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WO2024252183A1 - Single domain antibodies directed against human transmembrane- serine protease 2 (tmprss2) - Google Patents

Single domain antibodies directed against human transmembrane- serine protease 2 (tmprss2) Download PDF

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WO2024252183A1
WO2024252183A1 PCT/IB2024/000275 IB2024000275W WO2024252183A1 WO 2024252183 A1 WO2024252183 A1 WO 2024252183A1 IB 2024000275 W IB2024000275 W IB 2024000275W WO 2024252183 A1 WO2024252183 A1 WO 2024252183A1
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tmprss2
antibody
seq
vhh
cells
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PCT/IB2024/000275
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French (fr)
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Pierre Lafaye
Olivier Schwartz
Nell SAUNDERS
Julian BUCHRIESER
Eduard BAQUERO SALAZAR
Ignacio FERNANDEZ
Félix REY
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Institut Pasteur
Centre National De La Recherche Scientifique
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Publication of WO2024252183A1 publication Critical patent/WO2024252183A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)

Definitions

  • the invention relates to human coronavirus HKU1 and its receptor and antibodies that bind to its receptor.
  • BACKGROUND OF THE INVENTION [0002]
  • HKU1, 229E, NL63 and OC43 circulate worldwide causing common colds 1 .
  • Coronavirus spike proteins bind to cellular receptors to enable infection 2 .
  • NL63 uses angiotensin-converting enzyme 2 (ACE2) for viral entry 3
  • 229E uses human aminopeptidase-N 4 .
  • HKU1 and OC43 bind cells through 9-O-acelytated sialic acid but their protein receptors remain unknown 5 .
  • transmembrane-serine protease 2 (TMPRSS2) can cleave coronavirus spike and prime their fusion 6-10 .
  • TMPRSS2 transmembrane-serine protease 2
  • HKU1 was first identified in Hong Kong in 2005 11 , in an elderly patient with severe pneumonia. HKU1 usually causes common cold and benign respiratory symptoms, but complications include severe lower respiratory infections, particularly in young children, the elderly and immunocompromised individuals 12 . It is estimated that 70% of children seroconvert before the age of 6 years 13 .
  • HKU1A and B spikes display an overall amino-acid (aa) identity of 85% (Fig.1a), while B and C spikes share 99% identity.
  • conserveed regions in the HKU1A and B spikes include a putative RBD with a Receptor Binding Motif (RBM) 16,17 , S1/S2 and S2/S2’ cleavage sites 18 (Fig.1a).
  • Both HKU1 and OC43 spikes bind to 9-O-acetylated ⁇ 2,8 linked disialoside on target cells 5,19 . Their protein receptors have not been identified. After receptor binding, the coronavirus spike can be cleaved at the S2’ site by membrane bound proteases, such as TMPRSS2, or by endosomal cathepsins, releasing the fusion peptide present in the S2 domain and activating the spike for membrane fusion 2 .
  • TMPRSS2 belongs to the type II transmembrane serine protease (TTSP) family that comprises 19 cell-surface enzymes 20,21 .
  • TTSP are involved in many processes, including epithelial homeostasis, extracellular matrix degradation, hormone and growth factor activation, initiation of proteolytic cascades through cleavage of membrane cellular proteins 20 .
  • TTSP are synthesized as single-chain proenzymes that require proteolytic activation 20 .
  • TMPRSS2 undergoes autocleavage, its two subunits remain attached by a disulfide bond 22 .
  • TMPRSS2 is expressed in nasal, bronchial and small airways tissues, and more particularly in ciliated cells that are the target of HKU1 23,24 .
  • TMPRSS2 is involved in the activation of different viruses.
  • TMPRSS2 is overexpressed in prostate cancer and that its expression level correlated with cancer progression.
  • TMPRSS2 has been shown to activate PAR-2 and hepatocyte growth factor (HGF)/c-Met signalling pathways and to downregulate E-cadherin expression in prostate cancer cells.
  • HGF hepatocyte growth factor
  • TMPRSS2 has also been described to activate TTSP matriptase and degradation of extracellular matrix (ECM) laminin ⁇ 1 and nidogen-1 in vitro and in a xenograft mouse model of prostate cancer, promoting prostate cancer tumor growth and metastasis.
  • ECM extracellular matrix
  • the invention encompasses a TMPRSS2 antagonist and methods of making and using the antagonist.
  • the antagonist is an antibody.
  • the antibody comprises a VHH (also called nanobody).
  • the invention encompasses an antibody directed against the biologically active ectodomain of TMPRSS2.
  • the antibody comprises a VHH.
  • the VHH is A01 (SEQ ID NO: 19), A07 (SEQ ID NO: 20), C11 (SEQ ID NO: 21), D01 (SEQ ID NO: 22), or F05 (SEQ ID NO: 23).
  • binding of the antibody to TMPRSS2 reduces the catalytic activity of TMPRSS2.
  • the antibody is directed against amino acids 275-473 of TMPRSS2.
  • the antibody binds to a polypeptide comprising the amino acid sequence SEQ ID NO: 29.
  • the antibody binds to an epitope that comprises one or more amino acid residues selected from the group consisting of V275, Q276, V278, H279, V280, C281, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, E389, K390, T393, V415, Y416, D417, N418, L419, D435, S436, C437, Q438, G439, A441, T459, S460, W461, G462, S463, G464, C465, R470, G472 and V473 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24.
  • the VHH comprises the following CDR domains: CDR1: SG(S/F)(P/T)L(E/D)(H/Y)YDI(I/Y/G) (SEQ ID NO: 1); CDR2: SSIT(T/A)SGGRTNYADSVKG (SEQ ID NO: 2); and CDR3: A(G/A)(K/R)(V/I)GGRRNW(I/V)APLNG(Y/F)ENA(Y/L) (SEQ ID NO: 3).
  • the VHH comprises the following CDR domains: CDR1 selected from: SGSPLEHYDII (SEQ ID NO: 5), SGFTLDYYDIY (SEQ ID NO: 6), and SGSTLEHYDIG (SEQ ID NO: 7); CDR2 selected from: SSITTSGGHTNYADSVKG (SEQ ID NO: 10), SSITTSGGRTNYADSVKG (SEQ ID NO: 11), and SSITASGGRTNYADSVKG (SEQ ID NO: 12); and CDR3 selected from: AGRVGGRRNWIVPLDGYDNAY (SEQ ID NO: 15), AAKVGGRRNWIAPLNGYENAL (SEQ ID NO: 16), and AGKIGGRRNWVAPLDGFENAY (SEQ ID NO: 17).
  • CDR1 selected from: SGSPLEHYDII (SEQ ID NO: 5), SGFTLDYYDIY (SEQ ID NO: 6), and SGSTLEHYDIG (SEQ ID NO: 7
  • CDR2 selected from
  • the antibody binds to an epitope that comprises one or more amino acid residues selected from the group consisting of R150, Y152, Q159, Y161, K166, S167, W168, H169, S204, G205, S206, T207, P367, P369, G370, M371, M372, L373, Q374, P375, I404, I405, E406, T407, Q408, N411, I420, T421, P422, M424, I425, I456, N476, M478, V479, T481 and D482 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24.
  • the invention encompasses variants of any of the antibodies of the invention, wherein the antibody retains the same binding as the unchanged antibody. In various embodiments, the variant antibody has 1, 2, 3, 4, or 5 changes. [0019] In various embodiments, the antibody is a monomer. In various embodiments, the antibody is a multimer. In various embodiments, the antibody is a fusion protein, particularly a fusion protein with Fc. [0020] In various embodiments, the antibody is in a combination with another antibody. [0021] The invention encompasses methods of inhibiting binding to TMPRSS2 comprising contacting an TMPRSS2 antagonist of the invention, such as an antibody directed against the biologically active ectodomain of TMPRSS2, with a cell expressing TMPRSS2.
  • the invention encompasses methods of treating a viral infection comprising administering an TMPRSS2 antagonist of the invention, such as an antibody directed against the biologically active ectodomain of TMPRSS2, to an infected patient.
  • an TMPRSS2 antagonist of the invention such as an antibody directed against the biologically active ectodomain of TMPRSS2, to an infected patient.
  • the invention encompasses a method of detecting TMPRSS2 comprising contacting an antibody directed against the biologically active ectodomain of TMPRSS2 with a biological sample containing TMPRSS2 and detecting the resultant immunological complexes.
  • the invention encompasses the use of an antibody of the invention for detecting TMPRSS2.
  • the invention encompasses a method of diagnosing prostate cancer comprising contacting the antibody of the invention with a biological sample containing TMPRSS2 and detecting the resultant immunological complexes.
  • the invention encompasses the use of an antibody of the invention for diagnosing prostate cancer.
  • BRIEF DESCRIPTION OF THE DRAWINGS [0025] Figures 1-4: TMPRSS2 expression enables HKU1 spike mediated fusion.
  • Figure 1.293T cells expressing either GFP 1-10 or GFP11 were transfected with HKU1 spike and TMPRSS2 or pEmpty (ctrl), fusion was quantified by measuring the GFP area 20 hours post- transfection. Data are mean ⁇ SD of 3–6 independent experiments.
  • TMPRSS2 was transfected either in donor cells expressing GFP1-10 and HKU1A spike or in acceptor cells expressing GFP-11 and fusion was quantified by measuring the GFP area 20 hours post-transfection. Scale bar: 400 ⁇ m. Data are mean ⁇ SD of 3–6 independent experiments. One Way ANOVA with Tukey’s multiple comparisons * p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • Figure 3. TMPRSS2 was knocked-down in Caco2 acceptor cells expressing GFP11. Caco2 were mixed with 293T donor cells expressing GFP1-10 and HKU1A spike. Left panel: Relative expression of TMPRSS2.
  • TMPRSS2 acts as a receptor for HKU1 spike, enabling endosomal entry but not surface entry.
  • Figure 5. TMPRSS2 is composed of a Transmembrane Domain (TMD), a class A LDL receptor (LDLR), a Scavenger Receptor Cysteine-2 rich domain (SRCRD) and a serine peptidase.
  • TMD Transmembrane Domain
  • LDLR class A LDL receptor
  • SRCRD Scavenger Receptor Cysteine-2 rich domain
  • SA Scavenger Receptor Cysteine-2 rich domain
  • TMPRSS2 precursor undergoes autocleavage at the R255-L256 peptide bond, resulting in an active protease. The R255Q mutant does not autocleave.
  • FIG. 7 Western blot of 293T cells transfected with HKU1A spike and/or the indicated TMPRSS2 mutants. Membranes were probed for S1, S2, TMPRSS2 and actin. Representative blot of 3. Molecular weights: KDa.
  • Figure 8.293T cells expressing either GFP 1-10 or GFP11 were transfected with HKU1 spike and mutant TMPRSS2, fusion was quantified by measuring the GFP area 20 hours post-transfection and normalized to the fusion induced by WT TMPRSS2.
  • Data are mean ⁇ SD of 3 independent experiments.
  • Statistical analysis One Way ANOVA with Dunett’s multiple comparisons compared to WT TMPRSS2 using the non-normalized data.
  • Figure 9.293T cells expressing mutant TMPRSS2 were infected by Luc-encoding HKU1 spike pseudotyped lentiviral particles. Luminescence was read 48 hours post infection.
  • FIG. 11-17 HKU1 spike binds to TMPRSS2 through its RBD, W515 and R517 are essential for this interaction.
  • Figure 11.293T cells were transfected with TMPRSS2 mutants and incubated overnight in the presence or absence of 10 ⁇ M of Camostat. a. TMPRSS2 levels were assessed by flow cytometry on permeabilized cells. One representative experiment of 3 is shown.
  • FIG. 13 Binding of indicated soluble biotinylated trimeric spike on immobilized WT TMPRSS2 was measured by ELISA. Mean of 2 independent experiments [0041] Figure 14. Binding of S441A TMPRSS2 to RBD coated receptors was quantified by BLI. One representative experiment of 3 is shown. [0042] Figure 15.293T cells expressing either GFP 1-10 or GFP11 were transfected with TMPRSS2 and HKU1 WT spike and W515A and R517A mutant spike, fusion was quantified by measuring the GFP area 24 hours post-transfection.
  • FIG.293T cells expressing TMPRSS2 were infected by Luc-encoding WT, W515A and R517A mutant HKU1 spike pseudotyped lentiviral particles. Luminescence was read 48 hours post infection. Dotted line indicates the background. Data are mean ⁇ SD of 3 independent experiments.
  • Figure 18-21 TMPRSS2 targeting VHH inhibit HKU1/TMPRSS2 interaction.
  • Figure 18. Soluble TMPRSS2 was incubated for 15 minutes with VHHs, before adding the fluorogenic substrate Boq-QAR-AMC.
  • Figures 22-23 [0051] Figure 22.293 T cells were transfected with plasmids encoding for HKU1A or HKU1B spikes and stained 24 h later with mAb10, a pan-anti-coronavirus spike antibody. [0052] Data are representative of 3 independent experiments. Figure 23. 293T cells expressing either GFP 1-10 or GFP11 were transfected with HKU1 spike and the indicated constructs, fusion was quantified by measuring the GFP area 20 hours post-transfection.
  • Figures 24-26 [0054] Figure 24.293T cells were transfected with TMPRSS2 WT and mutants, the catalytic activity was assessed 24 hours post-transfection using BoC-QAR-AMC fluorogenic substrate. [0055] Figure 25.293T cells expressing ACE2 and mutant TMPRSS2 were infected by Luc- encoding SARS-CoV-2 spike pseudotyped lentiviral particles. Luminescence was read 48 hours post infection. Data are mean ⁇ SD of 4 independent experiments. Ratio paired t-test compared to the control condition **p ⁇ 0.01.
  • Figure 26.293T cells expressing WT or S441A TMPRSS2 were incubated for 2 hours with indicated drug, before infection by Luc-encoding HKU1B spike pseudotyped lentiviral particles. Luminescence was read 48 hours post infection. Left panel: effect of Camostat mesylate on HKU1B entry. Right panel: effect of SB412512 on HKU1B entry. Data are mean ⁇ SD of 4-6 independent experiments. [0057] Figures 27-30.293T cells were transfected with TMPRSS2 mutants and incubated overnight in the presence or absence of 10 ⁇ M of Camostat. Binding of indicated soluble biotinylated trimeric spike was measured by flow cytometry.
  • FIG. 27 % of binding soluble biotinylated trimeric HKU1A spike to TMPRSS2.
  • Figure 28 % of binding soluble biotinylated trimeric HKU1B spike to TMPRSS2.
  • Figure 29 % of binding soluble biotinylated trimeric SARS-CoV-2 spike to TMPRSS2.
  • Figure 30 % of binding soluble biotinylated trimeric SARS-CoV-2 spike to TMPRSS2.
  • FIG. 31 Binding of soluble biotinylated spike on immobilized ACE2 was measured by ELISA.
  • Figure 32 Binding of ACE2 to RBD coated receptors was quantified by BLI.
  • Figure 33 Binding of 600nM TMPRSS2 to RBD coated receptors or empty sensors was quantified by BLI.
  • Figure 34 Binding of TMPRSS2 to RBD coated receptors was quantified by BLI, at different concentrations. Black: fitting of the experimental curves. Left panel: HKU1A. Right panel: HKU1B.
  • Figure 35.293T were transfected with HKU1A or B spike, expression was measured by flow cytometry by using mab10.
  • Figure 36 Binding of TMPRSS2 to HKU1B R517A RBD coated receptors was quantified by BLI, at different concentrations.
  • Figure 37 Binding of VHH on TMPRSS2 was measured by BLI.
  • Figure 38 Soluble TMPRSS2 was incubated for 15 minutes with 1uM Camostat, before adding the fluorogenic substrate Boq-QAR-AMC. The initial rate of the reaction is plotted.
  • Figure 39.293T cells were transfected with TMPRSS2 S441A, binding to VHH (0.5uM) was assessed by flow cytometry.
  • Figure 40.293T cells either GFP 1-10 or GFP11 were transfected with TMPRSS2 and HKU1 spike in the presence of indicated amount of VHH, fusion was quantified by measuring the GFP area 20 hours post-transfection. Data were normalized to the non-treated condition for each spike.
  • Figure 41.293T were transfected with S441A TMPRSS2.24 hours post-transfection, they were incubated with indicated amount of VHH for 2 hours and infected by Luc-encoding HKU1 spike pseudotyped viruses. Luminescence was read 48 hours post infection.
  • FIG. 42 Crystal structure of A07 in complex with S441A TMPRSS2.
  • Figure 43.293T cells WT or stably expressing TMPRSS2, R255Q TMPRSS2 or S441A TMPRSS2 were stained with a commercial antibody or VHH-A01-Fc and secondary Goat anti-Human alexa-647.
  • Figure 44 U2OS cells and A549 cells WT or stably expressing TMPRSS2 or S441A TMPRSS2 were stained with VHH-A01-Fc and secondary Goat anti-Human alexa-647.
  • Figure 45 Figure 45.
  • Caco-2 WT, TMPRSS2 knock-out, TMPRSS2 and S441A TMPRSS2 cells were stained for TMPRSS2 using primary VHH-A01-Fc and secondary antibody Goat anti-Human Alexa- 647. Cells were analyzed by flow cytometry.
  • Figure 46 Caco-2 cells were pre-treated with indicated concentration of monomeric VHHs for 30 min and then infected, in presence of the VHHs, with SARS-CoV-2 D614G strain at the same MOI. Cells were fixed 24h post infection and stained for SARS-CoV-2 N and analyzed by flow cytometry.
  • Figure 47 Effect of the anti-TMPRSS2 VHH (A07) on HKU1 infection.
  • FIG. 54 Binding of the VHH-A07-Fc and VHH-A01-Fc to TMPRSS2 from different animals (Homo sapiens, Mesocricretus auratus, Macaca, Mustela furo, Mus musculus, Bos taurus, Gorilla gorilla) by flow cytometry.
  • Figure 54 Binding of the VHH-A07-Fc and VHH-A01-Fc to TMPRSS2 naturally expressed at surface of CaCo2 cells (intestinal cells).
  • Figure 55 Binding of the VHH-A07-Fc and VHH-A01-Fc to TMPRSS2 naturally expressed at surface of LNCap cells (human tumoral prostatic cells).
  • TMPRSS2 was identified as a high-affinity receptor for HKU1.
  • TMPRSS2 is a receptor for hCoV-HKU1.
  • TMPRSS2 triggers cell- cell fusion and viral entry and binds with high affinity to both HKU1A and B spikes.
  • the enzymatic activity of TMPRSS2 is required for HKU1-dependent cell-cell fusion but not for entry of HKU1 viral pseudotypes.
  • Viral entry is enhanced by Camostat in cells expressing the wild-type protease and is more efficient with catalytically inactive TMPRSS2 mutants.
  • a cathepsin inhibitor decreased infection mediated by the catalytically inactive TMPRSS2, but not by the wild-type protease.
  • TMPRSS2 directly binds to HKU1 but not to SARS-CoV-2 and OC43 spikes.
  • a conserved groove in a region of HKU1A and B spikes composed of amino acid 505, 515, 517-521 and 528 has been proposed to be involved in binding to an unknown receptor 16,17 . It is shown that the 515 and 517 residues of the spike are essential for binding to TMPRSS2, viral fusion and entry, thus adding evidence that this region may be part of the RBM.
  • the 508-520 aa region of HKU1 is deleted in OC43 (ProteinBlast), which may help explain why OC43 does not use TMPRSS2 as a receptor.
  • Anti-TMPRSS2 VHH were isolated that inhibited binding to HKU1 spikes and prevented cell- cell fusion and viral entry. These VHH antibodies confirmed the role of TMPRSS2 as a HKU1 receptor and provide useful tools to interfere with TMPRSS2 function. [0092] As TMPRSS2 also plays a role in the development of certain cancers 33 , these VHH antibodies may target tumor cells or inhibit infection of viruses relying on this enzyme, as alternative to small molecule inhibitors 34 .
  • TMPRSS2 Human coronavirus receptors that have been identified so far and allow productive viral entry are cell surface proteases. TMPRSS2 and other TTSP are known to prime human coronaviruses for fusion by cleaving their spikes, generally after viral binding to target cells. Of note, Omicron strains rely less on TMPRSS2 than previous SARS-CoV-2 lineages, reflecting a constant adaptation of coronaviruses to their hosts 35 .
  • TMPRSS2 also acts as a direct receptor for HKU1.
  • ACE2 binding is an ancestral and evolvable trait of sarbecoviruses 36 .
  • TMPRSS2 binding may also represent another parameter of coronavirus evolution. Whether coronaviruses have lost affinity or sensitivity to TMPRSS2 during evolution, or whether HKU1 has gained affinity for TMPRSS2 remains to be determined.
  • TMPRSS2 ANTAGONISTS [0095] The invention encompasses antagonists of TMPRSS2.
  • TMPRSS2 has the sequence MALNSGSPPAIGPYYENHGYQPENPYPAQPTVVPTVYEVHPAQYYPSPVPQYAPRVLTQASNPVVCTQ PKSPSGTVCTSKTKKALCITLTLGTFLVGAALAAGLLWKFMGSKCSNSGIECDSSGTCINPSNWCDGV SHCPGGEDENRCVRLYGPNFILQVYSSQRKSWHPVCQDDWNENYGRAACRDMGYKNNFYSSQGIVDDS GSTSFMKLNTSAGNVDIYKKLYHSDACSSKAVVSLRCIACGVNLNSSRQSRIVGGESALPGAWPWQVS LHVQNVHVCGGSIITPEWIVTAAHCVE
  • the antagonist is an antibody, such as a VHH.
  • the invention further encompasses the use of these antagonists to inhibit binding to TMPRSS2, such as the binding of HKU1 to host cells expressing TMPRSS2.
  • SINGLE DOMAIN VHH ANTIBODIES [0097]
  • the invention encompasses antibodies, particularly single domain VHH antibodies that bind to TMPRSS2.
  • a VHH antibody is an antibody that comprises the variable domain of an antibody that contains two heavy chains and lacks the two light chains usually found in antibodies.
  • the single domain VHH antibodies of the invention specifically bind to TMPRSS2.
  • the VHH has a Kd between 0.1-100 nM or 0.8-33nM.
  • the invention encompasses the following single domain VHH antibodies that bind to TMPRSS2: A01, A07, C11, D01, and F05. [0100] The invention encompasses a VHH comprising CDR1, CDR2, and CDR3 from any of A01, A07, C11, D01, and F05.
  • the invention encompasses a VHH comprising a CDR1 selected from: SGFSLDYYAIG (SEQ ID NO: 4), SGSPLEHYDII (SEQ ID NO: 5), SGFTLDYYDIY (SEQ ID NO: 6), SGSTLEHYDIG (SEQ ID NO: 7), and SGFTLDYYAIG (SEQ ID NO: 8) or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR1, preferably those that are not conserved between these sequences.
  • the invention encompasses a VHH comprising a CDR2 selected from: SCIGSSGDKTNYADSVKG (SEQ ID NO: 9), SSITTSGGHTNYADSVKG (SEQ ID NO: 10), SSITTSGGRTNYADSVKG (SEQ ID NO: 11), SSITASGGRTNYADSVKG (SEQ ID NO: 12), and SCISSSGDSIKYVDSVKG (SEQ ID NO: 13) or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR2, preferably those that are not conserved between these sequences.
  • a CDR2 selected from: SCIGSSGDKTNYADSVKG (SEQ ID NO: 9), SSITTSGGHTNYADSVKG (SEQ ID NO: 10), SSITTSGGRTNYADSVKG (SEQ ID NO: 11), SSITASGGRTNYADSVKG (SEQ ID NO: 12), and SCISSSGDSIKYVDSVKG (SEQ ID NO:
  • the invention encompasses a VHH comprising a CDR3 selected from: AAESALYSDCTEEQNPMLYDY (SEQ ID NO: 14), AGRVGGRRNWIVPLDGYDNAY (SEQ ID NO: 15), AAKVGGRRNWIAPLNGYENAL (SEQ ID NO: 16), AGKIGGRRNWVAPLDGFENAY (SEQ ID NO: 17), and AADALGSGCLTGNYDY (SEQ ID NO: 18) or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR3, preferably those that are not conserved between these sequences.
  • a CDR3 selected from: AAESALYSDCTEEQNPMLYDY (SEQ ID NO: 14), AGRVGGRRNWIVPLDGYDNAY (SEQ ID NO: 15), AAKVGGRRNWIAPLNGYENAL (SEQ ID NO: 16), AGKIGGRRNWVAPLDGFENAY (SEQ ID NO: 17), and AADA
  • the invention encompasses a VHH comprising a CDR1 selected from: SGFSLDYYAIG (SEQ ID NO: 4), SGSPLEHYDII (SEQ ID NO: 5), SGFTLDYYDIY (SEQ ID NO: 6), SGSTLEHYDIG (SEQ ID NO: 7), and SGFTLDYYAIG (SEQ ID NO: 8) or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR1 , preferably those that are not conserved between these sequences; a CDR2 selected from: SCIGSSGDKTNYADSVKG (SEQ ID NO: 9), SSITTSGGHTNYADSVKG (SEQ ID NO: 10), SSITTSGGRTNYADSVKG (SEQ ID NO: 11), SSITASGGRTNYADSVKG, (SEQ ID NO: 12) and SCISSSGDSIKYVDSVKG (SEQ ID NO: 13) or an amino acid sequence having up to two amino acid sequence
  • the invention encompasses a VHH comprising a CDR1 selected from: SGSPLEHYDII (SEQ ID NO: 5), SGFTLDYYDIY (SEQ ID NO: 6), and SGSTLEHYDIG (SEQ ID NO: 7), or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR1, preferably those that are not conserved between these sequences; a CDR2 selected from: SSITTSGGHTNYADSVKG (SEQ ID NO: 10), SSITTSGGRTNYADSVKG (SEQ ID NO: 11), and SSITASGGRTNYADSVKG (SEQ ID NO: 12), or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR2, preferably those that are not conserved between these sequences; and a CDR3 selected from: AGRVGGRRNWIVPLDGYDNAY (SEQ ID NO: 15), AAKVG
  • the invention encompasses a VHH comprising a CDR1 selected from: SGFSLDYYAIG (SEQ ID NO: 4) and SGFTLDYYAIG (SEQ ID NO: 8), or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR1, preferably those that are not conserved between these sequences; a CDR2 selected from: SCIGSSGDKTNYADSVKG (SEQ ID NO: 9) and SCISSSGDSIKYVDSVKG (SEQ ID NO: 13), or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR2, preferably those that are not conserved between these sequences; and a CDR3 selected from: AAESALYSDCTEEQNPMLYDY (SEQ ID NO: 14) and AADALGSGCLTGNYDY (SEQ ID NO: 18), or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions
  • the invention encompasses a VHH comprising a CDR1 selected from: SGSPLEHYDII (SEQ ID NO: 5), SGFTLDYYDIY (SEQ ID NO: 6), and SGSTLEHYDIG (SEQ ID NO: 7); a CDR2 selected from: SSITTSGGHTNYADSVKG (SEQ ID NO: 10), SSITTSGGRTNYADSVKG (SEQ ID NO: 11), and SSITASGGRTNYADSVKG (SEQ ID NO: 12); and a CDR3 selected from: AGRVGGRRNWIVPLDGYDNAY (SEQ ID NO: 15), AAKVGGRRNWIAPLNGYENAL (SEQ ID NO: 16), and AGKIGGRRNWVAPLDGFENAY (SEQ ID NO: 17).
