSTG (SEQ ID NO: 3)) was introduced at the N-terminus to ensure the protein secretion to the extracellular medium, and His(x8) and Strep tags were added in-tandem at the C-terminus, following a Thrombin cleavage site.

[0381] The plasmid was transiently transfected into Expi293F™ cells (Thermo-Fischer) using FectoPro® DNA transfection reagent (PolyPlus) according to the manufacturer's instructions, and 5 pM kifunensine was added to the culture medium immediately after transfection. The cells were incubated 5 days at 37 °C and harvested by centrifugation. The supernatant was concentrated and recombinant ACE2 was purified by affinity chromatography on a Streptactin column (IBA) and by size-exclusion chromatography (SEC) on a Superdex200 column (Cytiva) equilibrated in 10 mM Tris, 100 mM NaCI. ACE2 was digested over-night at room temperature with Thrombin (1 U per 100 pg of recombinant protein) to remove the His and Strep tags. The proteolysis was stopped with 1 mM PMSF (phenylmethylsulfonyl fluoride) and the untagged ACE2 tags was recovered in the flow-through after injection in a Streptactin column. A final SEC was performed, after which untagged ACE2 was concentrated and stored at -80 °C.

[0382] For competition experiments, ACE2 peptidase domain fused to a human Fc from IgGl

[0018] was used (sACE2-Fc). For the measurement of the affinity between VHH B07-Fc and sACE2 by interferometry (BLI), it was used a His-tagged sACE2 (sACE2-His) that was expressed as indicated above and purified by IMAC and size-exclusion chromatography.

[0383] Plasmids and transfection

[0384] Mouse ACE2 plasmids was purchased from addgene (pscALPSpuro-MmACE2, #158087). Hamster ACE2 pcDNA3.1 hygro plasmid was synthetized by GeneArt (ThermoFisher). Bat ACE2 plasmid was a gift of N. Jouvenet (Institut Pasteur, Paris, France). The construct human ACE2 (hACE2) was obtained by substitution of the sequence encoding mCardinal from mCardinal-Cl plasmid (addgene, #54799) with hACE2 from the pLenti6-attB-hACE2-BSD (Buchrierser, 2020). Polymorphism substitutions were generated by site-directed mutagenesis using the Q5-site directed mutagenesis kit (New England Biolabs) according to the manufacturer's instructions. The mutants were all verified by sequencing (Eurofins). For transient expression, HEK293 (2xl05) cells were cotransfected with WT or mutant ACE2 vectors and with EGFP-N1 (Clontech, Palo Alto, Calif.), in a 5:1 ratio. Viruses

[0385] SARS-CoV-2 delta (B.l.617.2; GISAID ID: EPI_ISL_2029113), and omicron BA.l (GISAID ID: EPI_ISL_6794907) and BQ.1.1 (GISAID ID: EPI_ISL_15731523) were previously described [4, 5, 19-21]. XBB.1.5 was isolated from a nasopharyngeal swab of an individual of Hopital Europeen Georges Pompidou (HEGP; Assistance Publique, Hopitaux de Paris) (GISAID ID: EPI_ISL_16353849) (Bruel, medRxiv, 2023, May30). The laboratory of Virology of HEGP sequenced the swabs. The patient provided informed consent for the use of the biological materials. The XBB.1.16.1 strain (hCoV-19 / France / GES-IPP07712 / 2023), the EG.5.1.3 strain (hCoV-19 / France / BRE-IPP15906 / 2023) and the BA.2.86.1 strain (hCoV-19 / France / IDF- IPP17625 / 2023) were supplied by the National Reference Centre for Respiratory Viruses hosted by Institut Pasteur (Paris, France) and headed by Dr Etienne Simon-Loriere (Planas et al., https: / / doi.org / 10.1101 / 2023.ll.20.567873). The human sample from which strain hCoV- 19 / France / GES-IPP07712 / 2023 was isolated has been provided by Dr Vanessa COCQUERELLE from Laboratory Deux Rives. The human sample from which strain hCoV-19 / France / BRE-IPP15906 / 2023 was isolated has been provided by Dr F. Kerdavid from Laboratoire Alliance Anabio, Melesse. The human sample from which hCoV-19 / France / IDF-IPP17625 / 2023 was isolated was provided by Dr Aude LESENNE from Cerballiance, Lisses (France). Titration of viral stocks was performed on Vero E6 cells, with a limiting dilution technique enabling the calculation of the median tissue culture infectious dose or on S-Fuse cells. In replication experiments, XBB.1.16.1 (hCoV-19 / France / GES-IPP07712 / 2023), XBB.1.5 (hCoV- 19 / France / PDL-IPP58867 / 2022), BQ.1.1 (hCoV-19 / France / IDF-IPP50823 / 2022) and BA.l (hCoV- 19 / France / PDL-IPP46934i / 2021) were supplied by the National Reference Centre for Respiratory Viruses hosted by Institut Pasteur (Paris, France). The human samples from which strains were isolated has been provided from the Laboratoire Deux Rives, CH Laval and Laboratoire des centres de sante et d'hopitaux d'IDF. Viral productions were performed on Vero-TMPRSS2 cells and titration of viral stocks was performed on vero E6 cells. Viral concentrations were determined through Plaque Assay and the titers were quantified as plaque forming units (pfu) per milliliter.

[0386] The sequence of the viral stocks was verified by RNAseq by the National Reference Centre for Respiratory Viruses hosted by Institut Pasteur (Paris, France). All work with infectious virus was performed in biosafety level 3 containment laboratories at Institut Pasteur.

[0387] Cell lines

[0388] HEK293-ACE2 (human embryonic kidney cell line) were generated by transfection and cultured for several weeks in Blasticidin (lO pg / ml). U2OS (human osteosarcoma cell line) and A549 (human lung epithelial cell line) cells stably expressing ACE2, and U2OS-ACE2 cells stably expressing the GFP split system (GFP1- 10 and GFP11; S-Fuse cells) were previously described

[0022] . These cells were cultured in DMEM or in F- 12K Nutrient Mixture Media (A549) supplemented with 10% FBS, and 1% penicillin / streptavidin. Blasticidin (10 pg / mL) and puromycin (1 pg / mL) were used to select for ACE2 and GFP split transgenes expression, respectively.

[0389] Vero E6 (Vero 76, clone E6, Vero E6, ATCC® CRL-1586TM, simian kidney cell line) was obtained from ATCC (USA) and cultured in DMEM medium (Gibco) containing 10 % (v / v) fetal bovine serum (FBS, ThermoFisher Scientific) and penicillin / streptavidin (ThermoFisher Scientific). IGROV-1 cells were from the NCI-60 cell line panel

[0021] and cultured in RPMI medium (Gibco) containing 10% FBS and penicillin / streptavidin. Vero E6 and IGROV-1 endogenously express ACE2.

[0390] Absence of mycoplasma contamination was confirmed in all cell lines with the Mycoalert Mycoplasma Detection Kit (Lonza). All cell lines were cultured at 37°C and 5 % CO2.

[0391] Animal immunization and library construction

[0392] All immunization processes were executed according to the French legislation and in compliance with the European Communities Council Directives (2010 / 63 / UE, French Law 2013-118, February 6, 2013). The Animal Experimentation Ethics Committee of Pasteur Institute (CETEA 89) approved this study (2020- 27412). It was subcutaneously injected an adult alpaca at days 0, 21, and 28 with approximately 150 pg of ACE2 mixed with Freund complete adjuvant for the first immunization and with Freund incomplete adjuvant for the following immunizations. A blood sample of about 250 ml of the immunized animal was collected and Peripheral Blood Mononuclear cells (PBMCs) were isolated on a ficoll discontinuous gradient.

[0393] Briefly, the vhh genes isolated from PBMCs were then cloned into the phagemid vector pHEN6 by using primers contained enzymatic Sfil and Notl restriction sites at the 5' and 3' ends, respectively. The size of the library was estimated at about 8xl07cfu. Phage display protocol was performed as described in Lafaye et al.

[0023] . 1012phage-VHH diluted in PBS were used to perform three rounds of panning by using sACE2 coated on Nunc Immunotubes (Maxisorp) tubes (10 pg / ml). To increase the stringency, different blocking buffers for each panning were used: 2% skimmed milk as saturating agent for the first round; 5% BSA (Bovine Serum Albumin) and Odyssey blocking buffer (LI-COR Biosciences) diluted at % for the second and third one.

[0394] Following panning, phage clones were screened by standard phage-ELISA procedures using a HRP / anti- M13 monoclonal antibody conjugate. The DNA corresponding to the positive VHHs was sequenced by GATC Biotech. The sequences were processed with DNA strider and analyzed using ClustalW2-Multiple Sequence Alignment of EMBL-EBL VHH production

[0395] The pHEN6 vector allows the periplasmic expression and purification of selected VHHs with a c-myc tag and His tag at the C terminal end in frame with the VHH. Transformed E. co / / TGI cells expressed VHHs in the periplasm after induction with IPTG (1 mM) for 16h at 30°C. After centrifugation, cells were resuspended in PBS then lysed by polymixin B sulfate (1 mg / ml) for lh at 4°C, the periplasmic extract being obtained after centrifugation. Purified VHHs were obtained by His pure Cobalt sepharose beads (Thermo) according to the manufacturer's instructions.

[0396] VHH-Fc production

[0397] The coding sequences of the selected VHHs were digested with Ncol and Notl and subcloned into pFuse- hulgG-Fc2 digested with Ncol and Notl

[0024] . E. coli XLl-Blue transformants were obtained on 2YT agar plates containing zeocin 25pg / ml. The plasmids coding for the recombinant proteins were purified with Nucleobond Xtra Midi Plus EF (Macherey Nagel), transiently transfected in Expi293™ cells (ThermoFisher Scientific) using Fectro PRO DNA transfection reagent (Polyplus), according to the manufacturer's instructions. Cells were incubated at 37°C for 5 days and then the cultures were centrifuged. Proteins were purified from the supernatants by affinity chromatography using a HiTrap protein A HP (Cytiva), followed by SEC on a Superdex 75 column (Cytiva) equilibrated in PBS. Peaks corresponding to the dimeric VHH-Fc proteins were concentrated and stored at -80°C until used.

[0398] Measurement of the affinity between sACE2 and VHH by interferometry

[0399] Equilibrium binding was performed via BLI-analysis using an Octet HTX instrument (Fortebio, Reading, UK). VHH-His (5 pg / ml) diluted into Fortebio's PBS working buffer were immobilized onto Ni-NTA biosensors (Fortebio), which were pre-equilibrated in the same buffer. Following a baseline, VHH-coated sensors were incubated with various concentrations of sACE2 (0.3; 0.625; 1.25; 2.5; 5; 10; 20 pg / ml) for 10 min to reach equilibrium. Subsequently, biosensors were put in working buffer for 10 min to initiate dissociation. All incubations were performed at a temperature of 30°C under continuous shaking (1,000 rpm). Data were analyzed using the Octet Software 11.0. Binding curves from the association and dissociation steps were fitted using a 1:1 binding model to determine KD.app values.

[0400] To measure B07-Fc binding on human or hamster ACE2, sACE2-His (1 or 2 pg / ml)-coated HIS2 sensors (sartorius) were incubated with various concentration of VHH-B07-Fc: from 0.08 nM to 62 nM (human ACE2) or from 18 to 595 nM (hamster ACE2).

[0401] To measure competitive binding to sACE2 protein between the different VHHs by BLI, sACE2 fused to the human Fc domain was immobilized onto Anti-hlgG Fc Capture (AHC) Biosensors (Sartorius) at 5 pg / ml for 10 minutes. A baseline step was realized by dipping sensors into the working buffer. Then, a first VHH was applied at 5 pg / ml, in order to bind the immobilized sACE2 and reach saturation. The sensor was then dipped into a well containing a mixture of the same VHH with the competitor one added at the same concentration (5 pg / ml).

[0402] Measurement of VHH binding on ACE2 expressing cells by flow cytometry

[0403] Cells (2xl05) were incubated for 60 min at 4°C with 10 pg / ml VHHs or 0,1 pg / ml VHH-Fc, washed twice with 2% FBS-PBS, incubated for 60 min at 4°C with anti-myc antibody (9E10, Santa cruz biotech), washed twice with 2% FBS-PBS, and incubated for 60 min at 4°C with 488-conjugated anti-mouse antibody (Invitrogen) (for VHHs detection), or with the alexa-Fluor-488-conjugated anti-human IgG antibody (Invitrogen) (for VHH-Fc detection). The stained cells were washed, fixed in PFA 1.5% and analyzed on Attune flow cytometer NxT flow cytometer (Thermo Fisher) and Kaluza software. Background was subtracted using the fluorescence intensity obtained on the parental cells (HEK293, A549, U2OS) or using a VHH control (a nti-lgE or anti-IgM VHHs) as negative control.