  • AGRVGGRRNWIVPLDGYDNAY SEQ ID NO: 15
  • AAKVGGRRNWIAPLNGYENAL SEQ ID NO: 16
  • AGKIGGRRNWVAPLDGFENAY S
  • the single domain VHH antibody can bind to the biologically active ectodomain of TMPRSS2. In some embodiments, the single domain VHH antibody can bind to TMPRSS2 and reduce its catalytic activity. In preferred embodiments, the single domain VHH antibody is A07, C11, or D01. [0109] In some embodiments the single domain VHH antibody comprises the following CDR domains: CDR1: SGFSLDYYAIG (SEQ ID NO: 4); CDR2: SCIGSSGDKTNYADSVKG (SEQ ID NO: 9); and CDR3: AAESALYSDCTEEQNPMLYDY (SEQ ID NO: 14).
  • the single domain VHH antibody comprises the following CDR domains: CDR1: SGSPLEHYDII (SEQ ID NO: 5); CDR2: SSITTSGGHTNYADSVKG (SEQ ID NO: 10); and CDR3: AGRVGGRRNWIVPLDGYDNAY (SEQ ID NO: 15). [0111] In some embodiments the single domain VHH antibody comprises the following CDR domains: CDR1: SGFTLDYYDIY (SEQ ID NO: 6); CDR2: SSITTSGGRTNYADSVKG (SEQ ID NO: 11); and CDR3: AAKVGGRRNWIAPLNGYENAL (SEQ ID NO: 16).
  • the single domain VHH antibody comprises the following CDR domains: CDR1: SGSTLEHYDIG (SEQ ID NO: 7); CDR2: SSITASGGRTNYADSVKG (SEQ ID NO: 12); and CDR3: AGKIGGRRNWVAPLDGFENAY (SEQ ID NO: 17).
  • the single domain VHH antibody comprises the following CDR domains: CDR1: SGFTLDYYAIG (SEQ ID NO: 8); CDR2: SCISSSGDSIKYVDSVKG (SEQ ID NO: 13); and CDR3: AADALGSGCLTGNYDY (SEQ ID NO: 18).
  • the single domain VHH antibody comprises the following CDR domains: CDR1: SG(S/F)(P/T)L(E/D)(H/Y)YDI(I/Y/G) (SEQ ID NO : 1); CDR2: SSIT(T/A)SGGRTNYADSVKG (SEQ ID NO: 2); and CDR3: A(G/A)(K/R)(V/I)GGRRNW(I/V)APLNG(Y/F)ENA(Y/L) (SEQ ID NO: 3).
  • the VHH antibody contains 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 A01, A07, C11, D01, and F05.
  • the VHH antibody comprises a CDR1 selected from: SGFSLDYYAIG (SEQ ID NO: 4), SGSPLEHYDII (SEQ ID NO: 5), SGFTLDYYDIY (SEQ ID NO: 6), SGSTLEHYDIG (SEQ ID NO: 7), and SGFTLDYYAIG (SEQ ID NO: 8); a CDR2 selected from: SCIGSSGDKTNYADSVKG (SEQ ID NO: 9), SSITTSGGHTNYADSVKG (SEQ ID NO: 10), SSITTSGGRTNYADSVKG (SEQ ID NO: 11), SSITASGGRTNYADSVKG (SEQ ID NO: 12), and SCISSSGDSIKYVDSVKG (SEQ ID NO: 13); and a CDR3 selected from: AAESALYSDCTEEQNPMLYDY (SEQ ID NO: 14), AGRVGGRRNWIVPLDGYDNAY (SEQ ID NO: 15), AAKVGGRRNWIAPLNGY
  • the VHH antibody contains an amino acid sequence that has at least 100, 105, 110, 115, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, or 130, amino acids identical to the amino acid sequence of A01, A07, C11, D01, or F05.
  • the VHH antibody comprises a CDR1 selected from: SGFSLDYYAIG (SEQ ID NO: 4), SGSPLEHYDII (SEQ ID NO: 5), SGFTLDYYDIY (SEQ ID NO: 6), SGSTLEHYDIG (SEQ ID NO: 7), and SGFTLDYYAIG (SEQ ID NO: 8); a CDR2 selected from: SCIGSSGDKTNYADSVKG (SEQ ID NO: 9), SSITTSGGHTNYADSVKG (SEQ ID NO: 10), SSITTSGGRTNYADSVKG (SEQ ID NO: 11), SSITASGGRTNYADSVKG (SEQ ID NO: 12), and SCISSSGDSIKYVDSVKG (SEQ ID NO: 13); and a CDR3 selected from: AAESALYSDCTEEQNPMLYDY (SEQ ID NO: 14), AGRVGGRRNWIVPLDGYDNAY (SEQ ID NO: 15), AAKVGGRRNWIAPLNGY
  • the antibody is directed against residues 275-473 of TMPRSS2.
  • the antibody directed against residues 275-473 of TMPRSS2 is a VHH antibody (e.g., A07).
  • the antibody binds to an epitope that comprises one or more amino acid residues selected from the group consisting of V275, Q276, V278, H279, V280, C281, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, E389, K390, T393, V415, Y416, D417, N418, L419, D435, S436, C437, Q438, G439, A441, T459, S460, W461, G462, S463, G464, C465, R470, G472 and V473 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24.
  • the antibody binds to an epitope that comprises at least 10, at least 20, at least 30, at least 40 of the amino acid residues selected from the group consisting of V275, Q276, V278, H279, V280, C281, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, E389, K390, T393, V415, Y416, D417, N418, L419, D435, S436, C437, Q438, G439, A441, T459, S460, W461, G462, S463, G464, C465, R470, G472 and V473 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24.
  • the antibody binds to an epitope that comprises the amino acid residues V275, Q276, V278, H279, V280, C281, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, E389, K390, T393, V415, Y416, D417, N418, L419, D435, S436, C437, Q438, G439, A441, T459, S460, W461, G462, S463, G464, C465, R470, G472 and V473 and optionally of one or more of G385, A386, D440, K467, and Y474 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24.
  • the antibody binds to an epitope that consists of the amino acid residues V275, Q276, V278, H279, V280, C281, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, E389, K390, T393, V415, Y416, D417, N418, L419, D435, S436, C437, Q438, G439, A441, T459, S460, W461, G462, S463, G464, C465, R470, G472 and V473 and optionally of one or more of G385, A386, D440, K467, and Y474 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24.
  • the antibody binds to an epitope that comprises at least 10, at least 20, at least 30 of the amino acid residues selected from the group consisting for V275, Q276, V278, H279, V280, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, E389, T393, V415, Y416, D417, N418, L419, S436, C437, G439, A441, T459, S460, W461, G462, S463 and R470, and optionally of one or more of C281, G385, A386, K390, D435, Q438, D440, G464, C465, K467, G472, V473 and Y474 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24.
  • the antibody binds to an epitope that comprises the amino acid residues V275, Q276, V278, H279, V280, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, E389, T393, V415, Y416, D417, N418, L419, S436, C437, G439, A441, T459, S460, W461, G462, S463 and R470, and optionally of one or more of C281, G385, A386, K390, D435, Q438, D440, G464, C465, K467, G472, V473 and Y474 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24.
  • the antibody binds to an epitope that consists of the amino acid residues V275, Q276, V278, H279, V280, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, E389, K390, T393, V415, Y416, D417, N418, L419, D435, S436, C437, Q438, G439, A441, T459, S460, W461, G462, S463, G464, C465, R470, G472 and V473 and optionally of one or more of C281, G385, A386, D440, K467, and Y474 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24.
  • the antibody binds to an epitope that comprises one or more amino acid residues selected from the group consisting of R150, Y152, Q159, Y161, K166, S167, W168, H169, S204, G205, S206, T207, P367, P369, G370, M371, M372, L373, Q374, P375, I404, I405, E406, T407, Q408, N411, I420, T421, P422, M424, I425, I456, N476, M478, V479, T481 and D482 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24.
  • the antibody binds to an epitope that comprises at least 10, at least 20, at least 30 amino acid residues selected from the group consisting of R150, Y152, Q159, Y161, K166, S167, W168, H169, S204, G205, S206, T207, P367, P369, G370, M371, M372, L373, Q374, P375, I404, I405, E406, T407, Q408, N411, I420, T421, P422, M424, I425, I456, N476, M478, V479, T481 and D482 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24.
  • the antibody binds to an epitope that comprises the amino acid residues R150, Y152, Q159, Y161, K166, S167, W168, H169, S204, G205, S206, T207, P367, P369, G370, M371, M372, L373, Q374, P375, I404, I405, E406, T407, Q408, N411, I420, T421, P422, M424, I425, I456, N476, M478, V479, T481 and D482 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24.
  • the antibody binds to an epitope that consists of amino acid residues R150, Y152, Q159, Y161, K166, S167, W168, H169, S204, G205, S206, T207, P367, P369, G370, M371, M372, L373, Q374, P375, I404, I405, E406, T407, Q408, N411, I420, T421, P422, M424, I425, I456, N476, M478, V479, T481 and D482 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24.
  • the epitope may be a continuous or discontinuous epitope.
  • fusion protein comprising a single domain VHH antibody of the invention and a second polypeptide or protein that is not a VHH is provided.
  • a "fusion protein” refers to a polypeptide having two 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 two portions and the linker will be in reading frame with each other. Preferably, the two portions of the polypeptide are obtained from heterologous or different polypeptides.
  • one of the portions is a single domain VHH antibody of the invention.
  • the single domain VHH antibody may be directly fused or linked via a linker moiety to the other elements of the fusion protein.
  • 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: 37), (Gly)5 (SEQ ID NO: 38), (Gly)4Ser (SEQ ID NO: 39), (Gly)4(Ser)(Gly)4 (SEQ ID NO: 40), or combinations thereof or a multimer thereof (for example a dimer, a trimer, or a tetramer, or greater).
  • a suitable linker may be (GGGGS)3 (SEQ ID NO: 41).
  • Alternative linkers include (Ala) 3 (His) 6 (SEQ ID NO: 42) or multimers thereof.
  • the second polypeptide or protein is selected from a Fab, Fc, F(ab’)2 (including chemically linked F(ab’)2 chains), Fab’, scFv (including multimer forms thereof, i.e. di-scFv, or tri-scFv), sdAb, or BiTE (bi-specific T-cell engager).
  • the second polypeptide is a Fc fragment of a mammalian immunoglobulin.
  • the Fc fragment of a mammalian immunoglobulin has the sequence EPKTPKPQPAAARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSPGK (SEQ ID NO: 25) [0140]
  • the fusion protein with the Fc has a Kd between 0.01-100 nM.
  • the fusion protein is the VHH A01-Fc having the amino acid sequence SEQ ID NO: 26.
  • the fusion protein is the VHH A07-Fc having the amino acid sequence SEQ ID NO: 27.
  • MULTIMERIC VHH ANTIBODIES Multimeric VHH antibodies are also provided.
  • the multimeric VHH antibody comprises at least one VHH of the invention, preferably at least two VHHs of the invention. Each VHH present in a multimeric VHH antibody may be in the form of a fusion protein or may not be in the form of a fusion protein.
  • the multimeric VHH antibody is a dimer.
  • the dimer comprises two copies of the same VHH antibody (homodimer). In some embodiments the dimer comprises one copy of each of two different VHH antibodies (heterodimer). [0145] In some embodiments the multimeric VHH antibody is a trimer. In some embodiments the trimer comprises two copies of the same VHH antibody and a third copy of a different VHH antibody (heterotrimer type 1). In some embodiments the trimer comprises one copy of each of three different VHH antibodies (heterotrimer type 2). In some embodiments the trimer comprises three copies of the same VHH antibody (homotrimer). [0146] Each VHH present in the multimeric VHH antibody may be the same as at least one other VHH in the multimeric VHH antibody.
  • each VHH present in the multimeric VHH antibody may be different than all other VHH antibodies present in the multimeric VHH antibody.
  • the multimeric VHH antibodies are Fc fusion proteins. In such embodiment the Fc portion may be responsible for linking the VHHs together into the multimeric form.
  • the VHH antibodies may be linked together via one or more type of linker, such as a Gly-Ser linker.
  • linker such as a Gly-Ser linker linker.
  • the dimeric VHH antibody is a homodimer. In other embodiments, the dimeric VHH antibody is a heterodimer. Mixtures comprising VHH antibody homodimers, mixtures comprising VHH antibody heterodimers, and mixtures comprising VHH antibody homodimers and heterodimers are also provided.
  • the invention encompasses a combination of a VHH antibody according to the invention with at least another antibody. In one embodiment, the other antibody is an antibody, especially a VHH antibody, directed against ACE2. In various embodiment, the combination is in a multimeric form.
  • the invention encompasses methods of making variants of A01, A07, C11, D01, and F05.
  • the DNA sequence encoding A01, A07, C11, D01, or F05 is changed by one or more nucleotides encoding one or more amino acids of the VHH to generate a variant of A01, A07, C11, D01, or F05.
  • a variant of A07 has at least one amino acid different than A07.
  • a variant of A01, A07, C11, D01, and F05 has at least one amino acid different than A01, A07, C11, D01, and F05, respectively.
  • a variant can contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9 ,10, 11, 12, 13, 14, 15, 20, 25, 30, 40, or 50, etc., amino acids different than A01, A07, C11, D01, and/or F05.
  • a variant can contain an amino acid sequence that has at least 100, 105, 110, 115, 120, 121, 122, 123, 124, 125, 126, 127, 128, or 129 amino acids identical to the amino acid sequence of A01, A07, C11, D01, and/or F05.
  • the variant can be generated in a recombinant vector comprising a DNA sequence encoding A01, A07, C11, D01, or F05.
  • the vector is an expression vector.
  • an isolated nucleic acid sequence that encodes the antibody of the invention, especially the single domain VHH antibody or a fusion protein comprising a single domain VHH antibody of the invention is provided.
  • the single domain VHH antibody binds to the catalytic domain of TMPRSS2.
  • the single domain VHH antibody is a dimer of VHH.
  • the VHH is selected from A07, C11 and D01.
  • the VHH is selected from A01 and F05.
  • the VHH binds to the residues 275-473, around TMPRSS2 catalytic site.
  • recombinant vectors comprising the isolated nucleic acid sequence of the invention.
  • 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 VHH antibody, either in vitro or in vivo.
  • the expression vector is pHEN6, pET23, pET22 or pASK-IBA2.
  • the expression vector encodes a protease cleavage site, such as TEV cleave site, inserted between the single domain VHH antibody protein coding sequence and a protein purification Tag, such as polyHis tag or strep tag.
  • a protein purification Tag such as polyHis tag or strep tag.
  • the expression vector encodes a His tag or a strep tag.
  • a TEV cleavage site is positioned to remove the His tag, for example, after purification.
  • 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 sequence enabling conditional expression, such as sequences of the Cre/Lox system or analogue systems.
  • the expression vector is a plasmid, a phage for bacterium introduction, a YAC able to transform yeast, a viral vector, or any expression vector.
  • An expression vector as defined herein is chosen to enable the production of an antibody of the invention, especially a single domain VHH antibody of the invention or a fusion protein comprising a single domain VHH antibody of the invention, either in vitro or in vivo.
  • 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.
  • 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. [0166] 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.
  • methods of producing the antibody of the invention especially single domain VHH antibody or the fusion protein comprising a single domain VHH antibody are provided.
  • the methods comprise culturing the recombinant cell comprising the isolated nucleic acid sequence that encodes the antibody of the invention, especially the single domain VHH antibody or the fusion protein comprising a single domain VHH antibody under conditions sufficient for production of the antibody of the invention, especially the single domain VHH antibody.
  • compositions of an antibody according to the invention especially an isolated single domain VHH antibody according to the invention, a monomeric fusion polypeptide according to the invention, or a multimeric VHH antibody according to the invention, especially a dimeric VHH antibody according to the invention.
  • the pharmaceutical composition may comprise one or a plurality of single domain VHH antibody according to the invention; and/or one or a plurality of monomeric fusion polypeptide according to the invention; and/or one or a plurality of multimeric VHH antibody according to the invention, especially one or a plurality of dimeric VHH antibody according to the invention.
  • the VHH is selected from A07, C11 and D01. [0170] In some embodiments, the VHH is selected from A01 and F05. [0171] In some embodiments, the antibody is VHH A01-Fc. [0172] In some embodiments, the antibody is VHH A07-Fc. [0173] Preferably, the antibody is selected from the group consisting of the VHH A07 having the amino acid sequence SEQ ID NO: 20, the VHH C11 having the amino acid sequence SEQ ID NO: 21, the VHH D01 having the amino acid sequence SEQ ID NO: 22, and the VHH A07-Fc having the amino acid sequence SEQ ID NO: 27.
  • 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.
  • 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.
  • the composition can be an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
  • the pharmaceutical composition preferably comprises a dose of 100 ⁇ g to 1g, for example a dose of at least 100 ⁇ g, 250 ⁇ g, 500 ⁇ g, 1mg, 2mg, 5mg, 10mg, 25mg, 50mg, 100mg, 250mg, or 500mg.
  • the invention encompasses methods for inhibiting coronavirus binding to host cells with an isolated single domain VHH antibody according to the invention, a monomeric fusion polypeptide according to the invention, or a multimeric VHH antibody according to the invention, especially a dimeric VHH antibody according to the invention.
  • the method comprises contacting an isolated single domain VHH antibody according to the invention with host cells, prior to or at the same time that the cells are contacted with a coronavirus.
  • the single domain VHH antibody reduces binding of the coronavirus to the host cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.
  • the coronavirus is SARS-CoV-2 or HKU1.
  • the method utilizes a single domain VHH antibody that binds to the catalytic domain of TMPRSS2.
  • the single domain VHH antibody is a dimer of VHH.
  • the VHH is selected from A07, C11 and D01.
  • the VHH binds to the residues 275-473, around TMPRSS2 catalytic site.
  • the invention encompasses preventing or treating viral infections mediated by TMPRSS2, including SARS-CoV2 infection and especially HKU1 infection, by administering an antibody according to the invention, especially an isolated single domain VHH antibody according to the invention, a monomeric fusion polypeptide of the invention, or a multimeric VHH antibody of the invention, especially a dimeric VHH antibody of the invention.
  • the VHH is selected from A07, C11 and D01.
  • the VHH is selected from A01 and F05.
  • the antibody is VHH A01-Fc.
  • the antibody is VHH A07-Fc.
  • the antibody is selected from the group consisting of the VHH A07 having the amino acid sequence SEQ ID NO: 20, the VHH C11 having the amino acid sequence SEQ ID NO: 21, the VHH D01 having the amino acid sequence SEQ ID NO: 22, and the VHH A07-Fc having the amino acid sequence SEQ ID NO: 27. More preferably, the antibody is the VHH A07-Fc having the amino acid sequence SEQ ID NO: 27.
  • the viral infection is an Influenza A and B virus infection, a metapneumovirus or parainfluenzavirus infection, or a SARS, MERS, or 229E coronavirus infection.
  • a pharmaceutical composition comprising an antibody according to the invention, especially an isolated single domain VHH antibody of the invention, a monomeric fusion polypeptide of the invention, or a multimeric VHH antibody of the invention, especially a dimeric VHH antibody of the invention is administered to a patient having a viral infection.
  • an antibody according to the invention, a monomeric fusion polypeptide according to the invention, or a multimeric VHH antibody according to the invention, especially a dimeric VHH antibody according to the invention is administered to a subject that has been tested positive for a coronavirus, including SARS-CoV-2, and especially HKU1, but has not yet manifested symptoms of infection.
  • administering the pharmaceutical composition to the subject reduces the coronavirus viral load of the subject.
  • a pharmaceutical composition of the invention is administered to a patient that has manifested symptoms of a coronavirus infection, including of SARS-CoV2 infection, and especially of HKU1 infection.
  • 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.
  • 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.
  • the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
  • an injectable composition for example as a sterile aqueous dispersion, preferably isotonic.
  • the daily dosage of the active agent will be from 0.01mg/kg body weight, typically around 1mg/kg, 2mg/kg or up to 4mg/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 composition is administered to a subject that has been tested positive for prostate cancer. In some embodiments, administering the pharmaceutical composition to the subject inhibits the growth of the prostate cancer of the subject.
  • the invention encompasses preventing or treating an HKU1 infection, by administering a TMPRSS2 antagonist. METHODS OF DETECTION [0199]
  • methods for detection of TMPRSS2 in a biological sample are provided. The methods may comprise providing an antibody according to the invention, especially a single domain VHH antibody according to this disclosure; providing a biological sample; contacting the single domain VHH antibody with the biological sample; and visualizing the antigen-antibody complexes formed.
  • the methods comprise an ELISA, LuLisa, lateral flow immunoassay, bead-based immunoassay, or multiplex bead-based immunoassay.
  • the single domain VHH antibody is a dimer of VHH.
  • the VHH is selected from A07, C11 and D01.
  • the VHH is selected from A01 and F05.
  • the antibody is VHH A01-Fc.
  • the antibody is VHH A07-Fc.
  • the antibody is selected from the group consisting of the VHH A01 having the amino acid sequence SEQ ID NO: 19, the VHH F05 having the amino acid sequence SEQ ID NO: 23 and the VHH A01-Fc having the amino acid sequence SEQ ID NO: 26. More preferably, the antibody is the VHH A01-Fc having the amino acid sequence SEQ ID NO: 26.
  • the method for detection of TMPRSS2 in a biological sample comprises providing a first single domain VHH antibody according to this disclosure, attached to a solid support; providing a biological sample from a subject; contacting the solid support with the biological sample under conditions sufficient to allow formation of first antigen-antibody complexes between TMPRSS2 in the biological sample and the VHH antibody attached to the solid support to form first antigen- antibody complexes; contacting the solid support with a second single domain VHH antibody according to any this disclosure under conditions sufficient to allow formation of second antigen- antibody complexes between TMPRSS2 and the second single domain VHH antibody; and visualizing the second antigen-antibody complexes.
  • the second single domain VHH antibody according to the invention is labeled and visualizing the second antigen-antibody complexes comprises visualizing the label.
  • the method utilizes a single domain VHH antibody that binds to the catalytic domain of TMPRSS2.
  • the VHH binds to the residues 275-473, around TMPRSS2 catalytic site.
  • the method utilizes a single domain VHH antibody that further comprises a label.
  • the method utilizes a single domain VHH antibody that is covalently attached to a substrate.
  • the single domain VHH antibodies can be used in any suitable assay format known in the art that is designed to utilize antibodies. In particular by an immunoassay, such as an immunoenzymatic method (e.g., ELISA).
  • the invention encompasses a composition comprising a single domain VHH antibody for detection of TMPRSS2 in a biological sample.
  • the single domain VHH antibody according to the invention is useful for the direct detection of TMPRSS2; the detection of TMPRSS2 can be carried out by an appropriate technique, in particular EIA, ELISA, RIA, immunofluorescence, luminescence in a biological sample.
  • the single domain VHH antibody is attached to an appropriate support, in particular a microplate or a bead.
  • the method for the detection of TMPRSS2 in a biological sample comprises providing a single domain VHH antibody of this disclosure; providing a biological sample from a patient; contacting said single domain VHH antibody with said biological sample; and visualizing the antigen-antibody complexes formed.
  • the method comprises an ELISA.
  • the protein-antibody complexes are detected with a second single domain VHH antibody that binds to TMPRSS2.
  • the second single domain VHH antibody comprises a label selected from a chemiluminescent label, an enzyme label, a fluorescence label, and a radioactive (e.g., iodine) label.
  • Preferred labels include a fluorescent label, such as FITC, a chromophore label, an affinity- ligand label, an enzyme label, such as alkaline phosphatase or a luciferase, horseradish peroxidase, or ⁇ 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 nanocry
  • Preferred labels include suitable enzymes such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, 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, methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow, Cascade Blue, Texas Red, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, fluorescent lanthanide complexes such as those
  • the method comprises comparing the results obtained with a positive and/or negative control.
  • the method comprises quantification of the TMPRSS2 detected.
  • METHODS OF DIAGNOSING PROSTATE CANCER [0221] Several reports have shown that TMPRSS2 is overexpressed in prostate cancer and that its expression level correlated with cancer progression.
  • the invention encompasses methods for diagnosing prostate cancer and/or following cancer progression and/or response to treatment.
  • the method comprises providing a biological sample from a patient, contacting the sample with an antibody, especially a VHH antibody, of the invention, and detecting the complexes formed between the antibody, especially the VHH antibody, and TMPRSS2.
  • the VHH is selected from A07, C11 and D01.
  • the VHH is selected from A01 and F05.
  • the antibody is VHH A01-Fc.
  • the antibody is VHH A07-Fc.
  • the antibody is selected from the group consisting of the VHH A01 having the amino acid sequence SEQ ID NO: 19, the VHH F05 having the amino acid sequence SEQ ID NO: 23 and the VHH A01-Fc having the amino acid sequence SEQ ID NO: 26.
  • the antibody is the VHH A01-Fc having the amino acid sequence SEQ ID NO: 26.
  • multiple samples provided from the same patient are taken over time and the expression of TMPRSS2 is quantitated over time.
  • samples from the same patient are provided before, during and/or after a cancer treatment and the expression of TMPRSS2 is quantitated to determine the effect of the treatment on TMPRSS2 expression.
  • MATERIAL AND METHODS EXAMPLE 1. Sequence alignments [0230] Alignments were performed using Protein Blast with default settings (NCBI). Alignment figures were done using the seqvisr package in R (Github: doi.
  • EXAMPLE 2 Plasmids [0231] Codon-optimized HKU1A (RefSeq: YP_173238.1) and B isolate N5 (UniProtKB/Swiss-Prot: Q0ZME7.1) full spikes were ordered as a synthetic gene (GeneArt, Thermo Fisher Scientific) and were cloned into a phCMV backbone (GeneBank: AJ318514), by replacing the VSV-G gene.
  • pQCXIP-BSR-GFP11 and pQCXIP-GFP1-10 were a kind gift from Yutaka Hata (Kodaka M, Yang Z, Nakagawa K, Maruyama J, Xu X, Sarkar A, Ichimura A, Nasu Y, Ozawa T, Iwasa H et al (2015) A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells. Exp Cell Res 336: 171–181) (Addgene plasmid #68716 and #68715).