[0404] To perform competition experiments of soluble spike binding an increasing concentration of VHH (between 0.01 to 100 pg / ml) were incubated with HEK293-ACE2 cells in RPMI / 10% FBS. After 30 min at 37°C, 5 pg / ml, or different doses (5, 8, 10 pg / ml), of soluble trimeric spike (Wuhan) (Sokal et al, 2021) was added for 30 min at 37°C. Then, immuno-staining with 5 pg / ml anti-spike monoclonal antibody mAb48 (Planchais et al, 2022) and alexa-Fluor-488-conjugated anti-human IgG antibody (Invitrogen) was performed at 4°C. Fixed cells (PFA 4%) were analyzed on Attune flow cytometer NxT flow cytometer (Thermo Fisher) and analyzed as described above.

[0405] Measurement of the effect of VHH on sACE2 enzymatic activity

[0406] Human ACE2 activity was evaluated using SensoLyte 390 ACE2 Activity Assay Kit (AnaSpec, cat. no. 72086) according to the manufacturer's protocol. The activity was measured on purified sACE2 (10 nM). B07, B09, B10 were added at different concentrations before activity measurement. The protease activity of ACE2 is indicated by an increase of the fluorescence after peptide cleavage.

[0407] To measure ACE2 activity on cells, 2.5xl04HEK293-ACE2 cells per well (96-well black plate, Greiner- BioOne) were incubated with B07, B09, B10 (>20 pM) or the control inhibitor DX600 (lpM) in 40 pl complete medium without phenol red (Gibco) before adding 40 pl of substrat diluted in PBS (SensoLyte 390 ACE2 Activity Assay kit). Peptide cleavage was measure in the supernatant on Tecan SPARK plate reader.

[0408] Measurement of the effect of VHH on SARS-CoV-2 induced cell-cell fusion U2OS-ACE2 GFP1-10 and GFP11 cells, also termed S-Fuse cells, become GFP+ when they are productively infected by SARS-CoV-2. Cells were mixed (ratio 1:1) (U2OS-ACE2 GFP1-10 (104) and GFP11 cells (104)) and plated in a pCIear 96-well plate (Greiner Bio-One). Serial dilution of VHHs were incubated with the cells at 37°C and infected with a SARS-CoV-2 strain. Eighteen hours later, cells were fixed with 4% paraformaldehyde (PFA), washed and stained with Hoechst (dilution 1:10000, Invitrogen). The extent of fusion was then quantified by measuring the number of object GFP+ with an Opera Phenix high-content confocal microscope (PerkinElmer) and Harmony software version 4.9 (PerkinElmer). The percentage of inhibition was calculated using the number of syncytia as value with the following formula: 100 x (1 - (value with VHH - value in 'non-infected') / (value in 'control VHH' - value in 'non-infected')). Inhibition activity of each VHH was expressed as the IC50. It was previously reported that the neutralization assay with the S-Fuse system is not affected by differences in fusogenicity between variants [6].

[0409] SARS-CoV-2 Viral replication

[0410] For the evaluation of SARS-CoV-2 replication, the number of SARS-CoV-2 copies was measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Individual VHHs were pre-incubated with Vero E6 cells (ten specified concentrations) 2 hours prior infection. Each plate included PBS (2 pl) and remdesivir (25 pM; SelleckChem) controls. After the pre-incubation time, the cells were exposed to the different variants of SARS-CoV-2 (at a multiplicity of infection between 0.07 and 0.33 PFU / Vero E6 cells). After a one-hour adsorption at 37°C, the supernatant was aspirated and replaced with 2% FBS / DMEM media containing the respective compounds at the indicated concentrations. The cells were then incubated at 37°C for 48 to 66 hours. Supernatants were collected and heat inactivated at 80°C for 20 minutes. The Luna Universal One-Step RT-qPCR Kit (New England Biolabs) was used for the detection of genomic SARS-CoV-2 RNA through RT-qPCR using a thermocycler (QuantStudio 6 thermocycler; Applied Biosystems). Specific primers targetingthe N-region viral gene: Forward: 5' TAATCAGACAAGGAACTGATTA (SEQ ID NO. 53) Reverse: 5' CGAAGGTGTGACTTCCATG (SEQ ID NO. 54) were used. Cycle threshold (Ct) were determined by the second derivative maximum method in the QuantStudio software. The quantity of viral genomes is expressed as Ct and was normalized against the Ct values of the negative (PBS) and positive controls (remdesivir 25 pM). Curve fits and IC50 values were obtained in Prism using the variable Hill slope model.

[0411] Cell viability assay

[0412] Cell viability assays were performed in compound-treated cells using the CellTiter-Glo assay according to the manufacturer's instructions (Promega) and a Berthold Centro XS LB960 luminometer. 3,000 cells / well of Vero E6 were seeded in white with clear bottom 384-well plates. The following day, compounds were added at concentrations indicated. PBS only and 10 pM camptotecin (Sigma-Aldrich) controls were added in each plate. After 48 h incubation, 10 pl of CellTiter Gio reagent was added in each well and the luminescence was recorded using a luminometer (Berthold Technologies) with 0.5 sec integration time. Raw data were normalized against appropriate negative (0 %) and positive controls (100 %) and are expressed in % of viral replication inhibition or % of cytotoxicity (Fig. 32).

[0413] Immunofluorescence imaging of primary human nasal epithelium cells and K18 mouse tissues.

[0414] MucilAir™, reconstructed human nasal epithelial cells (hNECs), previously differentiated for 4 weeks, were cultured in 700 pL MucilAir™ media on the basal side of the air / liquid interface (ALI) cultures as described (Saunders et al., 2023). K18 mouse nasal respiratory epithelium, olfactory epithelium and lung bronchioli were extracted and post-fixed overnight in 4% PFA at 4°C. For imaging, cells were fixed on the apical and basal sides with 4% PFA for 15 minutes. Cells were stained with anti-ACE2 VHH-B07-Fc at 6 pg / ml revealed with a Goat anti-Human Alexa Fluor-488 secondary Ab (Jackson Immuno Research Laboratories), Pha lloid in Atto 565 (Sigma-Aldrich) (F-actin staining) or a-tubulin (ThermoFisher Scientific) revealed with a Goat anti-Rabbit Alexa Fluor 555 secondary Ab (Invitrogen), and DAPI (ThermoFisher Scientific, lmg / ml) and imaged using the LSM-700 confocal microscope (Zeiss) as described (Saunders et al., 2023) (Bolland, W. et al. 2024). Results not shown.

[0415] SARS-CoV-2 infection and treatment in hACE2 K18 mouse model

[0416] B6.Cg-Tg(K18-ACE2)2Prlmn / J mice (stock #034860) were imported from The Jackson Laboratory and bred at the Institut Pasteur under strict specific pathogen-free conditions. Infection studies were performed on 10 to 13 wk-old male and female mice, in animal biosafety level 3 (BSL-3) facilities at the Institut Pasteur. All animals were handled in strict accordance with good animal practice. Animal work was approved by the Animal Experimentation Ethics Committee (CETEA 89) of the Institut Pasteur (project dap 210050), and authorized by the French Ministry of Research (under project 31816) before the experiments were initiated. Twenty-four hours before infection, anesthetized (ketamine / xylazine) mice were administered intra-nasally (i.n.) with either 0.9% NaCI (40pl), control anti-LDL VHH (7mg / kg in 40 pl) or B07-Fc VHH (7mg / kg in 50 pl). On the next day, they were again anesthetized (ketamine / xylazine) and inoculated i.n. with 1 x 105PFU of SARS-CoV-2 XBB.1.5 (10 pl / nostril). On day 3 post-infection, mice were euthanized by ketamine / xylazine overdose. The trachea and upper lung lobes were fixed by submersion in 10% phosphate buffered formalin for 24-36 hours prior to removal from the BSL3 for processing and transfer in 70% ethanol. Lower lung lobes (and nasal turbinates in a subset of mice) were dissected and frozen at -80°C. Samples were homogenized in 400 pl of cold PBS using lysing matrix M (MP Biomedical) and a MP Biomedical FastPrep 24 Tissue Homogenizer. Viral RNA was extracted using an extraction robot IDEAL-32 (I Dsolutions) and the NucleoMag Pathogen extraction kit (Macherey Nagel). Viral RNA quantification was performed by quantitative reverse transcription PCR (RT-qPCR) using the IP4 set of primers and probe (nCoV_IP4-14059 Fw GGTAACTGGTATGATTTCG (SEQ ID NO: 55) and nCoV_IP4-14146 Rv CTGGTCAAGGTTAATATAGG (SEQ ID NO: 56) giving a 107bp product, and nCoV_IP4-14084 Probe(+)TCATACAAACCACGCCAGG [5']Hex [3']BHQ-1) (SEQ ID NO: 57) and the Luna Universal Probe One- Step RT-qPCR Kit (NEB). Serial dilutions of a titrated viral stock were analyzed simultaneously to express viral loads as PFU equivalents (eqPFU) per gram of tissue.

[0417] Measurement of VHH concentration in lungs homogeneate by ELISA sACE2 (2pg / mL) were coated on polystyrene plate (ThermoScientific) over night at 4°C. After washing with PBS / Tween 0.1%, plates were incubated with soluble B07-Fc (from 0.07 to 10 ng / ml) or homogeneate of lower lung lobes in PBS 0.5% Gelatin, 0.1% Tween-20 (PGT solution) for 90 min at 37°C. A negative control (sACE2 alone) was prepared under the same conditions. Then, plates were incubated at 37°C with Peroxydase Affinipure rabbit anti Human IgG (Jackson ImmunoResearch, 1 / 1000) in PTG for 45 min and treated with ortho-phenylenediamine solution containing 0.1% hydrogen peroxid (H2O2) for 5 min. The reaction was stopped by adding chlorohydric acid 3M (HCI) and the absorbance of the wells was measured at 492X in a spectrophotometer-microplate reader (Sunrise, Tecan).

[0418] SARS-CoV-2 infection and treatment in Syrian golden hamster

[0419] The animals used were male Syrian golden hamsters (Mesocricetus auratus, strain RjHan:AURA), 6 week of age (weight between 70-90 grams), obtained from Janvier Laboratories. Golden hamsters were housed and manipulated in class III safety cabinets in the Institut Pasteur animal facilities accredited by the French Ministry of Agriculture. Animal work was approved by the Animal Experimentation Ethics Committee (CETEA) of the Institut Pasteur (project dap 210011) and authorized by the French legislation (project #21045) in compliance with the European Communities Council Directives (2010 / 63 / UE, French Law 2013- 118, February 6, 2013) and according to the regulations of Institut Pasteur Animal Care Committees before the experiments were initiated. Anesthetized animals received intranasal inoculation (5 mg / kg) of VHH- B07-Fc or VHH-ctl-Fc. Twenty-four hours after inoculation, hamsters were infected intranasally (i.n.) with 104 PFU of SARS-CoV-2 XBB.1.16.1 as previously described (Planchais et al., 2022).

[0420] All hamsters were followed-up daily. At day 3 post-infection, animals were euthanized with an excess of anesthetics (ketamine, xylazine and laocaine) (AVMA Guidelines 2020), and samples collected. 100 mg lung fragments were ground in Lysing Matrix X tubes (MP Biomedicals) using a grinder at a speed of 6.5 m / s for 30 seconds and centrifuged at 2,000 g for 10 minutes (4°C) to clarify the supernatant. For the evaluation of the number of SARS-CoV-2 copies in tissues by RT-qPCR, 100 pl of each grinding solution was mixed with 500 pL of Trizol to deactivate virulence. The total RNA was then extracted using the Direct-zol RNA MiniPrep Kit (Zymo Research) and quantified. The presence of genomic SARS-CoV-2 RNA in these samples was evaluated by one-step RT-qPCR in a final volume of 5 pl per reaction in 384- well PCR plates using a thermocycler (QuantStudio 6 thermocycler; Applied Biosystems). The primers used targeted N-region viral gene: Forward: 5' TAATCAGACAAGGAACTGATTA (SEQ ID NO: 53) Reverse: 5' CGAAGGTGTGACTTCCATG (SEQ ID NO: 54). Viral load quantification (expressed as RNA copy number / g lung) was assessed by linear regression using a standard curve of seven known quantities of RNA transcripts containing the N-region viral gene. All collected data were plotted and analyzed using GraphPad Prism software. Mann-Whitney tests were performed for statistical analysis.