  • pCSDest-TMPRSS2 was a kind gift from Roger Reeves (Edie S, Zaghloul NA, Leitch CC, Klinedinst DK, Lebron J, Thole JF, McCallion AS, Katsanis N, Reeves RH (2016) Survey of human chromosome 21 gene expression effects on early development in Danio Rerio. G38: 2215–2223)) (Addgene plasmid # 53887). All mutation in the HKU1 spike and in TMPRSS2 were introduced using the NEB Q5 Side-Directed mutagenesis kit. Plasmids were sequenced using the primers described (Sup).
  • phCMV-HKU1-S-mNeonGreen and pCDEST-TMPRSS2-mScarlet-I was generated by Gibson assembly. Codon-optimized synthetic genes coding for the TMPRSS2 from Mus musculus (Mouse C57BL/6 - UniPropt: Q3UKE3), Mustela furo (Ferret – UniProt: A0A8U0SMZ2), Mesocricetus auratus (Syrian Hamster Isoform X1 – UniProt: A0A1U8BWQ2), Gorilla (Gorilla-gorilla-gorilla - XP_055229600.1), Bos taurus (Bos-taurus - XP_015329141.1) and macaque (Macaque – UniProt: F6SVR2) with an Nterminal cMYC-tag were ordered to GeneArt (Thermo Fisher Scientific) and cloned into a phCMV back
  • pCAGGS-based expression vectors N-terminal MYC- epitope tagged TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS10, TMPRSS11A, TMPRSS11B, TMPRSS11D, TMPRSS11E, TMPRSS11F and TMPRSS13 were a kind gift from Stefan Pöhlmann (Hoffmann, M. et al. Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity. EBioMedicine 65, 103255 (2021)). EXAMPLE 3.
  • Spikes were stabilized by mutating the furin cleavage site ( 756 RRKRR 760 (SEQ ID NO: 44)> 756 GGSGS 760 (SEQ ID NO: 45) in HKU1A; 752 RRKRR 756 (SEQ ID NO: 44)> 752 GGSGS 756 (SEQ ID NO: 45) in HKU1B;), two residues in the S2 subunit ( 1071 AL 1072 > 1071 PP 1072 in HKU1A; 1067 NL 1068 > 1067 PP 1068 in HKU1B) and adding a Foldon trimerization motif at the C-terminus.
  • the ectodomain from the Wuhan SARS-CoV-2 Spike was cloned into pcDNA3.1(+) and was stabilized with 6 proline mutations (F817P, A892P, A899P, A942P, K986P, V987P), as reported.Hsieh, C.-L. et al. Structure-based design of prefusion-stabilized SARS-CoV-2 spikes. Science 369, 1501-1505 (2020).
  • the furin site was also replaced as above ( 682 RRAR 685 (SEQ ID NO: 30)> 682 GSAS 685 (SEQ ID NO: 31)), a C-terminal Foldon motif was introduced, as well as Hisx8, Strep, and Avi tags.
  • RBDs (residues 323-609 for HKU1A; 323-607 for HKU1B; 331-528 for SARS-CoV-2 Wuhan) were cloned into pCAGGS (HKU1) or pcDNA3.1(+) (SARS-CoV-2), following a murine immunoglobulin kappa signal peptide, and upstream of a thrombin cleavage site and in-tandem Hisx8, Strep and Avi-tags.
  • the WT TMPRSS2 ectodomain (residues 107-492) followed by C-terminal tags (8xHis-tag and AviTag) was synthesized and cloned into pcDNA3.1/Zeo(+) expression vector (Thermo Fisher Scientific).
  • the TMPRSS2 ectodomain with the S441A mutation was cloned into a modified pMT/BiP plasmid (Invitrogen; hereafter termed pT350), which translates the protein in frame with an enterokinase cleavage site and a double strep-tag at the C-terminal end.
  • the ACE2 peptidase domain (residues 19-615) was cloned in pcDNA3.1(+) with a murine immunoglobulin kappa signal peptide and a C-terminal thrombin cleavage site followed by a Strep-tag.
  • the coding sequences of the selected VHHs in the vector pHEN6 were subcloned into a bacterial expression vector pET23 encoding a C terminal His-tag using NcoI and NotI restriction sites.
  • the spike proteins were purified from culture supernatants by high-performance chromatography using the Ni Sepharose Excel Resin according to the manufacturer’s instructions (GE Healthcare), and dialyzed against PBS using Slide-A-Lyzer dialysis cassettes (Thermo Fisher Scientific).
  • the RBDs and ACE2 were purified on a Streptactin column (IBA). Eluted fractions were analyzed by SDS- PAGE and those containing bands of the expected molecular weight were pooled, concentrated and further purified by size-exclusion chromatography (Superdex 20010/300 column (Cytiva)).
  • AviTagged SARS-CoV-2 tri-S was biotinylated using Enzymatic Protein Biotinylation Kit (Sigma- Aldrich).
  • HKU1a-CoV, HKU1b-CoV and OC43-CoV spike proteins were biotinylated using EZ- LinkTM Sulfo-NHS-biotinylation kit (Thermo Fisher Scientific).
  • the pT350 plasmid encoding S441A TMPRSS2 was used to perform a stable transfection on Drosophila S2 cells with the pCoPuro plasmid for puromycin selection.
  • the cell line was selected and maintained in serum-free insect cell medium (HyClone, GE Healthcare) containing 7 ⁇ g/ml puromycin and 1% penicillin/streptomycin.
  • the cells were grown in spinner flasks until the density reached 10 7 cells/mL, at which point the protein expression was induced with 4 ⁇ M CdCl2. After 6 days, the culture was centrifuged and the supernatant was concentrated and used for affinity purification using a Streptactin column (IBA). The eluate was concentrated and applied onto a Superdex 20016/60 column (Cytiva) equilibrated with 10 mM Tris-HCl (pH 8.0), 100 mM NaCl. [0238] E.
  • coli BL21pLysS cells were transformed with the plasmids encoding the different VHHs, which were expressed in the cytoplasm after overnight induction with 0.5 mM IPTG at 16° C.
  • the cultures were centrifuged, the bacterial pellets were resuspended in 40 mL of lysis buffer (20 mM Tris-HCl, 200 mM NaCl, 20 mM imidazole, pH 8.0) containing complete protease inhibitor cocktail (Roche) and they were frozen at -80 °C until used.
  • HEK293T (293T), U2OS and Caco2/TC7 were cultured in DMEM with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS).
  • LNCaP cells were cultured in RPMI-1640 with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS).
  • GFP-split cells were cultured with 1 ⁇ g/ml of puromycin (InvivoGen).
  • Cells stably expressing TMPRSS2 were cultured with 200 ⁇ g/ml hygromycin. All cells lines were either purchased from ATCC or were kind donations from members of the Institut Pasteur and were routinely screened for mycoplasma.
  • GFP-split fusion assay For cell–cell fusion assays, 293T cell lines stably expressing GFP1-10 and GFP11 were co- cultured at a 1:1 ratio (6 ⁇ 10 4 cells/well) and were transfected in suspension with 100 ng of DNA with Lipofectamine 2000 (Thermo) in a 96-well plate (uClear, #655090) (10% spike, indicated amount of TMPRSS2 adjusted to 100 ng DNA with pQCXIP-Empty).
  • 293T GFP-split cells 1-10 and 11 were transfected separately in suspension with 500ng of DNA (10% spike, indicated amout of TMPRSS2 adjusted to 500ng with Empty) for 30 min at 37°C. Cells were washed twice and acceptor and donor cells were mixed and seeded at 6 ⁇ 10 4 cells/well.
  • Caco2 GFP11 cells were transfected with control siRNA-directed against Luciferase: 5′- CGUACGCGGAAUACUUCGA-3′ (SEQ ID NO: 32) or siGENOME Human TMPRSS2 SMARTpool (#7113 - Horizon Discovery) at 50nM using LipofectamineTM RNAiMAX (Thermo Fisher Scientific) in a 6 well dish for 48h.293T GFP1-10 cells were transfected with 10% of spike protein in a 6 well dish for 24 hours. Caco2 KD cells and 293T spike cells were then mixed at a 1:1.5 ratio and plated on a 96 well plate. The remaining Caco2 cells were used for RNA extraction for qPCR.
  • RNA Extraction, Reverse Transcription, and qPCR [0241] 48h post siRNA transfection, total of 5x10 5 caco-2 cells were lysed using RLT buffer (QIAGEN) supplemented with 10 ⁇ L of ⁇ -mercaptoethanol. RNA extraction was performed using the RNeasy plus mini kit (QIAGEN) according to the manufacturer's protocol. Reverse transcription was performed using SuperScript II (Thermo Fisher Scientific) according to the manufacturer's protocol.
  • qPCR was performed using iTaq universal SYBR green supermix (Bio Rad) on a QuantStudio 6 Real- Time PCR machine (Thermo Fisher Scientific). The following primers were used: ⁇ -Tubulin forward: 5’ – CTTCGGCCAGATCTTCAGAC – 3’ (SEQ ID NO: 33), reverse: 5’ – AGAGAGTGGGTCAGCTGGAA – 3’ (SEQ ID NO: 34); TMPRSS2 forward: 5’ – GGGGATACAAGCTGGGGTTC – 3’ (SEQ ID NO: 35), reverse: 5’ – GATTAGCCGTCTGCCCTCAT – 3’ (SEQ ID NO: 36).
  • EXAMPLE 7 EXAMPLE 7.
  • Pseudotype generation and infection were produced by transfection of 293T cells as previously described 38 . Briefly, cells were cotransfected with plasmids encoding for lentiviral proteins, a luciferase reporter, and the HKU1 spike plasmid. Pseudotyped virions were harvested at days 2 and 3 after transfection. Production efficacy was assessed by measuring infectivity or p24 concentration. Infection was performed in suspension using 10ng of p24 per well and 2x10 ⁇ 4 cells in 100uL in a 96 well plate.
  • EXAMPLE 8 Flow cytometry [0243] For Spike binding, 293T cells transfected with TMPRSS2 in the presence or absence of camostat for 24 h were stained with soluble biotinylated spike diluted in MACS buffer (PBS, 5g/L BSA, 2mM EDTA) at 2 ⁇ g/ml for 30 min at 4°C.
  • TMPRSS2 was assessed on fixed cells by staining intracellularly with rabbit anti- TMPRSS2 (Atlas HPA035787), for 30 minutes at RT in PBS/BSA/Azide/0.05% Saponin.
  • ELISA assay [0244] ELISAs were performed as previously described in Mouquet, H. et al. Memory B cell antibodies to HIV-1 gp140 cloned from individuals infected with clade A and B viruses. PLoS One 6, e24078 (2011).
  • BLI assay Affinity of recombinant Spikes, RBDs and VHHs towards purified ectodomains of S441A TMPRSS2 or ACE2 was assessed in real-time using a bio-layer interferometry Octet-Red384 device (Pall ForteBio). Nickel-NTA capture sensors (Sartorius) were loaded for 10 min at 1,000 rpm shaking speed with the Wuhan RBD at 100 nM, or the HKU1A/B RBDs at 200 nM, or each VHH at 100 nM in PBS.
  • the sensors were then blocked with PBS containing BSA at 0.2 mg/mL (assay buffer) and were incubated at 1,000 rpm with two-fold serially diluted concentrations (400 nM to 3.12 nM) of S441A TMPRSS2 or ACE2 ectodomains in assay buffer. Association and dissociation were monitored for 240 s and 180 s, respectively. A sample reference measurement was recorded from a sensor loaded with either RBD or VHH and dipped in the assay buffer. Association and dissociation profiles were fitted assuming a 1:1 binding model. Experiments to identify anti-TMPRSS2 VHHs that block binding to the HKU1 RBD were performed by immobilizing the HKU1B RBD on Nickel-NTA capture sensors.
  • Enzymatic activity was measured using BOC-QAR-AMC, a substrate of TMPRSS2 that fluoresces when cleaved.
  • TMPRSS2 mutants cells were transfected in a black bottom 96 well plate as described above. After 24 hours, media was replaced with 100uL SVF free, phenol red free media, containing 100 ⁇ M of fluorogenic substrate. Indicated concentration of inhibitors were added.
  • the following antibodies were diluted in WB buffer (PBS, 1% BSA, 0.05% Tween, 0.01% Na Azide): rabbit anti-human TMPRSS2 (Atlas antibodies cat# HPA035787, 1:1,000), rabbit anti-human actin (abcam, 60008-1-Ig, 1:10,000), rabbit anti-HKU1 S1 polyclonal antibody (Thermofisher Scientific Cat #PA5-120768, 1:2,000) and rabbit anti-HKU1 S2 polyclonal antibody (Thermofischer Scientific #PA5-120769, 1:1,000). Species- specific secondary DyLight-coupled antibodies were used (1:10,000) and proteins were revealed using a Licor Imager. Images were processed using Image Studio Lite software.
  • TMPRSS2 triggers spike-dependent fusion
  • HKU1A isolated N1 - NCBI:txid443239
  • B isolated N5 - NCBI:txid443241
  • HKU1A spike-expressing 293T donor cells were mixwd with Caco-2 acceptor cells, that naturally express TMRPSS2. Caco-2 fused with spike expressing cells, whereas silencing of TMPRSS2 using siRNA significantly reduced fusion (Fig.3).
  • HKU1 does not grow in any cell line tested up to date but can be amplified in primary differentiated human airway epithelial cultures 24,29 . No access to an infectious HKU1 strain was available.
  • TMPRSS2 lentiviral particles pseudotyped with HKU1 spikes were generated.
  • the viral pseudotype strategy has been successfully used to study SARS-CoV-2 entry and to identify ACE2 as a receptor for this virus 30 .
  • HKU1A and B pseudoviruses were unable to infect wild-type (WT) 293T cells.
  • Transient expression of TMPRSS2 allowed for efficient viral entry (Fig.4). Altogether, these results indicate that TMPRSS2 is necessary for HKU1A and B spike-mediated cell-cell fusion and viral entry.
  • EXAMPLE 15 EXAMPLE 15.
  • Inactive TMPRSS2 mutants allow HKU1 infection
  • two well-characterized TMPRSS2 mutants R255Q and S441A (Fig.5). were generated.
  • R255Q is mutated in its auto- cleavage site resulting in an immature inactive TMPRSS2, while S441A is mutated in its catalytic site.
  • S441A is mutated in its catalytic site.
  • R255Q and S441A were correctly expressed in 293T cells but lacked catalytic activity, measured with a substrate generating a fluorescent signal upon cleavage 31 ( Figure 24).
  • the cleavage profile of TMPRSS2 and HKU1 spike was generated by western blot (Fig.7).
  • WT TMPRSS2 When expressed alone, WT TMPRSS2 was cleaved while the mutants were not. When spike was expressed, the overall levels of TMPRSS2 were reduced, probably because of the co-transfection procedure, but a similar profile of TMPRSS2 processing was observed. Without TMPRSS2, the spike was partially cleaved into S1 and S2 subunits (Fig.7), most likely at the polybasic furin cleavage S1/S2 site. WT TMPRSS2, but not the R255Q and S441A mutants, generated additional cleavage bands in the spike, including a 100 kDa band below the S2 band, which likely corresponds to the S2’ fragment.
  • TMPRSS2 had slightly different levels of expression, when assessed by flow cytometry. Thus, the amounts of plasmid were adjusted to reach similar levels.
  • the catalytically inactive TMPRSS2 mutants barely induced cell-cell fusion with either HKU1A or B spikes (Fig.8), confirming the need for spike cleavage for cell-cell fusion.
  • the TMPRSS2 mutants readily allowed infection of pseudotypes bearing HKU1A or B spikes and were even more efficient than the WT protease, with a 2-10 increase in infection (Fig.9).
  • TMPRSS2 mutants behave as expected regarding the SARS- CoV-2 spike (D614G ancestral strain), which requires the enzymatic activity of the protease for infectivity enhancement 30 .
  • WT TMPRSS2 increased SARS-CoV-2 pseudotype entry by 8-fold, whereas this was not the case for R255Q and S441A mutants ( Figure 25).
  • the catalytic activity of TMPRSS2 is not required for HKU1-mediated viral entry, strongly suggesting that TMPRSS2 acts as a receptor for HKU1.
  • Coronavirus spike can either be processed by proteases at the surface of target cells, allowing for membrane fusion or by cathepsins in the endosome, allowing for entry after internalization 32 .
  • TMPRSS2 catalytic activity was not required for HKU1 entry, the cytoplasmic access route of pseudoviruses in cells expressing WT or S441A TMPRSS2 was examined. To this aim, infections were performed in presence of SB412515, a cathepsin L inhibitor, or Camostat mesylate, a TMPRSS2 inhibitor.
  • SB412515 reduced the entry of HKU1A pseudoviruses in the S441A TMPRSS2 cells and not the WT TMPRSS2 cells, indicating that viral entry occurs through endosomes with the catalytically inactive TMPRSS2 and at the surface with the WT protease.
  • Camostat increased HKU1A entry in WT TMPRSS2 cells, and not in S441A TMPRSS2 cells, suggesting that the chemical inhibition of TMPRSS2 stabilizes the protease at the surface and/or enhances endosomal entry.
  • HKU1B With HKU1B, Camostat and SB412515 had little or no effect on entry (Figure 26), suggesting that HKU1B priming might be different than that of HKU1A, although it still uses TMPRSS2 as a receptor. [0256] Taken together, these results show that TMPRSS2 is required for HKU1 infection and that its catalytical activity can be rescued by other proteases such as cathepsins. EXAMPLE 16. HKU1 spike binds to TMPRSS2 [0257] Recombinant soluble forms of HKU1A and B spikes were generated to investigate their binding to TMPRSS2 expressing cells (Fig.11-12, Figures 27-28).
  • Addition of Camostat increased binding to WT TMPRSS2 (Fig.12, Figures 27-28), indicating that the proteolytic activity of TMPRSS2 somehow interfered with the readout or decreased binding by degrading or shedding the bound spike.
  • SARS-CoV-2 spike binding to cells expressing ACE2 was detected, but not WT or mutant TMPRSS2 (Fig.12, Figure 29), highlighting the different behaviors of HKU1 and SARS-CoV- 2.
  • OC43 did not bind to cells expressing TMPRSS2 ( Figure 30).
  • the binding of recombinant soluble forms of TMPRSS2 and HKU1A or B spikes was demonstrated by ELISA, whereas it was not the case for the SARS-CoV-2 spike (Fig.13), indicating a direct interaction between TMPRSS2 and HKU1 spike.
  • soluble ACE2 bound to SARS- CoV-2 but not to HKU1 spike by ELISA and by BLI ( Figure 31-32).
  • the yield of soluble WT TMPRSS2 was low, limiting experiments, potentially due to TMPRSS2 autocleavage and stability.
  • a soluble TMPRSS2 S441A mutant was generated for further experiments.
  • affinity constants (Kd) of 93nM and 61nM were determined for HKU1A and B RBDs, respectively (Table 1, Table 2, Figure 34).
  • Table 1 Affinity (Kd) of the indicated RBD for TMPRSS2, ranges of affinity, association rate (kas) and dissociation rate (kdis) measured in 2-3 independent experiments.
  • NA denotes proteins for which interaction with the loaded sensor was below the interaction with an empty sensor.
  • Table 2 K d , k as and k dis of the indicated RBD for ACE2.
  • NA denotes proteins for which interaction with the loaded sensor was below the interaction with an empty sensor.
  • HKU1 spike receptor binding motif [0260] The residues W515 and R517 within HKU RBD have been reported to be critical for binding to a putative unknown cellular receptor 16 .
  • the W515A mutation abrogated interaction with TMPRSS2, reaching response levels comparable to those obtained with a sensor coated with the SARS-CoV-2 RBD, while the R517A mutation reduced binding by 3-fold (Fig.15, Figure 35, Table 1, Table 2).
  • VHH VHH
  • A01, F05, A07, C11 and D01 VHH
  • the VHH also recognized TMPRSS2 to various extents by flow cytometry ( Figure 39).
  • Three of them (A07, C11 and D01) reduced TMPRSS2 catalytic activity (Fig.18) and inhibited the HKU1 RBD–TMPRSS2 interaction measured by BLI (Fig. 19).
  • the same three VHH inhibited HKU1A and B spike – TMPRSS2 mediated cell-cell fusion (Fig.20).
  • TMPRSS2 S441A was expressed in S2 cells and purified as indicated above (Example 3. Protein production). The double Strep tag of the protein was removed by incubating the protein with 64 units of Enterokinase light chain (BioLabs) in 10 mM Tris, 100 mM NaCl, 2 mM CaCl2, pH 8.0, at room temperature, overnight.
  • the proteolysis reaction was buffer- exchanged into 10 mM Tris, 100 mM NaCl, pH 8.0, and subjected to another affinity purification, recovering the flow-through fraction containing the untagged TMPRSS2 S441A.
  • the protein was concentrated and its enzymatic deglycosylation with EndoD and EndoH was set up at room- temperature following overnight incubation with 1000 units of each glycosidase in 50 mM Na-acetate, 200 mM NaCl, pH 5.5.
  • the protein was further purified on a size exclusion chromatography (SEC) column Superdex 20016/60 (Cytiva) in 10 mM Tris, 100 mM NaCl, pH 8.0.
  • SEC size exclusion chromatography
  • the protein (at 68 ⁇ M) was incubated with VHH-A07 (102 ⁇ M) for 16 hours at 4 °C and the binding reaction was injected in a Superdex 20010/300 column (Cytiva) equilibrated with 10 mM Tris, 100 mM NaCl, pH 8.0. The eluted fractions were analyzed by SDS-PAGE and those corresponding to the complex were pooled, concentrated and used for crystallization. [0265] The crystallization trials were performed in 200 nanoliter sitting drops formed by mixing equal volumes of the protein and reservoir solution in the format of 96 Greiner plates, using a Mosquito robot.
  • Crystal appearance and growth were monitored by a Rock-Imager at the Core Facility for Protein Crystallization at Institut Pasteur in Paris, France (Weber, P. et al. High-throughput crystallization pipeline at the crystallography core facility of the Institut Pasteur. Molecules 24, 4451 (2019)).
  • the crystal used for data collection was grown in 0.05 M HEPES pH 7.0, 20% PEG 3350, 1% tryptone, 0.001% NaN 3 and frozen using the mother liquor containing 33% glycerol as cryo-buffer.
  • the X-ray diffraction data were collected at the SOLEIL synchrotron source (Saint Aubin, France), Proxima-1 beamline (Chavas, L. M. G. et al.
  • the high- resolution cut-off was based on the statistical indicator CC1/2 (Karplus, P. A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, 1030–1033 (2012)).
  • the phases were determined by molecular replacement using a TMPRSS2 structure (PDB: 7MEQ) and a VHH-A07 model generated using AlphaFold as search models in PHASER (McCoy, A. J. et al. Phaser crystallographic software. J Appl Crys- tallogr.40, 658–674 (2007)) from the PHENIX suite (Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution.
  • TMPRSS2 S441A (chain A) VHH-A07 (chain B) Crystal 1 – Alternate location (alt loc) A A LYS 342 NZ B VAL 100 O 2.637 A GLN 438 NE2 (alt loc A) B ARG 104 O 3.407 A GLY 439 N (alt loc A) B ARG 103 O 3.019 A SER 463 OG (alt loc A) B HIS 31 ND1 2.358 A ARG 470 NE B GLU 30 OE1 3.019 A ARG 470 NE B GLU 30 OE2 3.381 A ARG 470 NH2 B GLU 30 OE2 2.776 B HIS 31 ND1 A SER 463 OG (alt loc A) 2.358 B VAL 100 N A LYS
  • the epitope residues are the following: V275, Q276, V278, H279, V280, C281, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, E389, K390, T393, V415, Y416, D417, N418, L419, D435, S436, C437, Q438, G439, A441, T459, S460, W461, G462, S463, G464, K467, R470, G472, V473.
  • the paratope residues are the following: Q1, G26, S27, P28, E30, H31, Y32, D33, T53, S54, G55, G56, N74, G98, R99, V100, G101, G102, R103, R104, N105, W106, P109, D111, D114, N115, A116, Y117.
  • the epitope residues are the following: V275, Q276, V278, H279, V280, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, G385, A386, E389, K390, T393, V415, Y416, D417, N418, L419, S436, C437, G439, D440, A441, T459, S460, W461, G462, S463, C465, R470.
  • Crystal structure TMPRSS2 S441A+VHH A07 (Crystal form 2, PDB 8 S0N) [0275] Table 6. List of residues from cleaved form of TMPRSS2 and VHH-A07 that are buried at the interface of the complex (crystal form 2, PDB 8S0N).
  • the epitope residues are the following: V275, Q276, V278, H279, V280, C281, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, G385, E389, T393, V415, Y416, D417, N418, L419, S436, C437, G439, D440, A441, T459, S460, W461, G462, S463, C465, R470, G472, Y474. [0277] Crystals of TMPRSS2 with A01 were analyzed.
  • TMPRSS2 The structure of TMPRSS2 with the VHH A01 has also been studies. As it is shown below. [0279] Table 7. List of residues from TMPRSS2 (chain A) and VHH-A01 (chain U) that form polar contacts at the complex interface. The atoms involved and the distances between them are indicated.
  • the epitope residues are the following: [0285] R150, Y152, Q159, Y161, K166, S167, W168, H169, S204, G205, S206, T207, P367, P369, G370, M371, M372, L373, Q374, P375, I404, I405, E406, T407, Q408, N411, I420, T421, P422, M424, I425, I456, N476, M478, V479, T481, D482. EXAMPLE 20.
  • VHH-Fc Activities of VHH-Fc
  • U2OS cells stably expressing TMPRSS2 or an empty control construct were stained with VHH-A01-Fc (Fig.44).
  • Caco-2 cells endogenously express TMPRSS2.
  • 40 TMPRSS2 KO caco-2 cells were generated using CRISPR/Cas9.
  • WT and TMPRSS-2KO caco-2 cells were surface stained for TMPRSS2 using the dimerized VHH-A01-Fc (Fig.45).
  • VHH-A01-Fc stained WT caco-2 cells demonstrated by the shift in Alexa-647 MFI. This staining was significantly reduced in TMPRSS2-KO caco-2.
  • VHH-Fc VHH-A01-Fc and VHH-A07-Fc compared to VHHs A01 and A07 is summarized in the table below.
  • HEK 293T cells were transfected with plasmids expressing TMPRSS2-Scarlet-I.24h post transfection, binding of VHH-A01-Fc and VHH-A07-Fc was assessed on live cells by staining with a range of concentrations of anti-TMPRSS2 VHH A01-Fc or VHH A07-Fc (0,00032 ⁇ g ml ⁇ 1 to 5 ⁇ g ml ⁇ 1 ) for 30 min at 4 °C in MACS buffer, followed by staining with Alexa Fluor 647-conjugated Goat anti-Human Antibody (Thermo Fisher Scientific, A-21445, 1:500).
  • VHH-A07-Fc and VHH-A01-Fc both bind to TMPRSS2 but not to 9 other serine proteases (Fig.55).