[0421] Statistical analysis

[0422] Figures were generated using GraphPad Prism 10 (GraphPad software). Statistical analysis was conducted using GraphPad Prism 10. Statistical significance between different groups was calculated using the tests indicated in each figure legend. For immunofluorescence experiments on whole-mount preparations of mouse organs, following euthanasia, samples of nasal turbinates and lungs of K18-hACE2 mice were collected and fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 24h at 4°C. Then, nasal respiratory epithelium, olfactory epithelium and lung bronchioli were finely excised and postfixed in PBS containing 4% paraformaldehyde for 1 hour, rinsed in PBS and immersed in blocking buffer (20% normal goat serum (NGS) and 0.3% Triton X-100 in PBS) for 1 hour at room temperature, and then incubated overnight at 4 °C in primary antibodies (in PBS containing 2% bovine serum albumin (BSA)) : VHH-B07-Fc (at 6 pg / ml) and rat anti-atubulin (1 / 200; ThermoFisher Scientific). After rinsing in PBS, samples were incubated for 2 hours at room temperature in PBS-2% BSA with the appropriate secondary antibodies (goat anti-human Alexa Fluor 547 (Jackson Immuno Research Laboratories) ; donkey anti-rat Alexa-Fluor 488 (Invitrogen)), Pha lloid in Atto 647 (Sigma-Aldrich) (F-actin staining) and DAPI (TermoFisher Scientific, lmg / ml). Fluorescent immunolabellings were analyzed with a Zeiss LSM-700 confocal microscope. Results not shown.

[0423] Results

[0424] Example 1 - VHH production

[0425] It was immunized an alpaca with the soluble catalytic part of ACE2 (named ACE2 peptidase domain, or soluble ACE2, or sACE2) and a library containing about 8xl07different VHHs was constructed from cDNA encoding VHH domains isolated from lymphocytes. It was selected VHHs by phage display through three rounds of panning against sACE2, each with different buffer and washing conditions. In total, 192 individual clones were assayed by ELISA to test whether they could bind sACE2. Three specific VHHs were obtained, called B07 (SEQ ID NO: 4), B09 (SEQ ID NO: 5), and BIO (SEQ ID NO: 6). B07 and B09 sequences were really closed with only six differential residues including two in CDR1 and CDR2 variable loops. B07 and BIO were more distant, with eight different residues in CDR1 and CDR2, and a substantially different CDR3 sequence (Figure 1).

[0426] Example 2 - B07, B09, BIO interact with ACE2 in a nanomolar range

[0427] The different VHHs were expressed in fusion with a His tag at the C terminal end. It was characterized the interaction between VHH-His and sACE2 by determining association (kon) and dissociation (koff) rate constants using biolayer interferometry (BLI) technology (Figures 2-4). In this experiment, VHH-His was first immobilized on Ni-NTA biosensors and incubated with different concentrations of sACE2. An apparent KD value of 350 nM, 353 nM and 10 nM was measured for the B07, B09, and BIO, respectively (Figures 2-4), indicating that these VHHs bind sACE2 with good (B07, B09) and high (BIO) affinity.

[0428] To have an insight into the epitope recognized by the three VHHs, a binding competition assay was carried out using BLI system (Octet HTX, Sartorius) (Figure 5). B07 or B09 did not bind the immobilized ACE2 prebound to B07 (ACE2-B07) or B09 (ACE2-B09) (Figures 6-7), suggesting that these two VHHs recognize the same epitope on ACE2 surface. On the contrary, BIO bound ACE2-B07 or ACE2-B09, suggesting that this VHH recognize a different epitope than B07 and B09 (Figures 6-7). In agreement with this finding, the immobilized ACE2 pre-bound to BIO (ACE2-B10) did not impede the binding of B07 and B09 (Figure 8).

[0429] Example 3 - B07, B09, BIO bind ACE2 expressing cells

[0430] It was tested the ability of the three VHHs to bind ACE2 present at the surface of different cell lines, which express exogenous (HEK293-ACE2, A549-ACE2, U2OS-ACE2) or endogenous ACE2 (Vero E6, IGROV-1) (Figure 9). 10 pg / ml (667 nM) of each VHH were incubated with the different cell lines. VHHs are myc tagged, leading to detect their presence at the cell surface by flow cytometry after staining with anti-myc antibody. The three VHHs were specifically detected at the surface of all cell lines, revealing their interaction with membrane ACE2. BIO showed the highest effect in accordance with its high affinity for sACE2 defined by BLI (KD10 nM) (Figure 10).

[0431] Example 4 - B07, B09, B10 do not impact the enzymatic activity of sACE2 in vitro

[0432] It was determined whether B07, B09, B10 VHHs affected ACE2 function. ACE2 catalyzes the cleavage of small circulating peptides, thus playing a central role in the control of the renin-angiotensin system [9]. It was assayed the impact of 10 pM of each VHHs on the ability of sACE2 to cleave a fluorometric peptide (SensoLyte assay). Enzymatic cleavage leads the peptide to fluoresce, which is monitored at excitation / emission = 330 / 390 nm. No effect of the three VHHs on peptide cleavage was found (Figure 11), even at 40 pM of VHHs (Figure 13), contrary to the inhibitor control DX600 (Figure 12). This suggested that the interaction of B07, B09, BIO VHHs with sACE2 did not interfere with its enzymatic activity.

[0433] Example 5 - B07 and B09 neutralize different SARS-CoV-2 variants

[0434] It was examined whether the selected VHHs may inhibit the ability of SARS-CoV-2 infected cells to form syncytia using the S-Fuse assay

[0017] . S-Fuse cells (U2OS-ACE2 GFP1-10 and GFP11) were pre-incubated with serial dilution of VHHs and exposed to different SARS-CoV-2 strains (delta, omicron BA.l, Omicron BQ.1.1, Omicron XBB.1.5, XBB.1.16.1, EG.5.1.3; BA.2.86.1). Syncytia formation was quantified 18h after infection.