  • HEK 293T cells were transfected with plasmids expressing TMPRSS2 or 9 other Human serine proteases.24h post transfection, binding of VHH-A01-Fc and VHH-A07-Fc was assessed on live cells by staining with anti-TMPRSS2 VHH A01-Fc at 1 ⁇ g ml ⁇ 1 or VHH A07-Fc at 5 ⁇ g ml ⁇ 1 for 30 min at 4 °C in MACS buffer, followed by staining with Alexa Fluor 647-conjugated Goat anti- Human Antibody (Thermo Fisher Scientific, A-21445, 1:500).
  • VHH-A01-Fc binds to human TMPRSS2 but not to 6 other animal TMPRSS2 tested (Mesocricretus auratus, Macaca, Mustela furo, Mus musculus, Bos taurus, Gorilla gorilla (Fig.56). VHH-A07-Fc recognizes human, macaque, mouse and gorilla TMPRSS2 (Fig.56).
  • HEK 293T cells were transfected with plasmids expressing TMPRSS2 of different animal species.24h post transfection, binding of VHH-A01-Fc and VHH-A07-Fc to animal TMPRSS2 was assessed on live cells by staining with anti-TMPRSS2 VHH A01-Fc at 1 ⁇ g ml ⁇ 1 or VHH A07-Fc at 5 ⁇ g ml ⁇ 1 for 30 min at 4 °C in MACS buffer, followed by staining with Alexa Fluor 647-conjugated Goat anti- Human Antibody (Thermo Fisher Scientific, A-21445, 1:500).
  • VHH-A01-Fc does not inhibit TMPRSS2-HKU1 interactions nor the enzymatic activity.
  • VHH-A07-Fc inhibits HKU1 infection and TMPRSS2 enzymatic activity.
  • EXAMPLE 20 VHH A07 inhibits SARS-CoV-2 replication
  • Caco-2 cells were infected with SARS-CoV-2 in the presence of non-target, A01 and A07 monomeric VHHs (Fig.46). Both non-target and A01 did not inhibit SARS-CoV-2 infection, which is expected as they are unable to inhibit TMPRSS2 enzymatic activity (Fig.18).
  • VHH A07 which is capable of inhibiting TMPRSS2 enzymatic activity inhibited SARS-CoV-2 infection in a dose dependent manner.
  • EXAMPLE 21 Anti-TMPRSS2 VHH blocks HKU1 infection [0294] 259 HKU1 does not grow in any cell line tested up to date but viral amplification in human ciliated airway epithelial cell cultures has been reported 29,39,40 . HKU1 virus was isolated from a nasal swab of an individual suffering from a respiratory tract infection. To this end, primary human bronchial epithelial cells (HBE cells) differentiated at the air/liquid interface (ALI) for over 4 weeks were used.
  • HBE cells primary human bronchial epithelial cells differentiated at the air/liquid interface (ALI) for over 4 weeks were used.
  • Target cells were preincubated with A07 VHH and the spike intensity by immunofluorescence 48 h post-infection was measured (Fig.47).
  • the A07 VHH strongly reduced the appearance of infected cells, indicating that the spike binding and/or cleavage activities of TMPRSS2 are necessary for a productive HKU1 infection.
  • A07 covers the active site cleft and buries a large surface area (about 2600 ⁇ 2, ⁇ 1400 ⁇ 2 on the VHH and ⁇ 1200 ⁇ 2 on TMPRSS2), interacting with residues in exposed loops of the SP domain (loops 1, 2, 3, A, B, C, D), some also involved in the interaction with HKU1B RBD ( Figure 48).
  • Superimposing the RBD+TMPRSS2 S441A structure on the A07+TMPRSS2 S441A complex showed clashes between the VHH and the RBD ( Figure 49), explaining the blocking activity and providing further validation to the interaction site that we report here for the RBD.
  • the most notorious feature is the insertion of its long (21 residues) CDR3 in the substrate binding cleft in between the two lobes of the SP domain.
  • Superposing the complex with the peptide-bound structures of hepsin and TMPRSS13 shows that the side chain of R103A07 occupies the P1 position, making contacts with residues D435 T , S436 T and G464 T , which form the S1 site 18.
  • the TMPRSS2 S441A /A07 crystals displayed electron density for the TMPRSS2 LDLR-A domain, which was not resolved in the previous structure of TMPRSS2.
  • the LDLR-A domain includes a calcium-binding site formed by the side chains of D134 T , H138 T , D144 T , and E145 T , and the main chain carbonyl group of N131 T and V136 T .
  • the SP domain of cleaved TMPRSS2 (TMPRSS2-Cl) crystallized previously in complex with the Nafamostat inhibitor 18 superposes very well with its counterpart in the TMPRSS2 S441A /A07 crystals, with a root mean square deviation (RMSD) of 0.315 ⁇ for 1502 atoms in 236 residues.
  • RMSD root mean square deviation
  • TMPRSS2 Expression of TMPRSS2 was assessed on live cells by staining with anti-TMPRSS2 VHH A01-Fc at 1 ⁇ g ml ⁇ 1 for 30 min at 4 °C in MACS buffer, followed by staining with Alexa Fluor 647- conjugated Goat anti-Human Antibody (Thermo Fisher Scientific, A-21445, 1:500).
  • SEQUENCES Monomeric VHH sequences [0302] In the following sequences, CDR1 is in bold, CDR2 in bold and in italics and CDR3 in bold and underlined.
  • A01 (deposited as VHH TMPRSS2 A01/VHH-TMPRSS2-A01 at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES(CNCM) on June 1, 2023, under number I-5958) MAQVQLVESGGGLVQPGGSLRLSCVVSGFSLDYYAIGWFRQAPGKEREGVSCIGSSGDKTNYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAESALYSDCTEEQNPMLYDYWGQGTQVTVSS (SEQ ID NO: 19)
  • A07 (deposited as VHH TMPRSS2 A07/VHH-TMPRSS2-A07 at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES(CNCM) on June 1, 2023, under number I-5959) MAQVQLVESGGGLVQPGGSLRLSCTSSGSPLEHYDIIWFRQAPGREREGVSSITTSGGHTNYADSVKG RFTI
  • TMPRSS2 Activates the Human Coronavirus 229E for Cathepsin- Independent Host Cell Entry and Is Expressed in Viral Target Cells in the Respiratory Epithelium. Journal of Virology 87, 6150-6160 (2013). 7. Glowacka, I. et al. Evidence that TMPRSS2 Activates the Severe Acute Respiratory Syndrome Coronavirus Spike Protein for Membrane Fusion and Reduces Viral Control by the Humoral Immune Response. Journal of Virology 85, 4122-4134 (2011). 8. Shirato, K., Kawase, M. & Matsuyama, S. Wild-type human coronaviruses prefer cell-surface TMPRSS2 to endosomal cathepsins for cell entry.
  • TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes. Sci Immunol 5 (2020). 28. Kishimoto, M. et al. TMPRSS11D and TMPRSS13 Activate the SARS-CoV-2 Spike Protein. Viruses 13 (2021). 29. Dominguez, S. R. et al. Isolation, propagation, genome analysis and epidemiology of HKU1 betacoronaviruses. Journal of General Virology 95, 836-848 (2014). 30. Hoffmann, M. et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor.

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Abstract

The invention relates to the discovery that TMPRSS2 acts as a functional receptor for HKU1. TMPRSS2 triggers HKU1 spike-mediated cell-cell fusion and viral pseudotype infection. Catalytically inactive TMPRSS2 mutants do not cleave spike but allow infection, demonstrating that HKU1 binding and processing represent two distinct functions of the receptor. TMPRSS2 binds with high affinity to HKU1 receptor binding domain (RED), but neither to OC43 nor to SARS-CoV-2. Conserved amino acids were identified within the RED that are essential for binding to TMPRSS2 and infection. Anti- TMPRSS2 VHH antibodies were generated that inhibit HKU1 spike binding to TMPRSS2, fusion and infection.

Description

SINGLE DOMAIN ANTIBODIES DIRECTED AGAINST HUMAN TRANSMEMBRANE- SERINE PROTEASE 2 (TMPRSS2) FIELD OF THE INVENTION [0001] The invention relates to human coronavirus HKU1 and its receptor and antibodies that bind to its receptor. BACKGROUND OF THE INVENTION [0002] Four endemic seasonal human coronaviruses, HKU1, 229E, NL63 and OC43 circulate worldwide causing common colds1. Coronavirus spike proteins bind to cellular receptors to enable infection2. NL63 uses angiotensin-converting enzyme 2 (ACE2) for viral entry3, whereas 229E uses human aminopeptidase-N4. HKU1 and OC43 bind cells through 9-O-acelytated sialic acid but their protein receptors remain unknown5. After viral binding to target cells, transmembrane-serine protease 2 (TMPRSS2) can cleave coronavirus spike and prime their fusion6-10. [0003] HKU1 was first identified in Hong Kong in 200511, in an elderly patient with severe pneumonia. HKU1 usually causes common cold and benign respiratory symptoms, but complications include severe lower respiratory infections, particularly in young children, the elderly and immunocompromised individuals12. It is estimated that 70% of children seroconvert before the age of 6 years13. The global seroprevalence of HKU1 is similar to other HCoV and is between 75 and 95% depending on the study13,14 (M. White), while the incidence rate is around 2 per 100 persons per year15. Three main viral genotypes have been identified, HKU1A, B and C. HKU1A and B spikes display an overall amino-acid (aa) identity of 85% (Fig.1a), while B and C spikes share 99% identity. Conserved regions in the HKU1A and B spikes include a putative RBD with a Receptor Binding Motif (RBM)16,17, S1/S2 and S2/S2’ cleavage sites18 (Fig.1a). [0004] Both HKU1 and OC43 spikes bind to 9-O-acetylated α2,8 linked disialoside on target cells5,19. Their protein receptors have not been identified. After receptor binding, the coronavirus spike can be cleaved at the S2’ site by membrane bound proteases, such as TMPRSS2, or by endosomal cathepsins, releasing the fusion peptide present in the S2 domain and activating the spike for membrane fusion2. [0005] TMPRSS2 belongs to the type II transmembrane serine protease (TTSP) family that comprises 19 cell-surface enzymes20,21. TTSP are involved in many processes, including epithelial homeostasis, extracellular matrix degradation, hormone and growth factor activation, initiation of proteolytic cascades through cleavage of membrane cellular proteins20. TTSP are synthesized as single-chain proenzymes that require proteolytic activation20. TMPRSS2 undergoes autocleavage, its two subunits remain attached by a disulfide bond22. In the respiratory tract, TMPRSS2 is expressed in nasal, bronchial and small airways tissues, and more particularly in ciliated cells that are the target of HKU123,24. TMPRSS2 is involved in the activation of different viruses.39 [0006] Several reports have shown that TMPRSS2 is overexpressed in prostate cancer and that its expression level correlated with cancer progression. TMPRSS2 has been shown to activate PAR-2 and hepatocyte growth factor (HGF)/c-Met signalling pathways and to downregulate E-cadherin expression in prostate cancer cells. TMPRSS2 has also been described to activate TTSP matriptase and degradation of extracellular matrix (ECM) laminin β1 and nidogen-1 in vitro and in a xenograft mouse model of prostate cancer, promoting prostate cancer tumor growth and metastasis. In vivo using a mouse model with a targeted deletion of TMPRSS2, it was demonstrated that the activity of TMPRSS2 regulates prostate cancer cell invasion and metastasis to distant organs. In addition, by using a chemical inhibitor of TMPRSS2, prostate cancer metastasis was suppressed in vivo in this model. [0007] There is a need in the art for methods and compositions to interfere with infection by coronaviruses and for cancer treatment. The present invention fulfils this need. BRIEF SUMMARY OF THE INVENTION [0008] The invention encompasses a TMPRSS2 antagonist and methods of making and using the antagonist. In various embodiments, the antagonist is an antibody. In various embodiments, the antibody comprises a VHH (also called nanobody). [0009] The invention encompasses an antibody directed against the biologically active ectodomain of TMPRSS2. In various embodiments, the antibody comprises a VHH. [0010] In various embodiments, the VHH is A01 (SEQ ID NO: 19), A07 (SEQ ID NO: 20), C11 (SEQ ID NO: 21), D01 (SEQ ID NO: 22), or F05 (SEQ ID NO: 23). [0011] In various embodiments, binding of the antibody to TMPRSS2 reduces the catalytic activity of TMPRSS2. [0012] In various embodiments, the antibody is directed against amino acids 275-473 of TMPRSS2. [0013] In various embodiments, the antibody binds to a polypeptide comprising the amino acid sequence SEQ ID NO: 29. [0014] In various embodiments, the antibody binds to an epitope that comprises one or more amino acid residues selected from the group consisting of V275, Q276, V278, H279, V280, C281, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, E389, K390, T393, V415, Y416, D417, N418, L419, D435, S436, C437, Q438, G439, A441, T459, S460, W461, G462, S463, G464, C465, R470, G472 and V473 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24. [0015] In various embodiments, the VHH comprises the following CDR domains: CDR1: SG(S/F)(P/T)L(E/D)(H/Y)YDI(I/Y/G) (SEQ ID NO: 1); CDR2: SSIT(T/A)SGGRTNYADSVKG (SEQ ID NO: 2); and CDR3: A(G/A)(K/R)(V/I)GGRRNW(I/V)APLNG(Y/F)ENA(Y/L) (SEQ ID NO: 3). [0016] In various embodiments, the VHH comprises the following CDR domains: CDR1 selected from: SGSPLEHYDII (SEQ ID NO: 5), SGFTLDYYDIY (SEQ ID NO: 6), and SGSTLEHYDIG (SEQ ID NO: 7); CDR2 selected from: SSITTSGGHTNYADSVKG (SEQ ID NO: 10), SSITTSGGRTNYADSVKG (SEQ ID NO: 11), and SSITASGGRTNYADSVKG (SEQ ID NO: 12); and CDR3 selected from: AGRVGGRRNWIVPLDGYDNAY (SEQ ID NO: 15), AAKVGGRRNWIAPLNGYENAL (SEQ ID NO: 16), and AGKIGGRRNWVAPLDGFENAY (SEQ ID NO: 17). [0017] In various embodiments, the antibody binds to an epitope that comprises one or more amino acid residues selected from the group consisting of R150, Y152, Q159, Y161, K166, S167, W168, H169, S204, G205, S206, T207, P367, P369, G370, M371, M372, L373, Q374, P375, I404, I405, E406, T407, Q408, N411, I420, T421, P422, M424, I425, I456, N476, M478, V479, T481 and D482 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24. [0018] The invention encompasses variants of any of the antibodies of the invention, wherein the antibody retains the same binding as the unchanged antibody. In various embodiments, the variant antibody has 1, 2, 3, 4, or 5 changes. [0019] In various embodiments, the antibody is a monomer. In various embodiments, the antibody is a multimer. In various embodiments, the antibody is a fusion protein, particularly a fusion protein with Fc. [0020] In various embodiments, the antibody is in a combination with another antibody. [0021] The invention encompasses methods of inhibiting binding to TMPRSS2 comprising contacting an TMPRSS2 antagonist of the invention, such as an antibody directed against the biologically active ectodomain of TMPRSS2, with a cell expressing TMPRSS2. [0022] The invention encompasses methods of treating a viral infection comprising administering an TMPRSS2 antagonist of the invention, such as an antibody directed against the biologically active ectodomain of TMPRSS2, to an infected patient. [0023] The invention encompasses a method of detecting TMPRSS2 comprising contacting an antibody directed against the biologically active ectodomain of TMPRSS2 with a biological sample containing TMPRSS2 and detecting the resultant immunological complexes. The invention encompasses the use of an antibody of the invention for detecting TMPRSS2. [0024] The invention encompasses a method of diagnosing prostate cancer comprising contacting the antibody of the invention with a biological sample containing TMPRSS2 and detecting the resultant immunological complexes. The invention encompasses the use of an antibody of the invention for diagnosing prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS [0025] Figures 1-4: TMPRSS2 expression enables HKU1 spike mediated fusion. [0026] Figure 1.293T cells expressing either GFP 1-10 or GFP11 were transfected with HKU1 spike and TMPRSS2 or pEmpty (ctrl), fusion was quantified by measuring the GFP area 20 hours post- transfection. Data are mean ± SD of 3–6 independent experiments. Unpaired t-test compared to control condition. [0027] Figure 2. TMPRSS2 was transfected either in donor cells expressing GFP1-10 and HKU1A spike or in acceptor cells expressing GFP-11 and fusion was quantified by measuring the GFP area 20 hours post-transfection. Scale bar: 400 µm. Data are mean ± SD of 3–6 independent experiments. One Way ANOVA with Tukey’s multiple comparisons * p<0.05, **p<0.01, ***p<0.001. [0028] Figure 3. TMPRSS2 was knocked-down in Caco2 acceptor cells expressing GFP11. Caco2 were mixed with 293T donor cells expressing GFP1-10 and HKU1A spike. Left panel: Relative expression of TMPRSS2. Expression data were normalized to β-Tubulin endogenous control, and relative mRNA expression normalized to siCtrl condition (2−ΔΔCT) was plotted. Right panel: fusion was quantified by measuring the GFP area 20 hours post-transfection. Data are mean ± SD of 3–6 independent experiments. Unpaired t-test compared to control condition. [0029] Figure 4.293T cells expressing TMPRSS2 or pEmpty (ctrl), were infected by Luc-encoding HKU1 spike pseudotyped lentiviral particles. Luminescence was read 48 hours post infection. Data are mean ± SD of 3–6 independent experiments. Data are mean ± SD of 3–6 independent experiments. Unpaired t-test compared to control condition. [0030] Figures 5-10: Catalytically inactive TMPRSS2 acts as a receptor for HKU1 spike, enabling endosomal entry but not surface entry. [0031] Figure 5. TMPRSS2 is composed of a Transmembrane Domain (TMD), a class A LDL receptor (LDLR), a Scavenger Receptor Cysteine-2 rich domain (SRCRD) and a serine peptidase. [0032] Figure 6. TMPRSS2 precursor undergoes autocleavage at the R255-L256 peptide bond, resulting in an active protease. The R255Q mutant does not autocleave. Mutation of S441, one of the catalytic triad residues (H296, D345 and S441) results in a catalytically inactive TMPRSS2 protein (S441A). [0033] Figure 7. Western blot of 293T cells transfected with HKU1A spike and/or the indicated TMPRSS2 mutants. Membranes were probed for S1, S2, TMPRSS2 and actin. Representative blot of 3. Molecular weights: KDa. [0034] Figure 8.293T cells expressing either GFP 1-10 or GFP11 were transfected with HKU1 spike and mutant TMPRSS2, fusion was quantified by measuring the GFP area 20 hours post-transfection and normalized to the fusion induced by WT TMPRSS2. Left panel: HKU1A. Right panel: HKU1B. Data are mean ± SD of 3 independent experiments. Statistical analysis: One Way ANOVA with Dunett’s multiple comparisons compared to WT TMPRSS2 using the non-normalized data. [0035] Figure 9.293T cells expressing mutant TMPRSS2 were infected by Luc-encoding HKU1 spike pseudotyped lentiviral particles. Luminescence was read 48 hours post infection. Left panel: HKU1A. Right panel: HKU1B. Dotted line indicates the background. Data are mean ± SD of 3–6 independent experiments. Statistical analysis: Paired One Way ANOVA with Dunett’s multiple comparisons compared to WT TMPRSS2 using the non-normalized data. [0036] Figure 10.293T cells expressing WT or S441A TMPRSS2 were incubated for 2 hours with indicated drug, before infection by Luc-encoding HKU1A spike pseudotyped viruses. Luminescence was read 48 hours post infection. Left panel: effect of Camostat on HKU1A entry. Right panel: effect of SB412512 on HKU1A entry. Statistical analysis: Two-Way ANOVA with Dunett’s multiple comparisons compared to untreated using the non-normalized data * p<0.05, **p<0.01, ***p<0.001. [0037] Figure 11-17: HKU1 spike binds to TMPRSS2 through its RBD, W515 and R517 are essential for this interaction. [0038] Figure 11.293T cells were transfected with TMPRSS2 mutants and incubated overnight in the presence or absence of 10 µM of Camostat. a. TMPRSS2 levels were assessed by flow cytometry on permeabilized cells. One representative experiment of 3 is shown. [0039] Figure 12. Binding of indicated soluble biotinylated trimeric spike was measured by flow cytometry. One representative experiment of 3 is shown. [0040] Figure 13. Binding of indicated soluble biotinylated trimeric spike on immobilized WT TMPRSS2 was measured by ELISA. Mean of 2 independent experiments [0041] Figure 14. Binding of S441A TMPRSS2 to RBD coated receptors was quantified by BLI. One representative experiment of 3 is shown. [0042] Figure 15.293T cells expressing either GFP 1-10 or GFP11 were transfected with TMPRSS2 and HKU1 WT spike and W515A and R517A mutant spike, fusion was quantified by measuring the GFP area 24 hours post-transfection. [0043] Figure 16.293T cells expressing TMPRSS2 were infected by Luc-encoding WT, W515A and R517A mutant HKU1 spike pseudotyped lentiviral particles. Luminescence was read 48 hours post infection. Dotted line indicates the background. Data are mean ± SD of 3 independent experiments. [0044] Figure 17. Binding of TMPRSS2 to mutant RBD coated receptors was quantified by BLI. One representative experiment of 3 is shown. [0045] Figure 18-21: TMPRSS2 targeting VHH inhibit HKU1/TMPRSS2 interaction. [0046] Figure 18. Soluble TMPRSS2 was incubated for 15 minutes with VHHs, before adding the fluorogenic substrate Boq-QAR-AMC. The initial rate of the reaction at different VHH concentration is plotted. One representative experiment of 3 is shown. [0047] Figure 19. Soluble TMPRSS2-S441A was incubated for 15 minutes with 400 nM of VHH. Binding to HKU1B RBD coated receptor was measured by BLI. One representative experiment of 2 is shown. [0048] Figure 20.293T cells expressing either GFP 1-10 or GFP11 were transfected with TMPRSS2 and HKU1 spike in the presence of 1^M of VHH, fusion was quantified by measuring the GFP area 20 hours post-transfection. Data were normalized to the non-treated condition for each spike (dotted line). Data are mean ± SD of 4 independent experiments. One Way ANOVA with Dunett’s multiple comparisons compared to non-target VHH on log-transformed data *p<0.05, **p<0.01, ***p<0.001. [0049] Figure 21.293T were transfected with S441A TMPRSS2.24 hours post-transfection, they were incubated with 1^M VHH for 2 hours and infected by Luc-encoding HKU1 spike pseudotyped viruses. Luminescence was read 48 hours post infection. Data were normalized to the non-treated condition for each virus (dotted line). Data are mean ± SD of 3 independent experiments. One Way ANOVA with Dunett’s multiple comparisons compared to non-target VHH on log-transformed data *p<0.05, **p<0.01, ***p<0.001. [0050] Figures 22-23: [0051] Figure 22.293 T cells were transfected with plasmids encoding for HKU1A or HKU1B spikes and stained 24 h later with mAb10, a pan-anti-coronavirus spike antibody. [0052] Data are representative of 3 independent experiments.Figure 23. 293T cells expressing either GFP 1-10 or GFP11 were transfected with HKU1 spike and the indicated constructs, fusion was quantified by measuring the GFP area 20 hours post-transfection. [0053] Figures 24-26: [0054] Figure 24.293T cells were transfected with TMPRSS2 WT and mutants, the catalytic activity was assessed 24 hours post-transfection using BoC-QAR-AMC fluorogenic substrate. [0055] Figure 25.293T cells expressing ACE2 and mutant TMPRSS2 were infected by Luc- encoding SARS-CoV-2 spike pseudotyped lentiviral particles. Luminescence was read 48 hours post infection. Data are mean ± SD of 4 independent experiments. Ratio paired t-test compared to the control condition **p<0.01. [0056] Figure 26.293T cells expressing WT or S441A TMPRSS2 were incubated for 2 hours with indicated drug, before infection by Luc-encoding HKU1B spike pseudotyped lentiviral particles. Luminescence was read 48 hours post infection. Left panel: effect of Camostat mesylate on HKU1B entry. Right panel: effect of SB412512 on HKU1B entry. Data are mean ± SD of 4-6 independent experiments. [0057] Figures 27-30.293T cells were transfected with TMPRSS2 mutants and incubated overnight in the presence or absence of 10 µM of Camostat. Binding of indicated soluble biotinylated trimeric spike was measured by flow cytometry. % of cells binding to TMPRSS2 was quantified. Data are mean ± SD of 3-4 independent experiments. Two Way ANOVA with Dunett’s multiple comparisons compared to control cells with or without Camostat *p<0.05, **p<0.01, ***p<0.001. [0058] Figure 27. % of binding soluble biotinylated trimeric HKU1A spike to TMPRSS2. [0059] Figure 28. % of binding soluble biotinylated trimeric HKU1B spike to TMPRSS2. [0060] Figure 29. % of binding soluble biotinylated trimeric SARS-CoV-2 spike to TMPRSS2. [0061] Figure 30. % of binding soluble biotinylated trimeric OC43 spike to TMPRSS2. [0062] [0063] Figure 31. Binding of soluble biotinylated spike on immobilized ACE2 was measured by ELISA. [0064] Figure 32. Binding of ACE2 to RBD coated receptors was quantified by BLI. [0065] Figure 33. Binding of 600nM TMPRSS2 to RBD coated receptors or empty sensors was quantified by BLI. [0066] Figure 34. Binding of TMPRSS2 to RBD coated receptors was quantified by BLI, at different concentrations. Black: fitting of the experimental curves. Left panel: HKU1A. Right panel: HKU1B. [0067] Figure 35.293T were transfected with HKU1A or B spike, expression was measured by flow cytometry by using mab10. [0068] Figure 36. Binding of TMPRSS2 to HKU1B R517A RBD coated receptors was quantified by BLI, at different concentrations. [0069] Figure 37. Binding of VHH on TMPRSS2 was measured by BLI. [0070] Figure 38. Soluble TMPRSS2 was incubated for 15 minutes with 1uM Camostat, before adding the fluorogenic substrate Boq-QAR-AMC. The initial rate of the reaction is plotted. [0071] Figure 39.293T cells were transfected with TMPRSS2 S441A, binding to VHH (0.5uM) was assessed by flow cytometry. [0072] Figure 40.293T cells either GFP 1-10 or GFP11 were transfected with TMPRSS2 and HKU1 spike in the presence of indicated amount of VHH, fusion was quantified by measuring the GFP area 20 hours post-transfection. Data were normalized to the non-treated condition for each spike. [0073] Figure 41.293T were transfected with S441A TMPRSS2.24 hours post-transfection, they were incubated with indicated amount of VHH for 2 hours and infected by Luc-encoding HKU1 spike pseudotyped viruses. Luminescence was read 48 hours post infection. Data were normalized to the non-treated condition for each virus. [0074] Figure 42. Crystal structure of A07 in complex with S441A TMPRSS2. [0075] Figure 43.293T cells WT or stably expressing TMPRSS2, R255Q TMPRSS2 or S441A TMPRSS2 were stained with a commercial antibody or VHH-A01-Fc and secondary Goat anti-Human alexa-647. [0076] Figure 44. U2OS cells and A549 cells WT or stably expressing TMPRSS2 or S441A TMPRSS2 were stained with VHH-A01-Fc and secondary Goat anti-Human alexa-647. [0077] Figure 45. Caco-2 WT, TMPRSS2 knock-out, TMPRSS2 and S441A TMPRSS2 cells were stained for TMPRSS2 using primary VHH-A01-Fc and secondary antibody Goat anti-Human Alexa- 647. Cells were analyzed by flow cytometry. [0078] Figure 46. Caco-2 cells were pre-treated with indicated concentration of monomeric VHHs for 30 min and then infected, in presence of the VHHs, with SARS-CoV-2 D614G strain at the same MOI. Cells were fixed 24h post infection and stained for SARS-CoV-2 N and analyzed by flow cytometry. [0079] Figure 47. Effect of the anti-TMPRSS2 VHH (A07) on HKU1 infection. Infected cells were visualized with an anti-spike antibody and scored. Spike pixel intensity in 5 random fields per experiment was measured and normalized to the intensity in the infected but non-treated condition. Data are mean ± SD of 3 independent experiments for infected conditions, and mean of 2 for uninfected condition. Scale bar: 20 μm. Statistical analysis: Two-sided unpaired t-test compared to non-target VHH. [0080] Figure 48. Crystal structure of the VHH-A07 complexed to TMPRSS2S441A. Important structural elements are indicated in the VHH (CDR1A07, CDR2A07, CDR3A07), as well as in TMPRSS2S441A (loops B, C, D, 1, 2, 3). Residues from the catalytic triad are shown in purple. Subscripts in the labels identify the protein. The active site S441 was mutated to alanine in the crystallized construct, so it is annotated between parentheses. [0081] Figure 49. Superposition of the TMPRSS2S441A+RBD and TMPRSS2S441A+VHH-A07 complexes. For simplicity, TMPRSS2 from the complex with the RBD is not shown. [0082] Figure 50. Crystal structure of the ternary complex: HKU1 RBD-SD1/TMPRSS2S441A/VHH- A01. [0083] Figure 51. Dose response analysis of binding of the VHH-A07-Fc and VHH-A01-Fc to TMPRSS2. [0084] Figure 52. Binding of the VHH-A07-Fc and VHH-A01-Fc to different serine proteases (TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS10, TMPRSS11A, TMPRSS11B, TMPRSS11D, TMPRSS11E, TMPRSS11F, TMPRSS13). [0085] Figure 53. Binding of the VHH-A07-Fc and VHH-A01-Fc to TMPRSS2 from different animals (Homo sapiens, Mesocricretus auratus, Macaca, Mustela furo, Mus musculus, Bos taurus, Gorilla gorilla) by flow cytometry. [0086] Figure 54. Binding of the VHH-A07-Fc and VHH-A01-Fc to TMPRSS2 naturally expressed at surface of CaCo2 cells (intestinal cells). [0087] Figure 55. Binding of the VHH-A07-Fc and VHH-A01-Fc to TMPRSS2 naturally expressed at surface of LNCap cells (human tumoral prostatic cells). DETAILED DESCRIPTION OF THE INVENTION [0088] Using cell-cell fusion and viral pseudotype assays, as well as in vitro binding tests, TMPRSS2 was identified as a high-affinity receptor for HKU1. [0089] It was demonstrated that TMPRSS2 is a receptor for hCoV-HKU1. TMPRSS2 triggers cell- cell fusion and viral entry and binds with high affinity to both HKU1A and B spikes. The enzymatic activity of TMPRSS2 is required for HKU1-dependent cell-cell fusion but not for entry of HKU1 viral pseudotypes. Viral entry is enhanced by Camostat in cells expressing the wild-type protease and is more efficient with catalytically inactive TMPRSS2 mutants. A cathepsin inhibitor decreased infection mediated by the catalytically inactive TMPRSS2, but not by the wild-type protease. [0090] This indicates that after viral binding to TMPRSS2, viral particles can either fuse at the plasma membrane if the protease is active, or be internalized and processed in the endosomal compartment. TMPRSS2 directly binds to HKU1 but not to SARS-CoV-2 and OC43 spikes. The affinity of HKU1A and HKU1B (Kd = 61-93 nM) was slightly below that reported for the SARS-CoV- 2 interaction with ACE2. A conserved groove in a region of HKU1A and B spikes composed of amino acid 505, 515, 517-521 and 528 has been proposed to be involved in binding to an unknown receptor16,17. It is shown that the 515 and 517 residues of the spike are essential for binding to TMPRSS2, viral fusion and entry, thus adding evidence that this region may be part of the RBM. The 508-520 aa region of HKU1 is deleted in OC43 (ProteinBlast), which may help explain why OC43 does not use TMPRSS2 as a receptor. [0091] Anti-TMPRSS2 VHH were isolated that inhibited binding to HKU1 spikes and prevented cell- cell fusion and viral entry. These VHH antibodies confirmed the role of TMPRSS2 as a HKU1 receptor and provide useful tools to interfere with TMPRSS2 function. [0092] As TMPRSS2 also plays a role in the development of certain cancers33, these VHH antibodies may target tumor cells or inhibit infection of viruses relying on this enzyme, as alternative to small molecule inhibitors34. [0093] Since virus stocks of HKU1 infectious virus are not currently available, because the virus is difficult to isolate and amplify for reasons that remain to be elucidated24, single-cycle pseudovirus, was used, which allowed the clear demonstration of the role of TMPRSS2 at the viral entry step. [0094] Human coronavirus receptors that have been identified so far and allow productive viral entry are cell surface proteases. TMPRSS2 and other TTSP are known to prime human coronaviruses for fusion by cleaving their spikes, generally after viral binding to target cells. Of note, Omicron strains rely less on TMPRSS2 than previous SARS-CoV-2 lineages, reflecting a constant adaptation of coronaviruses to their hosts35. The invention provides the first evidence that TMPRSS2 also acts as a direct receptor for HKU1. ACE2 binding is an ancestral and evolvable trait of sarbecoviruses36. TMPRSS2 binding may also represent another parameter of coronavirus evolution. Whether coronaviruses have lost affinity or sensitivity to TMPRSS2 during evolution, or whether HKU1 has gained affinity for TMPRSS2 remains to be determined. These results highlight the critical role of TMPRSS2 and other proteases as a determinants of coronavirus tropism and pathogenesis37. TMPRSS2 ANTAGONISTS [0095] The invention encompasses antagonists of TMPRSS2. An antagonist of TMPRSS2 is a compound that blocks or dampens a biological response by binding to and blocking TMPRSS2; whereas agonist of TMPRSS2 is a compound that activates TMPRSS2 to produce a biological response. [0096] In some embodiment, TMPRSS2 has the sequence MALNSGSPPAIGPYYENHGYQPENPYPAQPTVVPTVYEVHPAQYYPSPVPQYAPRVLTQASNPVVCTQ PKSPSGTVCTSKTKKALCITLTLGTFLVGAALAAGLLWKFMGSKCSNSGIECDSSGTCINPSNWCDGV SHCPGGEDENRCVRLYGPNFILQVYSSQRKSWHPVCQDDWNENYGRAACRDMGYKNNFYSSQGIVDDS GSTSFMKLNTSAGNVDIYKKLYHSDACSSKAVVSLRCIACGVNLNSSRQSRIVGGESALPGAWPWQVS LHVQNVHVCGGSIITPEWIVTAAHCVEKPLNNPWHWTAFAGILRQSFMFYGAGYQVEKVISHPNYDSK TKNNDIALMKLQKPLTFNDLVKPVCLPNPGMMLQPEQLCWISGWGATEEKGKTSEVLNAAKVLLIETQ RCNSRYVYDNLITPAMICAGFLQGNVDSCQGDSGGPLVTSKNNIWWLIGDTSWGSGCAKAYRPGVYGN VMVFTDWIYRQMRADG (SEQ ID NO: 24) (NCBI Reference Sequence: NP_005647.3) or a 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 said sequence. In one embodiment, the antagonist is an antibody, such as a VHH. The invention further encompasses the use of these antagonists to inhibit binding to TMPRSS2, such as the binding of HKU1 to host cells expressing TMPRSS2. SINGLE DOMAIN VHH ANTIBODIES [0097] The invention encompasses antibodies, particularly single domain VHH antibodies that bind to TMPRSS2. A VHH antibody is an antibody that comprises the variable domain of an antibody that contains two heavy chains and lacks the two light chains usually found in antibodies. In various embodiments, the single domain VHH antibodies of the invention specifically bind to TMPRSS2. [0098] Preferably, the VHH has a Kd between 0.1-100 nM or 0.8-33nM. [0099] The invention encompasses the following single domain VHH antibodies that bind to TMPRSS2: A01, A07, C11, D01, and F05. [0100] The invention encompasses a VHH comprising CDR1, CDR2, and CDR3 from any of A01, A07, C11, D01, and F05. [0101] The invention encompasses a VHH comprising a CDR1 selected from: SGFSLDYYAIG (SEQ ID NO: 4), SGSPLEHYDII (SEQ ID NO: 5), SGFTLDYYDIY (SEQ ID NO: 6), SGSTLEHYDIG (SEQ ID NO: 7), and SGFTLDYYAIG (SEQ ID NO: 8) or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR1, preferably those that are not conserved between these sequences. [0102] The invention encompasses a VHH comprising a CDR2 selected from: SCIGSSGDKTNYADSVKG (SEQ ID NO: 9), SSITTSGGHTNYADSVKG (SEQ ID NO: 10), SSITTSGGRTNYADSVKG (SEQ ID NO: 11), SSITASGGRTNYADSVKG (SEQ ID NO: 12), and SCISSSGDSIKYVDSVKG (SEQ ID NO: 13) or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR2, preferably those that are not conserved between these sequences. [0103] The invention encompasses a VHH comprising a CDR3 selected from: AAESALYSDCTEEQNPMLYDY (SEQ ID NO: 14), AGRVGGRRNWIVPLDGYDNAY (SEQ ID NO: 15), AAKVGGRRNWIAPLNGYENAL (SEQ ID NO: 16), AGKIGGRRNWVAPLDGFENAY (SEQ ID NO: 17), and AADALGSGCLTGNYDY (SEQ ID NO: 18) or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR3, preferably those that are not conserved between these sequences. [0104] The invention encompasses a VHH comprising a CDR1 selected from: SGFSLDYYAIG (SEQ ID NO: 4), SGSPLEHYDII (SEQ ID NO: 5), SGFTLDYYDIY (SEQ ID NO: 6), SGSTLEHYDIG (SEQ ID NO: 7), and SGFTLDYYAIG (SEQ ID NO: 8) or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR1 , preferably those that are not conserved between these sequences; a CDR2 selected from: SCIGSSGDKTNYADSVKG (SEQ ID NO: 9), SSITTSGGHTNYADSVKG (SEQ ID NO: 10), SSITTSGGRTNYADSVKG (SEQ ID NO: 11), SSITASGGRTNYADSVKG, (SEQ ID NO: 12) and SCISSSGDSIKYVDSVKG (SEQ ID NO: 13) or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR2, preferably those that are not conserved between these sequences; and a CDR3 selected from: AAESALYSDCTEEQNPMLYDY (SEQ ID NO: 14), AGRVGGRRNWIVPLDGYDNAY (SEQ ID NO: 15), AAKVGGRRNWIAPLNGYENAL (SEQ ID NO: 16), AGKIGGRRNWVAPLDGFENAY (SEQ ID NO: 17), and AADALGSGCLTGNYDY (SEQ ID NO: 18) or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR3, preferably those that are not conserved between these sequences. [0105] The invention encompasses a VHH comprising a CDR1 selected from: SGSPLEHYDII (SEQ ID NO: 5), SGFTLDYYDIY (SEQ ID NO: 6), and SGSTLEHYDIG (SEQ ID NO: 7), or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR1, preferably those that are not conserved between these sequences; a CDR2 selected from: SSITTSGGHTNYADSVKG (SEQ ID NO: 10), SSITTSGGRTNYADSVKG (SEQ ID NO: 11), and SSITASGGRTNYADSVKG (SEQ ID NO: 12), or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR2, preferably those that are not conserved between these sequences; and a CDR3 selected from: AGRVGGRRNWIVPLDGYDNAY (SEQ ID NO: 15), AAKVGGRRNWIAPLNGYENAL (SEQ ID NO: 16), and AGKIGGRRNWVAPLDGFENAY (SEQ ID NO: 17), or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR3 preferably those that are not conserved between these sequences . [0106] The invention encompasses a VHH comprising a CDR1 selected from: SGFSLDYYAIG (SEQ ID NO: 4) and SGFTLDYYAIG (SEQ ID NO: 8), or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR1, preferably those that are not conserved between these sequences; a CDR2 selected from: SCIGSSGDKTNYADSVKG (SEQ ID NO: 9) and SCISSSGDSIKYVDSVKG (SEQ ID NO: 13), or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR2, preferably those that are not conserved between these sequences; and a CDR3 selected from: AAESALYSDCTEEQNPMLYDY (SEQ ID NO: 14) and AADALGSGCLTGNYDY (SEQ ID NO: 18), or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to the sequence of said CDR3, preferably those that are not conserved between these sequences. [0107] The invention encompasses a VHH comprising a CDR1 selected from: SGSPLEHYDII (SEQ ID NO: 5), SGFTLDYYDIY (SEQ ID NO: 6), and SGSTLEHYDIG (SEQ ID NO: 7); a CDR2 selected from: SSITTSGGHTNYADSVKG (SEQ ID NO: 10), SSITTSGGRTNYADSVKG (SEQ ID NO: 11), and SSITASGGRTNYADSVKG (SEQ ID NO: 12); and a CDR3 selected from: AGRVGGRRNWIVPLDGYDNAY (SEQ ID NO: 15), AAKVGGRRNWIAPLNGYENAL (SEQ ID NO: 16), and AGKIGGRRNWVAPLDGFENAY (SEQ ID NO: 17). [0108] In some embodiments, the single domain VHH antibody can bind to the biologically active ectodomain of TMPRSS2. In some embodiments, the single domain VHH antibody can bind to TMPRSS2 and reduce its catalytic activity. In preferred embodiments, the single domain VHH antibody is A07, C11, or D01. [0109] In some embodiments the single domain VHH antibody comprises the following CDR domains: CDR1: SGFSLDYYAIG (SEQ ID NO: 4); CDR2: SCIGSSGDKTNYADSVKG (SEQ ID NO: 9); and CDR3: AAESALYSDCTEEQNPMLYDY (SEQ ID NO: 14). [0110] In some embodiments the single domain VHH antibody comprises the following CDR domains: CDR1: SGSPLEHYDII (SEQ ID NO: 5); CDR2: SSITTSGGHTNYADSVKG (SEQ ID NO: 10); and CDR3: AGRVGGRRNWIVPLDGYDNAY (SEQ ID NO: 15). [0111] In some embodiments the single domain VHH antibody comprises the following CDR domains: CDR1: SGFTLDYYDIY (SEQ ID NO: 6); CDR2: SSITTSGGRTNYADSVKG (SEQ ID NO: 11); and CDR3: AAKVGGRRNWIAPLNGYENAL (SEQ ID NO: 16). [0112] In some embodiments the single domain VHH antibody comprises the following CDR domains: CDR1: SGSTLEHYDIG (SEQ ID NO: 7); CDR2: SSITASGGRTNYADSVKG (SEQ ID NO: 12); and CDR3: AGKIGGRRNWVAPLDGFENAY (SEQ ID NO: 17). [0113] In some embodiments the single domain VHH antibody comprises the following CDR domains: CDR1: SGFTLDYYAIG (SEQ ID NO: 8); CDR2: SCISSSGDSIKYVDSVKG (SEQ ID NO: 13); and CDR3: AADALGSGCLTGNYDY (SEQ ID NO: 18). [0114] In some embodiments the single domain VHH antibody comprises the following CDR domains: CDR1: SG(S/F)(P/T)L(E/D)(H/Y)YDI(I/Y/G) (SEQ ID NO : 1); CDR2: SSIT(T/A)SGGRTNYADSVKG (SEQ ID NO: 2); and CDR3: A(G/A)(K/R)(V/I)GGRRNW(I/V)APLNG(Y/F)ENA(Y/L) (SEQ ID NO: 3). [0115] In some embodiments, the VHH antibody contains 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 A01, A07, C11, D01, and F05. [0116] In some embodiments, the VHH antibody comprises a CDR1 selected from: SGFSLDYYAIG (SEQ ID NO: 4), SGSPLEHYDII (SEQ ID NO: 5), SGFTLDYYDIY (SEQ ID NO: 6), SGSTLEHYDIG (SEQ ID NO: 7), and SGFTLDYYAIG (SEQ ID NO: 8); a CDR2 selected from: SCIGSSGDKTNYADSVKG (SEQ ID NO: 9), SSITTSGGHTNYADSVKG (SEQ ID NO: 10), SSITTSGGRTNYADSVKG (SEQ ID NO: 11), SSITASGGRTNYADSVKG (SEQ ID NO: 12), and SCISSSGDSIKYVDSVKG (SEQ ID NO: 13); and a CDR3 selected from: AAESALYSDCTEEQNPMLYDY (SEQ ID NO: 14), AGRVGGRRNWIVPLDGYDNAY (SEQ ID NO: 15), AAKVGGRRNWIAPLNGYENAL (SEQ ID NO: 16), AGKIGGRRNWVAPLDGFENAY (SEQ ID NO: 17), and AADALGSGCLTGNYDY (SEQ ID NO: 18) and contains 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 A01 (SEQ ID NO: 19), A07 (SEQ ID NO: 20), C11 (SEQ ID NO: 21), D01 (SEQ ID NO: 22), or F05 (SEQ ID NO: 23). [0117] In some embodiments, the VHH antibody contains an amino acid sequence that has at least 100, 105, 110, 115, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, or 130, amino acids identical to the amino acid sequence of A01, A07, C11, D01, or F05. [0118] In some embodiments, the VHH antibody comprises a CDR1 selected from: SGFSLDYYAIG (SEQ ID NO: 4), SGSPLEHYDII (SEQ ID NO: 5), SGFTLDYYDIY (SEQ ID NO: 6), SGSTLEHYDIG (SEQ ID NO: 7), and SGFTLDYYAIG (SEQ ID NO: 8); a CDR2 selected from: SCIGSSGDKTNYADSVKG (SEQ ID NO: 9), SSITTSGGHTNYADSVKG (SEQ ID NO: 10), SSITTSGGRTNYADSVKG (SEQ ID NO: 11), SSITASGGRTNYADSVKG (SEQ ID NO: 12), and SCISSSGDSIKYVDSVKG (SEQ ID NO: 13); and a CDR3 selected from: AAESALYSDCTEEQNPMLYDY (SEQ ID NO: 14), AGRVGGRRNWIVPLDGYDNAY (SEQ ID NO: 15), AAKVGGRRNWIAPLNGYENAL (SEQ ID NO: 16), AGKIGGRRNWVAPLDGFENAY (SEQ ID NO: 17), and AADALGSGCLTGNYDY (SEQ ID NO: 18) and contains an amino acid sequence that has at least 100, 105, 110, 115, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, or 130, amino acids identical to the amino acid sequence of A01, A07, C11, D01, or F05. [0119] In some embodiments, the antibody is directed against residues 275-473 of TMPRSS2. In various embodiments, the antibody directed against residues 275-473 of TMPRSS2 is a VHH antibody (e.g., A07). [0120] In some embodiments, the antibody binds to an epitope that comprises one or more amino acid residues selected from the group consisting of V275, Q276, V278, H279, V280, C281, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, E389, K390, T393, V415, Y416, D417, N418, L419, D435, S436, C437, Q438, G439, A441, T459, S460, W461, G462, S463, G464, C465, R470, G472 and V473 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24. [0121] In some embodiments, the antibody binds to an epitope that comprises at least 10, at least 20, at least 30, at least 40 of the amino acid residues selected from the group consisting of V275, Q276, V278, H279, V280, C281, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, E389, K390, T393, V415, Y416, D417, N418, L419, D435, S436, C437, Q438, G439, A441, T459, S460, W461, G462, S463, G464, C465, R470, G472 and V473 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24. [0122] In some embodiments, the antibody binds to an epitope that comprises the amino acid residues V275, Q276, V278, H279, V280, C281, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, E389, K390, T393, V415, Y416, D417, N418, L419, D435, S436, C437, Q438, G439, A441, T459, S460, W461, G462, S463, G464, C465, R470, G472 and V473 and optionally of one or more of G385, A386, D440, K467, and Y474 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24. [0123] In some embodiments, the antibody binds to an epitope that consists of the amino acid residues V275, Q276, V278, H279, V280, C281, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, E389, K390, T393, V415, Y416, D417, N418, L419, D435, S436, C437, Q438, G439, A441, T459, S460, W461, G462, S463, G464, C465, R470, G472 and V473 and optionally of one or more of G385, A386, D440, K467, and Y474 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24. [0124] In some embodiments, the antibody binds to an epitope that comprises one or more amino acid residues selected from the group consisting of V275, Q276, V278, H279, V280, C281, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, G385, A386, E389, K390, T393, V415, Y416, D417, N418, L419, D435, S436, C437, Q438, G439, D440, A441, T459, S460, W461, G462, S463, G464, C465, K467, R470, G472, V473 and Y474 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24. [0125] In some embodiments, the antibody binds to an epitope that comprises at least 10, at least 20, at least 30 of the amino acid residues selected from the group consisting for V275, Q276, V278, H279, V280, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, E389, T393, V415, Y416, D417, N418, L419, S436, C437, G439, A441, T459, S460, W461, G462, S463 and R470, and optionally of one or more of C281, G385, A386, K390, D435, Q438, D440, G464, C465, K467, G472, V473 and Y474 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24. [0126] In some embodiments, the antibody binds to an epitope that comprises the amino acid residues V275, Q276, V278, H279, V280, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, E389, T393, V415, Y416, D417, N418, L419, S436, C437, G439, A441, T459, S460, W461, G462, S463 and R470, and optionally of one or more of C281, G385, A386, K390, D435, Q438, D440, G464, C465, K467, G472, V473 and Y474 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24. [0127] In some embodiments, the antibody binds to an epitope that consists of the amino acid residues V275, Q276, V278, H279, V280, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, E389, K390, T393, V415, Y416, D417, N418, L419, D435, S436, C437, Q438, G439, A441, T459, S460, W461, G462, S463, G464, C465, R470, G472 and V473 and optionally of one or more of C281, G385, A386, D440, K467, and Y474 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24. [0128] In some embodiments, the antibody binds to an epitope that comprises one or more amino acid residues selected from the group consisting of R150, Y152, Q159, Y161, K166, S167, W168, H169, S204, G205, S206, T207, P367, P369, G370, M371, M372, L373, Q374, P375, I404, I405, E406, T407, Q408, N411, I420, T421, P422, M424, I425, I456, N476, M478, V479, T481 and D482 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24. [0129] In some embodiments, the antibody binds to an epitope that comprises at least 10, at least 20, at least 30 amino acid residues selected from the group consisting of R150, Y152, Q159, Y161, K166, S167, W168, H169, S204, G205, S206, T207, P367, P369, G370, M371, M372, L373, Q374, P375, I404, I405, E406, T407, Q408, N411, I420, T421, P422, M424, I425, I456, N476, M478, V479, T481 and D482 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24. [0130] In some embodiments, the antibody binds to an epitope that comprises the amino acid residues R150, Y152, Q159, Y161, K166, S167, W168, H169, S204, G205, S206, T207, P367, P369, G370, M371, M372, L373, Q374, P375, I404, I405, E406, T407, Q408, N411, I420, T421, P422, M424, I425, I456, N476, M478, V479, T481 and D482 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24. [0131] In some embodiments, the antibody binds to an epitope that consists of amino acid residues R150, Y152, Q159, Y161, K166, S167, W168, H169, S204, G205, S206, T207, P367, P369, G370, M371, M372, L373, Q374, P375, I404, I405, E406, T407, Q408, N411, I420, T421, P422, M424, I425, I456, N476, M478, V479, T481 and D482 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24. [0132] [0133] The epitope may be a continuous or discontinuous epitope. Preferably, the epitope is discontinuous.The invention further encompasses the use of these antibodies, especially these VHH to inhibit binding to TMPRSS2. FUSION PROTEINS [0134] In another aspect a fusion protein comprising a single domain VHH antibody of the invention and a second polypeptide or protein that is not a VHH is provided. [0135] As used herein a "fusion protein" refers to a polypeptide having two 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 two portions and the linker will be in reading frame with each other. Preferably, the 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 VHH antibody of the invention. [0136] In the fusion protein of the present invention, the single domain VHH antibody may be directly fused or linked via a linker moiety to the other elements of the fusion protein. 