[0435] VHH Delta BA.l BQ.1.1 XBB.1.5 XBB.1.16.1 EG.5.1.3 BA.2.86.1

[0436] B07 29.53 19.65 10.18 9.002 5.44 3.20 1.45

[0437] B09 18.02 23.19 9.495 8.287 11.77 7.75 3.35

[0438] B10 -

[0439] Ctl -

[0440] IC50(pg / ml) Table 4 - Inhibition of fusion by VHHs after infection of S-Fuse cells with different SARS-CoV-2 variants

[0441] B07 and B09, which share a close sequence (Figure 1), inhibited syncytia formation induced with the 7 strains tested (Delta, BA.l, BQ.1.1, XBB.1.5, XBB.1.16.1, EG.5.1.3; BA.2.86.1) with similar IC50 (from 1.45 to 30 pg / ml - 96.7 to 1970 nM) (Figures 14 and 20). B10, which differed in its CDR3 (Figure 1), displayed no inhibition activities on the different strains (Figures 14 and 20), while having the highest ACE2 affinity (Figure 2). This result was in accordance with the fact that B10 targeted a different epitope than B07 and B09 (Figure 3).

[0442] Example 6 - The fusion of VHH to Fc fragment increases their neutralization efficacy

[0443] In order to stabilize the VHHs and increase their efficacy, dimeric VHHs by fusing the DNA fragment coding for the full length of VHH B07 to the DNA fragment coding for the human IgGl Fc were produced

[0018] . The B07-Fc obtained was tested as previously for their ability to bind membrane ACE2 on different cell lines (Figure 15), to interact with sACE2 in vitro (BLI) (Figure 16), to prevent the enzymatic activity of ACE2 in vitro (Figure 17) and to neutralize SARS-CoV-2 strains (Figure 18A-18D, Figure 25A-C).

[0444] B07-Fc was specifically detected at the surface of different cell lines expressing exogenous (HEK293-ACE2, A549-ACE2, U2OS-ACE2) or endogenous ACE2 (Vero E6, IGROV-1) by flow cytometry after staining with anti-IgG antibodies (Figure 15). B07-Fc bound sACE2 with higher affinity (X3500) compared to the monomeric B07 (Figure 2), with a measured apparent KD (KD app) of 0.11 nM (Figure 16). As seen with B07, B07-Fc did not affect the enzymatic peptide cleavage of ACE2 (Figure 17) and did not impact cell viability, even at high concentration (Figure 32).

[0445] B07-Fc inhibited syncytia formation induced with the 7 SARS-CoV-2 variants and sub-variants tested (Delta, BA.l, BQ.1.1, XBB.1.5, XBB.1.16.1, EG.5.1.3, BA.2.86.1) (Figure 18A-18D, Figure 25 A-C) with an IC502 log lower (from 0.08 to 0.23 pg / ml- 0.96 to 2.86 nM) than B07 (Figure 14).

[0446] These results suggested that the fusion of the VHH B07 to huFc increased its neutralizing efficacy without affecting its enzymatic activity. Therefore, such a VHH represented a good candidate for further in vivo studies.

[0447] IC50 Delta BA.l BQ.1.1 XBB.1.5 XBB.1.16.1 EG.5.1.3 BA.2.86.1 pg / ml 0.229 0.175 0.113 0.117 0.09 0.08 0.10 nM 2.86- 2.19- 1.42- 1.46- 1.19 0.96 1.26

[0448] Table 5 - Inhibition of fusion by B07-Fc after infection of S-Fuse cells with different SARS-CoV-2 variants B07-Fc also inhibited infection of Delta and Omicron variants (BA.l, BQ.1.1, XBB.1.5, XBB.1.16.1) with IC50 from 0.6 to 4.2 pg / ml (7.9 to 22.4 nM) (Fig. 26A-E).

[0449] IC50 Delta BA.l BQ.1.1 XBB.1.5 XBB.1.16.1 nM 10.23 51.55 22.37 16.70 7.86

[0450] Table 6 - Inhibition of SARS-CoV-2 replication by B07-Fc variants

[0451] Example 7 - VHHs B07, B09, B10 and B07-Fc do not impact the enzymatic activity of ACE2 in cellulo

[0452] No effect of the three VHHs on peptide cleavage was found in vitro, even at 40 pM of VHHs, contrary to the inhibitor control DX600 (IC50 = 0.54 pM) (example 4). This result was confirmed in cellulo by following peptide cleavage using HEK293-ACE2 live cells instead of soluble ACE2 (Fig. 19). This result suggested that the interaction of B07, B09, and B10 VHHs with sACE2, or cell surface ACE2, did not interfere with its enzymatic activity. As B07, B07-Fc has no effect on peptide cleavage using HEK293-ACE2 live cells (Fig. 40).

[0453] Example 8 - VHH mode of action: VHHs B07 and B09 neutralize SARS-CoV-2 variants with a competitive mechanism To assess whether B07 and B09 neutralized SARS-CoV-2 infection by inhibiting binding to the spike (S) protein, it was performed competition experiments in which binding of soluble S (Wuhan) on HEK293- ACE2 cells was measured in the presence of increasing concentration of VHHs B07 and B09. BIO was used as control. S binding was assessed after immuno-staining with an anti-S antibody and flow cytometry (Fig. 21). Pre-incubation of HEK293-ACE2 with VHHs B07 and B09 prevented soluble S binding, while BIO had no effect (Fig. 21). This suggested that B07 and B09 neutralized SARS-CoV-2 infection by inhibiting ACE2- spike interaction.

[0454] It was then performed competition experiments with B07 or B09 using increasing concentrations of S from 5 to 10 pg / ml (Fig. 22 and 23). The relationship between the observed IC50 value and tracer concentration (here S) in displacement experiments indicates whether binding inhibition is competitive (i.e. displaceable) or noncompetitive (Watson et al, 2005). The IC50 value is expected to increase linearly with S concentration for competitive inhibition (Watson et al., 2005). IC50 values yielded a linear regression with increasing concentration of S (Fig. 24), indicating that both B07 and B09 inhibited S binding through a competitive mechanism.

[0455] Example 9 - B07-Fc characterization: B07-Fc detects ciliated cells on primary hNECs

[0456] It was shown in Example 6 that B07-Fc was specifically detected at the surface of different cell lines expressing exogenous (HEK293-ACE2, A549-ACE2) or endogenous ACE2 (IGROV-1, Vero E6) by flow cytometry after staining with anti-IgG antibodies (Fig. 15). In addition, B07-Fc specifically stained primary human nasal epithelial cells (hNECs), as shown by confocal immunofluorescence imaging. This air-liquid culture system is an effective tool for studying SARS-CoV-2 infection (Bolland, 2023, doi.org:10.1101 / 2023.09.06.556548). As described (Lee et al, 2020; Wu et al, 2023), ACE2 co-localized with a-tubulin on the surface of motile cilia.

[0457] Example 10 -The VHH-B07-FC binding efficacy is found in most of hACE2 variants

[0458] It was evaluated whether human ACE2 (hACE2) polymorphism affect B07-Fc binding efficacy. It was constructed polymorphic human ACE2 variants by substituting residues located within the vicinity of S binding (S19P, I21T, K26R, T27A, H34N, E35K, E37K, K68E, M82I, P84T) in a myc-hACE2 expressing plasmid. It was tested their cell surface expression after anti-myc staining and their reactivity with B07-Fc by flow cytometry. None of the substitution altered the correct cell surface expression of myc-hACE2 constructs. Eight of ten constructs were correctly detected by B07-Fc, suggesting that hACE2 polymorphism might have little impact on B07-Fc antiviral efficacy. However, B07-Fc failed to detect hACE2-H34N and hACE2- E35K expressing cells (Fig. 27), revealing the importance of H34 and E35 for B07-Fc binding to hACE2. The substitution of E35 by an Alanine (E35A) abrogated B07-Fc binding, confirming that E35 may be part of the epitope (Fig. 39). Phylogenetic diversities in ACE2 might also influence B07-Fc binding.

[0459] B07-Fc poorly recognized hamster, mouse, and bat ACE2 expressing cells compared to hACE2 expressing cells (Fig. 28). BLI experiments confirmed that B07-Fc bound soluble hamster ACE2 with smaller avidity (3 log) compared to human ACE2 (Human ACE2 KDApp=0.11nM, Hamster ACE2 KDApp=65nM).

[0460] The presence of a Glutamine at position 34 (Q34) in these different species may explain this lower binding efficacy. Indeed, B07-Fc did not react with hACE2-H34Q. Therefore, the B07-Fc epitope, and therefore B07 epitope, seems critically dependent upon residues H34 and E35 of hACE2.

[0461] Example 11 - Fc-conjugated B07 dimers protect against Omicron infection in vivo

[0462] It was evaluated in vivo the prophylactic potential of neutralizing VHH B07-Fc using hACE2 transgenic mouse model (K18-hACE2) and XBB.1.5 Omicron variant infection. Mice treated intranasally with a single administration of VHH B07-Fc (7 mg / kg), or VHH-Fc control (7 mg / kg) were infected 24 h later intranasally with lxlO5PFU of XBB.1.5 variant. Animals were euthanized at day 3 post-infection and lung viral load was measured by RT-qPCR In 6 of the 10 B07-Fc-treated mice, lung viral load level was strongly reduced (> 3 log) or below the detection limit, suggesting that the lung had been protected from viral infection (Fig. 29). It was assessed by immunofluorescence the cytopathic effect induced by XBB.1.5 in the presence (B07-Fc) or absence (ctl-Fc) of B07-Fc. Nasal, respiratory and lung epithelia were stained at day 3 p.i. with phalloidin (to visualize F-actin), anti-alpha tubulin antibodies (to visualize cilia) and Dapi (to visualize the nucleus). Protected mice showed no tissue damage (disappearance of the ciliated structure) compared to control mice (ctl-Fc and uninfected K18-hACE2), corroborating the protector effect of B07-Fc. The dose of B07-Fc in the lung homogenate measured by ELISA was variable between treated mice, revealing probable heterogeneity in the diffusion of VHH in the lung. Notably, all mice in which the B07-Fc dose in the lungs was above 215 ng B07-Fc of lung extract had lung viral load below the limit of detection, while the mice with lower B07-Fc dose exhibited high lung viral load (Fig. 30). Therefore, it was concluded that B07-Fc, when used prophylactically in K18 mouse and when correctly delivered into the lungs, strongly protects against Omicron pulmonary infection.

[0463] Next, it was evaluated the prophylactic potential of VHH-B07-Fc using Syrian hamster preclinical model. VHH-B07-Fc was administered intranasally at 5 mg / kg and the hamsters were infected 24h later with lxlO4PFU of the XBB.1.16.1 variant. Lung viral load was modestly but significantly lower in hamster treated with B07-Fc compared with the control animals (3.49xl07vs 5.7xl08copies / g lung), indicating a lower protection potential of VHH-B07-Fc in this species compared with transgenic mice expressing human ACE2. (Fig. 31). The weak binding of B07-Fc with hamster ACE2 compared to hACE2 (Fig. 28) likely explained the low effectiveness of B07-Fc in hamster.

[0464] Example 12 - Characterization of the non-neutralizing VHH BlO-Fc

[0465] The BlO-Fc was tested for its ability to interact with sACE2 in vitro, to bind membrane ACE2 using different cell types, to prevent the enzymatic activity of ACE2 in vitro, and to neutralize SARS-CoV-2 variants.

[0466] As observed for B07-Fc, BlO-Fc bound sACE2 with higher avidity (x 700) compared to its monomeric homolog (Fig. 4), with a measured apparent KD (KD app) of 0.5 nM. BlO-Fc was specifically detected at the surface of different cell lines expressing exogenous (HEK293-ACE2, A549-ACE2) or endogenous ACE2 (IGROV-1, Vero E6) by flow cytometry after staining with anti-IgG antibodies (Fig. 34-36). BlO-Fc did not affect enzymatic peptide cleavage activity of sACE2 (Fig. 37). Finally, BlO-Fc did not inhibit syncytia formation induced with the 5 SARS-CoV-2 variants and sub-variants tested (BA.l, BQ.1.1, XBB.1.16.1, EG.5.1.3, BA.2.86.1) (Fig. 38).