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: 37), (Gly)5 (SEQ ID NO: 38), (Gly)4Ser (SEQ ID NO: 39), (Gly)4(Ser)(Gly)4 (SEQ ID NO: 40), or combinations thereof or a multimer thereof (for example a dimer, a trimer, or a tetramer, or greater). For example, a suitable linker may be (GGGGS)3 (SEQ ID NO: 41). Alternative linkers include (Ala)3(His)6 (SEQ ID NO: 42) or multimers thereof. Also included is a linker sequence which has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, using the default parameters of the BLAST computer program. [0137] In some embodiments of the fusion protein, the second polypeptide or protein is selected from a Fab, Fc, F(ab’)2 (including chemically linked F(ab’)2 chains), Fab’, scFv (including multimer forms thereof, i.e. di-scFv, or tri-scFv), sdAb, or BiTE (bi-specific T-cell engager). [0138] In some embodiments the second polypeptide is a Fc fragment of a mammalian immunoglobulin. [0139] In some embodiments the Fc fragment of a mammalian immunoglobulin has the sequence EPKTPKPQPAAARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 25) [0140] Preferably, the fusion protein with the Fc has a Kd between 0.01-100 nM. [0141] In some embodiment, the fusion protein is the VHH A01-Fc having the amino acid sequence SEQ ID NO: 26. [0142] In some embodiment, the fusion protein is the VHH A07-Fc having the amino acid sequence SEQ ID NO: 27. MULTIMERIC VHH ANTIBODIES [0143] Multimeric VHH antibodies are also provided. The multimeric VHH antibody comprises at least one VHH of the invention, preferably at least two VHHs of the invention. Each VHH present in a multimeric VHH antibody may be in the form of a fusion protein or may not be in the form of a fusion protein. [0144] In some embodiments the multimeric VHH antibody is a dimer. In some embodiments the dimer comprises two copies of the same VHH antibody (homodimer). In some embodiments the dimer comprises one copy of each of two different VHH antibodies (heterodimer). [0145] In some embodiments the multimeric VHH antibody is a trimer. In some embodiments the trimer comprises two copies of the same VHH antibody and a third copy of a different VHH antibody (heterotrimer type 1). In some embodiments the trimer comprises one copy of each of three different VHH antibodies (heterotrimer type 2). In some embodiments the trimer comprises three copies of the same VHH antibody (homotrimer). [0146] Each VHH present in the multimeric VHH antibody may be the same as at least one other VHH in the multimeric VHH antibody. Alternatively, each VHH present in the multimeric VHH antibody may be different than all other VHH antibodies present in the multimeric VHH antibody. [0147] In certain embodiments the multimeric VHH antibodies are Fc fusion proteins. In such embodiment the Fc portion may be responsible for linking the VHHs together into the multimeric form. [0148] In certain embodiments the VHH antibodies may be linked together via one or more type of linker, such as a Gly-Ser linker. [0149] When the VHH-Fc fusion polypeptides are expressed in a suitable recombinant cell type dimeric VHH antibodies will be formed. In some embodiments a single VHH-Fc fusion polypeptide is expressed, and homodimers are formed. In other embodiments a plurality of VHH-Fc fusion polypeptides are expressed, and a mixture of homodimers and heterodimers are formed. Therefore, in some embodiments the dimeric VHH antibody is a homodimer. In other embodiments, the dimeric VHH antibody is a heterodimer. Mixtures comprising VHH antibody homodimers, mixtures comprising VHH antibody heterodimers, and mixtures comprising VHH antibody homodimers and heterodimers are also provided. [0150] The invention encompasses a combination of a VHH antibody according to the invention with at least another antibody. In one embodiment, the other antibody is an antibody, especially a VHH antibody, directed against ACE2. In various embodiment, the combination is in a multimeric form. METHODS OF MAKING VARIANTS [0151] The invention encompasses methods of making variants of A01, A07, C11, D01, and F05. In some embodiments, the DNA sequence encoding A01, A07, C11, D01, or F05 is changed by one or more nucleotides encoding one or more amino acids of the VHH to generate a variant of A01, A07, C11, D01, or F05. [0152] Thus, a variant of A07 has at least one amino acid different than A07. A variant of A01, A07, C11, D01, and F05 has at least one amino acid different than A01, A07, C11, D01, and F05, respectively. A variant can contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9 ,10, 11, 12, 13, 14, 15, 20, 25, 30, 40, or 50, etc., amino acids different than A01, A07, C11, D01, and/or F05. A variant can contain an amino acid sequence that has at least 100, 105, 110, 115, 120, 121, 122, 123, 124, 125, 126, 127, 128, or 129 amino acids identical to the amino acid sequence of A01, A07, C11, D01, and/or F05. [0153] The variant can be generated in a recombinant vector comprising a DNA sequence encoding A01, A07, C11, D01, or F05. Preferably, the vector is an expression vector. NUCLEIC ACIDS AND CELLS [0154] In another aspect an isolated nucleic acid sequence that encodes the antibody of the invention, especially the single domain VHH antibody or a fusion protein comprising a single domain VHH antibody of the invention is provided. [0155] In some embodiments the single domain VHH antibody binds to the catalytic domain of TMPRSS2. [0156] In some embodiments the single domain VHH antibody is a dimer of VHH. [0157] In some embodiments, the VHH is selected from A07, C11 and D01. [0158] In some embodiments, the VHH is selected from A01 and F05. [0159] In some embodiments, the VHH binds to the residues 275-473, around TMPRSS2 catalytic site. [0160] Also provided are recombinant vectors comprising the isolated nucleic acid sequence of the invention. 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 VHH antibody, either in vitro or in vivo. [0161] In one embodiment, the expression vector is pHEN6, pET23, pET22 or pASK-IBA2. In one embodiment, the expression vector encodes a protease cleavage site, such as TEV cleave site, inserted between the single domain VHH 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. [0162] 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 sequence enabling conditional expression, such as sequences of the Cre/Lox system or analogue systems. [0163] In various embodiments, the expression vector is a plasmid, a phage for bacterium introduction, a YAC able to transform yeast, a viral vector, or any expression vector. An expression vector as defined herein is chosen to enable the production of an antibody of the invention, especially a single domain VHH antibody of the invention or a fusion protein comprising a single domain VHH antibody of the invention, either in vitro or in vivo. [0164] 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. [0165] 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. [0166] 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. [0167] In another aspect methods of producing the antibody of the invention, especially single domain VHH antibody or the fusion protein comprising a single domain VHH antibody are provided. The methods comprise culturing the recombinant cell comprising the isolated nucleic acid sequence that encodes the antibody of the invention, especially the single domain VHH antibody or the fusion protein comprising a single domain VHH antibody under conditions sufficient for production of the antibody of the invention, especially the single domain VHH antibody. PHARMACEUTICAL COMPOSITIONS, USES, AND METHODS [0168] Also provided are pharmaceutical compositions of an antibody according to the invention, especially an isolated single domain VHH antibody according to the invention, a monomeric fusion polypeptide according to the invention, or a multimeric VHH antibody according to the invention, especially a dimeric VHH antibody according to the invention. The pharmaceutical composition may comprise one or a plurality of single domain VHH antibody according to the invention; and/or one or a plurality of monomeric fusion polypeptide according to the invention; and/or one or a plurality of multimeric VHH antibody according to the invention, especially one or a plurality of dimeric VHH antibody according to the invention. [0169] In some embodiments, the VHH is selected from A07, C11 and D01. [0170] In some embodiments, the VHH is selected from A01 and F05. [0171] In some embodiments, the antibody is VHH A01-Fc. [0172] In some embodiments, the antibody is VHH A07-Fc. [0173] Preferably, the antibody is selected from the group consisting of the VHH A07 having the amino acid sequence SEQ ID NO: 20, the VHH C11 having the amino acid sequence SEQ ID NO: 21, the VHH D01 having the amino acid sequence SEQ ID NO: 22, and the VHH A07-Fc having the amino acid sequence SEQ ID NO: 27. More preferably, the antibody is the VHH A07-Fc having the amino acid sequence SEQ ID NO: 27. [0174] 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. [0175] 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. [0176] The composition can be an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic. [0177] The pharmaceutical composition preferably comprises a dose of 100µg to 1g, for example a dose of at least 100µg, 250 µg, 500 µg, 1mg, 2mg, 5mg, 10mg, 25mg, 50mg, 100mg, 250mg, or 500mg. METHODS OF INHIBITING BINDING [0178] The invention encompasses methods for inhibiting coronavirus binding to host cells with an isolated single domain VHH antibody according to the invention, a monomeric fusion polypeptide according to the invention, or a multimeric VHH antibody according to the invention, especially a dimeric VHH antibody according to the invention. [0179] In one embodiment, the method comprises contacting an isolated single domain VHH antibody according to the invention with host cells, prior to or at the same time that the cells are contacted with a coronavirus. Preferably, the single domain VHH antibody reduces binding of the coronavirus to the host cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. [0180] In various embodiments, the coronavirus is SARS-CoV-2 or HKU1. [0181] In some embodiments the method utilizes a single domain VHH antibody that binds to the catalytic domain of TMPRSS2. [0182] In some embodiments the single domain VHH antibody is a dimer of VHH. [0183] In some embodiments, the VHH is selected from A07, C11 and D01. [0184] In some embodiments, the VHH binds to the residues 275-473, around TMPRSS2 catalytic site. METHODS OF PREVENTING OR TREATING VIRAL INFECTIONS [0185] The invention encompasses preventing or treating viral infections mediated by TMPRSS2, including SARS-CoV2 infection and especially HKU1 infection, by administering an antibody according to the invention, especially an isolated single domain VHH antibody according to the invention, a monomeric fusion polypeptide of the invention, or a multimeric VHH antibody of the invention, especially a dimeric VHH antibody of the invention. [0186] In some embodiments, the VHH is selected from A07, C11 and D01. [0187] In some embodiments, the VHH is selected from A01 and F05. [0188] In some embodiments, the antibody is VHH A01-Fc. [0189] In some embodiments, the antibody is VHH A07-Fc. [0190] Preferably, the antibody is selected from the group consisting of the VHH A07 having the amino acid sequence SEQ ID NO: 20, the VHH C11 having the amino acid sequence SEQ ID NO: 21, the VHH D01 having the amino acid sequence SEQ ID NO: 22, and the VHH A07-Fc having the amino acid sequence SEQ ID NO: 27. More preferably, the antibody is the VHH A07-Fc having the amino acid sequence SEQ ID NO: 27. [0191] In various embodiments, the viral infection is an Influenza A and B virus infection, a metapneumovirus or parainfluenzavirus infection, or a SARS, MERS, or 229E coronavirus infection. [0192] In various embodiments of the methods, a pharmaceutical composition comprising an antibody according to the invention, especially an isolated single domain VHH antibody of the invention, a monomeric fusion polypeptide of the invention, or a multimeric VHH antibody of the invention, especially a dimeric VHH antibody of the invention is administered to a patient having a viral infection. [0193] In some embodiments, an antibody according to the invention, a monomeric fusion polypeptide according to the invention, or a multimeric VHH antibody according to the invention, especially a dimeric VHH antibody according to the invention is administered to a subject that has been tested positive for a coronavirus, including SARS-CoV-2, and especially HKU1, but has not yet manifested symptoms of infection. In some embodiments of the methods administering the pharmaceutical composition to the subject reduces the coronavirus viral load of the subject. [0194] In some embodiments, a pharmaceutical composition of the invention is administered to a patient that has manifested symptoms of a coronavirus infection, including of SARS-CoV2 infection, and especially of HKU1 infection. 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. [0195] 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. [0196] For administration to mammals, and particularly humans, it is expected that the daily dosage of the active agent will be from 0.01mg/kg body weight, typically around 1mg/kg, 2mg/kg or up to 4mg/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. [0197] In some embodiments of the methods, the pharmaceutical composition is administered to a subject that has been tested positive for prostate cancer. In some embodiments, administering the pharmaceutical composition to the subject inhibits the growth of the prostate cancer of the subject. [0198] The invention encompasses preventing or treating an HKU1 infection, by administering a TMPRSS2 antagonist. METHODS OF DETECTION [0199] In another aspect, methods for detection of TMPRSS2 in a biological sample are provided. The methods may comprise providing an antibody according to the invention, especially a single domain VHH antibody according to this disclosure; providing a biological sample; contacting the single domain VHH antibody with the biological sample; and visualizing the antigen-antibody complexes formed. In some embodiments the methods comprise an ELISA, LuLisa, lateral flow immunoassay, bead-based immunoassay, or multiplex bead-based immunoassay. [0200] In some embodiments the single domain VHH antibody is a dimer of VHH. [0201] In some embodiments, the VHH is selected from A07, C11 and D01. [0202] In some embodiments, the VHH is selected from A01 and F05. [0203] In some embodiments, the antibody is VHH A01-Fc. [0204] In some embodiments, the antibody is VHH A07-Fc. [0205] Preferably, the antibody is selected from the group consisting of the VHH A01 having the amino acid sequence SEQ ID NO: 19, the VHH F05 having the amino acid sequence SEQ ID NO: 23 and the VHH A01-Fc having the amino acid sequence SEQ ID NO: 26. More preferably, the antibody is the VHH A01-Fc having the amino acid sequence SEQ ID NO: 26. [0206] In some embodiments the method for detection of TMPRSS2 in a biological sample comprises providing a first single domain VHH antibody according to this disclosure, attached to a solid support; providing a biological sample from a subject; contacting the solid support with the biological sample under conditions sufficient to allow formation of first antigen-antibody complexes between TMPRSS2 in the biological sample and the VHH antibody attached to the solid support to form first antigen- antibody complexes; contacting the solid support with a second single domain VHH antibody according to any this disclosure under conditions sufficient to allow formation of second antigen- antibody complexes between TMPRSS2 and the second single domain VHH antibody; and visualizing the second antigen-antibody complexes. In some embodiments, the second single domain VHH antibody according to the invention is labeled and visualizing the second antigen-antibody complexes comprises visualizing the label. [0207] In some embodiments the method utilizes a single domain VHH antibody that binds to the catalytic domain of TMPRSS2. [0208] In some embodiments, the VHH binds to the residues 275-473, around TMPRSS2 catalytic site. [0209] In some embodiments the method utilizes a single domain VHH antibody that further comprises a label. [0210] In some embodiments the method utilizes a single domain VHH antibody that is covalently attached to a substrate. [0211] A skilled artisan will appreciate that the single domain VHH antibodies can be used in any suitable assay format known in the art that is designed to utilize antibodies. In particular by an immunoassay, such as an immunoenzymatic method (e.g., ELISA). [0212] The invention encompasses a composition comprising a single domain VHH antibody for detection of TMPRSS2 in a biological sample. [0213] The single domain VHH antibody according to the invention is useful for the direct detection of TMPRSS2; the detection of TMPRSS2 can be carried out by an appropriate technique, in particular EIA, ELISA, RIA, immunofluorescence, luminescence in a biological sample. [0214] In one embodiment, the single domain VHH antibody is attached to an appropriate support, in particular a microplate or a bead. [0215] In one embodiment, the method for the detection of TMPRSS2 in a biological sample comprises providing a single domain VHH antibody of this disclosure; providing a biological sample from a patient; contacting said single domain VHH antibody with said biological sample; and visualizing the antigen-antibody complexes formed. Preferably, the method comprises an ELISA. [0216] Preferably, the protein-antibody complexes are detected with a second single domain VHH antibody that binds to TMPRSS2. [0217] Preferably, the second single domain VHH antibody comprises a label selected from a chemiluminescent label, an enzyme label, a fluorescence label, and a radioactive (e.g., iodine) label. [0218] Preferred labels include a fluorescent label, such as FITC, a chromophore label, an affinity- ligand label, an enzyme label, such as alkaline phosphatase or a luciferase, horseradish peroxidase, or β 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. [0219] Preferred labels include suitable enzymes such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, 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, 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 include 14C, 123I, 124I, 125I, 32P, 33P, 35S, or 3H. [0220] Preferably, the method comprises comparing the results obtained with a positive and/or negative control. In various embodiments, the method comprises quantification of the TMPRSS2 detected. METHODS OF DIAGNOSING PROSTATE CANCER [0221] Several reports have shown that TMPRSS2 is overexpressed in prostate cancer and that its expression level correlated with cancer progression. [0222] The invention encompasses methods for diagnosing prostate cancer and/or following cancer progression and/or response to treatment. In various embodiments, the method comprises providing a biological sample from a patient, contacting the sample with an antibody, especially a VHH antibody, of the invention, and detecting the complexes formed between the antibody, especially the VHH antibody, and TMPRSS2. Such methods can be used to quantitate the expression of TMPRSS2, for example, by comparing the level of complexes to positive and/or negative controls. [0223] In some embodiments, the VHH is selected from A07, C11 and D01. [0224] In some embodiments, the VHH is selected from A01 and F05. [0225] In some embodiments, the antibody is VHH A01-Fc. [0226] In some embodiments, the antibody is VHH A07-Fc. [0227] Preferably, the antibody is selected from the group consisting of the VHH A01 having the amino acid sequence SEQ ID NO: 19, the VHH F05 having the amino acid sequence SEQ ID NO: 23 and the VHH A01-Fc having the amino acid sequence SEQ ID NO: 26. More preferably, the antibody is the VHH A01-Fc having the amino acid sequence SEQ ID NO: 26. [0228] In various embodiments, multiple samples provided from the same patient are taken over time and the expression of TMPRSS2 is quantitated over time. [0229] In various embodiments, samples from the same patient are provided before, during and/or after a cancer treatment and the expression of TMPRSS2 is quantitated to determine the effect of the treatment on TMPRSS2 expression. EXAMPLES MATERIAL AND METHODS EXAMPLE 1. Sequence alignments [0230] Alignments were performed using Protein Blast with default settings (NCBI). Alignment figures were done using the seqvisr package in R (Github: doi. org / 10.5281/zenodo.6583981). EXAMPLE 2. Plasmids [0231] Codon-optimized HKU1A (RefSeq: YP_173238.1) and B isolate N5 (UniProtKB/Swiss-Prot: Q0ZME7.1) full spikes were ordered as a synthetic gene (GeneArt, Thermo Fisher Scientific) and were cloned into a phCMV backbone (GeneBank: AJ318514), by replacing the VSV-G gene. pQCXIP-Empty control plasmid was previously described (Buchrieser J, Degrelle SA, Couderc T, Nevers Q, Disson O, Manet C, Donahue DA, Porrot F, Hillion KH, Perthame E et al (2019) IFITM proteins inhibit placental syncytiotrophoblast formation and promote fetal demise. Science 365: 176– 180). pQCXIP-BSR-GFP11 and pQCXIP-GFP1-10 were a kind gift from Yutaka Hata (Kodaka M, Yang Z, Nakagawa K, Maruyama J, Xu X, Sarkar A, Ichimura A, Nasu Y, Ozawa T, Iwasa H et al (2015) A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells. Exp Cell Res 336: 171–181) (Addgene plasmid #68716 and #68715). pCSDest-TMPRSS2 was a kind gift from Roger Reeves (Edie S, Zaghloul NA, Leitch CC, Klinedinst DK, Lebron J, Thole JF, McCallion AS, Katsanis N, Reeves RH (2018) Survey of human chromosome 21 gene expression effects on early development in Danio Rerio. G38: 2215–2223)) (Addgene plasmid # 53887). All mutation in the HKU1 spike and in TMPRSS2 were introduced using the NEB Q5 Side-Directed mutagenesis kit. Plasmids were sequenced using the primers described (Sup). phCMV-HKU1-S-mNeonGreen and pCDEST-TMPRSS2-mScarlet-I was generated by Gibson assembly. Codon-optimized synthetic genes coding for the TMPRSS2 from Mus musculus (Mouse C57BL/6 - UniPropt: Q3UKE3), Mustela furo (Ferret – UniProt: A0A8U0SMZ2), Mesocricetus auratus (Syrian Hamster Isoform X1 – UniProt: A0A1U8BWQ2), Gorilla (Gorilla-gorilla-gorilla - XP_055229600.1), Bos taurus (Bos-taurus - XP_015329141.1) and macaque (Macaque – UniProt: F6SVR2) with an Nterminal cMYC-tag were ordered to GeneArt (Thermo Fisher Scientific) and cloned into a phCMV backbone (GeneBank: AJ318514) by replacing the VSV-G gene. pCAGGS-based expression vectors N-terminal MYC- epitope tagged TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS10, TMPRSS11A, TMPRSS11B, TMPRSS11D, TMPRSS11E, TMPRSS11F and TMPRSS13 were a kind gift from Stefan Pöhlmann (Hoffmann, M. et al. Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity. EBioMedicine 65, 103255 (2021)). EXAMPLE 3. Protein production [0232] Construct design [0233] Spike, RBD, TMPRSS2 and ACE2 constructs were obtained from Genscript as codon- optimized synthetic genes. Ectodomains from HKU1A (residues 14-1281) and B (residues 14-1276) were cloned into pcDNA3.1(+), downstream of a murine Ig kappa signal peptide (METDTLLLWVLLLWVPGSTG (SEQ ID NO: 43)) and upstream of a thrombin cleavage site followed by a His-tag. Spikes were stabilized by mutating the furin cleavage site (756RRKRR760 (SEQ ID NO: 44)>756GGSGS760(SEQ ID NO: 45) in HKU1A; 752RRKRR756(SEQ ID NO: 44)>752GGSGS756(SEQ ID NO: 45) in HKU1B;), two residues in the S2 subunit (1071AL1072>1071PP1072 in HKU1A; 1067NL1068>1067PP1068 in HKU1B) and adding a Foldon trimerization motif at the C-terminus. The ectodomain from the Wuhan SARS-CoV-2 Spike (residues 1-1208) was cloned into pcDNA3.1(+) and was stabilized with 6 proline mutations (F817P, A892P, A899P, A942P, K986P, V987P), as reported.Hsieh, C.-L. et al. Structure-based design of prefusion-stabilized SARS-CoV-2 spikes. Science 369, 1501-1505 (2020). The furin site was also replaced as above (682RRAR685 (SEQ ID NO: 30)>682GSAS685(SEQ ID NO: 31)), a C-terminal Foldon motif was introduced, as well as Hisx8, Strep, and Avi tags. [0234] The RBDs (residues 323-609 for HKU1A; 323-607 for HKU1B; 331-528 for SARS-CoV-2 Wuhan) were cloned into pCAGGS (HKU1) or pcDNA3.1(+) (SARS-CoV-2), following a murine immunoglobulin kappa signal peptide, and upstream of a thrombin cleavage site and in-tandem Hisx8, Strep and Avi-tags. The WT TMPRSS2 ectodomain (residues 107-492) followed by C-terminal tags (8xHis-tag and AviTag) was synthesized and cloned into pcDNA3.1/Zeo(+) expression vector (Thermo Fisher Scientific). The TMPRSS2 ectodomain with the S441A mutation was cloned into a modified pMT/BiP plasmid (Invitrogen; hereafter termed pT350), which translates the protein in frame with an enterokinase cleavage site and a double strep-tag at the C-terminal end. The ACE2 peptidase domain (residues 19-615) was cloned in pcDNA3.1(+) with a murine immunoglobulin kappa signal peptide and a C-terminal thrombin cleavage site followed by a Strep-tag. The coding sequences of the selected VHHs in the vector pHEN6 were subcloned into a bacterial expression vector pET23 encoding a C terminal His-tag using NcoI and NotI restriction sites. [0235] Protein expression and purification [0236] RBD and ACE2-encoding plasmids were transiently transfected into Expi293FTM cells (Thermo-Fischer) using FectoPro ^ DNA transfection reagent (PolyPlus). Spike encoding plasmids were transiently transfected into Expi293FTM cells (Thermo-Fischer) using polyethylenimine (PEI) precipitation method, as previously described. Lorin et al., J Immunol Methods 2015 Jul;422:102-10. After 5 days at 37 °C, cells were harvested by centrifugation and the supernatants were concentrated. The spike proteins were purified from culture supernatants by high-performance chromatography using the Ni Sepharose Excel Resin according to the manufacturer’s instructions (GE Healthcare), and dialyzed against PBS using Slide-A-Lyzer dialysis cassettes (Thermo Fisher Scientific). The RBDs and ACE2 were purified on a Streptactin column (IBA). Eluted fractions were analyzed by SDS- PAGE and those containing bands of the expected molecular weight were pooled, concentrated and further purified by size-exclusion chromatography (Superdex 20010/300 column (Cytiva)). AviTagged SARS-CoV-2 tri-S was biotinylated using Enzymatic Protein Biotinylation Kit (Sigma- Aldrich). HKU1a-CoV, HKU1b-CoV and OC43-CoV spike proteins were biotinylated using EZ- Link™ Sulfo-NHS-biotinylation kit (Thermo Fisher Scientific). [0237] The pT350 plasmid encoding S441A TMPRSS2 was used to perform a stable transfection on Drosophila S2 cells with the pCoPuro plasmid for puromycin selection. The cell line was selected and maintained in serum-free insect cell medium (HyClone, GE Healthcare) containing 7 µg/ml puromycin and 1% penicillin/streptomycin. For protein production, the cells were grown in spinner flasks until the density reached 107 cells/mL, at which point the protein expression was induced with 4 µM CdCl2. After 6 days, the culture was centrifuged and the supernatant was concentrated and used for affinity purification using a Streptactin column (IBA). The eluate was concentrated and applied onto a Superdex 20016/60 column (Cytiva) equilibrated with 10 mM Tris-HCl (pH 8.0), 100 mM NaCl. [0238] E. coli BL21pLysS cells were transformed with the plasmids encoding the different VHHs, which were expressed in the cytoplasm after overnight induction with 0.5 mM IPTG at 16° C. The cultures were centrifuged, the bacterial pellets were resuspended in 40 mL of lysis buffer (20 mM Tris-HCl, 200 mM NaCl, 20 mM imidazole, pH 8.0) containing complete protease inhibitor cocktail (Roche) and they were frozen at -80 °C until used. On the purification day, the resuspended pellets were thawed, sonicated (15 minutes, 9s on-pulse, 5s off-pulse), centrifuged and loaded onto a HisTrap column. Bound proteins were eluted with a linear gradient of buffer B (20 mM Tris-HCl, 200 mM NaCl, 500 mM imidazole, pH 8.0) and analyzed by SDS-PAGE. Fractions with higher purity were pooled, concentrated and further purified by SEC on a Superdex 7516/60 column (Cytiva) pre- equilibrated in 10 mM Tris-HCl, 100 mM NaCl, pH 8.0. EXAMPLE 3. Cells [0239] HEK293T (293T), U2OS and Caco2/TC7 were cultured in DMEM with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS). LNCaP cells were cultured in RPMI-1640 with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS). GFP-split cells were cultured with 1 µg/ml of puromycin (InvivoGen). Cells stably expressing TMPRSS2 were cultured with 200 µg/ml hygromycin. All cells lines were either purchased from ATCC or were kind donations from members of the Institut Pasteur and were routinely screened for mycoplasma. EXAMPLE 4. GFP-split fusion assay [0240] For cell–cell fusion assays, 293T cell lines stably expressing GFP1-10 and GFP11 were co- cultured at a 1:1 ratio (6 × 104 cells/well) and were transfected in suspension with 100 ng of DNA with Lipofectamine 2000 (Thermo) in a 96-well plate (uClear, #655090) (10% spike, indicated amount of TMPRSS2 adjusted to 100 ng DNA with pQCXIP-Empty). For Acceptor-Donor experiments, 293T GFP-split cells 1-10 and 11 were transfected separately in suspension with 500ng of DNA (10% spike, indicated amout of TMPRSS2 adjusted to 500ng with Empty) for 30 min at 37°C. Cells were washed twice and acceptor and donor cells were mixed and seeded at 6 × 104 cells/well. For Caco2 KD experiments, Caco2 GFP11 cells were transfected with control siRNA-directed against Luciferase: 5′- CGUACGCGGAAUACUUCGA-3′ (SEQ ID NO: 32) or siGENOME Human TMPRSS2 SMARTpool (#7113 - Horizon Discovery) at 50nM using Lipofectamine™ RNAiMAX (Thermo Fisher Scientific) in a 6 well dish for 48h.293T GFP1-10 cells were transfected with 10% of spike protein in a 6 well dish for 24 hours. Caco2 KD cells and 293T spike cells were then mixed at a 1:1.5 ratio and plated on a 96 well plate. The remaining Caco2 cells were used for RNA extraction for qPCR. For all experiments described above, at 20 h post-transfection, images covering 90% of the well surface, were acquired per well on an Opera Phenix High-Content Screening System (PerkinElmer). The GFP area was quantified on Harmony High-Content Imaging and Analysis Software. EXAMPLE 5. RNA Extraction, Reverse Transcription, and qPCR [0241] 48h post siRNA transfection, total of 5x105 caco-2 cells were lysed using RLT buffer (QIAGEN) supplemented with 10 μL of β-mercaptoethanol. RNA extraction was performed using the RNeasy plus mini kit (QIAGEN) according to the manufacturer's protocol. Reverse transcription was performed using SuperScript II (Thermo Fisher Scientific) according to the manufacturer's protocol. qPCR was performed using iTaq universal SYBR green supermix (Bio Rad) on a QuantStudio 6 Real- Time PCR machine (Thermo Fisher Scientific). The following primers were used: β-Tubulin forward: 5’ – CTTCGGCCAGATCTTCAGAC – 3’ (SEQ ID NO: 33), reverse: 5’ – AGAGAGTGGGTCAGCTGGAA – 3’ (SEQ ID NO: 34); TMPRSS2 forward: 5’ – GGGGATACAAGCTGGGGTTC – 3’ (SEQ ID NO: 35), reverse: 5’ – GATTAGCCGTCTGCCCTCAT – 3’ (SEQ ID NO: 36). EXAMPLE 7. Pseudotype generation and infection [0242] Pseudotyped viruses were produced by transfection of 293T cells as previously described 38. Briefly, cells were cotransfected with plasmids encoding for lentiviral proteins, a luciferase reporter, and the HKU1 spike plasmid. Pseudotyped virions were harvested at days 2 and 3 after transfection. Production efficacy was assessed by measuring infectivity or p24 concentration. Infection was performed in suspension using 10ng of p24 per well and 2x10^4 cells in 100uL in a 96 well plate. The next day 100uL of media was added.48 hours post-infection, 100uL of media was carefully removed, and 100uL of Bright-Glo™ lysis buffer (ProMega) was added. After 10 minutes, 150uL of lysate was transferred to a white plate, and luminescence was acquired using the EnSpire (PerkinElmer). EXAMPLE 8. Flow cytometry [0243] For Spike binding, 293T cells transfected with TMPRSS2 in the presence or absence of camostat for 24 h were stained with soluble biotinylated spike diluted in MACS buffer (PBS, 5g/L BSA, 2mM EDTA) at 2 µg/ml for 30 min at 4°C. The cells were then washed twice with PBS and then incubated with Alexa Fluor 647-conjugated streptavidin (Thermo Fisher Scientific, 1:400) for 30 min at 4°C. Finally, the cells were washed once with PBS and then fixed with 4% paraformaldehyde. The results were acquired using an Attune Nxt Flow Cytometer (Life Technologies). Transfection efficiency for TMPRSS2 was assessed on fixed cells by staining intracellularly with rabbit anti- TMPRSS2 (Atlas HPA035787), for 30 minutes at RT in PBS/BSA/Azide/0.05% Saponin. For the spike, transfection efficiency was measured at the surface on live cells using mab10 diluted in MACS buffer for 30 minutes at 4°C, a human secondary IgG. mab10 is an antibody generated during the early stages of the epidemic from a patient infected with the Wuhan strain by the Mouquet lab at Institut Pasteur, which cross-reacts with HKU1. EXAMPLE 9. ELISA assay [0244] ELISAs were performed as previously described in Mouquet, H. et al. Memory B cell antibodies to HIV-1 gp140 cloned from individuals infected with clade A and B viruses. PLoS One 6, e24078 (2011). Briefly, high-binding 96-well ELISA plates (Costar; Corning) were coated overnight with 250 ng/well of purified TMPRSS2. After washings with 0.05% Tween 20-PBS (washing buffer), the plates were blocked for 2 h with 2% BSA, 1 mM EDTA, 0.05% Tween 20-PBS (Blocking buffer), washed, and incubated with serially diluted soluble biotinylated spike proteins. Recombinant spike proteins were tested at 10 µg/ml, and 7 consecutive 1:2 dilutions in PBS. After washings, the plates were revealed by incubation for 1 h with HRP-conjugated streptavidin (BD Biosciences) in blocking buffer and by adding 100 µl of HRP chromogenic substrate (ABTS solution, Euromedex) after washing steps. Optical densities were measured at 405 nm (OD405nm) and background values given by incubation of PBS alone in coated wells were subtracted. Experiments were performed using HydroSpeed microplate washer and Sunrise microplate absorbance reader (Tecan). EXAMPLE 10. BLI assay [0245] Affinity of recombinant Spikes, RBDs and VHHs towards purified ectodomains of S441A TMPRSS2 or ACE2 was assessed in real-time using a bio-layer interferometry Octet-Red384 device (Pall ForteBio). Nickel-NTA capture sensors (Sartorius) were loaded for 10 min at 1,000 rpm shaking speed with the Wuhan RBD at 100 nM, or the HKU1A/B RBDs at 200 nM, or each VHH at 100 nM in PBS. The sensors were then blocked with PBS containing BSA at 0.2 mg/mL (assay buffer) and were incubated at 1,000 rpm with two-fold serially diluted concentrations (400 nM to 3.12 nM) of S441A TMPRSS2 or ACE2 ectodomains in assay buffer. Association and dissociation were monitored for 240 s and 180 s, respectively. A sample reference measurement was recorded from a sensor loaded with either RBD or VHH and dipped in the assay buffer. Association and dissociation profiles were fitted assuming a 1:1 binding model. Experiments to identify anti-TMPRSS2 VHHs that block binding to the HKU1 RBD were performed by immobilizing the HKU1B RBD on Nickel-NTA capture sensors. They were blocked in assay buffer and dipped into solutions containing a pre-incubated mixture of TMPRSS2 S441A (200 nM) and a VHH (400 nM). The signal corresponding to the association was recorded. EXAMPLE 11. Enzymatic activity [0246] Enzymatic activity was measured using BOC-QAR-AMC, a substrate of TMPRSS2 that fluoresces when cleaved. For TMPRSS2 mutants, cells were transfected in a black bottom 96 well plate as described above. After 24 hours, media was replaced with 100uL SVF free, phenol red free media, containing 100 µM of fluorogenic substrate. Indicated concentration of inhibitors were added. When assay was performed with soluble TMPRSS2, 60nM of soluble protein was added to the well, and mixed with VHH VHHs. They were incubated 15 minutes, before adding 100 µM of fluorogenic substrate. EXAMPLE 12. Western Blot [0247] Cells were lysed in TXNE buffer (1% Triton X-100, 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1X Roche cOMPLETE protease inhibitors) for 30 min on ice. Equal amounts (10μg) of cell lysates were analyzed by Western blot. The following antibodies were diluted in WB buffer (PBS, 1% BSA, 0.05% Tween, 0.01% Na Azide): rabbit anti-human TMPRSS2 (Atlas antibodies cat# HPA035787, 1:1,000), rabbit anti-human actin (abcam, 60008-1-Ig, 1:10,000), rabbit anti-HKU1 S1 polyclonal antibody (Thermofisher Scientific Cat #PA5-120768, 1:2,000) and rabbit anti-HKU1 S2 polyclonal antibody (Thermofischer Scientific #PA5-120769, 1:1,000). Species- specific secondary DyLight-coupled antibodies were used (1:10,000) and proteins were revealed using a Licor Imager. Images were processed using Image Studio Lite software. EXAMPLE 13. Statistical analysis [0248] Flow cytometry data were analyzed with FlowJo v10 software (Tristar). Calculations were all performed with Microsoft Excel 365. GraphPad Prism 9 was used to generate figures and for statistical analysis. Statistical significance between different conditions was calculated using the tests indicated in the corresponding figure legends. RESULTS EXAMPLE 14. TMPRSS2 triggers spike-dependent fusion [0249] To investigate the effect of TMPRSS2 on HKU1 spike mediated fusion, plasmids encoding for the HKU1A (isolate N1 - NCBI:txid443239) and B (Isolate N5 - NCBI:txid443241) spikes were generated. In 293T cells that do not express TMPRSS2, transient transfection of spike led to surface expression of the two proteins, as assessed by flow cytometry using Mab10, a pan S2 coronavirus antibody25 (Figure 22). To study cell-cell fusion, a GFP-Split based model was used in which fused cells become GFP+ (Fig.1)26. HKU1A and B spikes did not induce fusion alone but were highly fusogenic when co-expressed with TMPRSS2 (Fig.1, Figure 22). Fluorescent TMPRSS2- mNeonGreen and HKU1A Spike-mScarlet-I were generated to follow fusion in real-time by video microscopy. Cells expressing spike formed syncytia with cells expressing TMPRSS2 in less than one hour, indicating that this process is rapid. As a control of specificity, a panel of 9 other surface or intracellular proteases were tested, including the coronavirus receptors APN, DPP4 and ACE2, as well as TMPRSS4 and TMPRSS11D, that have been reported to cleave the SARS-CoV-2 spikes27,28. None of the tested proteases triggered cell-cell fusion (Figure 23). By expressing TMPRSS2 either on donor cells (spike-transfected) or acceptor cells, it was observed that the protease had to be on acceptor cells, opposite of the spike to induce high levels of fusion (Fig.2). The low fusion observed when they were both expressed on donor cells is likely due to transfection leakage. [0250] It was then investigated whether endogenous levels of TMPRSS2 were sufficient to induce HKU1 spike-dependent fusion. HKU1A spike-expressing 293T donor cells were mixwd with Caco-2 acceptor cells, that naturally express TMRPSS2. Caco-2 fused with spike expressing cells, whereas silencing of TMPRSS2 using siRNA significantly reduced fusion (Fig.3). [0251] HKU1 does not grow in any cell line tested up to date but can be amplified in primary differentiated human airway epithelial cultures24,29. No access to an infectious HKU1 strain was available. To further investigate the role of TMPRSS2 in HKU1 entry, lentiviral particles pseudotyped with HKU1 spikes were generated. The viral pseudotype strategy has been successfully used to study SARS-CoV-2 entry and to identify ACE2 as a receptor for this virus30. HKU1A and B pseudoviruses were unable to infect wild-type (WT) 293T cells. Transient expression of TMPRSS2 allowed for efficient viral entry (Fig.4). Altogether, these results indicate that TMPRSS2 is necessary for HKU1A and B spike-mediated cell-cell fusion and viral entry. EXAMPLE 15. Inactive TMPRSS2 mutants allow HKU1 infection [0252] To further analyze the role of TMPRSS2 in HKU1 spike fusion, two well-characterized TMPRSS2 mutants, R255Q and S441A (Fig.5). were generated. R255Q is mutated in its auto- cleavage site resulting in an immature inactive TMPRSS2, while S441A is mutated in its catalytic site. As expected, R255Q and S441A were correctly expressed in 293T cells but lacked catalytic activity, measured with a substrate generating a fluorescent signal upon cleavage31 (Figure 24). The cleavage profile of TMPRSS2 and HKU1 spike was generated by western blot (Fig.7). When expressed alone, WT TMPRSS2 was cleaved while the mutants were not. When spike was expressed, the overall levels of TMPRSS2 were reduced, probably because of the co-transfection procedure, but a similar profile of TMPRSS2 processing was observed. Without TMPRSS2, the spike was partially cleaved into S1 and S2 subunits (Fig.7), most likely at the polybasic furin cleavage S1/S2 site. WT TMPRSS2, but not the R255Q and S441A mutants, generated additional cleavage bands in the spike, including a 100 kDa band below the S2 band, which likely corresponds to the S2’ fragment. [0253] The wild type and mutant TMPRSS2 had slightly different levels of expression, when assessed by flow cytometry. Thus, the amounts of plasmid were adjusted to reach similar levels. The catalytically inactive TMPRSS2 mutants barely induced cell-cell fusion with either HKU1A or B spikes (Fig.8), confirming the need for spike cleavage for cell-cell fusion. [0254] In striking contrast, the TMPRSS2 mutants readily allowed infection of pseudotypes bearing HKU1A or B spikes and were even more efficient than the WT protease, with a 2-10 increase in infection (Fig.9). It was validated that TMPRSS2 mutants behave as expected regarding the SARS- CoV-2 spike (D614G ancestral strain), which requires the enzymatic activity of the protease for infectivity enhancement30. In 293T-ACE2 cells, WT TMPRSS2 increased SARS-CoV-2 pseudotype entry by 8-fold, whereas this was not the case for R255Q and S441A mutants (Figure 25). Thus, the catalytic activity of TMPRSS2 is not required for HKU1-mediated viral entry, strongly suggesting that TMPRSS2 acts as a receptor for HKU1. [0255] Coronavirus spike can either be processed by proteases at the surface of target cells, allowing for membrane fusion or by cathepsins in the endosome, allowing for entry after internalization32. As TMPRSS2 catalytic activity was not required for HKU1 entry, the cytoplasmic access route of pseudoviruses in cells expressing WT or S441A TMPRSS2 was examined. To this aim, infections were performed in presence of SB412515, a cathepsin L inhibitor, or Camostat mesylate, a TMPRSS2 inhibitor. SB412515 reduced the entry of HKU1A pseudoviruses in the S441A TMPRSS2 cells and not the WT TMPRSS2 cells, indicating that viral entry occurs through endosomes with the catalytically inactive TMPRSS2 and at the surface with the WT protease. Conversely, Camostat increased HKU1A entry in WT TMPRSS2 cells, and not in S441A TMPRSS2 cells, suggesting that the chemical inhibition of TMPRSS2 stabilizes the protease at the surface and/or enhances endosomal entry. With HKU1B, Camostat and SB412515 had little or no effect on entry (Figure 26), suggesting that HKU1B priming might be different than that of HKU1A, although it still uses TMPRSS2 as a receptor. [0256] Taken together, these results show that TMPRSS2 is required for HKU1 infection and that its catalytical activity can be rescued by other proteases such as cathepsins. EXAMPLE 16. HKU1 spike binds to TMPRSS2 [0257] Recombinant soluble forms of HKU1A and B spikes were generated to investigate their binding to TMPRSS2 expressing cells (Fig.11-12, Figures 27-28). HKU1 spike bound weakly to WT TMPRSS2 and more strongly to TMPRSS2 S441A and R255Q. Addition of Camostat increased binding to WT TMPRSS2 (Fig.12, Figures 27-28), indicating that the proteolytic activity of TMPRSS2 somehow interfered with the readout or decreased binding by degrading or shedding the bound spike. Alternatively, WT TMPRSS2’s turnover might be faster than the mutants. As expected, with a soluble SARS-CoV-2 spike, binding to cells expressing ACE2 was detected, but not WT or mutant TMPRSS2 (Fig.12, Figure 29), highlighting the different behaviors of HKU1 and SARS-CoV- 2. OC43 did not bind to cells expressing TMPRSS2 (Figure 30). [0258] The binding of recombinant soluble forms of TMPRSS2 and HKU1A or B spikes was demonstrated by ELISA, whereas it was not the case for the SARS-CoV-2 spike (Fig.13), indicating a direct interaction between TMPRSS2 and HKU1 spike. Conversely, soluble ACE2 bound to SARS- CoV-2 but not to HKU1 spike by ELISA and by BLI (Figure 31-32). The yield of soluble WT TMPRSS2 was low, limiting experiments, potentially due to TMPRSS2 autocleavage and stability. A soluble TMPRSS2 S441A mutant was generated for further experiments. Due to the low yield of soluble WT TMPRSS2, we generated a soluble S441A mutant that could be obtained in amounts that allowed biophysical experiments. We expressed the RBD (residues 323-609 for HKU1A; 323-607 for HKU1B; 331-528 for SARS-CoV-2) to measure their affinity for S441A TMPRSS2 by biolayer interferometry (BLI). In these experiments, the interaction of TMPRSS2 with SARS-CoV-2 RBD was lower than the interaction of TMPRSS2 with an unloaded sensor (Figure 33). Using different S441A TMPRSS2 concentrations, affinity constants (Kd) of 93nM and 61nM were determined for HKU1A and B RBDs, respectively (Table 1, Table 2, Figure 34). [0259] Table 1: Affinity (Kd) of the indicated RBD for TMPRSS2, ranges of affinity, association rate (kas) and dissociation rate (kdis) measured in 2-3 independent experiments. NA denotes proteins for which interaction with the loaded sensor was below the interaction with an empty sensor. Table 2: Kd, kas and kdis of the indicated RBD for ACE2. NA denotes proteins for which interaction with the loaded sensor was below the interaction with an empty sensor. EXAMPLE 17. HKU1 spike receptor binding motif [0260] The residues W515 and R517 within HKU RBD have been reported to be critical for binding to a putative unknown cellular receptor16. We produced recombinant HKU1B RBD with the W515A or R517A mutations. The W515A mutation abrogated interaction with TMPRSS2, reaching response levels comparable to those obtained with a sensor coated with the SARS-CoV-2 RBD, while the R517A mutation reduced binding by 3-fold (Fig.15, Figure 35, Table 1, Table 2). Expression plasmids for the two W515A or R517A mutants in the HKU1A and B background were generated. The mutants were correctly expressed at the cell surface, as assessed by flow cytometry (Figures 35- 36) but they lost their cell-cell fusion properties (Fig.15). Their ability to trigger viral pseudotype infection was decreased by 2 to 3 logs (Fig.15). Therefore, the conserved W515 and R517 residues within the HKU1A and B RBD are critical for binding to TMPRSS2, viral fusion and entry. EXAMPLE 18. Anti-TMPRSS2 VHH block spike binding [0261] There are no currently available anti-TMPRSS2 monoclonal antibodies that interfere with the function of the protein, as most of the existing ones are directed towards cytosolic fragments of the protein. An alpaca was immunized with soluble S4441A TMPRSS2 in order to produce VHH.5 VHH (A01, F05, A07, C11 and D01) were isolated that bound with an affinity in the nM range to TMPRSS2 (Figure 37). The VHH also recognized TMPRSS2 to various extents by flow cytometry (Figure 39). Three of them (A07, C11 and D01) reduced TMPRSS2 catalytic activity (Fig.18) and inhibited the HKU1 RBD–TMPRSS2 interaction measured by BLI (Fig. 19). The same three VHH inhibited HKU1A and B spike – TMPRSS2 mediated cell-cell fusion (Fig.20). Their effect on viral entry was examined independently of their inhibition of the catalytic activity of TMPRSS2. They reduced pseudotype infection of 293T cells expressing S441A TMPRSS2 (Fig.21) in a dose dependent manner (Figure 39and Figure 40). The two other VHH (A01 and F05), despite an efficient binding to TMPRSS2, did not interfere with its enzymatic activity and proviral roles (Fig.18-21 and Fig.37-41), suggesting that they bind to regions different from that of three active VHH and that are not involved in HKU1 binding. [0262] Table 3: Affinity of the VHH for TMPRSS2 and sum-up of their effect [0263] VHH-A01did not bind to TMPRSS4 and TMPRSS11D, as assessed by flow cytometry. EXAMPLE 19. Crystals of TMPRSS2 with VHH [0264] For structural studies, TMPRSS2 S441A was expressed in S2 cells and purified as indicated above (Example 3. Protein production). The double Strep tag of the protein was removed by incubating the protein with 64 units of Enterokinase light chain (BioLabs) in 10 mM Tris, 100 mM NaCl, 2 mM CaCl2, pH 8.0, at room temperature, overnight. The proteolysis reaction was buffer- exchanged into 10 mM Tris, 100 mM NaCl, pH 8.0, and subjected to another affinity purification, recovering the flow-through fraction containing the untagged TMPRSS2 S441A. The protein was concentrated and its enzymatic deglycosylation with EndoD and EndoH was set up at room- temperature following overnight incubation with 1000 units of each glycosidase in 50 mM Na-acetate, 200 mM NaCl, pH 5.5. The protein was further purified on a size exclusion chromatography (SEC) column Superdex 20016/60 (Cytiva) in 10 mM Tris, 100 mM NaCl, pH 8.0. Then, the protein (at 68 µM) was incubated with VHH-A07 (102 µM) for 16 hours at 4 °C and the binding reaction was injected in a Superdex 20010/300 column (Cytiva) equilibrated with 10 mM Tris, 100 mM NaCl, pH 8.0. The eluted fractions were analyzed by SDS-PAGE and those corresponding to the complex were pooled, concentrated and used for crystallization. [0265] The crystallization trials were performed in 200 nanoliter sitting drops formed by mixing equal volumes of the protein and reservoir solution in the format of 96 Greiner plates, using a Mosquito robot. Crystal appearance and growth were monitored by a Rock-Imager at the Core Facility for Protein Crystallization at Institut Pasteur in Paris, France (Weber, P. et al. High-throughput crystallization pipeline at the crystallography core facility of the Institut Pasteur. Molecules 24, 4451 (2019)).The crystal used for data collection was grown in 0.05 M HEPES pH 7.0, 20% PEG 3350, 1% tryptone, 0.001% NaN3 and frozen using the mother liquor containing 33% glycerol as cryo-buffer. [0266] The X-ray diffraction data were collected at the SOLEIL synchrotron source (Saint Aubin, France), Proxima-1 beamline (Chavas, L. M. G. et al. PROXIMA-1 beamline for macromolecular crystallography measurements at Synchrotron SOLEIL. J. Syn- chrotron Radiat.28, 970–976 (2021)), at wavelength of 0.9786Å, with a Pilatus Eiger X 16 M detector (Dectris). Diffraction data were processed using XDS (Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr.66, 125–132 (2010) and scaled and merged with AIMLESS (Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr.69, 1204–1214 (2013)). The high- resolution cut-off was based on the statistical indicator CC1/2 (Karplus, P. A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, 1030–1033 (2012)). The phases were determined by molecular replacement using a TMPRSS2 structure (PDB: 7MEQ) and a VHH-A07 model generated using AlphaFold as search models in PHASER (McCoy, A. J. et al. Phaser crystallographic software. J Appl Crys- tallogr.40, 658–674 (2007)) from the PHENIX suite (Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr.66, 213–221 (2010)). The ASU was found to contain one 1:1 complex, which was refined by iterative rounds of phenix.refine and Coot (Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol Crystallogr.60, 2126– 2132 (2004)). [0267] Crystals of TMPRSS2 with A07 were analyzed. A preliminary structure shows that A07 binds to the residues 275-473, around TMPRSS2 catalytic site. [0268] Table 3. List of residues from TMPRSS2 (chain A) and VHH-A07 (chain B) that form polar contacts at the complex interface. The atoms involved and the distances between them are indicated. Hydrogen bonds Salt bridges B:GLN 3[ NE2] 3.60 A:ASP 417[ O ] B:ARG 101[ NH1] 3.24 A:GLU 299[ OE1] B:GLN 