[0467] Example 13 - Comparison with commercial anti ACE2 antibody

[0468] VHHs against ACE2 represent useful tools to answer fundamental questions regarding the SARS-CoV-2 entry process. VHHs BlO-Fc and B07-Fc bind ACE2 with high efficacy (KD app. < 0.5 nM). Compared to commercial mAb targeting ACE2 (AF 933 R&D system), BIO exhibited a similar efficacy as the commercial antibody, while B07-Fc and BlO-Fc bound ACE2 expressing HEK cells with a much higher efficacy (xlOOO) (IC50 mAB AF933: 10.1 pg / ml ; IC50 B07-Fc : 21.8ng / ml ; IC50 BlO-Fc : 56.7ng / ml) (Fig. 33). These antibodies may allow the detection of ACE2 in locations in primary cells not accessible to conventional antibodies. Furthermore, B10 and BlO-Fc, which are not neutralizing, represent promising tools to track ACE2 in SARS-CoV-2 infected cells.

[0469] Example 14 - ACE2 down modulation

[0470] Preliminary results on ACE2 modulation showed only a moderate down modulation of ACE2 in the presence of B07-Fc. According these preliminary results, only 25% of ACE2 disappear from the cell surface after 16h.

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Claims

CLAIMS1. An isolated anti-ACE2 single domain antibody comprising the following complementary determining regions (CDR):- CDR1 having the amino acid sequence selected from SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 and variants thereof having up to 6 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11;- CDR2 having the amino acid sequence selected from SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17 and variants thereof having up to 5 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17; and- CDR3 having the amino acid sequence selected from SEQ ID NO: 18 and SEQ ID NO: 19 and variants thereof having up to 2 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 18 or SEQ ID NO: 19; or a polypeptide comprising thereof.

2. The isolated anti-ACE2 single domain antibody according to claim 1, wherein:- CDR1 has the amino acid sequence selected from SEQ ID NO: 7 and variants thereof having up to 2 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 7;- CDR2 has the amino acid sequence selected from SEQ ID NO: 12 and variants thereof having up to 2 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 12; and- CDR3 has the amino acid sequence selected from SEQ ID NO: 18 and SEQ ID NO: 19 and variants thereof having up to 2 amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 18 or SEQ ID NO: 19.

3. The isolated anti-ACE2 single domain antibody according to claim 1 or 2, wherein:- CDR1 has the amino acid sequence selected from SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11;- CDR2 has the amino acid sequence selected from SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17; and- CDR3 has the amino acid sequence selected from SEQ ID NO: 18 and SEQ ID NO: 19.

4. The isolated anti-ACE2 single domain antibody according to any one of claims 1 to 3, wherein the single domain antibody is a camelid single domain antibody (VHH).

5. The isolated anti-ACE2 single domain antibody according to any one of claims 1 to 4, wherein the anti- ACE2 single domain antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

6. The isolated anti-ACE2 single domain antibody according to any one of claims 1 to 5, wherein the antiACE? single domain antibody has a SARS-CoV-2 neutralization titer of less than 50 pg / ml.

7. A multimeric single domain antibody comprising at least one anti-ACE2 single domain antibody according to any one of claims 1 to 6 linked to at least one other single domain antibody.

8. The multimeric single domain antibody according to claim 7, wherein the multimeric single domain antibody comprises at least 2, 3 or 4 anti-ACE2 single domain antibodies according to any one of claims 1 to 6.

9. The multimeric single domain antibody according to claim 7 or 8, wherein the multimeric single domain antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 45, 46 and 47 or that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 45, 46 and 47.

10. A fusion protein comprising the anti-ACE2 single domain antibody according to any one of claims 1 to 6, or a multimeric single domain antibody according to any one of claims 7 to 9 covalently linked to at least a second polypeptide that is not a single domain antibody.

11. The fusion protein according to claim 10 wherein the second polypeptide is a human Fc fragment which is fused at the C-terminus of the single domain antibody or of the multimeric single domain antibody.

12. The fusion protein according to claim 10 or 11, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 32, 33 and 34 or that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 32, 33 and 34.

13. The multimeric single domain antibody according to any one of claims 7 to 9 or the fusion protein according to any one of claims 10 to 12, wherein the multimeric single domain antibody or the fusion protein has a SARS-CoV-2 neutralization titer of less than 0.5 pg / ml.

14. A pharmaceutical composition comprising the anti-ACE2 single domain antibody according to any one of claims 1 to 6, the multimeric single domain antibody according to any one of claims 7 to 9 and / or the fusion protein according to any one of claims 10 to 12 and a pharmaceutically acceptable carrier, diluent, adjuvant or buffer solution.

15. The anti-ACE2 single domain antibody according to any one of claims 1 to 6, the multimeric single domain antibody according to any one of claims 7 to 9, the fusion protein according to any one of claims 10 to 12 and / or the pharmaceutical composition according to claim 14 for use as medicament.

16. The anti-ACE2 single domain antibody according to any one of claims 1 to 6, the multimeric single domain antibody according to any one of claims 7 to 9, the fusion protein according to any one of claims 10 to 12 and / or the pharmaceutical composition according to claim 14 for use in the treatment of a disease caused by a virus having ACE2 as receptor, preferably a disease selected from the group consisting of SARS, or COVID-19.

17. A detection agent comprising the anti-ACE2 single domain antibody according to any one of claims 1 to 6, the multimeric single domain antibody according to any one of claims 7 to 9 or the fusion protein according to any one of claims 10 to 12 and a label.

18. An in vitro method for detection of a ACE2 in a sample, comprising: providing a detection agent comprising the anti-ACE2 single domain antibody according to any one of claims 1 to 6, the multimeric single domain antibody according to any one of claims 7 to 9 or the fusion protein according to any one of claims 10 to 12; providing a sample; contacting the detection agent with the sample; and visualizing the antigen-detection agent complexes formed.

19. An isolated nucleic acid sequence that encodes the anti-ACE2 single domain antibody according to any one of claims 1 to 6, the multimeric single domain antibody according to any one of claims 7 to 9 or the fusion protein according to any one of claims 10 to 12.

20. A recombinant vector comprising the nucleic acid sequence according to claim 19.

21. A recombinant cell comprising the nucleic acid sequence according to claim 19 or the recombinant vector according to claim 20.

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