3[ NE2] 3.42 A:ASP 417[ OD2] B:ARG 105[ NH1] 3.22 A:ASP 435[ OD1] B:HIS 33[ ND1] 2.42 A:SER 463[ OG ] B:ARG 105[ NH1] 3.73 A:ASP 435[ OD2] B:THR 54[ OG1] 3.61 A:GLN 438[ OE1] B:ARG 105[ NH2] 3.20 A:ASP 435[ OD2] B:SER 56[ OG ] 2.84 A:GLN 438[ OE1] B:GLU 32[ OE1] 3.96 A:ARG 470[ NE ] B:ARG 101[ NH1] 3.24 A:GLU 299[ OE1] B:GLU 32[ OE2] 2.86 A:ARG 470[ NE ] B:VAL 102[ N ] 3.00 A:LYS 340[ O ] B:GLU 32[ OE2] 2.74 A:ARG 470[ NH2] B:ARG 105[ N ] 3.23 A:SER 460[ O ] B:ARG 105[ NH1] 3.22 A:ASP 435[ OD1] B:ARG 105[ NH1] 3.19 A:SER 436[ OG ] B:ARG 105[ NH2] 3.20 A:ASP 435[ OD2] B:ARG 105[ NH2] 2.83 A:GLY 464[ O ] B:ARG 106[ NH1] 2.90 A:HIS 296[ O ] B:ARG 106[ NH1] 3.23 A:CYS 297[ O ] B:ASN 107[ N ] 3.10 A:VAL 280[ O ] B:ASN 107[ ND2] 3.13 A:THR 393[ OG1] B:ASN 107[ ND2] 3.01 A:HIS 279[ O ] B:ASN 117[ ND2] 3.22 A:ASP 338[ OD1] B:ASN 117[ ND2] 2.92 A:SER 339[ OG ] B:GLU 32[ O ] 3.72 A:SER 463[ OG ] B:GLU 32[ OE2] 2.86 A:ARG 470[ NE ] B:GLU 32[ OE2] 2.74 A:ARG 470[ NH2] B:VAL 102[ O ] 2.62 A:LYS 342[ NZ ] B:ARG 105[ O ] 3.02 A:GLY 439[ N ] [0269] Table 4. List of residues from TMPRSS2 and VHH-A07 that are buried at the interface of the complex. VHH-A07 residues at TMPRSS2 S441A the interface residues at the interface Q3 V275 G28 Q276 S29 V278 P30 H279 E32 V280 H33 C281 Y34 H296 D35 C297 T54 E299 T55 K300 S56 P301 G57 L302 H59 D338 N76 S339 G100 K340 R101 T341 V102 K342 G103 E389 G104 K390 R105 T393 R106 V415 N107 Y416 W108 D417 P111 N418 N117 L419 A118 D435 Y119 S436 C437 Q438 G439 A441 T459 S460 W461 G462 S463 G464 C465 R470 G472 V473 Crystal structure TMPRSS2 S441A+VHH A07 (Crystal form 1, PDB 8 S0L) Further studies have been carried on the structure of TMPRSS2 with A07 and it was found that the crystal had a cleaved form of TMPRSS2 (mature) and also the immature form (uncleaved). [0270] Table 5. List of residues from cleaved form of TMPRSS2 and VHH-A07 that are buried at the interface of the complex (crystal form 1, PDB 8S0L). [0271] TMPRSS2 S441A (chain A) VHH-A07 (chain B) Crystal 1 – Alternate location (alt loc) A A LYS 342 NZ B VAL 100 O 2.637 A GLN 438 NE2 (alt loc A) B ARG 104 O 3.407 A GLY 439 N (alt loc A) B ARG 103 O 3.019 A SER 463 OG (alt loc A) B HIS 31 ND1 2.358 A ARG 470 NE B GLU 30 OE1 3.019 A ARG 470 NE B GLU 30 OE2 3.381 A ARG 470 NH2 B GLU 30 OE2 2.776 B HIS 31 ND1 A SER 463 OG (alt loc A) 2.358 B VAL 100 N A LYS 340 O 2.928 B ARG 103 N A SER 460 O 3.270 B ARG 103 NH1 (alt loc A) A ASP 435 OD1 (alt loc A) 3.138 B ARG 103 NH2 (alt loc A) A ASP 435 OD2 (alt loc A) 3.166 B ARG 103 NH2 (alt loc A) A GLY 464 O (alt loc A) 2.980 B ARG 104 NH1 A CYS 297 O 3.237 B ASN 105 N A VAL 280 O 3.138 B ASN 105 ND2 (alt loc A) A HIS 279 O 2.945 B ASN 115 ND2 A ASP 338 OD1 3.121 B ASN 115 ND2 A SER 339 OG 3.159 TMPRSS2 S441A (chain A) VHH-A07 (chain B) Crystal 1 - Alternate location (alt loc) B A LYS 342 NZ B VAL 100 O 2.637 A SER 463 OG (alt loc B) B THR 53 OG1 2.656 A ARG 470 NE B GLU 30 OE1 3.019 A ARG 470 NE B GLU 30 OE2 3.381 A ARG 470 NH2 B GLU 30 OE2 2.776 B THR 53 OG1 A SER 463 OG (alt loc B) 2.656 B VAL 100 N A LYS 340 O 2.928 B ARG 103 N A SER 460 O 3.270 B ARG 104 NH1 A CYS 297 O 3.237 B ASN 105 N A VAL 280 O 3.138 B ASN 115 ND2 A ASP 338 OD1 3.121 B ASN 115 ND2 A SER 339 OG 3.159 [0272] In the alternate conformation A (cleaved form of TMPRSS2), the paratope residues (from VHH-A07) are the following: Q1, G26, S27, P28, E30, H31, Y32, D33, T52, T53, S54, G55, G56, H57, N74, G98, R99, V100, G101, G102, R103, R104, N105, W106, P109, D111, D114, N115, A116, Y117. The epitope residues (from cleaved form of TMPRSS2) are the following: V275, Q276, V278, H279, V280, C281, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, E389, K390, T393, V415, Y416, D417, N418, L419, D435, S436, C437, Q438, G439, A441, T459, S460, W461, G462, S463, G464, K467, R470, G472, V473. [0273] In the alternate conformation B (uncleaved form of TMPRSS2), the paratope residues (from VHH-A07 (chain B)) are the following: Q1, G26, S27, P28, E30, H31, Y32, D33, T53, S54, G55, G56, N74, G98, R99, V100, G101, G102, R103, R104, N105, W106, P109, D111, D114, N115, A116, Y117. The epitope residues (from uncleaved form of TMPRSS2 (chain A)) are the following: V275, Q276, V278, H279, V280, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, G385, A386, E389, K390, T393, V415, Y416, D417, N418, L419, S436, C437, G439, D440, A441, T459, S460, W461, G462, S463, C465, R470. [0274] Crystal structure TMPRSS2 S441A+VHH A07 (Crystal form 2, PDB 8 S0N) [0275] Table 6. List of residues from cleaved form of TMPRSS2 and VHH-A07 that are buried at the interface of the complex (crystal form 2, PDB 8S0N). TMPRSS2 S441A (chains A and C) VHH-A07 (chain B and D) Crystal 2 A LYS 342 NZ B VAL 100 O 2.372 A SER 463 OG B THR 53 OG1 2.752 A ARG 470 NE B GLU 30 OE2 3.212 A ARG 470 NH2 B GLU 30 OE2 2.447 B THR 53 OG1 A SER 463 OG 2.752 B VAL 100 N A LYS 340 O 3.086 B ARG 103 N A HIS 296 NE2 3.142 B ARG 104 N A HIS 296 NE2 3.241 B ARG 104 NH1 A CYS 297 O 3.049 B ARG 104 NH1 A GLU 299 O 3.232 B ARG 104 NH2 A LYS 300 O 3.136 B ASN 105 N A VAL 280 O 3.443 B ASN 105 ND2 A HIS 279 O 3.471 [0276] In this crystal structure, it was found that the paratope residues (from VHH-A07 (chain B)) are the following: Q1, G26, S27, P28, E30, H31, Y32, D33, T53, S54, R99, V100, G101, G102, R103, R104, N105, W106, I107, P109, D111, D114, N115, A116, Y117. The epitope residues (from uncleaved form of TMPRSS2 (chain A)) are the following: V275, Q276, V278, H279, V280, C281, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, G385, E389, T393, V415, Y416, D417, N418, L419, S436, C437, G439, D440, A441, T459, S460, W461, G462, S463, C465, R470, G472, Y474. [0277] Crystals of TMPRSS2 with A01 were analyzed. [0278] The structure of TMPRSS2 with the VHH A01 has also been studies. As it is shown below. [0279] Table 7. List of residues from TMPRSS2 (chain A) and VHH-A01 (chain U) that form polar contacts at the complex interface. The atoms involved and the distances between them are indicated. [0280] List of polar interactions: RBD (chains A and D) TMPRSS2 S441A (chains B and E) VHH-A01 (chain U and V) B ARG 150 NH1 U ASP 30 O 3.068 B TYR 152 OH U ASP 105 OD2 2.535 B LYS 166 NZ U ASP 73 OD1 2.330 B TRP 168 N U SER 54 O 3.103 B THR 207 N U ASP 56 OD2 3.146 B GLY 370 N U ALA 101 O 3.119 B LEU 373 N U LEU 102 O 2.602 B GLN 408 N U ASP 116 OD1 3.155 B GLN 408 NE2 U PRO 112 O 3.500 U TYR 31 OH B ASP 482 OD2 2.725 U TYR 32 OH B PRO 422 O 2.688 U TYR 117 OH B ILE 420 O 3.278 [0281] The paratope residues (from VHH-A01 (chain U)) are the following: [0282] V2, G26, F27, D30, Y31, Y32, S53, S54, G55, D56, K57, R72, D73, N74, S100, A101, L102, Y103, S104, D105, N111, P112, M113, L114, Y115, D116, Y117, W118. [0283] [0284] The epitope residues (from TMPRSS2 (chain B)) are the following: [0285] R150, Y152, Q159, Y161, K166, S167, W168, H169, S204, G205, S206, T207, P367, P369, G370, M371, M372, L373, Q374, P375, I404, I405, E406, T407, Q408, N411, I420, T421, P422, M424, I425, I456, N476, M478, V479, T481, D482. EXAMPLE 20. Activities of VHH-Fc [0286] U2OS cells stably expressing TMPRSS2 or an empty control construct were stained with VHH-A01-Fc (Fig.44). [0287] Caco-2 cells endogenously express TMPRSS2.40 TMPRSS2 KO caco-2 cells were generated using CRISPR/Cas9. WT and TMPRSS-2KO caco-2 cells were surface stained for TMPRSS2 using the dimerized VHH-A01-Fc (Fig.45). VHH-A01-Fc stained WT caco-2 cells, demonstrated by the shift in Alexa-647 MFI. This staining was significantly reduced in TMPRSS2-KO caco-2. [0288] The activity of the VHH-Fc VHH-A01-Fc and VHH-A07-Fc compared to VHHs A01 and A07 is summarized in the table below. Works Specificity Specifici Enzymati Inhibition for Work to ot ty to Inhibition Kd her uman other c activity of HKU1 Cytomet s in IF h species of HKU1 TM inhibitio pseudotype ry TTSP PRSS2 n infection infection A01_m onome 0.78 ++ ++ Specific to N ND (- ric nM TMPRSS2 D - - expected) A07_m onome 13.3 + ND Specific to nM TMP ND ++ ++ ++ ric RSS2 A01_Fc ND ++ ++ ND Specific to ND (- human - - expected) Recognizes A07_Fc ND + ND ND human and ++ ++ ND (++ other species expected) [0289] VHH-A07-Fc and VHH-A01-Fc both bind efficiently to TMPRSS2 at a dose of 0.2-1µg/ml (Fig.54). HEK 293T cells were transfected with plasmids expressing TMPRSS2-Scarlet-I.24h post transfection, binding of VHH-A01-Fc and VHH-A07-Fc was assessed on live cells by staining with a range of concentrations of anti-TMPRSS2 VHH A01-Fc or VHH A07-Fc (0,00032 µg ml−1 to 5 µg ml−1) for 30 min at 4 °C in MACS buffer, followed by staining with Alexa Fluor 647-conjugated Goat anti-Human Antibody (Thermo Fisher Scientific, A-21445, 1:500). [0290] VHH-A07-Fc and VHH-A01-Fc both bind to TMPRSS2 but not to 9 other serine proteases (Fig.55). HEK 293T cells were transfected with plasmids expressing TMPRSS2 or 9 other Human serine proteases.24h post transfection, binding of VHH-A01-Fc and VHH-A07-Fc was assessed on live cells by staining with anti-TMPRSS2 VHH A01-Fc at 1 µg ml−1 or VHH A07-Fc at 5 µg ml−1 for 30 min at 4 °C in MACS buffer, followed by staining with Alexa Fluor 647-conjugated Goat anti- Human Antibody (Thermo Fisher Scientific, A-21445, 1:500). [0291] VHH-A01-Fc binds to human TMPRSS2 but not to 6 other animal TMPRSS2 tested (Mesocricretus auratus, Macaca, Mustela furo, Mus musculus, Bos taurus, Gorilla gorilla (Fig.56). VHH-A07-Fc recognizes human, macaque, mouse and gorilla TMPRSS2 (Fig.56). HEK 293T cells were transfected with plasmids expressing TMPRSS2 of different animal species.24h post transfection, binding of VHH-A01-Fc and VHH-A07-Fc to animal TMPRSS2 was assessed on live cells by staining with anti-TMPRSS2 VHH A01-Fc at 1 µg ml−1 or VHH A07-Fc at 5 µg ml−1 for 30 min at 4 °C in MACS buffer, followed by staining with Alexa Fluor 647-conjugated Goat anti- Human Antibody (Thermo Fisher Scientific, A-21445, 1:500). [0292] VHH-A01-Fc does not inhibit TMPRSS2-HKU1 interactions nor the enzymatic activity. VHH-A07-Fc inhibits HKU1 infection and TMPRSS2 enzymatic activity. EXAMPLE 20. VHH A07 inhibits SARS-CoV-2 replication [0293] Caco-2 cells were infected with SARS-CoV-2 in the presence of non-target, A01 and A07 monomeric VHHs (Fig.46). Both non-target and A01 did not inhibit SARS-CoV-2 infection, which is expected as they are unable to inhibit TMPRSS2 enzymatic activity (Fig.18). In contrast, VHH A07 which is capable of inhibiting TMPRSS2 enzymatic activity inhibited SARS-CoV-2 infection in a dose dependent manner. EXAMPLE 21. Anti-TMPRSS2 VHH blocks HKU1 infection [0294] 259 HKU1 does not grow in any cell line tested up to date but viral amplification in human ciliated airway epithelial cell cultures has been reported29,39,40. HKU1 virus was isolated from a nasal swab of an individual suffering from a respiratory tract infection. To this end, primary human bronchial epithelial cells (HBE cells) differentiated at the air/liquid interface (ALI) for over 4 weeks were used. [0295] Immunofluorescence of HBE cells with the anti-TMPRSS2 VHH-A01-Fc revealed a preferential staining of ciliated cells, with a positive signal accumulating at the cilia. Then the virus was amplified from the clinical sample by one passage on HBE cells. It was observed an increase of HKU1 viral RNA in apical culture supernatants, with concentrations peaking at 5x106 viral RNA copies/μL at 2-3 days post-infection (dpi). [0296] Metagenomic sequencing of the viral supernatant identified a HKU1 genotype 269 B, and no other virus was detected. Target cells were preincubated with A07 VHH and the spike intensity by immunofluorescence 48 h post-infection was measured (Fig.47). The A07 VHH strongly reduced the appearance of infected cells, indicating that the spike binding and/or cleavage activities of TMPRSS2 are necessary for a productive HKU1 infection. EXAMPLE 22. VHH A07 inserts its CDR3 into the TMPRSS2 substrate-binding groove. [0297] It was crystallized the complex between TMPRSS2S441A and the inhibitory VHH A07 (Figure 48) and determined its structure to 1.8 Å resolution. A07 covers the active site cleft and buries a large surface area (about 2600 Å2, ~1400 Å2 on the VHH and ~1200 Å2 on TMPRSS2), interacting with residues in exposed loops of the SP domain (loops 1, 2, 3, A, B, C, D), some also involved in the interaction with HKU1B RBD (Figure 48). Superimposing the RBD+TMPRSS2S441A structure on the A07+TMPRSS2S441A complex showed clashes between the VHH and the RBD (Figure 49), explaining the blocking activity and providing further validation to the interaction site that we report here for the RBD. [0298] VHH A07 contacts TMPRSS2 almost exclusively through its CDRs. The most notorious feature is the insertion of its long (21 residues) CDR3 in the substrate binding cleft in between the two lobes of the SP domain. Superposing the complex with the peptide-bound structures of hepsin and TMPRSS13 shows that the side chain of R103A07 occupies the P1 position, making contacts with residues D435T, S436T and G464T, which form the S1 site 18. [0299] The TMPRSS2S441A/A07 crystals displayed electron density for the TMPRSS2 LDLR-A domain, which was not resolved in the previous structure of TMPRSS2. The LDLR-A domain includes a calcium-binding site formed by the side chains of D134T, H138T, D144T, and E145T, and the main chain carbonyl group of N131T and V136T. The SP domain of cleaved TMPRSS2 (TMPRSS2-Cl) crystallized previously in complex with the Nafamostat inhibitor 18, superposes very well with its counterpart in the TMPRSS2S441A/A07 crystals, with a root mean square deviation (RMSD) of 0.315 Å for 1502 atoms in 236 residues. In this structure, the residues immediately downstream the autocleavage site (I256T and V257T) are found in an internal pocket where the free amino group of I256T forms a salt-bridge with the side chain of D440T. This interaction can only be established after cleavage of the protease and is a characteristic feature of serine proteases in the active conformation. This observation indicates that TMPRSS2S441A in the crystals of the complex with A07 underwent cleavage by a contaminating protease. The crystallographic data further indicated that the maturation cleavage did not occur in 100% of the molecules forming the crystal. Another conformation was detected, for which the high-resolution diffraction allowed the refinement of an atomic model. In this second model, the loop bearing the cleavage site was disordered, with the first residue with clear electron density after the cleavage site being G259T, as expected for an uncleaved form - or zymogen - of TMPRSS2S441A. It was confirmed that this is indeed the case by crystallizing the TMPRSS2S441A/A07 complex under different conditions, which yielded crystals that diffracted to 2.4 Å resolution. The residues I256T and V257T were not visible in the structure determined from these new crystals, in which TMPRSS2S441A showed no evidence of proteolysis. The resulting model aligned very well with the second conformation described above, supporting the hypothesis that it corresponds to the TMPRSS2 zymogen. Furthermore, this structure has the characteristic “zymogen triad” initially observed for chymotrypsinogen, D194-H40-S3231, corresponding to D440-H279-S272 in TMPRSS2.Upon activation, D440 is released from this triad to make the salt-bridge/hydrogen bond with the newly formed N-terminus at I256. This rearrangement leads to formation of the oxyanion hole required for cleavage of the scissile peptide bond of the substrate. Proteases that do not have a zymogen triad, such as the tissue-type plasminogen activator, have a high level of catalytic activity in the zymogen form. EXAMPLE 22. VHH A01-Fc recognizes TMPRSS2 naturally expressed at the surface of intestinal cells (Caco2 cells, Fig.54) and human tumoral prostatic cells (LNCaps cells, Fig.55). [0300] Expression of TMPRSS2 was assessed on live cells by staining with anti-TMPRSS2 VHH A01-Fc at 1 µg ml−1 for 30 min at 4 °C in MACS buffer, followed by staining with Alexa Fluor 647- conjugated Goat anti-Human Antibody (Thermo Fisher Scientific, A-21445, 1:500). SEQUENCES [0301] Monomeric VHH sequences [0302] In the following sequences, CDR1 is in bold, CDR2 in bold and in italics and CDR3 in bold and underlined. A01 (deposited as VHH TMPRSS2 A01/VHH-TMPRSS2-A01 at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES(CNCM) on June 1, 2023, under number I-5958) MAQVQLVESGGGLVQPGGSLRLSCVVSGFSLDYYAIGWFRQAPGKEREGVSCIGSSGDKTNYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAESALYSDCTEEQNPMLYDYWGQGTQVTVSS (SEQ ID NO: 19) A07 (deposited as VHH TMPRSS2 A07/VHH-TMPRSS2-A07 at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES(CNCM) on June 1, 2023, under number I-5959) MAQVQLVESGGGLVQPGGSLRLSCTSSGSPLEHYDIIWFRQAPGREREGVSSITTSGGHTNYADSVKG RFTISRDNAKNVVYLQMNSLKPEDTAVYYCAGRVGGRRNWIVPLDGYDNAYWGQGTQVTVSS (SEQ ID NO: 20) C11 (deposited as VHH TMPRSS2 C11/VHH-TMPRSS2-C11 at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES(CNCM) on June 1, 2023, under number I-5960) MAQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYDIYWFRQAPGKEREGVSSITTSGGRTNYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAKVGGRRNWIAPLNGYENALWGKGTLVTVSS (SEQ ID NO: 21) D01 (deposited as VHH TMPRSS2 D01/VHH-TMPRSS2-D01 at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES(CNCM) on June 1, 2023, under number I-5961) MAEVQLVESGGGLVQPGGPLRLSCASSGSTLEHYDIGWFRQVPGGLREGVSSITASGGRTNYADSVKG RFTISRDNGKNAVYLQMNSLKPEDTAVYYCAGKIGGRRNWVAPLDGFENAYWGQGTQVTVSS (SEQ ID NO: 22) F05 (deposited as VHH TMPRSS2 F05/VHH-TMPRSS2-F05 at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES(CNCM) on June 1, 2023, under number I-5962) MAEVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSGDSIKYVDSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYQCAADALGSGCLTGNYDYWGQGTRVTVSS (SEQ ID NO: 23) [0303] Dimeric VHH sequences: >TMPR-A1-Fc-human (VHH-A01-Fc) MAQVQLVESGGGLVQPGGSLRLSCVVSGFSLDYYAIGWFRQAPGKEREGVSCIGSSGDKTNYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAESALYSDCTEEQNPMLYDYWGQGTRVTVSSEPKTPK PQPAAARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 26) >TMPR-A7-Fc human (VHH-A07-Fc) MAQVQLVESGGGLVQPGGSLRLSCTSSGSPLEHYDIIWFRQAPGREREGVSSITTSGGHTNYADSVKD RFTISRDNAKNVVYLQMNSLKPEDTAVYYCAGRVGGRRNWIVPLDGYDNAYWGQGTQVTVSSEPKTPK PQPAAARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 27) [0304] TMPRSS2 Sequences: Sequence of TMPRSS2 - (Addgene plasmid # 53887) gene used for plasmid transfection in cells MALNSGSPPAIGPYYENHGYQPENPYPAQPTVVPTVYEVHPAQYYPSPVPQYAPRVLTQASNPVVCTQ PKSPSGTVCTSKTKKALCITLTLGTFLVGAALAAGLLWKFMGSKCSNSGIECDSSGTCINPSNWCDGV SHCPGGEDENRCVRLYGPNFILQVYSSQRKSWHPVCQDDWNENYGRAACRDMGYKNNFYSSQGIVDDS GSTSFMKLNTSAGNVDIYKKLYHSDACSSKAVVSLRCIACGVNLNSSRQSRIVGGESALPGAWPWQVS LHVQNVHVCGGSIITPEWIVTAAHCVEKPLNNPWHWTAFAGILRQSFMFYGAGYQVEKVISHPNYDSK TKNNDIALMKLQKPLTFNDLVKPVCLPNPGMMLQPEQLCWISGWGATEEKGKTSEVLNAAKVLLIETQ RCNSRYVYDNLITPAMICAGFLQGNVDSCQGDSGGPLVTSKNNIWWLIGDTSWGSGCAKAYRPGVYGN VMVFTDWIYRQMRADG* (SEQ ID NO: 24) Sequence of soluble TMPRSS2 used for enzymatic activity tests: IgK SP - ectodomain res. 106-492 - GSG - thrombin cleavage site – 8xHIS - GTG - AVITAG - Stop MGWSCIILFLVATATGVHSWKFMGSKCSNSGIECDSSGTCINPSNWCDGVSHCPGGEDENRCVRLYGP NFILQVYSSQRKSWHPVCQDDWNENYGRAACRDMGYKNNFYSSQGIVDDSGSTSFMKLNTSAGNVDIY KKLYHSDACSSKAVVSLRCIACGVNLNSSRQSRIVGGESALPGAWPWQVSLHVQNVHVCGGSIITPEW IVTAAHCVEKPLNNPWHWTAFAGILRQSFMFYGAGYQVEKVISHPNYDSKTKNNDIALMKLQKPLTFN DLVKPVCLPNPGMMLQPEQLCWISGWGATEEKGKTSEVLNAAKVLLIETQRCNSRYVYDNLITPAMIC AGFLQGNVDSCQGDSGGPLVTSKNNIWWLIGDTSWGSGCAKAYRPGVYGNVMVFTDWIYRQMRADGGS GLVPRGSHHHHHHHHGTGGLNDIFEAQKIEWHE*(SEQ ID NO: 28) Sequence of the soluble S441A TMPRSS2 used for BLI: Sequence after enterokinase cleavage (used for immunization): KFMGSKCSNSGIECDSSGTCINPSNWCDGVSHCPGGEDENRCVRLYGPNFILQVYSSQRKSWHPVCQD DWNENYGRAACRDMGYKNNFYSSQGIVDDSGSTSFMKLNTSAGNVDIYKKLYHSDACSSKAVVSLRCI ACGVNLNSSRQSRIVGGESALPGAWPWQVSLHVQNVHVCGGSIITPEWIVTAAHCVEKPLNNPWHWTA FAGILRQSFMFYGAGYQVEKVISHPNYDSKTKNNDIALMKLQKPLTFNDLVKPVCLPNPGMMLQPEQL CWISGWGATEEKGKTSEVLNAAKVLLIETQRCNSRYVYDNLITPAMICAGFLQGNVDSCQGDAGGPLV TSKNNIWWLIGDTSWGSGCAKAYRPGVYGNVMVFTDWIYRQMRADGGPFEDDDDKAGWSHPQFEKGGG SGGGSGGGSWSHPQFEK (SEQ ID NO: 29) REFERENCES 1. 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J. et al. Structure and activity of human TMPRSS2 protease implicated in SARS- CoV-2 activation. Nature Chemical Biology 18, 963-971 (2022). 23. Lukassen, S. et al. SARS-CoV-2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial transient secretory cells. The EMBO Journal 39, e105114 (2020). 24. Pyrc, K. et al. Culturing the Unculturable: Human Coronavirus HKU1 Infects, Replicates, and Produces Progeny Virions in Human Ciliated Airway Epithelial Cell Cultures. Journal of Virology 84, 11255-11263 (2010). 25. Planchais, C. et al. Potent human broadly SARS-CoV-2–neutralizing IgA and IgG antibodies effective against Omicron BA.1 and BA.2. Journal of Experimental Medicine 219 (2022). 26. Buchrieser, J. et al. Syncytia formation by SARS-CoV-2-infected cells. EMBO J 39, e106267 (2020). 27. Zang, R. et al. TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes. Sci Immunol 5 (2020). 28. Kishimoto, M. et al. 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Claims

CLAIMS 1. An antibody directed against the biologically active ectodomain of TMPRSS2. 2. The antibody according to claim 1, wherein the antibody binds to a polypeptide comprising the amino acid sequence SEQ ID NO: 29. 3. The antibody according to claim 1 or 2, wherein the antibody comprises a VHH. 4. The antibody according to any one of claims 1 to 3, wherein the binding of the antibody to TMPRSS2 reduces the catalytic activity of TMPRSS2. 5. The antibody according to any one of claims 1 to 4, wherein the antibody is directed against amino acids 275-473 of TMPRSS2. 6. The antibody according to any one of claims 1 to 5, wherein the antibody binds to an epitope that comprises one or more amino acid residues selected from the group consisting of V275, Q276, V278, H279, V280, C281, H296, C297, E299, K300, P301, L302, D338, S339, K340, T341, K342, E389, K390, T393, V415, Y416, D417, N418, L419, D435, S436, C437, Q438, G439, A441, T459, S460, W461, G462, S463, G464, C465, R470, G472 and V473 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24. 7. The antibody according to any one of claims 1 to 6, wherein the VHH comprises the following CDR domains: CDR1: SG(S/F)(P/T)L(E/D)(H/Y)YDI(I/Y/G) (SEQ ID NO: 1); CDR2: SSIT(T/A)SGGRTNYADSVKG (SEQ ID NO: 2); and CDR3: A(G/A)(K/R)(V/I)GGRRNW(I/V)APLNG(Y/F)ENA(Y/L) (SEQ ID NO: 3). 8. The antibody according to any one of claims 1 to 7, wherein the VHH comprises the following CDR domains: CDR1 selected from: SGSPLEHYDII (SEQ ID NO: 5), SGFTLDYYDIY (SEQ ID NO: 6), and SGSTLEHYDIG (SEQ ID NO: 7); CDR2 selected from: SSITTSGGHTNYADSVKG (SEQ ID NO : 10), SSITTSGGRTNYADSVKG (SEQ ID NO : 11), and SSITASGGRTNYADSVKG (SEQ ID NO : 12); and CDR3 selected from: AGRVGGRRNWIVPLDGYDNAY (SEQ ID NO : 15), AAKVGGRRNWIAPLNGYENAL (SEQ ID NO : 16), and AGKIGGRRNWVAPLDGFENAY (SEQ ID NO : 17). 9. The antibody according to any one of claims 1 to 8, wherein the VHH is A07 (SEQ ID NO: 20), C11 (SEQ ID NO: 21), or D01 (SEQ ID NO: 22). 10. The antibody according to any one of claims 1 to 9, wherein the antibody binds to an epitope that comprises one or more amino acid residues selected from the group consisting of R150, Y152, Q159, Y161, K166, S167, W168, H169, S204, G205, S206, T207, P367, P369, G370, M371, M372, L373, Q374, P375, I404, I405, E406, T407, Q408, N411, I420, T421, P422, M424, I425, I456, N476, M478, V479, T481 and D482 of TMPRSS2 when numbered in accordance with SEQ ID NO: 24. 11. The antibody according to any one of claims 1 to 10, wherein the VHH is A01 (SEQ ID NO: 19) or F05 (SEQ ID NO: 23). 12. A variant antibody of the antibody of any of claims 4 to 10, wherein the VHH retains the same binding as the unchanged antibody. 13. The variant antibody of claim 12, having 1, 2, 3, 4, or 5 changes. 14. The antibody of any of claims 1 to 13, wherein the antibody is a monomer. 15. The antibody of any of claims 1 to 14, wherein the antibody is a multimer. 16. The antibody of any of claims 1 to 15, wherein the antibody is a fusion protein. 17. The antibody of any of claims 1 to 16, wherein the antibody is a fusion protein with Fc. 18. The antibody of any of claims 1 to15, wherein the antibody is in a combination with another antibody. 19. A method of inhibiting binding to TMPRSS2 comprising contacting the antibody of any of claims 1 to 18 with a cell expressing TMPRSS2. 20. A method of treating a viral infection comprising administering the antibody of any of claims 1 to 18 to an infected patient. 21. A method of detecting TMPRSS2 comprising contacting the antibody of any of claims 1 to 18 with a biological sample containing TMPRSS2 and detecting the resultant immunological complexes. 22. A method of diagnosing prostate cancer comprising contacting the antibody of any of claims 1 to 18 with a biological sample from a patient and detecting the resultant immunological complexes. 23. Use of the antibody of any of claims 1 to 18 for diagnosing prostate cancer.
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